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ease the production of artemisinin was reported using Glomus mosseae and Bacillus subtilis [105]. Although clear evidence for the effect of this symbiosis on the enhancement of artemisinin production is still unknown, Arora et al. [104] suggested that it might be due to improved growth and nutrient status of the plant. Heterologous production Metabolic engineering of several platforms, such as Nicotiana benthamiana or chloroplasts, has been conducted. Although ADS and CYP71AV1 were introduced into N. benthamiana, the production of artemisinic acid 12-β-diglucoside, instead of artemisinic acid, was detected at 39.5 mg/kg fresh weight (FW) [106]. The production yield of artemisinic acid in tobacco chloroplasts was also very low (0.1 mg/g FW) [107]. The production of plant natural compounds in microorganisms is an alternative approach with several advantages. The metabolic pathways in microorganisms could be modified to produce various types of natural compounds, including isoprenoids, alkaloids, and phenylpropanoids. Microorganisms can grow rapidly, allowing shorter production time compared with the biosynthesis of desired natural compounds in plants. Scaling up production to industrial scale is also possible [108].

ld be modified to produce various types of natural compounds, including isoprenoids, alkaloids, and phenylpropanoids. Microorganisms can grow rapidly, allowing shorter production time compared with the biosynthesis of desired natural compounds in plants. Scaling up production to industrial scale is also possible [108]. The production of artemisinin precursors in microorganisms was first reported in 2003. Martin et al. [109] expressed entire genes encoding the MVA pathway from yeast Saccharomyces cerevisiae in Escherichia coli to increase the intracellular concentration of FPP. To prevent the rapid loss of highly volatile amorpha-4,11-diene during culturing, the culture media was overlaid with dodecane to trap amorpha-4,11-diene, referred to as a two-phase partitioning bioreactor. As a result, they recovered the volatilized amorpha-4,11-diene, improving production titers from 24 mg/L to approximately 500 mg/L in a fed-batch bioreactor [110]. The coexpression of MevT operon with extra copies of HMGR reduced the accumulation of toxic HMG-CoA and increased production of mevalonate by threefold [111]. The replacement of lac by lacUV5 promoter with a codon-optimized MevT and an additional copy of MK also led to the increase in artemisinin production [112]. Tsuruta et al. [113] succeeded in enhancing amorpha-4,11-diene production in E. coli up to 27.4 g/L by replacing yeast HMGS and HMGR with the equivalent enzymes from gram-positive bacteria Staphylococcus aureus.

codon-optimized MevT and an additional copy of MK also led to the increase in artemisinin production [112]. Tsuruta et al. [113] succeeded in enhancing amorpha-4,11-diene production in E. coli up to 27.4 g/L by replacing yeast HMGS and HMGR with the equivalent enzymes from gram-positive bacteria Staphylococcus aureus. Engineering of the MEP pathway and membrane efflux transporters to improve the production of amorpha-4,11-diene in E. coli has been reported as well [114–117]. However, there are many issues regarding the expression of membrane-bound cytochrome P450s in this bacterium posing a limitation on the production of the subsequent oxidized compounds. To overcome these problems, Chang et al. [118] engineered the N-terminal transmembrane domain of the codon-optimized CYP71AV1 and coexpressed it with CPR from A. annua. As a result, production of artemisinic acid (105 mg/L) in this E. coli strain was obtained. Two years later, the same group replaced CYP71AV1 by engineered P450 from gram-positive bacteria Bacillus megaterium (P450BM3) and could produce artemisinic-11S,12-epoxide at higher than 250 mg/L successfully [119]. From this finding, a novel semi-biosynthetic route for the production of artemisinin stemming from the cleavage of this epoxide followed by several oxidation steps was proposed.

Introduction As a worldwide disease, malaria has been one of the main cause of illness and death in humans for over a century, especially in sub-Saharan Africa and Southeast Asia. More than 200 million cases of malaria are reported every year; in 2015, there were 214 million cases and 438,000 related deaths [1]. This disease is caused by five species of Plasmodium parasites: P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi. Among these, P. falciparum is the major cause of malaria infection in Africa, and P. vivax is the most widely distributed malaria-causing parasite globally [1]. Several antimalarial drugs have been developed since the seventeenth century. However, malaria-causing parasites have developed resistance to these conventional drugs, leading to treatment failure.

r cause of malaria infection in Africa, and P. vivax is the most widely distributed malaria-causing parasite globally [1]. Several antimalarial drugs have been developed since the seventeenth century. However, malaria-causing parasites have developed resistance to these conventional drugs, leading to treatment failure. In response to the urgent need for new antimalarial drugs, Chinese scientists Professor Youyou Tu and her research group discovered artemisinin, the most effective antimalarial drug derived from Artemisia annua in 1971 [2]. Artemisinin is a sesquiterpene lactone with an endoperoxide bridge, which is necessary for antimalarial activity during multiple stages of parasite development [3–7]. Owing to its rapid action and high effectiveness against malaria, the combination of artemisinin derivatives and other antimalarial drugs, so-called artemisinin-based combination therapies (ACTs), has been recommended as the first-line treatments against malaria since 2006 [8]. ACTs have become the most powerful strategy to prevent malaria and related deaths. Professor Tu was then awarded the Nobel Prize in Physiology or Medicine in 2015 for the discovery of this effective antimalarial compound.

herapies (ACTs), has been recommended as the first-line treatments against malaria since 2006 [8]. ACTs have become the most powerful strategy to prevent malaria and related deaths. Professor Tu was then awarded the Nobel Prize in Physiology or Medicine in 2015 for the discovery of this effective antimalarial compound. The demand for ACTs increases dramatically each year; yet, the production yield of artemisinin from A. annua is very low and varies widely from 0.01 to 2 % dry weight [9]. Alternative approaches, including plant breeding technologies, synthetic biology, and total and semi-syntheses of artemisinin, have been investigated to enhance the production and reduce the cost of this compound. In addition, the recent emergence of artemisinin-resistant Plasmodium parasites has also become a new challenge to scientists in the elucidation of the mechanism of resistance and identification of the new strategies for malaria treatment. In this review, we summarize recent studies on the enhancement of artemisinin production and on artemisinin biosynthetic genes in other Artemisia species, conducted in our laboratory. In addition, the current understanding of the mode of action of artemisinin against malaria-causing parasites and, in turn, the mechanism of resistance of the parasites to this compound are also presented. Finally, the current situation of malaria infection and future directions, including ongoing studies on antimalarial drug development, are discussed.

ing of the mode of action of artemisinin against malaria-causing parasites and, in turn, the mechanism of resistance of the parasites to this compound are also presented. Finally, the current situation of malaria infection and future directions, including ongoing studies on antimalarial drug development, are discussed. Discovery of artemisinin Before the discovery of artemisinin, powder derived from cinchona tree bark had been used to treat malaria since the seventeenth century. The active compound from this plant, quinine, was first isolated in 1820 and was used as the only effective antimalarial compound until the 1920s. The quinine derivative chloroquine was developed as a new effective antimalarial drug once quinine-resistant Plasmodium strains appeared. During that time, the insecticide DDT was widely used to control the spread of infected mosquitoes as well. However, in the 1960s, increasing of chloroquine-resistant Plasmodium strains and DDT-resistant mosquitoes became a critical sign of the failure of malaria prevention and treatment [10].

smodium strains appeared. During that time, the insecticide DDT was widely used to control the spread of infected mosquitoes as well. However, in the 1960s, increasing of chloroquine-resistant Plasmodium strains and DDT-resistant mosquitoes became a critical sign of the failure of malaria prevention and treatment [10]. In response to the urgent need for new antimalarial drugs, the Chinese government launched a national project against malaria called Project 523 in 1967 [2]. The group, led by Professor Youyou Tu, investigated more than 2000 Chinese herbs used as traditional Chinese medicines to treat fever. Among these herbs, an extract from A. annua showed highly effective inhibition against growth of malaria-causing parasites. The active antimalarial components were then extracted from the leaves of mature plants in 1971 [2, 10–12]. After purification, the active antimalarial compound, named qinghaosu or artemisinin, was obtained as colorless needle-like crystals. Its stereochemistry and chemical and X-ray crystal structures were determined and reported several years later [2, 10, 11, 13]. Clinical trials involving either a non-toxic A. annua extract or pure artemisinin have been conducted since 1972 by several groups, and all patients in these trials quickly recovered from the disease [11, 12]. These results clearly indicated that artemisinin is an effective antimalarial compound with rapid action and low toxicity.

als involving either a non-toxic A. annua extract or pure artemisinin have been conducted since 1972 by several groups, and all patients in these trials quickly recovered from the disease [11, 12]. These results clearly indicated that artemisinin is an effective antimalarial compound with rapid action and low toxicity. Despite showing effective antimalarial activity, the low solubility of artemisinin in both oil and water becomes a therapeutic limitation of this compound. To address this problem, many scientists have developed semi-synthetic drugs and synthesized artemisinin derivatives with higher solubility. Some of these artemisinin derivatives, which have been used until the present, include dihydroartemisinin, artemether, and artesunate [14]. In addition, the combination of artemisinin or its derivatives with other conventional antimalarial drugs greatly increased the parasite clearance rate in patients and was first recommended as a new strategy for malaria treatment in 1984 [15]. This strategy, known as ACT, has been recommended by the World Health Organization (WHO) as a first-line treatment for malaria to prevent recurrence and development of resistance in malaria-causing parasites, whereas the monotherapy is considered as an inappropriate treatment [2, 8, 13, 14, 16].

treatment in 1984 [15]. This strategy, known as ACT, has been recommended by the World Health Organization (WHO) as a first-line treatment for malaria to prevent recurrence and development of resistance in malaria-causing parasites, whereas the monotherapy is considered as an inappropriate treatment [2, 8, 13, 14, 16]. Biosynthesis of artemisinin and expression pattern of artemisinin biosynthetic genes in A. annua A precursor of artemisinin, farnesyl pyrophosphate (FPP, C15), is synthesized from two C-5 isoprenoid units derived from the cytosolic mevalonate (MVA) pathway and one isoprenoid unit derived from the non-mevalonate (MEP or DXP) pathway [17, 18]. FPP is cyclized to amorpha-4,11-diene by amorpha-4,11-diene synthase (ADS) [19–21] via the generation of bisabolyl and 4-amorphenyl cation intermediates [22, 23] (Fig. 1). The following step is the oxidation of amorpha-4,11-diene to artemisinic alcohol by amorpha-4,11-diene 12-monooxygenase (CYP71AV1) [24]. This enzyme also catalyzes the oxidation of artemisinic alcohol to artemisinic aldehyde and artemisinic acid. In addition, alcohol dehydrogenase 1 (ADH1) and aldehyde dehydrogenase 1 (ALDH1) also show specific oxidation activity on artemisinic alcohol into artemisinic aldehyde and on artemisinic aldehyde into artemisinic acid, respectively [25, 26]. Artemisinic acid was thought to be the last precursor of artemisinin. However, it has been revealed that this compound is converted non-enzymatically into arteannuin B and related compounds, rather than artemisinin [27]. The next step of artemisinin biosynthesis is the reduction of artemisinic aldehyde into dihydroartemisinic aldehyde by artemisinic aldehyde Δ11(13) reductase (DBR2) [28]. Then, ALDH1 oxidizes dihydroartemisinic aldehyde into dihydroartemisinic acid, which is converted non-enzymatically into artemisinin [26, 29], as shown in Fig. 1. Rydén et al. [30] discovered dihydroartemisinic aldehyde reductase 1 (RED1), which reduces dihydroartemisinic aldehyde into dihydroartemisinic alcohol. Although the role of RED1 in artemisinin biosynthesis is still unclear, it has been suggested that the silencing of RED1 might increase the production of artemisinin in A. annua.Fig. 1 Summary of artemisinin biosynthesis, transgenic approaches to enhance artemisinin production, and artemisinin mode of action. The enzymes responsible for each reaction are indicated next to the arrows. Suppression of competing pathways and artemisinin activity and its targets are shown in bold.

sinin in A. annua.Fig. 1 Summary of artemisinin biosynthesis, transgenic approaches to enhance artemisinin production, and artemisinin mode of action. The enzymes responsible for each reaction are indicated next to the arrows. Suppression of competing pathways and artemisinin activity and its targets are shown in bold. Transgenic approaches regulating artemisinin production are shown in black boxes. Cyclization mechanism of FPP to generate amorpha-4,11-diene is highlighted in gray. Full names of intermediates and enzymes involved in the pathway are as follows: HMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme A, G3P glycerol-3-phosphate, DXP 1-deoxy-d-xylulose 5-phosphate, MEP 2C-methyl-d-erythritol 4-phosphate, CDP-ME 4-diphosphocytidyl-2C-methyl d-erythritol, CDP-MEP CDP-ME 2-phosphate, MEC-PP 2C-methyl-d-erythritol 2,4-cyclodiphosphate, HMB-PP (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, IPP isopentenyl pyrophosphate, DMAPP dimethylallyl pyrophosphate, atoB (ERG10) acetoacetyl-CoA thiolase, HMGS (ERG13) HMG-CoA synthase, HMGR HMG-CoA reductase, MK (EGR12) mevalonate kinase, PMK (ERG8) phosphomevalonate kinase, MVD1 (ERG19) mevalonate pyrophosphate decarboxylase, dxs DXP synthase, dxr DXP reductase, ispD CDP-ME synthase, ispE CDP-ME kinase, ispF MEC-PP synthase, ispG HMB-PP synthase, ispH HMB-PP reductase, IDI IPP isomerase, FPS (ispA) farnesyl pyrophosphate (FPP) synthase, SQS (ERG9) squalene synthase, ADS amorpha-4,11-diene synthase, CYP71AV1 amorpha-4,11-diene 12-monooxygenase, CPR cytochrome P450 reductase, ADH1 alcohol dehydrogenase 1, ALDH1 aldehyde dehydrogenase 1, DBR2 artemisinic aldehyde Δ11(13) reductase, RED1 dihydroartemisinic aldehyde reductase 1 (color figure online)

thase, SQS (ERG9) squalene synthase, ADS amorpha-4,11-diene synthase, CYP71AV1 amorpha-4,11-diene 12-monooxygenase, CPR cytochrome P450 reductase, ADH1 alcohol dehydrogenase 1, ALDH1 aldehyde dehydrogenase 1, DBR2 artemisinic aldehyde Δ11(13) reductase, RED1 dihydroartemisinic aldehyde reductase 1 (color figure online) Artemisinin is produced mainly in glandular secretory trichomes (GSTs) and its accumulation level declines as plants mature. Olofsson et al. [31] showed that GSTs of A. annua are found in all aerial tissues of plants, but not in roots or hairy roots. The density of GSTs is highest in flower buds and young leaves and decreases as leaves age.

d mainly in glandular secretory trichomes (GSTs) and its accumulation level declines as plants mature. Olofsson et al. [31] showed that GSTs of A. annua are found in all aerial tissues of plants, but not in roots or hairy roots. The density of GSTs is highest in flower buds and young leaves and decreases as leaves age. The expression pattern of genes involved in the artemisinin biosynthetic pathway has been investigated extensively for over a decade. The expression of genes in the upstream pathway shows no correlation with the density of GSTs or the accumulation levels of artemisinin intermediates [32]. In contrast, the expression of genes in the downstream pathway is consistent with the density of GSTs in each tissue. The expression of ADS is highest in GSTs, high in flower buds and young leaves, low in stems, negligible in old leaves and hairy roots, and not detected in roots [31, 33–37]. CYP71AV1, DBR2, and ALDH1 showed similar expression patterns: highest in GSTs and very low in stems and roots. In hairy roots, the expression levels of CYP71AV1 and DBR2 are relatively low, but the expression of ALDH1 is negligible [24, 26, 28, 31]. The expression levels of CYP71AV1 and DBR2 in leaves and flowers show similar patterns, as they are high in leaf primordia and flower buds but decrease as leaves and flowers develop [38–40]. The expression pattern of ALDH1 in leaves at different stages is similar to those of CYP71AV1 and DBR2 [31]. Although there is no report on the expression level of ALDH1 during different stages of flowering, this gene shows higher expression in flowers than in leaves [26, 31]. The expression of RED1 is relatively low in flower buds, young leaves, and stems. In contrast, the expression of this gene is much higher in old leaves and roots than in young leaves [30, 31]. Interestingly, the expression of RED1 is approximately 50-fold higher in hairy roots compared with old leaves. Nevertheless, the function of RED1 in hairy roots has not been established [31].

eaves, and stems. In contrast, the expression of this gene is much higher in old leaves and roots than in young leaves [30, 31]. Interestingly, the expression of RED1 is approximately 50-fold higher in hairy roots compared with old leaves. Nevertheless, the function of RED1 in hairy roots has not been established [31]. The expression levels of ADS and ALDH1, as well as their enzymatic activities in high-artemisinin-producing and low-artemisinin-producing A. annua cultivars, show no differences. Even though the expression levels of CYP71AV1 in these two cultivars are similar, CYP71AV1 in a high-artemisinin-producing cultivar shows lower enzyme activity, which is suitable for the change in metabolic flux to dihydro-analogues and artemisinin production [41]. In contrast, the activity of DBR2 in both cultivars shows no significant difference, but the gene encoding this enzyme shows considerably higher expression levels in high-artemisinin-producing cultivars than in low-artemisinin-producing cultivars [42].

etabolic flux to dihydro-analogues and artemisinin production [41]. In contrast, the activity of DBR2 in both cultivars shows no significant difference, but the gene encoding this enzyme shows considerably higher expression levels in high-artemisinin-producing cultivars than in low-artemisinin-producing cultivars [42]. Mode of action of artemisinin Before artemisinin can exert its action, the endoperoxide bridge has to be activated to generate the free radical species. Two activation pathways of artemisinin have been suggested, namely the mitochondrial and heme-mediated degradation pathways [43]. Mitochondria-activated artemisinin is involved in lipid peroxidation inducing cytotoxicity via the generation of reactive oxygen species (ROS) and depolarization of the parasite mitochondrial and plasma membranes [43–47]. In the heme-mediated pathway, two activation models (i.e., a reductive scission model and an open peroxide model) have been proposed, both of which lead to the generation of an active carbon-centered radical [48]. Even though the non-heme Fe2+ ion was suggested to bind and activate artemisinin [7], recent studies showed that heme plays a predominant role in artemisinin activation rather than the Fe2+ ion [5]. In Plasmodium spp., heme is produced via endogenous heme biosynthesis at the early ring stage and via hemoglobin digestion at the trophozoite stage. However, the level of heme biosynthesized endogenously in the parasites is much lower than its production via hemoglobin digestion, suggesting that hemoglobin-derived heme plays a major role in artemisinin activation [5, 49]. Recently, Xie et al. [50] reported that falcipains FP2a and FP3 (two main cysteine protease hemoglobinases) are also involved in the potential activation of artemisinin at an early ring stage.

duction via hemoglobin digestion, suggesting that hemoglobin-derived heme plays a major role in artemisinin activation [5, 49]. Recently, Xie et al. [50] reported that falcipains FP2a and FP3 (two main cysteine protease hemoglobinases) are also involved in the potential activation of artemisinin at an early ring stage. After hemoglobin digestion, the heme detoxification protein (HDP) can trigger the conversion of free heme to hemozoin, which is essential for parasite survival [4, 51]. However, the formation of the artemisinin-free heme complex shows an inhibitory effect on this conversion [51]. A translationally controlled tumor protein (PfTCTP) was also reported as a potential target of artemisinin, as it could form a covalent bond with this protein, resulting in protein malfunction [52, 53]. Eckstein-Ludwig et al. [54] showed that artemisinin specifically mediated the inhibition of PfATP6, an orthologous sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), outside the food vacuole. Recently, five enzymes involved in the key metabolic pathways of the parasite were also reported as potential targets of artemisinin, namely ornithine aminotransferase (OAT), pyruvate kinase (PyrK), l-lactate dehydrogenase (LDH), spermidine synthase (SpdSyn), and S-adenosylmethionine synthetase (SAMS). All of them are covalently modified by the interaction with artemisinin, resulting in the irreversible malfunction of enzyme activities [5].

emisinin, namely ornithine aminotransferase (OAT), pyruvate kinase (PyrK), l-lactate dehydrogenase (LDH), spermidine synthase (SpdSyn), and S-adenosylmethionine synthetase (SAMS). All of them are covalently modified by the interaction with artemisinin, resulting in the irreversible malfunction of enzyme activities [5]. Enhancement of artemisinin production The demand for artemisinin increases every year. Even though total synthesis of artemisinin from commercially available chemicals or semi-synthesis from its intermediates have been reported, all of those methods are costly and require several synthesis steps [55, 56]. In this review, we summarize recent studies regarding four approaches to enhance the production of artemisinin: (1) plant breeding technologies, (2) overexpression of genes involved in the artemisinin biosynthetic pathway, (3) direct or indirect upregulation of artemisinin biosynthesis, and (4) heterologous production.

In this review, we summarize recent studies regarding four approaches to enhance the production of artemisinin: (1) plant breeding technologies, (2) overexpression of genes involved in the artemisinin biosynthetic pathway, (3) direct or indirect upregulation of artemisinin biosynthesis, and (4) heterologous production. Plant breeding technologies Conventional plant breeding techniques to select high-artemisinin-producing cultivars have been used for decades. These techniques include cultivation of A. annua and collection of cultivars with the desired properties. At present, a robust hybrid A. annua is generated from the combination of high-artemisinin-producing and vigorous cultivars to increase the production yield of artemisinin to more than 2 % dry weight [57–59]. Recently, an alternative approach to increase the production of artemisinin from the cultivation of high-artemisinic acid or dihydroartemisinic acid-producing cultivars was proposed, since a method for the semi-synthesis of artemisinin from these two precursors has been developed [56, 60].

than 2 % dry weight [57–59]. Recently, an alternative approach to increase the production of artemisinin from the cultivation of high-artemisinic acid or dihydroartemisinic acid-producing cultivars was proposed, since a method for the semi-synthesis of artemisinin from these two precursors has been developed [56, 60]. Scientists at the University of York used advanced breeding techniques to evaluate the distribution of traits that contribute to artemisinin yield [61]. From the screening of 23,000 strains, they succeeded in identifying genes and molecular markers for fast-track breeding, enabling the construction of a detailed genetic map of A. annua with nine linkage groups. The established quantitative trait loci (QTL) map is also applicable for rapid identification of A. annua parental lines with useful traits for plant breeding. Two hybrids, called Hybrid 1209r Shennong and Hybrid 8001r Zenith, were developed with high artemisinin productivity of up to 36.3 and 54.5 kg/ha, respectively. The diallel cross approach to determine the combining ability of the robust parental lines for the production of artemisinin high-yielding A. annua hybrids was also developed by the same group and showed consistent results with the QTL-based molecular breeding approach [62].

of up to 36.3 and 54.5 kg/ha, respectively. The diallel cross approach to determine the combining ability of the robust parental lines for the production of artemisinin high-yielding A. annua hybrids was also developed by the same group and showed consistent results with the QTL-based molecular breeding approach [62]. Hairy root culture is another method to enhance the production of secondary (specialized) metabolites, owing to its rapid growth capabilities [63]. Transformation protocols to obtain hairy roots containing artemisinin from this plant have been reported [64, 65]. In our laboratory, we also attempted to establish the conditions for A. annua hairy root cultivation. However, we still could not detect even trace amounts of artemisinin or its intermediates from the extract of hairy root cultures by GC–MS (unpublished data). Artemisinin biosynthetic genes are highly expressed in trichomes but almost negligible in root tissue [31, 33–40], suggesting that the production of this compound by hairy root cultures could be somewhat difficult. Therefore, the most suitable conditions for hairy root cultures to enhance production of artemisinin must be investigated. In addition, the identification of artemisinin production from root extracts requires extreme care, and NMR spectroscopic and mass spectrometric analyses are required.

could be somewhat difficult. Therefore, the most suitable conditions for hairy root cultures to enhance production of artemisinin must be investigated. In addition, the identification of artemisinin production from root extracts requires extreme care, and NMR spectroscopic and mass spectrometric analyses are required. Overexpression of genes involved in artemisinin biosynthetic pathway Metabolic engineering of A. annua by overexpressing genes involved in artemisinin biosynthesis has been given more attention during the last 20 years. To obtain successful transformants, several parameters for Agrobacterium tumefaciens-mediated transformation, such as the concentration of antibiotics, method and duration of co-cultivation, and phytohormones supplied for plant regeneration, have been optimized [66–71]. Among various explants available for transformation, stem internodes and young inflorescence seem to be the most appropriate [70–72]. Phytohormones α-naphthaleneacetic acid and 6-benzylaminopurine are crucial for GST development in young leaves, and root generation also affects GST size [73]. Recently, Kiani et al. [72] developed miniprep methods using A. tumefaciens- and Agrobacterium rhizogenes-mediated transformation. This method exhibits higher transformation rates with faster development of transformants within 3–4 weeks compared with other methods.

aves, and root generation also affects GST size [73]. Recently, Kiani et al. [72] developed miniprep methods using A. tumefaciens- and Agrobacterium rhizogenes-mediated transformation. This method exhibits higher transformation rates with faster development of transformants within 3–4 weeks compared with other methods. The overexpression of several genes involved in artemisinin biosynthesis in A. annua has been evaluated. Overexpression of farnesyl pyrophosphate synthase (FPS) increased artemisinin production up to 2- to 3.6-fold higher than that in the control [74, 75]. Overexpressing CYP71AV1 and its redox partner cytochrome P450 reductase (CPR) in artemisinin biosynthesis could increase artemisinin content in planta by 38 % [76]. Xiang et al. [77] generated dxr- and CYP71AV1/CPR-overexpressing A. annua and found that both transformants increased the production of artemisinin. The overexpression of DBR2 increased the production of artemisinin as well as its precursor dihydroartemisinic acid, up to twofold, compared with non-transgenic plants. It also increased production of artemisinic acid up to 5.48- to 9.06-fold and arteannuin B up to twofold [78]. The reason why overexpression of DBR2 enhanced biosynthesis of artemisinic acid and arteannuin B has not been revealed. However, Yuan et al. [78] hypothesized that excess dihydroartemisinic acid might be converted into artemisinic acid in planta.

isinic acid up to 5.48- to 9.06-fold and arteannuin B up to twofold [78]. The reason why overexpression of DBR2 enhanced biosynthesis of artemisinic acid and arteannuin B has not been revealed. However, Yuan et al. [78] hypothesized that excess dihydroartemisinic acid might be converted into artemisinic acid in planta. Overexpression of multiple genes involved in artemisinin biosynthesis could greatly increase the production of artemisinin in planta. Chen et al. [79] showed that the co-overexpression of FPS, CYP71AV1, and CPR increased artemisinin levels in A. annua up to 3.6-fold. The co-overexpression of HMGR and FPS increased production of artemisinin up to 1.8-fold higher than that in the control [80]. Alam et al. [81, 82] co-overexpressed HMGR and ADS in A. annua and found greatly increased artemisinin levels, up to 7.65-fold, in this transgenic line. Suppressing the expression of genes involved in the pathways competing with artemisinin biosynthesis is another approach to enhance artemisinin content in planta. Zhang et al. [83] used RNAi techniques to suppress the expression of SQS, the first committed gene in sterol biosynthesis. The suppression of this gene enhanced the production of artemisinin up to 3.14-fold.

e pathways competing with artemisinin biosynthesis is another approach to enhance artemisinin content in planta. Zhang et al. [83] used RNAi techniques to suppress the expression of SQS, the first committed gene in sterol biosynthesis. The suppression of this gene enhanced the production of artemisinin up to 3.14-fold. Direct or indirect upregulation of artemisinin biosynthesis The effect of several stresses on production of artemisinin in A. annua has been analyzed since the 1990s. These stresses usually lead to the generation of ROS (required for the last non-enzymatic step in artemisinin biosynthesis) or upregulate the expression of artemisinin biosynthetic genes [84–87]. Details of the stresses placed on artemisinin production have been summarized previously [88, 89], and the appropriate cultivation conditions of A. annua were suggested [9].

ROS (required for the last non-enzymatic step in artemisinin biosynthesis) or upregulate the expression of artemisinin biosynthetic genes [84–87]. Details of the stresses placed on artemisinin production have been summarized previously [88, 89], and the appropriate cultivation conditions of A. annua were suggested [9]. Some transcription factors upregulated the expression of artemisinin biosynthetic genes and promoted production of artemisinin in A. annua. The WRKY1 transcription factor is thought to bind to the W-box cis-acting elements of promoters to promote gene expression. It is also involved in the regulation of plant defense responses and developmental and physiological processes. Ma et al. [33] showed that the transcript levels of HMGR, ADS, CYP71AV1, and DBR2 were induced in transient AaWRKY1-overexpressing leaves. Furthermore, the specific overexpression of this transcription factor in GSTs increased transcript levels of CYP71AV1 up to 33-fold, compared with the wild type [90]. AaORA, one of the APETALA2/ethylene response factor (AP2/ERF) transcription factor involved in plant responses to biotic and abiotic stresses, showed a similar expression pattern to those of ADS, CYP71AV1, and DBR2. The overexpression of this transcription factor led to the upregulation of the expression levels of ADS, CYP71AV1, and DBR2in planta and promoted artemisinin production [91]. Yu et al. [92] also reported the enhancement of artemisinin production via overexpression of two transcription factors from the same family, AaERF1 and AaERF2, which bind to the promoter regions of ADS and CYP71AV1. Another transcription factor that positively regulates the biosynthesis of artemisinin is a basic helix-loop-helix (bHLH) transcription factor, involved in metabolic regulation of various hormones, developmental processes, and regulation of light signaling, iron and phosphate homeostasis, and various abiotic stresses [93]. Recently, Zhang et al. [94] reported that a basic leucine zipper transcription factor (AabZIP1) binds to the ABA-responsive elements (ABRE) of ADS and CYP71AV1 promoters and upregulates the expression of ADS, CYP71AV1, DBR2, and ALDH1.

light signaling, iron and phosphate homeostasis, and various abiotic stresses [93]. Recently, Zhang et al. [94] reported that a basic leucine zipper transcription factor (AabZIP1) binds to the ABA-responsive elements (ABRE) of ADS and CYP71AV1 promoters and upregulates the expression of ADS, CYP71AV1, DBR2, and ALDH1. Several phytohormones upregulating artemisinin biosynthesis have been reported. Treatment with salicylic acid upregulates the expression of HMGR and ADS, as well as induces ROS generation, driving the conversion of dihydroartemisinic acid into artemisinin [95]. Methyl jasmonate (MeJA) promotes the formation of GSTs and enhances the expression of several genes involved in the artemisinin biosynthetic pathway and related transcription factors (ORA and ERF1), leading to the enhancement of artemisinin production [96–98]. This phytohormone also regulates trichome-specific fatty acyl-CoA reductase 1 (TFAR1), ABCG transporter unigenes (AaABCG6 and AaABCG7), and allene oxide cyclase (AaAOC) [96, 99, 100]. TFAR1 is involved in the formation of cuticular wax during GST expansion in A. annua. AaABCG6 and AaABCG7 are ATP-binding cassette transporter G, involved in the development of trichome cuticle and may share a common regulatory system with ADS and CYP71AV1. AaAOC is involved in JA biosynthesis. The expression of this gene may be upregulated by treatment with not only MeJA but also ABA and ethylene [100]. The overexpression of the ABA receptor, AaPYL9, also improves the sensitivity of ABA and promotes artemisinin biosynthesis after ABA treatment [99, 101].

DS and CYP71AV1. AaAOC is involved in JA biosynthesis. The expression of this gene may be upregulated by treatment with not only MeJA but also ABA and ethylene [100]. The overexpression of the ABA receptor, AaPYL9, also improves the sensitivity of ABA and promotes artemisinin biosynthesis after ABA treatment [99, 101]. The enhancement of artemisinin production can be achieved by increased GST density. Singh et al. [102] reported that the expression of bgl1, encoding β-glucosidase from Trichoderma reesei, in A. annua improved the density of GSTs in flowers up to 66 % and increased the production of artemisinin up to five-fold compared with the control. The expression of rolB and rolC of A. rhizogenes also increases GST density and upregulates the expression of ADS, CYP71AV1, ALDH1, and TFAR1. Artemisinin content is then increased 2- to 9-fold and 4-fold in rolB- and rolC-expressing plants, respectively [103]. Co-cultivation of an endophytic fungus Piriformospora indica and a nitrogen-fixing bacterium Azotobacter chroococcum with A. annua increases artemisinin content up to 70 % [104]. This dual symbiosis also shows a positive effect on plant height, dry weight, and leaf yield. Another example of using symbiosis to increase the production of artemisinin was reported using Glomus mosseae and Bacillus subtilis [105]. Although clear evidence for the effect of this symbiosis on the enhancement of artemisinin production is still unknown, Arora et al. [104] suggested that it might be due to improved growth and nutrient status of the plant.

gram-positive bacteria Bacillus megaterium (P450BM3) and could produce artemisinic-11S,12-epoxide at higher than 250 mg/L successfully [119]. From this finding, a novel semi-biosynthetic route for the production of artemisinin stemming from the cleavage of this epoxide followed by several oxidation steps was proposed. Yeast is another attractive host for the production of artemisinin precursors as it produces FPP for sterol biosynthesis via the MVA pathway. Since the MVA pathway in S. cerevisiae has been characterized, ADS was introduced into this yeast, and an amorpha-4,11-diene-producing yeast strain was generated successfully [120]. While there are many issues concerning the expression of cytochrome P450s in E. coli, the expression of this gene in yeast is much more feasible. Therefore, CYP71AV1 and CPR were coexpressed, and all genes involved in the MVA pathway were upregulated either directly or indirectly. The competing pathway (sterol biosynthetic pathway) was also downregulated using a methionine-repressible promoter to improve the production of artemisinic acid in the yeast expression system. As a result, this transgenic yeast strain produced artemisinic acid at up to 100 mg/L [121, 122]. Several factors were further optimized for the production of artemisinic acid in an industrial fermenter. For example, the carbon source for growing yeast in a fermenter was switched from glucose to galactose, and the oxygen transfer rate was controlled. With this development, called the galactose fed-batch process controlled by the DO-stat algorithm, the artemisinic acid titer increased to 2.5 g/L [123].

strial fermenter. For example, the carbon source for growing yeast in a fermenter was switched from glucose to galactose, and the oxygen transfer rate was controlled. With this development, called the galactose fed-batch process controlled by the DO-stat algorithm, the artemisinic acid titer increased to 2.5 g/L [123]. Despite conferring a higher production yield of artemisinic acid, the use of galactose is costly and not applicable, especially in developing countries. Thus, lower-cost chemicals are needed as carbon sources. Yeast with GAL1, GAL7, GAL10, and GAL80 deletions was generated to exclude the use of galactose, and ethanol was alternatively used as a carbon source. Two additional copies of truncated HMGR (tHMG1) were integrated into this yeast strain. As a result, the production of amorpha-4,11-diene was increased up to more than 40 g/L [124]. Further development was performed by the introduction of artemisinin biosynthetic genes, CYP71AV1, CPR, ADH1, and ALDH1, to oxidize amorpha-4,11-diene into artemisinic acid. Cytochrome  b5 (CYB5) was also introduced into this strain as it can accelerate cytochrome P450 reactions [125]. High-level production of artemisinic acid, at 25 g/L, was thereby achieved. The semi-synthesis of artemisinin from artemisinic acid was also optimized, and the overall yield after purification increased to 40–45 % [126, 127]. A potent coupled chromatography–crystallization method to purify artemisinin was then developed, and the recovery yield of this antimalarial compound from the reaction mixture increased to 61.5 %, with 99 % purity [128]. All of the transgenes and modifications to several heterologous hosts mentioned here are summarized in Table 1.Table 1 Heterologous production of artemisinin intermediates

inin was then developed, and the recovery yield of this antimalarial compound from the reaction mixture increased to 61.5 %, with 99 % purity [128]. All of the transgenes and modifications to several heterologous hosts mentioned here are summarized in Table 1.Table 1 Heterologous production of artemisinin intermediates Host No. Transgenes or modifications Product Yield References N. benthamiana 1 P 35S-tHMGR-FPS-ADS, P 35S-CYP71AV1 Artemisinic acid 12-β-diglucoside 39.5 mg/kg FW [106] Tobacco chloroplasts 2 P rrn16S-atoB-HMGS-HMGR-MK-PMK-MVD1, P psbA-E. coli IDI-FPS-ADS-CYP71AV1-AaCPR Artemisinic acid 0.1 mg/g FW [107] E. coli 3 P lac-MevT a, P lac-MBIS b, P trc-ADS Amorpha-4,11-diene 24 mg/L [109] 4 Same as 3 but overlaid with dodecane Amorpha-4,11-diene 500 mg/L [110] 5 P BAD-MevT, P BAD-tHMGR1 Mevalonate Threefold from CTc [111] 6 P lacUV5-MevT (codon opt.)-MBIS, P trc-ERG12 (codon opt.)-ADS Amorpha-4,11-diene 293 mg/L [112] 7 P lacUV5-MevT (codon opt.) with HMGS and HMGR from S. aureus, P lac-MBIS, P lac-ADS Amorpha-4,11-diene 27.4 g/L [113] 8 P BAD-dxs-IDI-ispDF, ADS with Δpts and optimized medium Amorpha-4,11-diene 182 mg/L [114] 9 P TM2-galP-glk, P T7-dxs-IDI-ispA-ADS Amorpha-4,11-diene 201.2 mg/L [115] 10 AcrB, TolC (x2), ADS (codon opt.) Amorpha-4,11-diene 404.83 mg/L [116] 11 P BAD-dxs-IDI-ispDF, P araBAD-ADS, P TM1-macAB-TolC Amorpha-4,11-diene ~30 mg/L/OD [117] 12 Same as 3 with CYP71AV1 (codon opt., engineered N-terminal transmembrane)-AaCPR Artemisinic acid 105 mg/L [118] 13 Same as 12 but replaced CYP71AV1 with P450 BM3 Artemisinic-11S,12-epoxide 250 mg/L [119]

ene 404.83 mg/L [116] 11 P BAD-dxs-IDI-ispDF, P araBAD-ADS, P TM1-macAB-TolC Amorpha-4,11-diene ~30 mg/L/OD [117] 12 Same as 3 with CYP71AV1 (codon opt., engineered N-terminal transmembrane)-AaCPR Artemisinic acid 105 mg/L [118] 13 Same as 12 but replaced CYP71AV1 with P450 BM3 Artemisinic-11S,12-epoxide 250 mg/L [119] S. cerevisiae 14 P GAL1-ADS Amorpha-4,11-diene 600 μg/L [120] 15 P GAL1-tHMGR P GAL1-upc2-1 erg9::P MET3-ERG9 P GAL1-tHMGR P GAL1-ERG20, P GAL1-ADS P GAL10-CYP71AV1 P GAL1-AaCPR Artemisinic acid 100 mg/L [121, 122] 16 Same as 15 with optimized culture condition Artemisinic acid 2.5 g/L [123] 17 gal80Δ::nat r MAT a erg9Δ∷kan r P MET3-ERG9, leu2-3,112∷HIS P GAL1-MVD1 P GAL10-ERG8 his3Δ1∷HIS P GAL1-ERG12 P GAL10-ERG10ade1Δ∷P GAL1-tHMG1 P GAL10-IDI1 ADE1 ura3-52∷P GAL1-tHMG1 P GAL10-ERG13 URA3trp1-289∷P GAL1-tHMG1 P GAL10-ERG20 TRP1 [pAM322] Amorpha-4,11-diene 41 g/L [124] 18 Same as 17. but replaced gal80Δ::nat r with gal1Δ gal7Δ gal10Δ::hphA Amorpha-4,11-diene 37 g/L [124] 19 gal1Δ,gal10Δ,gal7Δ::P GAL3-CPR1natA, erg9Δ::dsdAP CTR3- ERG9, leu2-3,112::kanAP GAL7-AaCYB5P GAL1-ERG19P GAL10- ERG8, ade1Δ::P GAL1-tHMG1P GAL10-IDI1_ADE1, his3Δ1::hphAP GAL7-AaALDH1P GAL1-ERG12P GAL10-ERG10, ura3-52:: P GAL1-tHMG1P GAL10-ERG13hisG, trp1-289:: P GAL1- tHMG1P GAL10-ERG20TRP1, ndt80Δ::P TDH1-HEM1HIS3PPGK1-CTT1, gal80Δ::URA3P GAL7-AaADH1, [pAM552: 2μ-LEU2d P GAL1-ADS P GAL10-CYP71AV1] Artemisinic acid 25 g/L [126, 127] a MevT operon consists of atoB-HMGS-tHMGR b MBIS operon consists ofERG12-ERG8-MVD1-IDI-ispA cProduction yield as compared to control (CT)

S. cerevisiae 14 P GAL1-ADS Amorpha-4,11-diene 600 μg/L [120] 15 P GAL1-tHMGR P GAL1-upc2-1 erg9::P MET3-ERG9 P GAL1-tHMGR P GAL1-ERG20, P GAL1-ADS P GAL10-CYP71AV1 P GAL1-AaCPR Artemisinic acid 100 mg/L [121, 122] 16 Same as 15 with optimized culture condition Artemisinic acid 2.5 g/L [123] 17 gal80Δ::nat r MAT a erg9Δ∷kan r P MET3-ERG9, leu2-3,112∷HIS P GAL1-MVD1 P GAL10-ERG8 his3Δ1∷HIS P GAL1-ERG12 P GAL10-ERG10ade1Δ∷P GAL1-tHMG1 P GAL10-IDI1 ADE1 ura3-52∷P GAL1-tHMG1 P GAL10-ERG13 URA3trp1-289∷P GAL1-tHMG1 P GAL10-ERG20 TRP1 [pAM322] Amorpha-4,11-diene 41 g/L [124] 18 Same as 17. but replaced gal80Δ::nat r with gal1Δ gal7Δ gal10Δ::hphA Amorpha-4,11-diene 37 g/L [124] 19 gal1Δ,gal10Δ,gal7Δ::P GAL3-CPR1natA, erg9Δ::dsdAP CTR3- ERG9, leu2-3,112::kanAP GAL7-AaCYB5P GAL1-ERG19P GAL10- ERG8, ade1Δ::P GAL1-tHMG1P GAL10-IDI1_ADE1, his3Δ1::hphAP GAL7-AaALDH1P GAL1-ERG12P GAL10-ERG10, ura3-52:: P GAL1-tHMG1P GAL10-ERG13hisG, trp1-289:: P GAL1- tHMG1P GAL10-ERG20TRP1, ndt80Δ::P TDH1-HEM1HIS3PPGK1-CTT1, gal80Δ::URA3P GAL7-AaADH1, [pAM552: 2μ-LEU2d P GAL1-ADS P GAL10-CYP71AV1] Artemisinic acid 25 g/L [126, 127] a MevT operon consists of atoB-HMGS-tHMGR b MBIS operon consists ofERG12-ERG8-MVD1-IDI-ispA cProduction yield as compared to control (CT) Artemisinin biosynthetic genes in non-artemisinin-producing Artemisia species Some studies reported that artemisinin is produced in other Artemisia species [129–134]. However, we attempted to isolate artemisinin from other Artemisia species but failed to detect any trace amounts of artemisinin or its intermediates (unpublished data). Thus, we analyzed the expression of genes highly homologous to artemisinin biosynthetic genes in these species. Firstly, we selected A. afra and A. absinthium as they are widely cultivated in Africa and exhibit anti-plasmodial activity [135–138]. Putative ADS orthologs were not expressed in either A. afra or A. absinthium [139]. However, we detected the expression of putative CYP71AV1 orthologs in both species. Functional analysis revealed that these orthologous enzymes show similar catalytic activities to their correspondent in A. annua on the oxidation of amorpha-4,11-diene into artemisinic acid [139]. We also detected the expression of DBR2 ortholog in A. absinthium, and the encoded enzyme showed comparable activity to that of A. annua DBR2 [140]. In addition, we showed that this plant can convert the fed artemisinin intermediates into the following products along the biosynthetic pathway of artemisinin [140]. Our findings suggest that ADS might be a limiting factor for the production of artemisinin in planta, and A. absinthium could be an alternative host for artemisinin production. The introduction of ADS into A. absinthium might lead to the generation of artemisinin-producing A. absinthium, which could be used as an alternative approach to produce artemisinin in other Artemisia species. To prove this hypothesis, this research is now ongoing in our laboratory.

ive host for artemisinin production. The introduction of ADS into A. absinthium might lead to the generation of artemisinin-producing A. absinthium, which could be used as an alternative approach to produce artemisinin in other Artemisia species. To prove this hypothesis, this research is now ongoing in our laboratory. Next challenge: artemisinin-resistant Plasmodium parasites Artemisinin is the most effective antimalarial drug and has been used as an ACT to treat malaria for over a decade. However, the emergence of artemisinin-resistant Plasmodium parasites in Southeast Asia, prolonging the parasite clearance rate in patients, has been reported recently and has become a critical issue [141–144]. No correlation between resistance and other previously proposed candidate targets of artemisinin (PfATP6 and PfTCTP) was detected [145]. However, it has been suggested that the resistance occurs predominantly during the early ring stage of parasite development as a result of the multiple forms of mutations in the PF3D7_1343700 kelch propeller domain (K13-propeller) on chromosome 13 [146–155]. K13-propeller mutations lead to the increase of phosphatidylinositol-3-kinase (PfPI3K), which is required for the mediation of cell signaling and survival [156, 157], and prolong parasite development at the ring stage when the activation level of artemisinin is rather low [5, 7, 158]. The B subfamily of ABC transporters, known as multidrug resistance proteins (MDR), also promotes artemisinin resistance. In artemether–lumefantrine post-treatment infections, alleles of Pfmdr1 tended to have 86N, 184F, and 1246D, rather than the common YYY haplotype, and increased the number of treatment failures [159]. The deletion of Pfmdr5 induced greater sensitivity to artemisinin treatment, suggesting that this gene might contribute to artemisinin resistance as well [160].

infections, alleles of Pfmdr1 tended to have 86N, 184F, and 1246D, rather than the common YYY haplotype, and increased the number of treatment failures [159]. The deletion of Pfmdr5 induced greater sensitivity to artemisinin treatment, suggesting that this gene might contribute to artemisinin resistance as well [160]. Current situation of malaria infection and ongoing studies on antimalarial drug development Since ACTs have become the major treatment for malaria and strict preventive measures against parasite-infected mosquitoes have been implemented, the malaria-related mortality rate and case incidence have decreased gradually during the past 10 years [1]. Although artemisinin-resistant Plasmodium parasites have emerged and show a significant delay in clearance rate, the response of dihydroartemisinin against either wild-type parasites or mutants exhibits similar Km values suggesting that dihydroartemisinin does not lose its activity against the mutants [161]. Extending the treatment courses could be an effective strategy to clear resistant parasite infection. However, the parasites can still develop complete resistance against artemisinin-based treatment at any point in the future. In addition, the proportion of malaria-infected patients is concentrated in countries with low national income levels. Among these, more than 68 million infected children do not receive any ACTs [1]. Therefore, large amounts of low-cost artemisinin for ACTs, by either increasing the cultivation of high-artemisinin-producing A. annua plants or developing cheaper synthetic biological processes in the long term, are required to prevent any further development of parasites and meet the demand of ACTs worldwide. Moreover, novel effective antimalarial treatments must be developed continually. Recently, low-cost plant-based artemisinin combination therapy (pACT) has driven attention on the production of no semi-synthetic artemisinin in planta as this treatment showed higher antimalarial activity, and the synergistic effect of artemisinin and the plant matrix overcame resistance to artemisinin [162–169]. Several scientists have also focused on the investigation of novel potential drug targets [170–180] and on the synthesis of novel antimalarial compounds including artemisinin hybrids [181–186]. Still, further studies on these avenues are required.

sinin and the plant matrix overcame resistance to artemisinin [162–169]. Several scientists have also focused on the investigation of novel potential drug targets [170–180] and on the synthesis of novel antimalarial compounds including artemisinin hybrids [181–186]. Still, further studies on these avenues are required. Conclusion Several approaches to enhance the production of artemisinin have been investigated for over a decade. As a result, the availability of artemisinin for ACTs is increasing, and the number of malaria-related deaths is decreasing gradually. Although artemisinin is still effective against malaria-causing parasites, the emergence of artemisinin-resistant strains has posed a new challenge to scientists worldwide. Therefore, elucidating the mode of action of artemisinin and the mechanism of resistance against this compound in Plasmodium parasites is important for further development of antimalarial drugs. We hope that the current understanding of artemisinin as summarized in this review will provide clues for further investigation and development of antimalarial treatments to overcome artemisinin resistance in Plasmodium parasites in the future. This work was supported by a Grant-in-Aid for Scientific Research (23780104) and a research grant from the Shorai Foundation for Science and Technology to H.S.; Frontier Research Base for Global Young Researcher, Osaka University from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) to E.O.F.; and the Monbukagakusho Scholarship to P.M.

ific Research (23780104) and a research grant from the Shorai Foundation for Science and Technology to H.S.; Frontier Research Base for Global Young Researcher, Osaka University from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) to E.O.F.; and the Monbukagakusho Scholarship to P.M. Conflict of interest The authors declare that they have no conflict of interest.

Introduction Naturally occurring chemicals represent a treasure trove of compounds which hold promise as the seeds of discovery for drugs and medicines and which may facilitate the elucidation of structure and function investigations of bioactivity [1]. Ōmura’s research group at the Kitasato Institute is a global pioneer in the search for bioactive agents that may be of use in developing drugs and medicines to fight to infection and combat tropical diseases (such as the filariases, malaria, trypanosomiasis, etc.), all originating from microbial metabolites. At present, 483 new compounds have been discovered, 26 of which have become useful, widely used agents in human and animal health, including the ground-breaking avermectins [2].

which the mixture was incubated for 30 min at room temperature. Luminescence was measured with an EnSpire multi-plate reader (Perkin Elmer, Foster City, CA, USA). The experiments were repeated three times. Each IC50 was calculated using a four-parameter logistic model (Prism 5.0, GraphPad Software, San Diego, CA, USA). Formalin test ICR male mice (5 weeks of age, 8 mice per group) were orally administered water, 350 mg/kg EFE, or 700 mg/kg Ephedra Herb extract for 3 days. On the third day, paw-licking was induced in the mice by intraplantar injection of 20 μl of 2.5 % formalin 6 h after extract/water administration. After the injection, the mice were individually placed into a glass cage, in which the amount of time that the animal spent licking the injected paw was measured as an indicator of pain. Paw-licking was recorded for 30 min in two phases, the first phase (0–5 min) and second phase (15–30 min). The protocol for animal experiments was approved by the Ethics Review Committee for Animal Experimentation of the National Institute of Health Sciences.

o infection and combat tropical diseases (such as the filariases, malaria, trypanosomiasis, etc.), all originating from microbial metabolites. At present, 483 new compounds have been discovered, 26 of which have become useful, widely used agents in human and animal health, including the ground-breaking avermectins [2]. Malaria is one of the world’s worst health and socioeconomic problems, causing widespread death, disease, disability, and economic loss. Infection arises when a protozoal parasite of the Plasmodium genus is transmitted to humans via the bites of blood-feeding mosquitoes. Plasmodium falciparum parasites cause the most deadly form of the disease, which can cause death in a few days, especially if cerebral malaria develops. Generally, most deaths occur in children under 5 years old, although deaths have been reduced markedly by recent global initiatives to tackle the disease [3–5]. Commonly used drugs to combat malaria include quinine, chloroquine, mefloquine, halofantrine, and sulfadoxine/pyrimethamine (Fig. 1). However, drug resistance in parasites has usually developed quickly, rendering many of these drugs useless, preventing effective treatment and hindering disease elimination efforts. In 1972, Professor Tu Youyou discovered artemisinin to be the active ingredient in the plant Artemisia annua, which was commonly used in China to treat fever. Artemisinin derivatives became the most effective therapeutic drugs against malaria [6]. The World Health Organization (WHO) recommends artemisinin-based combination therapies (ACTs) for malaria treatment [7], a multidrug approach requiring the use of artemisinin together with other drugs to help offset the pace of drug resistance to artemisinin developing and spreading. ACTs are already compromised because the safety of artemisinin with regard to use during first trimester pregnancy is yet to be established and, worse, resistance to artemisinin derivatives developed almost immediately in locations along the Thai–Cambodian border [8–11]. Therefore, inexpensive and potent antimalarial drugs, especially those that have different modes of action, are urgently required on a probably continuing basis due to the ability of the malaria parasites to quickly develop drug resistance.Fig. 1 Therapeutic drugs for malaria

ons along the Thai–Cambodian border [8–11]. Therefore, inexpensive and potent antimalarial drugs, especially those that have different modes of action, are urgently required on a probably continuing basis due to the ability of the malaria parasites to quickly develop drug resistance.Fig. 1 Therapeutic drugs for malaria Many of the therapies currently in development use known antimalarial pharmacophores (e.g., aminoquinolines and/or peroxides), which have been chemically modified to overcome the failures of their predecessors [12]. Although these compounds have been important in the treatment of malaria, it would be highly advantageous to discover chemotypes with novel action mechanisms [13]. However, despite important advances in our understanding of the Plasmodium genome, the identification and validation of new drug targets have been challenging [14–16].

ese compounds have been important in the treatment of malaria, it would be highly advantageous to discover chemotypes with novel action mechanisms [13]. However, despite important advances in our understanding of the Plasmodium genome, the identification and validation of new drug targets have been challenging [14–16]. 16-Hydroxy-16,22-dihydroapparicine (1), a known 5-nor stemmadenine alkaloid, was identified at the Kitasato Institute as a main component of a leaf’s MeOH extract from the plant Tabernaemontana dichotoma, which displayed antimalarial properties. The potent antimalarial activity of the complex leaf extract against chloroquine-resistant Plasmodium falciparum (K1 strain) parasites in vitro, and its moderate selectivity (against MRC-5 strain human cells) are summarized in Table 1. Natural compound 1 was originally isolated from a leaf of Tabernaemontana dichotoma in 1984 by the Verpoorte group [17] (Fig. 2). The relative structural determination of 1 was based on detailed NMR study, yet the absolute stereochemistry was not determined. As 1 has the potential to contain antimalarial activity, we decided to attempt the total synthesis of 1 to confirm its stereochemistry and investigate its antimalarial effect.Table 1 Antimalarial activity and cytotoxicity of Tabernaemontana dichotoma extract IC50 (μg/mL) Antimalarial activity Cytotoxicity Selectivity index (SI) K1a FCR3b MRC-5 M/Kc M/Fd Tabernaemontana dichotoma MeOH extract 0.59 0.35 >25.0 >42.4 >71.4 Artemisinin 0.006 0.006 45.2 7528 7528 aChloroquine-resistant strain bChloroquine-sensitive strain cMRC-5/K1 dMRC-5/FCR3

16-Hydroxy-16,22-dihydroapparicine (1), a known 5-nor stemmadenine alkaloid, was identified at the Kitasato Institute as a main component of a leaf’s MeOH extract from the plant Tabernaemontana dichotoma, which displayed antimalarial properties. The potent antimalarial activity of the complex leaf extract against chloroquine-resistant Plasmodium falciparum (K1 strain) parasites in vitro, and its moderate selectivity (against MRC-5 strain human cells) are summarized in Table 1. Natural compound 1 was originally isolated from a leaf of Tabernaemontana dichotoma in 1984 by the Verpoorte group [17] (Fig. 2). The relative structural determination of 1 was based on detailed NMR study, yet the absolute stereochemistry was not determined. As 1 has the potential to contain antimalarial activity, we decided to attempt the total synthesis of 1 to confirm its stereochemistry and investigate its antimalarial effect.Table 1 Antimalarial activity and cytotoxicity of Tabernaemontana dichotoma extract IC50 (μg/mL) Antimalarial activity Cytotoxicity Selectivity index (SI) K1a FCR3b MRC-5 M/Kc M/Fd Tabernaemontana dichotoma MeOH extract 0.59 0.35 >25.0 >42.4 >71.4 Artemisinin 0.006 0.006 45.2 7528 7528 aChloroquine-resistant strain bChloroquine-sensitive strain cMRC-5/K1 dMRC-5/FCR3 Fig. 2 Structure of (15S,16S)-16-hydroxy-16,22-dihydroapparicine (1) In this review, the total synthesis, stereochemical determination, and antimalarial activity of 16-hydroxy-16,22-dihydroapparicine are discussed [18, 19].

Tabernaemontana dichotoma MeOH extract 0.59 0.35 >25.0 >42.4 >71.4 Artemisinin 0.006 0.006 45.2 7528 7528 aChloroquine-resistant strain bChloroquine-sensitive strain cMRC-5/K1 dMRC-5/FCR3 Fig. 2 Structure of (15S,16S)-16-hydroxy-16,22-dihydroapparicine (1) In this review, the total synthesis, stereochemical determination, and antimalarial activity of 16-hydroxy-16,22-dihydroapparicine are discussed [18, 19]. Naturally occurring compound 1 has the same framework as Apparicine (2), the first 5-nor stemmadenine alkaloid discovered, which was isolated from Aspidosperma dasycarpon more than 45 years ago [20, 21] (Fig. 3). There are currently 22 known 5-nor stemmadenine alkaloid compounds [22–32], with the compounds exhibiting a wide range of biological activity, including being antimicrobial [33–35] and antibacterial (antituberculoid) [32], as well as displaying opioid properties [36]. Consequently, these alkaloids are of considerable interest. The main structural feature of the alkaloids is the strained 1-azabicyclo[4.2.2]decane skeleton, including a single carbon connection, between the indole 3-position and aliphatic nitrogen moiety, which is a defining characteristic of these compounds. The relative stereochemistry of 2–5 has also been reported for conolidine (6), the completed asymmetric total synthesis being accomplished by Micalizio’s group [37].Fig. 3 Structure of apparicine (2) and related compounds

e 3-position and aliphatic nitrogen moiety, which is a defining characteristic of these compounds. The relative stereochemistry of 2–5 has also been reported for conolidine (6), the completed asymmetric total synthesis being accomplished by Micalizio’s group [37].Fig. 3 Structure of apparicine (2) and related compounds Proposed biosynthesis The special architecture involved, embodying a 1-azabicyclo[4.2.2]decane, is probably the result of the C-5 tryptamine atom being excised from the alkaloid stemmadenine by a retro-Mannich reaction. Some in vitro transformations of stemmadenine-type to 5-nor stemmadenine-type alkaloids have provided further support for this biogenetic model, which the following summarizes. Kutney and co-workers reported the biosynthesis of the 1-azabicyclo[4.2.2]decane structure in the 5-nor stemmadenine alkaloids 50 years ago, using incorporated radioisotope experiments on the plant Aspidosperma pyricollum. Later, Lim and co-workers [38] reported partial synthesis of the pseudo-aminal type indole alkaloids, such as apparicine (2), using Potier’s expected biomimetic oxidative transformation from pericine (7) (Scheme 1).Scheme 1 Biomimetic transformation

porated radioisotope experiments on the plant Aspidosperma pyricollum. Later, Lim and co-workers [38] reported partial synthesis of the pseudo-aminal type indole alkaloids, such as apparicine (2), using Potier’s expected biomimetic oxidative transformation from pericine (7) (Scheme 1).Scheme 1 Biomimetic transformation Synthesis studies Due to their unique structure and potentially useful biological activity, the total synthesis of 5-nor stemmadenine alkaloids has been reported by Bennasar et al. [39, 40], Micalizio [37], and Takayama [41] (Scheme 2). In addition, synthetic work on the 1-azabicyclo[4.2.2]decane skeleton core has been published by Joule [42, 43] and Weinreb’s group [44] (Scheme 3). A recent report of the total synthesis of (±)-apparicine (2) by Bennasar and co-workers [39, 40] detailed an approach which utilized an intramolecular Heck reaction. Micalizio and Takayama [37, 41] reported the total syntheses of conolidine (6), which could be derived from an iminium ion under intramolecular Mannich reaction. In addition, Micalizio and co-workers [37] clarified the absolute stereochemistry of 6.Scheme 2 Reported total synthesis of apparicine and conolidine Scheme 3 Reported synthetic study of the 1-azabicyclo[4.2.2]decane skeleton In 1977, Joule and co-workers [42, 43] reported the synthesis of apparicine, detailing an approach to 2 which utilizes an intramolecular Mannich cyclization to construct the 1-azabicyclo[4.2.2]decane skeleton. Weinreb’s group [44] reported the construction of a 4-cyclic compound 17 using nitrosoalkene and indole in 2014.

In 1977, Joule and co-workers [42, 43] reported the synthesis of apparicine, detailing an approach to 2 which utilizes an intramolecular Mannich cyclization to construct the 1-azabicyclo[4.2.2]decane skeleton. Weinreb’s group [44] reported the construction of a 4-cyclic compound 17 using nitrosoalkene and indole in 2014. Our synthetic approach used a distinctive reaction based on the hypothesis that the main structural feature of these alkaloids is the strained 1-azabicyclo[4.2.2]decane skeleton, including a single carbon connection between the indole 3-position and aliphatic nitrogen moiety, which is a gramine-type (or vinamidine-type) moiety (Fig. 4). This structure has a “push–pull” nature, which is stabilized by electron-donating or electron-withdrawing groups. For example, the aliphatic carbon–nitrogen bond of the gramine type (or vinamidine type) is easily cleaved by retro-Mannich reaction under acid [45], base [46–48], and thermal [49] conditions, and with various reagents (e.g., trialkylphosphine [50–55], Lewis acid [56], phthalimide [57], thiol [58, 59], and activated ester [60, 61]) to generate the indolinium cation. We, therefore, anticipated that the propendiamine moiety was an indicator of reactivity similar to the aminal, leading us to suppose the framework as a “pseudo-aminal type structure”.Fig. 4 Gramines (vinamidines) as versatile pseudo-aminal type compounds

and activated ester [60, 61]) to generate the indolinium cation. We, therefore, anticipated that the propendiamine moiety was an indicator of reactivity similar to the aminal, leading us to suppose the framework as a “pseudo-aminal type structure”.Fig. 4 Gramines (vinamidines) as versatile pseudo-aminal type compounds To complete the total synthesis of (15S*,16S*)-16-hydroxy-16,22-dihydroapparicine (1), we designed a novel phosphineimine-mediated cascade reaction, without any isolated unstable intermediate (Scheme 4). The cascade reaction sequence was: (1) Staudinger reaction of an azide 21 with triphenylphosphine to generate phosphineimine intermediate 20 [62]; (2) intramolecular N-allylation of phosphineimine transformed into aminophosphinium 19 [63–65]; (3) aza-Wittig reaction of 19 with formaldehyde; and (4) intramolecular Mannich reaction; nucleophilic attack might be performed from the indole 3-position to iminium cation 18. We needed to solve two challenging issues. Firstly, the N-allylation of the phosphineimine group; phosphineimine has relatively high nucleophilicity, while the leaving group involves sufficient electrophilicity. Secondly, the formation of iminium cation using the aminophosphonium salt; there was no reported generation of iminium cation using the aminophosphonium salt and aldehyde via the aza-Wittig reaction. We found a solitary instance of the aminophosphonium salt with excess DMF to generating formamidinium salt [66]. However, the potential reactivity of the aminophosphonium salt has never been investigated. If we could overcome these challenges, an aminophosphonium salt (such as 19) could become a useful reactant for the aza-Wittig reaction. The key precursor 21 could be prepared from diastereoselective methylation of 2-acylindole 22 with completion of the C-16 stereochemistry outcome of the Felkin–Anh transition state [67–71] (Scheme 5). Compound 22 could be constructed with the indole nucleophile and azidoaldehyde 23.Scheme 4 Designed novel phosphineimine-mediated cascade reaction

be prepared from diastereoselective methylation of 2-acylindole 22 with completion of the C-16 stereochemistry outcome of the Felkin–Anh transition state [67–71] (Scheme 5). Compound 22 could be constructed with the indole nucleophile and azidoaldehyde 23.Scheme 4 Designed novel phosphineimine-mediated cascade reaction Scheme 5 Retrosynthetic analysis of key intermediate To construct the C-15 stereocenter, we envisaged a remote stereocontrolled Michael reaction [72] of the α,β-unsaturated carboxamide 25 with the crotonic acid derivative. Synthesis of the azidoaldehyde 23 began from commercially available cis-butenediol, to afford (−)-25 [73] (Scheme 6). With the Michael accepter in hand, we attempted the remote stereocontrolled Michael reaction of (−)-25, with only minor success, (−)-25 appearing with no stereoselection and in low yield, along with γ-adduct as an undesired product. Subsequently, olefin isomerization afforded the unsaturated E-olefin 24 as a 1:1 diastereomixture (at C-15). Then, eight steps functionalization provided the azidoaldehyde (±)-23 in 38 % overall yield.Scheme 6 Synthesis of azidoaldehyde (23)

stereoselection and in low yield, along with γ-adduct as an undesired product. Subsequently, olefin isomerization afforded the unsaturated E-olefin 24 as a 1:1 diastereomixture (at C-15). Then, eight steps functionalization provided the azidoaldehyde (±)-23 in 38 % overall yield.Scheme 6 Synthesis of azidoaldehyde (23) With the azidoaldehyde (±)-23 and the N-phenylsulfonyl indole 27 [74–81] in hand, we examined the nucleophilic addition, the hydroxyindole (±)-28 being provided in 85 % yield as a single diastereomer (Scheme 7). Following the oxidation of (±)-28 to obtain the (±)-ketoindole, the N-phenylsulfonyl and pivaloyl groups were subsequently removed under basic solvolysis to provide the hydroxyketoindole (±)-22 in 87 % yield. Diastereoselective methylation of (±)-22 converted it to dihydroxyindole (±)-29 as a single diastereomer in excellent yield. The planar structure of (±)-29 was confirmed by HMQC and HMBC studies. We expected the stereoselectivity outcome to be the Felkin–Anh transition state and so sought a suitable leaving group on the allyl alcohol. We eventually discovered a 3-nitropyridyl group [82, 83] as an efficient leaving group, allowing conversion of the 3-nitropyridinylation of (±)-29 into the cascade reaction precursor (±)-21 in 93 % yield, using the process reported by Ballesteros and co-workers [84, 85]. We then attempted construction of the 1-azabicyclo[4.2.2]decane skeleton, including the pseudo-aminal moiety. The cascade reaction precursor (±)-21, with PPh3 at 60 °C, generated iminophosphorane, the reaction mixture subsequently being acidified using AcOH for activation of the 3-nitropyridyl group. Finally, formaldehyde and PPTS were added to the reaction mixture to convert the iminophosphonium cation, followed by a Mannich reaction to furnish (±)-1 in 88 % yield. The relative stereochemistry was confirmed by ROESY correlations (Fig. 5).Scheme 7 Synthesis of proposed hydroxyapparicine (1)

he 3-nitropyridyl group. Finally, formaldehyde and PPTS were added to the reaction mixture to convert the iminophosphonium cation, followed by a Mannich reaction to furnish (±)-1 in 88 % yield. The relative stereochemistry was confirmed by ROESY correlations (Fig. 5).Scheme 7 Synthesis of proposed hydroxyapparicine (1) Fig. 5 ROESY observations of synthetic (±)-(15S*,16S*)-1 Structure determination However, the spectral data of synthetic (±)-1 did not agree with that of naturally occurring 1 [17]. In particular, analysis of synthetic (±)-1, showed a ROESY relationship between H-18 or H-19 and 16-Me. Consequently, the relative stereochemistry of synthetic (±)-1 was determined to be a 15S*,16S*-configuration. Data of synthetic (±)-(15S*,16S*)-1 were then compared with naturally occurring compound (Table 2), with 1H and 13C NMR indicating differences of chemical shift (differences of all positions are shown in the experiment section). In 1H NMR, 16-Me and H-6α,β signals were registered more than 0.20 ppm and, furthermore, the 13C signals of the piperidine ring were greatly shifted from those seen in natural occurring 1. Therefore, we expected that the 16-Me group in naturally occurring 1 was on the opposite face for the tri-substituted exo-cyclic olefin. Accordingly, the relative stereochemistry was anticipated to be the 15S*,16R*-configuration.Table 2 Comparison of the NMR data of synthetic (±)-(15S*,16S*)-16-hydroxy-16,22-dihydroapparicine (1) with those reported for the natural product

Formalin test ICR male mice (5 weeks of age, 8 mice per group) were orally administered water, 350 mg/kg EFE, or 700 mg/kg Ephedra Herb extract for 3 days. On the third day, paw-licking was induced in the mice by intraplantar injection of 20 μl of 2.5 % formalin 6 h after extract/water administration. After the injection, the mice were individually placed into a glass cage, in which the amount of time that the animal spent licking the injected paw was measured as an indicator of pain. Paw-licking was recorded for 30 min in two phases, the first phase (0–5 min) and second phase (15–30 min). The protocol for animal experiments was approved by the Ethics Review Committee for Animal Experimentation of the National Institute of Health Sciences. Evaluation of anti-influenza activity Madin–Darby canine kidney (MDCK) cells (3 × 104 cells/100 μl) were incubated in 100 μl of 10 % FCS-minimal essential medium (MEM) in a 96-well plate for 24 h and washed with MEM. Next, the cells were incubated for 72 h at 37 °C in 100 μl of MEM or MEM containing a twofold serial dilution of 10 μM oseltamivir, 50 μg/ml EFE, or 50 μg/ml Ephedra Herb extract with or without 100 TCID50 of influenza virus A/WSN/33(H1N1). Living cells were then stained with crystal violet, after which the absorbance (560 nm) of each cell sample was quantified using a microplate reader. These experiments were performed externally by AVSS Corporation. Each IC50 was calculated using a four-parameter logistic model (Prism 5.0, GraphPad Software).

y occurring 1 was on the opposite face for the tri-substituted exo-cyclic olefin. Accordingly, the relative stereochemistry was anticipated to be the 15S*,16R*-configuration.Table 2 Comparison of the NMR data of synthetic (±)-(15S*,16S*)-16-hydroxy-16,22-dihydroapparicine (1) with those reported for the natural product Position 1HNMR 13CNMR Synthetic (±)-(15S*,16S*)-1 a Reported 1 b Δδ c Synthetic (±)-(15S*,16S*)-1 a Reported 1 b Δδ c δ H (int., mult, J in Hz) δ H (int., mult, J in Hz) δ C δ C NH 8.30 (br s) 9.10 (br s) −0.80 – – – 2 – – – 136.1 138.1 −2.0 3 3.04 (ddd, 14.0, 12.0, 7.0) 2.89–2.95 (m) – 46.8 48.4 −1.6 2.85 (dd, 14.0, 7.0) – 6 4.25 (d, 18.0) 3.95 (d, 17.5) 0.3 53.4 50.4 3 4.58 (d, 18.0) 4.73 (d, 17.5) −0.15 7 – – – 109.4 107.3 2.1 8 – – – 127.9 129.9 −2.0 9 7.44 (d, 7.0) 7.46 (br d, 8.0) −0.02 118.5 118.5 0 10 7.08 (ddd, 8.0, 7.0, 1.0) 7.18 (ddd, 8.0, 7.5, 1.0) −0.10 119.2 119.2 0 11 7.20 (ddd, 8.0, 7.0, 1.0) 7.08 (ddd, 8.0, 7.5, 1.0) 0.12 122.6 122.3 −0.3 12 7.32 (ddd, 7.0, 2.0, 1.0) 7.33 (br d, 8.0) −0.01 110.4 110.3 −0.1 13 – – – 135.3 135.2 0.1 14 1.87 (dddd, 14.0, 12.0, 7.0, 1.0) 2.01–2.22 (m)  - 25.0 23.4 1.6 2.22 (dddd, 14.0, 11.0, 7.0, 2.0) 15 3.35 (d, 7.0) 3.32 (dd, 3.5, 12.0) 0.02 44.0 43.2 0.8 16 – – – 76.2 74.5 1.7 18 1.75 (d, 8.6) 1.75 (ddd, 6.9, 2.5, 1.0) 0 13.7 13.8 −0.1 19 5.59 (br dq, 7.0, 1.0) 5.69 (q, 6.9) –0.10 122.0 124.9 −2.9 20 – – – 136.1 134.5 1.6 21 3.79 (br d, 16.0) 3.66 (br d, 17.0) 0.13   55.1 53.2 1.9 3.64 (br dq, 16.0, 2.0) 3.58 (br d, 17.0) 0.06 22 1.56 (s) 1.73 (s) –0.17 30.1 30.2 −0.1 aMeasured in CDCl3 (1H: 500 MHz, 13C: 125 MHz)

NH 8.30 (br s) 9.10 (br s) −0.80 – – – 2 – – – 136.1 138.1 −2.0 3 3.04 (ddd, 14.0, 12.0, 7.0) 2.89–2.95 (m) – 46.8 48.4 −1.6 2.85 (dd, 14.0, 7.0) – 6 4.25 (d, 18.0) 3.95 (d, 17.5) 0.3 53.4 50.4 3 4.58 (d, 18.0) 4.73 (d, 17.5) −0.15 7 – – – 109.4 107.3 2.1 8 – – – 127.9 129.9 −2.0 9 7.44 (d, 7.0) 7.46 (br d, 8.0) −0.02 118.5 118.5 0 10 7.08 (ddd, 8.0, 7.0, 1.0) 7.18 (ddd, 8.0, 7.5, 1.0) −0.10 119.2 119.2 0 11 7.20 (ddd, 8.0, 7.0, 1.0) 7.08 (ddd, 8.0, 7.5, 1.0) 0.12 122.6 122.3 −0.3 12 7.32 (ddd, 7.0, 2.0, 1.0) 7.33 (br d, 8.0) −0.01 110.4 110.3 −0.1 13 – – – 135.3 135.2 0.1 14 1.87 (dddd, 14.0, 12.0, 7.0, 1.0) 2.01–2.22 (m)  - 25.0 23.4 1.6 2.22 (dddd, 14.0, 11.0, 7.0, 2.0) 15 3.35 (d, 7.0) 3.32 (dd, 3.5, 12.0) 0.02 44.0 43.2 0.8 16 – – – 76.2 74.5 1.7 18 1.75 (d, 8.6) 1.75 (ddd, 6.9, 2.5, 1.0) 0 13.7 13.8 −0.1 19 5.59 (br dq, 7.0, 1.0) 5.69 (q, 6.9) –0.10 122.0 124.9 −2.9 20 – – – 136.1 134.5 1.6 21 3.79 (br d, 16.0) 3.66 (br d, 17.0) 0.13   55.1 53.2 1.9 3.64 (br dq, 16.0, 2.0) 3.58 (br d, 17.0) 0.06 22 1.56 (s) 1.73 (s) –0.17 30.1 30.2 −0.1 aMeasured in CDCl3 (1H: 500 MHz, 13C: 125 MHz) bMeasured in CDCl3 (1H: 300 MHz, 13C: 75 MHz) cΔδ (δSyn − δNat)

NH 8.30 (br s) 9.10 (br s) −0.80 – – – 2 – – – 136.1 138.1 −2.0 3 3.04 (ddd, 14.0, 12.0, 7.0) 2.89–2.95 (m) – 46.8 48.4 −1.6 2.85 (dd, 14.0, 7.0) – 6 4.25 (d, 18.0) 3.95 (d, 17.5) 0.3 53.4 50.4 3 4.58 (d, 18.0) 4.73 (d, 17.5) −0.15 7 – – – 109.4 107.3 2.1 8 – – – 127.9 129.9 −2.0 9 7.44 (d, 7.0) 7.46 (br d, 8.0) −0.02 118.5 118.5 0 10 7.08 (ddd, 8.0, 7.0, 1.0) 7.18 (ddd, 8.0, 7.5, 1.0) −0.10 119.2 119.2 0 11 7.20 (ddd, 8.0, 7.0, 1.0) 7.08 (ddd, 8.0, 7.5, 1.0) 0.12 122.6 122.3 −0.3 12 7.32 (ddd, 7.0, 2.0, 1.0) 7.33 (br d, 8.0) −0.01 110.4 110.3 −0.1 13 – – – 135.3 135.2 0.1 14 1.87 (dddd, 14.0, 12.0, 7.0, 1.0) 2.01–2.22 (m)  - 25.0 23.4 1.6 2.22 (dddd, 14.0, 11.0, 7.0, 2.0) 15 3.35 (d, 7.0) 3.32 (dd, 3.5, 12.0) 0.02 44.0 43.2 0.8 16 – – – 76.2 74.5 1.7 18 1.75 (d, 8.6) 1.75 (ddd, 6.9, 2.5, 1.0) 0 13.7 13.8 −0.1 19 5.59 (br dq, 7.0, 1.0) 5.69 (q, 6.9) –0.10 122.0 124.9 −2.9 20 – – – 136.1 134.5 1.6 21 3.79 (br d, 16.0) 3.66 (br d, 17.0) 0.13   55.1 53.2 1.9 3.64 (br dq, 16.0, 2.0) 3.58 (br d, 17.0) 0.06 22 1.56 (s) 1.73 (s) –0.17 30.1 30.2 −0.1 aMeasured in CDCl3 (1H: 500 MHz, 13C: 125 MHz) bMeasured in CDCl3 (1H: 300 MHz, 13C: 75 MHz) cΔδ (δSyn − δNat) To confirm this consideration, we set about the synthesis of 15S*,16R*-isomer. The disputed stereocenter was prepared from ketoindole and methyl anion via the Felkin–Anh transition state. Therefore, the R-configuration could be constructed with methylketone (±)-30 and indole nucleophile. We search and optimized nucleophilic addition using indole nucleophile. As a result, we found (t-butyldimethylsilyloxy)methyl (TBSOM) group [86–91] protected iodoindole as a suitable compound (Scheme 8). Hence, nucleophilic addition of 31 with (±)-30 was converted into (±)-32 in 97 % yield as a single diastereomer. The planar structure of (±)-32 was confirmed by 2D NMR study. Subsequently, global deprotection of (±)-32 obtained (±)-33 in excellent yields. Following the same reaction sequence as the synthesis of (±)-(15S*,16S*)-1 produced (±)-(15S*,16R*)-1. Characterization data provided for synthetic (±)-(15S*,16R*)-1 were fully consistent with the data for the naturally occurring compound reported by Verpoorte and co-workers [17] (Table 3). In addition, an NOE relationship was observed between H-14a and H-22 (i.e., 16-Me) (Fig. 6).Scheme 8 Total synthesis of (±)-(15S*,16R*)-1

terization data provided for synthetic (±)-(15S*,16R*)-1 were fully consistent with the data for the naturally occurring compound reported by Verpoorte and co-workers [17] (Table 3). In addition, an NOE relationship was observed between H-14a and H-22 (i.e., 16-Me) (Fig. 6).Scheme 8 Total synthesis of (±)-(15S*,16R*)-1 Table 3 Comparison of the NMR data of synthetic (±)-(15S*,16R*)-16-hydroxy-16,22-dihydroapparicine; (±)-(15S*,16R*)-1 with those reported for the natural product Position Synthetic (±)-(15S*,16R*)-1 a Reported 1 b Δδ c Synthetic (±)-(15S*,16R*)-1 a Reported 1 b Δδ c δ H (int., mult, J in Hz) δ H (int., mult, J in Hz) δ C δ C NH 8.42 (br s) 9.10 (br s) −0.68 – – – 2 – – – 138.2 138.1 0.1 3 2.89–2.98 (m) 2.89–2.95 (m) 0 48.4 48.4 0 6 4.77 (d, 17.2) 4.73 (d, 17.5) 0.04 50.3 50.4 −0.1 3.96 (d, 17.2) 3.95 (d, 17.5) 0.01 7 – – – 106.9 107.3 −0.4 8 – – – 128.6 129.9 −1.3 9 7.47 (d, 8.0) 7.46 (br d, 8.0) 0.01 118.5 118.5 0 10 7.18 (dd, 7.5, 7.5) 7.18 (ddd, 8.0, 7.5, 1.0) 0 119.2 119.2 0 11 7.08 (dd, 7.5, 7.5) 7.08 (ddd, 8.0, 7.5, 1.0) 0 122.4 122.3 0.1 12 7.31 (d, 8.0) 7.33 (br d, 8.0) −0.02 110.4 110.3 0.1 13 – – – 135.2 135.2 0 14 2.17 (m) 2.01–2.22 (m) – 23.2 23.4 0.2 2.02 (m) – 15 3.31 (dd, 3.2, 11.7) 3.32 (dd, 3.5, 12.0) −0.01 43.1 43.2 −0.1 16 – – – 74.5 74.5 0 18 1.75 (d, 8.6) 1.75 (ddd, 6.9, 2.5, 1.0) 0 13.8 13.8 0 19 5.67 (q, 6.9) 5.69 (q, 6.9) −0.02 125.2 124.9 0.3 20 – – – 134.1 134.5 −0.4 21 3.70 (d, 17.2) 3.66 (br d, 17.0) 0.04 53.1 53.2 0.1 3.52 (d, 16.6) 3.58 (br d, 17.0) −0.06 22 1.74 (s) 1.73 (s) 0.01 30.1 30.2 −0.1 aMeasured in CDCl3 (1H: 500 MHz, 13C: 125 MHz)

NH 8.42 (br s) 9.10 (br s) −0.68 – – – 2 – – – 138.2 138.1 0.1 3 2.89–2.98 (m) 2.89–2.95 (m) 0 48.4 48.4 0 6 4.77 (d, 17.2) 4.73 (d, 17.5) 0.04 50.3 50.4 −0.1 3.96 (d, 17.2) 3.95 (d, 17.5) 0.01 7 – – – 106.9 107.3 −0.4 8 – – – 128.6 129.9 −1.3 9 7.47 (d, 8.0) 7.46 (br d, 8.0) 0.01 118.5 118.5 0 10 7.18 (dd, 7.5, 7.5) 7.18 (ddd, 8.0, 7.5, 1.0) 0 119.2 119.2 0 11 7.08 (dd, 7.5, 7.5) 7.08 (ddd, 8.0, 7.5, 1.0) 0 122.4 122.3 0.1 12 7.31 (d, 8.0) 7.33 (br d, 8.0) −0.02 110.4 110.3 0.1 13 – – – 135.2 135.2 0 14 2.17 (m) 2.01–2.22 (m) – 23.2 23.4 0.2 2.02 (m) – 15 3.31 (dd, 3.2, 11.7) 3.32 (dd, 3.5, 12.0) −0.01 43.1 43.2 −0.1 16 – – – 74.5 74.5 0 18 1.75 (d, 8.6) 1.75 (ddd, 6.9, 2.5, 1.0) 0 13.8 13.8 0 19 5.67 (q, 6.9) 5.69 (q, 6.9) −0.02 125.2 124.9 0.3 20 – – – 134.1 134.5 −0.4 21 3.70 (d, 17.2) 3.66 (br d, 17.0) 0.04 53.1 53.2 0.1 3.52 (d, 16.6) 3.58 (br d, 17.0) −0.06 22 1.74 (s) 1.73 (s) 0.01 30.1 30.2 −0.1 aMeasured in CDCl3 (1H: 500 MHz, 13C: 125 MHz) bMeasured in CDCl3 (1H: 300 MHz, 13C: 75 MHz) cΔδ (δSyn − δNat) Fig. 6 NOE observations of synthetic (+)-(15S*,16R*)-1

NH 8.42 (br s) 9.10 (br s) −0.68 – – – 2 – – – 138.2 138.1 0.1 3 2.89–2.98 (m) 2.89–2.95 (m) 0 48.4 48.4 0 6 4.77 (d, 17.2) 4.73 (d, 17.5) 0.04 50.3 50.4 −0.1 3.96 (d, 17.2) 3.95 (d, 17.5) 0.01 7 – – – 106.9 107.3 −0.4 8 – – – 128.6 129.9 −1.3 9 7.47 (d, 8.0) 7.46 (br d, 8.0) 0.01 118.5 118.5 0 10 7.18 (dd, 7.5, 7.5) 7.18 (ddd, 8.0, 7.5, 1.0) 0 119.2 119.2 0 11 7.08 (dd, 7.5, 7.5) 7.08 (ddd, 8.0, 7.5, 1.0) 0 122.4 122.3 0.1 12 7.31 (d, 8.0) 7.33 (br d, 8.0) −0.02 110.4 110.3 0.1 13 – – – 135.2 135.2 0 14 2.17 (m) 2.01–2.22 (m) – 23.2 23.4 0.2 2.02 (m) – 15 3.31 (dd, 3.2, 11.7) 3.32 (dd, 3.5, 12.0) −0.01 43.1 43.2 −0.1 16 – – – 74.5 74.5 0 18 1.75 (d, 8.6) 1.75 (ddd, 6.9, 2.5, 1.0) 0 13.8 13.8 0 19 5.67 (q, 6.9) 5.69 (q, 6.9) −0.02 125.2 124.9 0.3 20 – – – 134.1 134.5 −0.4 21 3.70 (d, 17.2) 3.66 (br d, 17.0) 0.04 53.1 53.2 0.1 3.52 (d, 16.6) 3.58 (br d, 17.0) −0.06 22 1.74 (s) 1.73 (s) 0.01 30.1 30.2 −0.1 aMeasured in CDCl3 (1H: 500 MHz, 13C: 125 MHz) bMeasured in CDCl3 (1H: 300 MHz, 13C: 75 MHz) cΔδ (δSyn − δNat) Fig. 6 NOE observations of synthetic (+)-(15S*,16R*)-1 To clarify the cascade reaction mechanism, we attempted the experiment outlined in Scheme 9. At first, to provide the corresponding primary amine, a Staudinger reaction of (±)-34 with PPh3 was carried out under reflux condition to obtain the piperidine-indole (±)-37, without acidic activation of the 3-nitropyridinyl group. ESI mass-monitoring of the first reaction allowed phosphineimine 35 to be easily generated from (±)-34 and PPh3 without transformation into primary amine via solvolysis. In a time-dependent change, phosphineimine smoothly converted to the aminophosphonium cation 36. Though the 3-nitropyridinyl group was a low electrophile, it was unnecessary for acidic activation. We inferred that the 1,3-allylic strain [92] was a key component, occurring via the tri-substituted olefin. Therefore, the 3-nitropyridinyl group was located within close proximity of the phosphineimine group. Subsequent intramolecular Mannich reaction of piperidine-indole (±)-37 provided (±)-(15S*,16R*)-1 in 43 % yield, using formaldehyde and PPTS. We subsequently expected that the aza-Wittig reaction of 36 with formaldehyde could assist in generating the iminium cation precursor 38 in a cascade reaction.Scheme 9 Stepwise synthesis of (±)-(15S*,16R*)-1

nich reaction of piperidine-indole (±)-37 provided (±)-(15S*,16R*)-1 in 43 % yield, using formaldehyde and PPTS. We subsequently expected that the aza-Wittig reaction of 36 with formaldehyde could assist in generating the iminium cation precursor 38 in a cascade reaction.Scheme 9 Stepwise synthesis of (±)-(15S*,16R*)-1 Asymmetric total synthesis of 16-hydroxy-16,22-dihydroapparicine We achieved the total synthesis of racemic 16-hydroxy-16,22-dihydroapparicine (1) and determined the true relative stereochemistry of the naturally occurring compound. In the next stage, we established the absolute stereochemistry of 1. In order to accomplish asymmetric total synthesis, we used the chiral methylketone 30 (Scheme 10), which could be supplied from azidobutyrolactone 39, including the appropriate functional groups. If 39 formed acetylbutyrolactone 40, its acetyl and ester moiety could be transformed into E-ethylidene and azido groups, respectively. Acetylbutyrolactone 40 was, therefore, our key intermediate, with synthetic manners for related compounds having already been reported by Smith’s group and others [93–96]. We expected that 40 would involve a C-15 stereocenter being constructed by the intramolecular chirality transferring Michael reaction. We expected to perform via 5-exo-cyclization in the ketoester 41, which should be stereo-specifically constructed by the Baldwin rule [97] and Thorpe–Ingold effect [98, 99].Scheme 10 Asymmetric synthetic plan of (15S,16R)-1

15 stereocenter being constructed by the intramolecular chirality transferring Michael reaction. We expected to perform via 5-exo-cyclization in the ketoester 41, which should be stereo-specifically constructed by the Baldwin rule [97] and Thorpe–Ingold effect [98, 99].Scheme 10 Asymmetric synthetic plan of (15S,16R)-1 Synthesis of the optically pure tri-substituted 40 began from commercially available (−)-(R)-methyl lactate 42, which, after with four steps of preparation, provided the ketoester (+)-41 in excellent yield (Scheme 11). With the optically pure (+)-41 in hand, we attempted the intramolecular chirality transferring Michael reaction [100–104]. Through extensive optimization, we found a suitable condition to provide (+)-40 in 91 % yield as a single diastereomer, and assignment of the relative stereochemistry was derived from the coupling constants and NOE correlation between α and γ protons. The key factor of the intramolecular chirality transferring Michael reaction was the solvent’s effect; polar solvent was stabilized to the anticipated transition state. The acetyl group of (+)-40 converted into the ethylidene moiety along with the separable Z-isomer. The tri-substituted olefin moiety was determined to be of E-configuration by NOE correlation. The configuration of the C-3 stereocenter of (−)-43 was determined after simple modification; hydrogenation of (−)-43 obtained a single diastereomer, and the stereochemistry was confirmed to be S-configuration by NOE and ROESY correlation. Compound (−)-43 was transformed into primary alcohol 44 by stepwise preparation; at first, selective hydrolysis of the ethyl ester group under basic condition generated carboxylic acid, followed by the corresponding acid anhydride. The furnished carboxylic anhydride was immediately reduced to the desired (−)-44 in 82 % yield over the two steps [105, 106]. Subsequently, four steps functionalization provided the chiral methylketone (+)-30 in excellent yield without racemization. The optical purity of the (+)-30 (97 %ee) was confirmed by chiral HPLC analysis. The R-isomer of (−)-30 was prepared in the same asymmetric synthetic manner from (−)-(S)-methyl lactate.Scheme 11 Asymmetric synthesis of methylketone (+)-30

/33(H1N1). Living cells were then stained with crystal violet, after which the absorbance (560 nm) of each cell sample was quantified using a microplate reader. These experiments were performed externally by AVSS Corporation. Each IC50 was calculated using a four-parameter logistic model (Prism 5.0, GraphPad Software). Repeated oral dose toxicity assessment Special pathogen-free ICR mice (Crl:CD1) (5 weeks old) were obtained from Charles River Laboratories (Boston, MA, USA). The mice were kept in a laboratory animal facility with temperature and relative humidity maintained at 20–26 °C and 30–70 %, respectively, a 12 h-light–dark cycle, and 8–10 air charges per hour. The mice were housed in polycarbonate cages and offered CE-2 pellet feed (Nippon Formula Feed Mfg. Co., Ltd., Ehime, Japan) and groundwater that was disinfected with 0.5 % chlorine and filtered through a 5-μm filter. The mice were acclimated for 7 days before the start of the study. The mice were grouped into three groups: water, EFE, or Ephedra Herb extract. Each group included five male mice and five female mice.

provided the chiral methylketone (+)-30 in excellent yield without racemization. The optical purity of the (+)-30 (97 %ee) was confirmed by chiral HPLC analysis. The R-isomer of (−)-30 was prepared in the same asymmetric synthetic manner from (−)-(S)-methyl lactate.Scheme 11 Asymmetric synthesis of methylketone (+)-30 Finally, (+)-32 was exposed to the same procedure using (±)-(15S*,16R*)-16-hydroxy-16,22-dihydroapparicine 1 (Scheme 12). The cascade reaction precursor (−)-36 underwent the same cascade reaction condition as that for the synthesis of (±)-(15S*,16S*)-1, (±)-(15S*,16R*)-1 to give (+)-(15S,16R)-1. Characterization data proved that synthetic (+)-(15S,16R)-1 was fully consistent with the data for the natural compound, as reported by Verpoorte and co-workers [17]. The optical rotation of synthetic (+)-(15S,16R)-1, [α]D26 +112.2 (c 0.9, EtOH), compared well with the values reported for the natural sample, [α]D20 +129 (c 0.1, EtOH), and the optical rotation of synthetic (−)-(15R,16S)-1, [α]D26 −104.2 (c 0.1, EtOH), was prepared in an asymmetric synthetic manner. In addition, an NOE relationship was observed between H-14a and H-22 (i.e., 16-Me). Therefore, the C-16 stereochemistry was determined to be the R-configuration.Scheme 12 End game of the total synthesis of (+)-(15S, 16R)-1

tic (−)-(15R,16S)-1, [α]D26 −104.2 (c 0.1, EtOH), was prepared in an asymmetric synthetic manner. In addition, an NOE relationship was observed between H-14a and H-22 (i.e., 16-Me). Therefore, the C-16 stereochemistry was determined to be the R-configuration.Scheme 12 End game of the total synthesis of (+)-(15S, 16R)-1 Biological activity Naturally occurring and synthetic compounds were tested for antimalarial activity against Plasmodium falciparum parasites (chloroquine-resistant K1 strain and chloroquine-susceptible FCR3 strain) and for cytotoxicity (against human MCR-5 cells) [107–109], in comparison with the first-line antimalarial artemisinin.

vity Naturally occurring and synthetic compounds were tested for antimalarial activity against Plasmodium falciparum parasites (chloroquine-resistant K1 strain and chloroquine-susceptible FCR3 strain) and for cytotoxicity (against human MCR-5 cells) [107–109], in comparison with the first-line antimalarial artemisinin. The in vitro antimalarial activities and cytotoxicities of the naturally occurring and synthetic compounds are summarized in Table 1. As shown in Table 4, Tabernaemontana leaf extract (which includes (+)-(15S,16R)-16-hydroxy-16,22-dihydroapparicine) showed activity against both the chloroquine-resistant K1 strain and the chloroquine-sensitive FCR3 strains of Plasmodium falciparum (approximately 78-fold less potent than artemisinin, and with synthetic (±)-(15S*,16S*)-1 having no measurable impact on chloroquine-susceptible parasites). Synthetic (±)-(15S*,16S*)-1, (+)-(15S,16R)-1, (−)-1 displayed moderate to weak antimalarial activity (in the range of 9.0 to >12.5 μg/mL), while synthetic (−)-1 and intermediaries showed minimal impact (7.17 to >12.5 μg/mL). The cytotoxicities against human cells of all synthetic compounds were weak (IC50 of 17–75 μg/mL), on average similar to that of artemisinin.Table 4 Antimalarial activity of synthetic 1 and some intermediate compounds IC50 (μg/mL) Antimalarial activity Cytotoxicity Selectivity index (SI) K1a FCR3b MRC-5 M/Kc M/Fd

The in vitro antimalarial activities and cytotoxicities of the naturally occurring and synthetic compounds are summarized in Table 1. As shown in Table 4, Tabernaemontana leaf extract (which includes (+)-(15S,16R)-16-hydroxy-16,22-dihydroapparicine) showed activity against both the chloroquine-resistant K1 strain and the chloroquine-sensitive FCR3 strains of Plasmodium falciparum (approximately 78-fold less potent than artemisinin, and with synthetic (±)-(15S*,16S*)-1 having no measurable impact on chloroquine-susceptible parasites). Synthetic (±)-(15S*,16S*)-1, (+)-(15S,16R)-1, (−)-1 displayed moderate to weak antimalarial activity (in the range of 9.0 to >12.5 μg/mL), while synthetic (−)-1 and intermediaries showed minimal impact (7.17 to >12.5 μg/mL). The cytotoxicities against human cells of all synthetic compounds were weak (IC50 of 17–75 μg/mL), on average similar to that of artemisinin.Table 4 Antimalarial activity of synthetic 1 and some intermediate compounds IC50 (μg/mL) Antimalarial activity Cytotoxicity Selectivity index (SI) K1a FCR3b MRC-5 M/Kc M/Fd Tabernaemontana leaf extract 0.59 0.35 >25.0 >42.4 >71.4 Synthetic (±)-(15S*,16S*)-1 >12.5 ND 33.3 >2.7 – Synthetic (+)-(15S,16R)-1 9.00 8.37 51.2 5.7 6.1 Synthetic (−)-1 10.87 ND 75.2 6.9 – (−)-(15S,16R)-32 >12.5 >12.5 ND – – (−)-(15S,16R)-39 9.38 >12.5 54.0 5.8 <4.3 (−)-(15S,16R)-33 7.58 7.17 17.8 2.3 2.5 (−)-(15S,16R)-34 8.04 >12.5 >100.0 >12.4 8.0 (±)-(15S,16R)-37 8.98 >12.5 40.8 4.5 <3.3 Artemisinin 0.006 0.006 45.2 7528 7528 aChloroquine-resistant strain bChloroquine-sensitive strain cMRC-5/K1 dMRC-5/FCR3

Tabernaemontana leaf extract 0.59 0.35 >25.0 >42.4 >71.4 Synthetic (±)-(15S*,16S*)-1 >12.5 ND 33.3 >2.7 – Synthetic (+)-(15S,16R)-1 9.00 8.37 51.2 5.7 6.1 Synthetic (−)-1 10.87 ND 75.2 6.9 – (−)-(15S,16R)-32 >12.5 >12.5 ND – – (−)-(15S,16R)-39 9.38 >12.5 54.0 5.8 <4.3 (−)-(15S,16R)-33 7.58 7.17 17.8 2.3 2.5 (−)-(15S,16R)-34 8.04 >12.5 >100.0 >12.4 8.0 (±)-(15S,16R)-37 8.98 >12.5 40.8 4.5 <3.3 Artemisinin 0.006 0.006 45.2 7528 7528 aChloroquine-resistant strain bChloroquine-sensitive strain cMRC-5/K1 dMRC-5/FCR3 The IC50 value of synthetic (+)-(15S,16R)-1 proved to be significantly lower than the leaf extract containing naturally occurring (+)-(15S,16R)-16-hydroxy-16,22-dihydroapparicine.

Tabernaemontana leaf extract 0.59 0.35 >25.0 >42.4 >71.4 Synthetic (±)-(15S*,16S*)-1 >12.5 ND 33.3 >2.7 – Synthetic (+)-(15S,16R)-1 9.00 8.37 51.2 5.7 6.1 Synthetic (−)-1 10.87 ND 75.2 6.9 – (−)-(15S,16R)-32 >12.5 >12.5 ND – – (−)-(15S,16R)-39 9.38 >12.5 54.0 5.8 <4.3 (−)-(15S,16R)-33 7.58 7.17 17.8 2.3 2.5 (−)-(15S,16R)-34 8.04 >12.5 >100.0 >12.4 8.0 (±)-(15S,16R)-37 8.98 >12.5 40.8 4.5 <3.3 Artemisinin 0.006 0.006 45.2 7528 7528 aChloroquine-resistant strain bChloroquine-sensitive strain cMRC-5/K1 dMRC-5/FCR3 The IC50 value of synthetic (+)-(15S,16R)-1 proved to be significantly lower than the leaf extract containing naturally occurring (+)-(15S,16R)-16-hydroxy-16,22-dihydroapparicine. Conclusion We achieved the first total synthesis of (+)-(15S,16R)-16-hydroxy-16,22-dihydroapparicine (1) and the (−)-enantiomer and determined the absolute stereochemistry of naturally occurring 1. The synthesis involved a novel cascade reaction for efficient construction of the 1-azabicyclo[4.2.2]decane, including a pseudo-aminal moiety, via a Staudinger reaction, N-allylation, aza-Wittig reaction, and Mannich reaction. In addition, we developed a new method using diastereoselective 1,2-addition of methylketone, using N-TBSOM protecting the indole nucleophile and intramolecular chirality transferring Michael reaction with neighboring group participation. In particular, intramolecular chirality transferring Michael reaction proved to be a useful method for synthesis of the chiral tri-substituted butyrolactone. We established an effective enantioselective synthetic route for the production of pseudo-aminal alkaloids.

Michael reaction with neighboring group participation. In particular, intramolecular chirality transferring Michael reaction proved to be a useful method for synthesis of the chiral tri-substituted butyrolactone. We established an effective enantioselective synthetic route for the production of pseudo-aminal alkaloids. Synthetic (+)-(15S,16R)-1 exhibited moderate/weak antimalarial activity against chloroquine-resistant Plasmodium falciparum parasites and there is a possibility that the structurally unique compounds may be useful for the development of novel antimalarial drug candidates. This work was supported by a grant for the 21st Century COE Program; a Grant-in-Aid for Young Scientists (22790017) to T.H. from the Ministry of Education, Culture, Sports, Science and Technology (MEXT); and a Kitasato University Research Grant for Young Researchers to T.H. We also thank Dr. Kenichiro Nagai and Ms. Noriko Sato (School of Pharmacy, Kitasato University) for their contributions. We are grateful to Dr. Toh-Seok Kam (University of Malaya) for providing an authentic natural sample of 16-hydroxy-16,22-dihydroapparicine.

Introduction Ephedra Herb (EH) is officially defined as the terrestrial stem of Ephedra sinica Stapf, Ephedra intermedia Schrenk et C. A. Meyer, or Ephedra equisetina Bunge (Ephedraceae) in the Japanese Pharmacopoeia 16th edition (JP16) [1]. EH is a component of Kampo (Japanese traditional herbal medicine) formulae for the treatment of headaches, bronchial asthma, nasal inflammation, and the common cold, and is reported to have anti-inflammatory [2], antitussive [3], and anti-influenza activities [4]. Ephedrine alkaloids (EAs) were isolated as principal ingredients in EH by Prof. Nagayoshi Nagai in 1885 [5]. Miura [6] showed that ephedrine has mydriatic action in the rabbits. Then, Amatsu and Kubota [7] reported that ephedrine raised the blood pressure by contraction of the peripheral vessels following intravenous (i.v.) injection in dogs. Chen and Schmidt [8] found that ephedrine showed circulatory stimulatory effects when it was orally administered. Furthermore, MacDermot [9] revealed that the injection of ephedrine into patients with bronchial asthma showed beneficial effects. EAs have considerable pharmacological activities, and are believed to be the principal active ingredients in EH. The content of EAs in EH are regulated in the JP16. However, EAs are known to induce palpitation, hypertension, insomnia, and dysuria as major side effects. Therefore, the administration of EAs-containing drugs to patients with cardiovascular-related diseases is severely contraindicated.

cipal active ingredients in EH. The content of EAs in EH are regulated in the JP16. However, EAs are known to induce palpitation, hypertension, insomnia, and dysuria as major side effects. Therefore, the administration of EAs-containing drugs to patients with cardiovascular-related diseases is severely contraindicated. Previously, we found that EH extract impaired hepatocyte growth factor (HGF)-induced cancer cell motility, likely by suppressing the HGF-c-Met signaling pathway [10], since dysregulation of this pathway promotes tumor formation, growth, progression, metastasis, and therapeutic resistance [11, 12]. Therefore, EH may have applications in cancer therapy as a novel c-Met inhibitor. Recently, we revealed that herbacetin, a flavonoid aglycon in EH, inhibited HGF/c-Met/Akt signal and HGF-induced motility of human MDA-MB-231 breast cancer cells [13]. In addition, we found that herbacetin had analgesic effects in the formalin test [14]. These results indicate that some of the pharmacological effects of EH may not be due to EAs and, therefore, the prospect of preparing an EAs-free EH extract (EFE) as a new and potentially safer natural medicine without the side effects associated with EAs appealed to us.

Co., Ltd., Ehime, Japan) and groundwater that was disinfected with 0.5 % chlorine and filtered through a 5-μm filter. The mice were acclimated for 7 days before the start of the study. The mice were grouped into three groups: water, EFE, or Ephedra Herb extract. Each group included five male mice and five female mice. The dosages of EFE and Ephedra Herb extract were converted to 50-fold of the human maximum dose of Ephedra Herb extract, equivalent to 6 g of cut crude drug. The doses of EFE and Ephedra Herb extract were 632 mg/kg/day and 755 mg/kg/day, respectively. The mice were orally administered water, EFE, or Ephedra Herb extract once per day for 14 days. Clinical signs and mortality were assessed several times per day. Body weight, food consumption, and water consumption were measured twice per week throughout the experiment. After 14 days, all mice were anesthetized by isoflurane inhalation, after which blood samples were collected from the abdominal aorta.

had analgesic effects in the formalin test [14]. These results indicate that some of the pharmacological effects of EH may not be due to EAs and, therefore, the prospect of preparing an EAs-free EH extract (EFE) as a new and potentially safer natural medicine without the side effects associated with EAs appealed to us. Therefore, to achieve the aim of this present study, which was the production of a clinically useful EH extract with none of the side effects associated with EAs, we developed an efficient method for preparing EFE from EH extract. Furthermore, we clarified the chemical composition of the EFE and analyzed the herbacetin content as a candidate marker using LC–MS because EFE contains no EAs, which are markers for the quantitative assay of EH stipulated by the JP16. In addition, we examined its antiproliferative effects against the H1975 non-small cell lung cancer (NSCLC) cell line. Materials and methods Materials and reagents EH (JP16 grade) originally produced from E. sinica was purchased from Uchida Wakanyaku Co., Ltd. The authentic EAs used were: ephedrine, purchased from Dainippon Pharma Co., Ltd.; methylephedrine and pseudoephedrine, from Alps Pharmaceutical Ind., Co., Ltd.; and norephedrine from Tokyo Chemical Industry Co. Ltd. 6-Methoxykynurenic acid was purchased from Chemicia Scientific, LLC. trans-Cinnamic acid was purchased from Wako Co., while herbacetin 7-O-neohesperidoside and herbacetin 7-O-glucoside were isolated from EH [15]. Herbacetin and apigenin were purchased from ChromaDex Co. and Wako Co., respectively.

cal Industry Co. Ltd. 6-Methoxykynurenic acid was purchased from Chemicia Scientific, LLC. trans-Cinnamic acid was purchased from Wako Co., while herbacetin 7-O-neohesperidoside and herbacetin 7-O-glucoside were isolated from EH [15]. Herbacetin and apigenin were purchased from ChromaDex Co. and Wako Co., respectively. General procedures Unless otherwise noted, the following instruments and conditions were used. The 1H-NMR, 13C-NMR, and 2D-NMR spectra were recorded using an ECA-800 or ECA-600 spectrometer (JEOL), and chemical shifts were expressed in δ (ppm) with tetramethylsilane (TMS) as the reference standard. The LC/Orbitrap MS analysis was performed using an LC-20A UFLC system (Shimadzu) equipped with the LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). The UPLC/MS analysis was performed using a Xevo TQD UPLC/MS system (Waters). The LC/MS analysis was performed using an LC-20A UFLC system equipped with an LCMS-2020 mass spectrometer (Shimadzu). Selection of optimal cation exchange resin for EFE preparation Sample preparation EH (10 g) was extracted with hot water (100 ml) for 1 h at 95 °C. After filtration, the extract volume was adjusted to 100 ml with water and 2 ml was exposed to each cation exchange resin (2 ml) shown in Table 1, stirred for 10 s, and then the solution was left to stand for 1 h. The supernatant (0.5 ml) volume was adjusted to 5 ml with MeOH and subsequently used as the sample solution for the HPLC analysis.Table 1 Cation exchange resins

00 ml with water and 2 ml was exposed to each cation exchange resin (2 ml) shown in Table 1, stirred for 10 s, and then the solution was left to stand for 1 h. The supernatant (0.5 ml) volume was adjusted to 5 ml with MeOH and subsequently used as the sample solution for the HPLC analysis.Table 1 Cation exchange resins Resins Class Manufacturers A WK10 Weak Mitsubishi Chemical Co., Japan B WK11 Weak Mitsubishi Chemical Co., Japan C WK20 Weak Mitsubishi Chemical Co., Japan D WK40L Weak Mitsubishi Chemical Co., Japan E SK104 Strong Mitsubishi Chemical Co., Japan F SK110 Strong Mitsubishi Chemical Co., Japan G SK1B Strong Mitsubishi Chemical Co., Japan H UBK530 Strong Mitsubishi Chemical Co., Japan I UBK12 Strong Mitsubishi Chemical Co., Japan J PK216 Strong Mitsubishi Chemical Co., Japan K IR120B Strong Organo Co., Japan L FPC3500 Strong Organo Co., Japan M 1060H Strong Organo Co., Japan Quantitative analyses of ephedrine alkaloids The HPLC analysis was performed using the HITACHI HPLC system (pump, L-2130; degasser, L-2130; auto-sampler, L-2200; column oven, L-2300; and diode array detector, L-2450). The analytical conditions were as follows [1]: column, SHISEIDO AG 120 (4.6 mm i.d. × 150 mm; 5 μm, Shiseido, Tokyo); mobile phase, sodium lauryl sulfate (5 g) dissolved in acetonitrile (MeCN, 350 ml), followed by the addition of water (650 ml) and phosphoric acid (1 ml); flow rate, 0.8 ml/min; column oven temperature, 45 °C; injection volume, 10 μl; and monitoring wavelength, 210 nm.

120 (4.6 mm i.d. × 150 mm; 5 μm, Shiseido, Tokyo); mobile phase, sodium lauryl sulfate (5 g) dissolved in acetonitrile (MeCN, 350 ml), followed by the addition of water (650 ml) and phosphoric acid (1 ml); flow rate, 0.8 ml/min; column oven temperature, 45 °C; injection volume, 10 μl; and monitoring wavelength, 210 nm. Preparation of EFE EH (10 kg) was added to water (100 l), extracted at 95 °C for 1 h, and the extract was filtered through the SK-1B ion exchange resin (10 l, Mitsubishi Chemical Co.), which was treated with 1 M HCl (30 l) and water (100 l) prior to use, at a flow rate of 0.5 l/min, and then the resin was washed with water (10 l). The unadsorbed fraction (110 l) was adjusted to pH 5 with 5 % aqueous sodium bicarbonate (NaHCO3aq., 6 l). The solution was evaporated under reduced pressure to obtain EFE (1.2 kg, yield 12.0 %).

1 M HCl (30 l) and water (100 l) prior to use, at a flow rate of 0.5 l/min, and then the resin was washed with water (10 l). The unadsorbed fraction (110 l) was adjusted to pH 5 with 5 % aqueous sodium bicarbonate (NaHCO3aq., 6 l). The solution was evaporated under reduced pressure to obtain EFE (1.2 kg, yield 12.0 %). Analysis of the chemical composition of EFE Preparation of sample solution EH (200 g) was added to water (2000 ml), extracted at 95 °C for 1 h, filtered, and then the residue was washed with water (200 ml). The extract was centrifuged at 1800 × g for 10 min, and then half of the supernatant was concentrated under reduced pressure to obtain the EH extract (14.1 g), while the other half was passed through the SK-1B ion exchange resin (100 ml), which was treated with 1 M HCl (30 ml) and water (100 ml) prior to use, and then the resin was washed with water (100 ml). The unadsorbed fraction (1100 ml) was adjusted to pH 5 using 5 % NaHCO3aq. (60 ml), and then the solution was evaporated under reduced pressure to obtain EFE (11.8 g). Each extract batch was adjusted to a concentration of 5 mg/ml with 50 % aqueous MeOH (MeOHaq.), filtered using a 0.45-μm filter, and then subsequently used as the sample solutions.

0 ml) was adjusted to pH 5 using 5 % NaHCO3aq. (60 ml), and then the solution was evaporated under reduced pressure to obtain EFE (11.8 g). Each extract batch was adjusted to a concentration of 5 mg/ml with 50 % aqueous MeOH (MeOHaq.), filtered using a 0.45-μm filter, and then subsequently used as the sample solutions. Analysis of LC/Orbitrap MS The LC/Orbitrap MS analytical conditions were as follows: column, Inertsil ODS-3 (2.1 mm i.d. × 150 mm, 5 μm; GL Sciences); mobile phase, 0.1 % formic acid (HCOOH) in water (A)–0.1 % HCOOH in MeOH (B) in a gradient mode of 5 % B (0–10 min) → 75 % B (70 min) → 100 % B (80 min) → 100 % B (90 min) → 5 % B (90.01 min) → 5 % B (95 min); injection volume, 1 μl; flow rate, 0.2 ml/min; column oven temperature, 40 °C; and photodiode array (PDA) (200–400 nm). Furthermore, the MS conditions were: interface, electrospray ionization (ESI) positive/negative; source voltage, 4.0 kV; capillary voltage, 10 V; source temperature, 300 °C; sheath and auxiliary gas flow rates, 50 and 25, respectively; scan range, m/z 50–2000; and mass resolution, 30,000 full width.

(PDA) (200–400 nm). Furthermore, the MS conditions were: interface, electrospray ionization (ESI) positive/negative; source voltage, 4.0 kV; capillary voltage, 10 V; source temperature, 300 °C; sheath and auxiliary gas flow rates, 50 and 25, respectively; scan range, m/z 50–2000; and mass resolution, 30,000 full width. Analysis of UPLC/MS The UPLC analytical conditions were: column, Inertsil ODS-3 (2.1 mm i.d. × 150 mm, 5 μm; GL Sciences); mobile phase, 0.1 % HCOOH in water (A)–0.1 % HCOOH in MeOH (B) in a gradient mode of 5 % B (0 min) → 50 % B (40 min) → 100 % B (50 min) → 100 % B (55 min) → 5 % B (55.1 min) → 5 % B (60 min); injection volume, 1 μl; flow rate, 0.2 ml/min; and PDA (200–400 nm). In addition, the MS conditions were: interface, ESI positive/negative; capillary voltage, 4.5 kV; source and desolvation temperatures, 150 and 400 °C, respectively; desolvation gas flow, 800 l/h; cone voltage, 50 V; cone gas flow, 50 l/h; and scan range, m/z 100–1200. Synthesis of 6-hydroxykynurenic acid (2) 6-Methoxykynurenic acid (48.4 mg) was dissolved in ethylene glycol (5 ml) and potassium hydroxide (KOH, 1 g) was added, followed by refluxing for 4.5 h. The reaction mixture was carefully neutralized with HCl under cooling conditions, separated using LH-20, and preparative TLC was performed to obtain 2 (10.7 mg).

d (2) 6-Methoxykynurenic acid (48.4 mg) was dissolved in ethylene glycol (5 ml) and potassium hydroxide (KOH, 1 g) was added, followed by refluxing for 4.5 h. The reaction mixture was carefully neutralized with HCl under cooling conditions, separated using LH-20, and preparative TLC was performed to obtain 2 (10.7 mg). 6-Hydroxykynurenic acid (2): White crystal, 1H-NMR (800 MHz, DMSO-d6): δH 6.35 (1H, s, H-3), 7.31 (1H, d, J = 3.2 Hz, H-5), 7.05 (1H, dd, J = 8.8, 3.2 Hz, H-7), 7.77 (1H, d, J = 8.8 Hz, H-8), 9.73 (1H, br s), 11.2 (1H, s). 13C-NMR (200 MHz, DMSO-d6): δC 146.9 (C-2), 106.5 (C-3), 163.7 (C-4), 127.4 (C-4a), 107.5 (C-5), 153.8 (C-6), 122.1 (C-7), 121.3 (C-8), 123.5 (C-8a), 166.8 (COOH). ESI-MS: m/z 206 [M+H]+. Quantitative analysis of herbacetin 7-O-neohesperidoside Calibration curve The standard stock solution was prepared by accurately weighing an adequate amount of herbacetin 7-O-neohesperidoside and dissolving it in 50 % MeOH. The working standard solutions were prepared by diluting the stock solution with 50 % MeOH to give six graded concentrations of 0.1, 0.5, 1.0, 5.0, and 50.0 µg/ml. Each standard solution was analyzed in sextuplicate, and the regression equation was calculated in the form y = Ax + B. Sample preparation The EFE sample for the LC/MS analysis was prepared by adjusting the concentration to 1 mg/ml with 50 % MeOH and filtering with a 0.45-μm filter.

Quantitative analysis of herbacetin 7-O-neohesperidoside Calibration curve The standard stock solution was prepared by accurately weighing an adequate amount of herbacetin 7-O-neohesperidoside and dissolving it in 50 % MeOH. The working standard solutions were prepared by diluting the stock solution with 50 % MeOH to give six graded concentrations of 0.1, 0.5, 1.0, 5.0, and 50.0 µg/ml. Each standard solution was analyzed in sextuplicate, and the regression equation was calculated in the form y = Ax + B. Sample preparation The EFE sample for the LC/MS analysis was prepared by adjusting the concentration to 1 mg/ml with 50 % MeOH and filtering with a 0.45-μm filter. Quantitative analysis The chromatographic analytical conditions were: column, Inertsil ODS-3 (150 mm × 2.1 mm i.d., 5 μm; GL Sciences); mobile phase, 0.1 % HCOOH in water (A)–MeCN (B) in a gradient mode of 0 % B (0 min) → 40 % B (50 min) → 100 % B (60 min) → 100 % B (70 min) → 0 % B (70.01 min) → 0 % B (75 min); injection volume, 1 μl; and flow rate, 0.2 ml/min. The MS conditions were: interface, ESI negative; nebulizer gas flow, 1.5 l/min; drying gas flow, 10 l/min; curved desolvation line (CDL) and heat block temperature, 250 and 200 °C, respectively; detector and interface voltage, 120 and 4.5/−4.5 kV, respectively; interface current, 0.6 μA; and MS range, m/z 609 (selective ion monitoring, SIM). The EFE sample was analyzed in sextuplicate.

1.5 l/min; drying gas flow, 10 l/min; curved desolvation line (CDL) and heat block temperature, 250 and 200 °C, respectively; detector and interface voltage, 120 and 4.5/−4.5 kV, respectively; interface current, 0.6 μA; and MS range, m/z 609 (selective ion monitoring, SIM). The EFE sample was analyzed in sextuplicate. Quantitative analysis of herbacetin Acid hydrolysis of EFE HCl (6 M, 5.0 ml) was correctly added to EFE (5.0 mg) and the reaction mixture was heated at 70 °C for 6 h, followed by separation on a CHP 20P column (ϕ 2 × 20 cm). After washing with water, the MeOH eluted fraction was evaporated under reduced pressure, the residue was dissolved in MeOH, and the volume was adjusted to 5.0 ml. Then, 4.5 ml was added to 0.5 ml of apigenin solution (1 mg/ml, MeOH), followed by filtration with a 0.45-μm filter. Calibration curve The calibration curve for herbacetin was constructed similarly to that for herbacetin 7-O-neohesperidoside, with graded concentrations of 0.018, 0.045, 0.09, 0.18, 0.45, 0.9, and 1.8 μg/ml. Furthermore, apigenin was used as the internal standard at a final concentration of 100 µg/ml.

Quantitative analysis of herbacetin Acid hydrolysis of EFE HCl (6 M, 5.0 ml) was correctly added to EFE (5.0 mg) and the reaction mixture was heated at 70 °C for 6 h, followed by separation on a CHP 20P column (ϕ 2 × 20 cm). After washing with water, the MeOH eluted fraction was evaporated under reduced pressure, the residue was dissolved in MeOH, and the volume was adjusted to 5.0 ml. Then, 4.5 ml was added to 0.5 ml of apigenin solution (1 mg/ml, MeOH), followed by filtration with a 0.45-μm filter. Calibration curve The calibration curve for herbacetin was constructed similarly to that for herbacetin 7-O-neohesperidoside, with graded concentrations of 0.018, 0.045, 0.09, 0.18, 0.45, 0.9, and 1.8 μg/ml. Furthermore, apigenin was used as the internal standard at a final concentration of 100 µg/ml. Quantitative analysis The chromatographic analytical conditions were: column, Xbridge C18 (2.1 mm i.d. × 100 mm, 3.5 μm; Waters); mobile phase, 0.1 % HCOOH in water (A)–MeCN (B) in a gradient mode of 0 % B (0 min) → 40% B (50 min) → 100 % B (60 min) → 100 % B (70 min) → 0 % B (70.01 min) → 0 % B (75 min); injection volume, 1 μl; and flow rate, 0.2 ml/min. The MS analytical conditions were: interface, ESI negative; nebulizer and drying gas flow, 1.5 and 10 l/min, respectively; CDL and heat block temperature, 250 and 200 °C, respectively; detector and interface voltage, 120 and 4.5/−4.5 kV, respectively; interface current, 0.6 μA; and MS range, m/z 301 (SIM). The EFE sample was analyzed in sextuplicate.

e: interface, ESI negative; nebulizer and drying gas flow, 1.5 and 10 l/min, respectively; CDL and heat block temperature, 250 and 200 °C, respectively; detector and interface voltage, 120 and 4.5/−4.5 kV, respectively; interface current, 0.6 μA; and MS range, m/z 301 (SIM). The EFE sample was analyzed in sextuplicate. Antiproliferative effect The H1975 NSCLC cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were suspended at a density of 2 × 103 cells in 100 μl Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen Co.) containing 10 % fetal calf serum (FCS, Sigma-Aldrich), with or without 50–200 μg/ml of EH extract or EFE in each well of a 96-well plate and incubated at 37 °C for 72 h. To each well was added 10 μl of Cell Counting Kit-8 solution (Dojindo Co.), and after a 2-h incubation at 37 °C, the absorbance of the formazan generated in each well was measured at 450 nm using an iMark plate reader (Bio-Rad Laboratories, Inc.). The IC50 value was calculated using a four-parameter logistic model (Prism 5.0, GraphPad software).

l of Cell Counting Kit-8 solution (Dojindo Co.), and after a 2-h incubation at 37 °C, the absorbance of the formazan generated in each well was measured at 450 nm using an iMark plate reader (Bio-Rad Laboratories, Inc.). The IC50 value was calculated using a four-parameter logistic model (Prism 5.0, GraphPad software). Results and discussion We used cation exchange resins to remove EAs from the EH extract and efficiently prepared the EFE. The prototype EFEs were formulated using the 13 cation exchange resins shown in Table 1, and the residual EAs in each prototype were quantitatively determined. The results revealed that the residual ratios of EAs in the resins C-, F–K-, and M-treated prototypes were less than 1 % (Fig. 1) and, therefore, we focused on resin G (SK1B) considering cost efficiency, in preparing the EFE for practical clinical use. By using resin G, the yield of the EFE prepared for subsequent experiments from the EH extract was high (1.2 kg; y. 12.0 % from EH).Fig. 1 Residual ratio of ephedrine alkaloids (EAs) exposed to each cation exchange resin (%). Amounts of ephedrine and pseudoephedrine in Ephedra Herb (EH) corresponding to 100 % of the vertical axis were 74.0 and 22.1 mg, respectively

for subsequent experiments from the EH extract was high (1.2 kg; y. 12.0 % from EH).Fig. 1 Residual ratio of ephedrine alkaloids (EAs) exposed to each cation exchange resin (%). Amounts of ephedrine and pseudoephedrine in Ephedra Herb (EH) corresponding to 100 % of the vertical axis were 74.0 and 22.1 mg, respectively To determine the chemical composition of the prepared EFE, we analyzed the EH extract and EFE using LC/Orbitrap MS (Fig. 2). The chromatograms of both were very similar, but the peaks around retention times 6–10 min and peaks 1 and 2 of the EH extract disappeared from that of EFE, while the height of peak 3 was slightly decreased. This observation was also supported by the LC/Orbitrap MS analysis of the adsorbed fraction on the cation exchange resin: the peaks mentioned above were detected in TIC or PDA (254 nm) chromatograms (Fig. 1S). The peaks that disappeared around retention times 6–10 min were identified as ephedrine/pseudoephedrine, methylephedrine/pseudomethylephedrine, and norephedrine/pseudonorephedrine by comparing their quasi-molecular ion peaks (m/z 166.12, 180.14, and 152.11) and retention times with those of authentic standard compounds (Fig. 3). Next, we examined the chromatograms to identify peaks 1–3. Previously, Starratt and Caveney [16] isolated 6-methoxykynurenic acid (1) and 6-hydroxykynurenic acid (2) from the cation exchange resin-treated fraction of Ephedra pachyclada subsp. sinica. The accurate determination of the masses of peaks 1 and 2 using Orbitrap MS analysis suggest that their molecular formulae were C11H9O4N and C10H7O4N, respectively. Therefore, we speculated that these peaks were those of 1 and 2. Furthermore, the UPLC/MS analysis of the authentic sample of 1 showed a retention time and MS consistent with peak 1 (Fig. 2S). Therefore, we identified peak 1 as 6-methoxykynurenic acid (1, Fig. 4). Next, to identify peak 2, we synthesized 2 from the authentic sample of 1 using Kuo et al.’s method [17]. The product obtained was isolated and analyzed using NMR. The signal at δH 4.00 attributed to the 6-methoxy group in 1 had disappeared from the NMR spectrum of 2, indicating that it was a demethylated form of 1. The retention time and MS of 2 in the UPLC/MS analysis were the same as those of peak 2 (Fig. 3S). Therefore, we identified peak 2 as 6-hydroxykynurenic acid (2). Since peak 3 was hard to identify based on MS data, we separated the EH extract to identify it. Briefly, EH (100 g) was extracted with hot water (1 l) for 1.5 h.

retention time and MS of 2 in the UPLC/MS analysis were the same as those of peak 2 (Fig. 3S). Therefore, we identified peak 2 as 6-hydroxykynurenic acid (2). Since peak 3 was hard to identify based on MS data, we separated the EH extract to identify it. Briefly, EH (100 g) was extracted with hot water (1 l) for 1.5 h. The extract (13.6 g) was fractionated by HP-20, CHP-20P, and silica gel column chromatography to get the fraction rich in peak 3. The NMR spectral signals of the fraction were observed at δH 7.75 (1H, d, J = 16.0 Hz), δH 6.45 (1H, d, J = 16.0 Hz), indicating a trans-double bond; δH 7.55 (2H, m), δH 7.40 (3H, m), indicating monosubstituted benzene; and δC 169.5, indicating carbonyl carbon of carboxylic acid, which speculated that the compound was trans-cinnamic acid (3). The UPLC/MS analysis of the authentic standard trans-cinnamic acid sample gave a peak with the same retention time and MS as those of peak 3 (Fig. 4S). Therefore, we identified peak 3 as trans-cinnamic acid (3, Fig. 4).Fig. 2 LC/Orbitrap MS analyses of a Ephedra Herb (EH) extract and b ephedrine alkaloids-free EH extract (EFE): a photodiode array (PDA, 254 nm) and b total ion chromatogram (TIC). Peak 1 6-methoxykynurenic acid; peak 2 6-hydroxykynurenic acid; peak 3 trans-cinnamic acid Fig. 3 Extracted ion chromatograms of a Ephedra Herb (EH) extract and b ephedrine alkaloids-free EH extract (EFE) samples. m/z 166.12 [M+H]+, dl-ephedrine/dl-pseudoephedrine; m/z 180.14 [M+H]+, dl-methylephedrine/dl-pseudomethylephedrine; m/z 152.12 [M+H]+, dl-norephedrine/dl-pseudonorephedrine

The extract (13.6 g) was fractionated by HP-20, CHP-20P, and silica gel column chromatography to get the fraction rich in peak 3. The NMR spectral signals of the fraction were observed at δH 7.75 (1H, d, J = 16.0 Hz), δH 6.45 (1H, d, J = 16.0 Hz), indicating a trans-double bond; δH 7.55 (2H, m), δH 7.40 (3H, m), indicating monosubstituted benzene; and δC 169.5, indicating carbonyl carbon of carboxylic acid, which speculated that the compound was trans-cinnamic acid (3). The UPLC/MS analysis of the authentic standard trans-cinnamic acid sample gave a peak with the same retention time and MS as those of peak 3 (Fig. 4S). Therefore, we identified peak 3 as trans-cinnamic acid (3, Fig. 4).Fig. 2 LC/Orbitrap MS analyses of a Ephedra Herb (EH) extract and b ephedrine alkaloids-free EH extract (EFE): a photodiode array (PDA, 254 nm) and b total ion chromatogram (TIC). Peak 1 6-methoxykynurenic acid; peak 2 6-hydroxykynurenic acid; peak 3 trans-cinnamic acid Fig. 3 Extracted ion chromatograms of a Ephedra Herb (EH) extract and b ephedrine alkaloids-free EH extract (EFE) samples. m/z 166.12 [M+H]+, dl-ephedrine/dl-pseudoephedrine; m/z 180.14 [M+H]+, dl-methylephedrine/dl-pseudomethylephedrine; m/z 152.12 [M+H]+, dl-norephedrine/dl-pseudonorephedrine Fig. 4 Structures of compounds 1, 6-methoxykynurenic acid; 2, 6-hydroxykynurenic acid; 3, trans-cinnamic acid; 4, herbacetin

Fig. 3 Extracted ion chromatograms of a Ephedra Herb (EH) extract and b ephedrine alkaloids-free EH extract (EFE) samples. m/z 166.12 [M+H]+, dl-ephedrine/dl-pseudoephedrine; m/z 180.14 [M+H]+, dl-methylephedrine/dl-pseudomethylephedrine; m/z 152.12 [M+H]+, dl-norephedrine/dl-pseudonorephedrine Fig. 4 Structures of compounds 1, 6-methoxykynurenic acid; 2, 6-hydroxykynurenic acid; 3, trans-cinnamic acid; 4, herbacetin For the prepared EFE to satisfy quality standards for clinical use, a new stipulated quality control marker compound was required because EAs, the previously defined marker compounds for the quantitative assay of EH, are not present in EFE. Thus, we focused on herbacetin 7-O-neohesperidoside that was previously isolated from EH [15] and its aglycon, herbacetin, which have anticancer effects [13], and quantitatively determined their contents in EFE. First, we quantitatively analyzed herbacetin 7-O-neohesperidoside using the absolute calibration method. The quasi-molecular ion, m/z 609 [M–H]−, was detected using the SIM mode to obtain good linearity, accuracy, and precision in the concentration range of 1–50 μg/mL (Table 2). Subsequently, we analyzed herbacetin 7-O-neohesperidoside in EFE in sextuplicate using this condition (Fig. 5SA). The result revealed that the herbacetin 7-O-neohesperidoside content in EFE was 0.094 ± 0.009 %.Table 2 Quantitative analysis of herbacetin 7-O-neohesperidoside and herbacetin

range of 1–50 μg/mL (Table 2). Subsequently, we analyzed herbacetin 7-O-neohesperidoside in EFE in sextuplicate using this condition (Fig. 5SA). The result revealed that the herbacetin 7-O-neohesperidoside content in EFE was 0.094 ± 0.009 %.Table 2 Quantitative analysis of herbacetin 7-O-neohesperidoside and herbacetin Compound Monitoring ion (m/z) Retention time (min) Linear range (μg/ml) Regression equation R 2 Concentration (μg/ml) Accuracy (%) Precision (%) Herbacetin 7-O-neohesperidoside 609 31 1–50 y = 15413x + 3746 0.9997 1 −4.28 15.7 5 −22.6 16.2 50 −7.22 9.87 Herbacetin 301 30 0.018–1.8 y = 0.4416x − 0.0122 0.9999 0.018 −1.20 1.22 0.18 2.84 8.21 1.8 8.43 2.64

tion time (min) Linear range (μg/ml) Regression equation R 2 Concentration (μg/ml) Accuracy (%) Precision (%) Herbacetin 7-O-neohesperidoside 609 31 1–50 y = 15413x + 3746 0.9997 1 −4.28 15.7 5 −22.6 16.2 50 −7.22 9.87 Herbacetin 301 30 0.018–1.8 y = 0.4416x − 0.0122 0.9999 0.018 −1.20 1.22 0.18 2.84 8.21 1.8 8.43 2.64 Then, the total amount of herbacetin contained in the acid-hydrolyzed EFE was quantitatively determined using the internal reference method with apigenin (m/z 269 [M–H]−) as an internal standard. The quasi-molecular ion, m/z 301 [M–H]−, was detected using SIM to obtain good linearity, accuracy, and precision in the concentration range of 0.018–1.8 μg/ml (Fig. 5SB, Table 2). HCl (6 M) was added to the EFE sample, which was then stirred at 70 °C until the peaks corresponding to herbacetin 7-O-neohesperidoside and herbacetin 7-O-glucoside, which we previously isolated from EH, disappeared [14] in the LC/MS. Then, we analyzed the herbacetin content of the HCl-hydrolyzed EFE and found that it was 0.104 ± 0.002 %. These data showed that the sum of the herbacetin glycosides present in EFE was about 0.1 %, which appeared to be adequate for quantitative evaluation using HPLC. Herbacetin is a c-Met inhibitor found in EFE and is commercially available. These results suggest that it may be a suitable quality control marker compound for EFE.

data showed that the sum of the herbacetin glycosides present in EFE was about 0.1 %, which appeared to be adequate for quantitative evaluation using HPLC. Herbacetin is a c-Met inhibitor found in EFE and is commercially available. These results suggest that it may be a suitable quality control marker compound for EFE. Finally, to confirm whether the pharmacological effect was retained in the prepared EFE, we examined the effect of the EH extract and EFE on the growth of H1975 cells, and the result showed that they both prevented the proliferation of H1975 cells concentration-dependently (Fig. 5). The IC50 values of the EH extract and EFE were 88 and 76 μg/ml, respectively. These results revealed that the biological activity of EFE was retained and, therefore, the constituents that were removed during the manufacturing process did not affect its antiproliferative effect. Previously, we reported that EH impedes the HGF-induced motility of cancer cells by the inhibiting c-Met tyrosine kinase activity [10]. In addition, Tang et al. [18] reported that SU11274, a c-Met inhibitor, induced apoptosis of H1975 cells and inhibited their tumorigenesis in vivo. These reports suggest that EFE, as well as EH extract, suppressed the growth of H1975 cells through the inhibition of c-Met. Interestingly, EFE inhibited HGF-induced phosphorylation of c-Met and c-Met tyrosine kinase activity [19]. Therefore, EFE is expected to become a useful carcinostatic agent against cancer cells expressing c-Met. Furthermore, we have recently clarified that EFE shows analgesic and anti-influenza activity [19], suggesting that EFE has considerable therapeutic effects.Fig. 5 Ephedra Herb (EH) extract and ephedrine alkaloids-free EH extract (EFE) prevented proliferation of the H1975 non-small cell lung cancer (NSCLC) cell line

et. Furthermore, we have recently clarified that EFE shows analgesic and anti-influenza activity [19], suggesting that EFE has considerable therapeutic effects.Fig. 5 Ephedra Herb (EH) extract and ephedrine alkaloids-free EH extract (EFE) prevented proliferation of the H1975 non-small cell lung cancer (NSCLC) cell line It has been reported that therapeutic regimens using Kampo medicines reduce the drug cost for patients compared with therapy using modern medicines [20–22]. Therefore, the relatively low cost of Kampo prescriptions is expected to have a considerable positive economic impact on cancer therapy, which frequently involves the use of expensive patented medicines. In the present study, EFE showed antiproliferative effects against cancer cells. Therefore, the clinical use of EFE derived from EH could not only expand the application range of EH, but could also contribute to reducing or, at least, limiting the use of expense anticancer therapies. Although EFE originated from EH, it is now a distinct medicine from EH. Therefore, drug approval for the clinical use of EFE as a new material will be required under the present drug regulation guidelines of Japan. However, no newly produced compounds were detected following the process used for EFE production from EH extract, which has been widely prescribed to Japanese patients for a long time. In addition, EAs, which are the main substance responsible for the side effects of EH, were effectively removed from EFE. Therefore, EFE is expected to be safer than EH. Future strategies for further developing EFE for clinical use involve the standardization of its quality. An efficient procedure for EFE production and quantitative determination of a marker compound, herbacetin, were demonstrated in this study, which could contribute to overcoming the limitations associated with the use of this herbal product.

her developing EFE for clinical use involve the standardization of its quality. An efficient procedure for EFE production and quantitative determination of a marker compound, herbacetin, were demonstrated in this study, which could contribute to overcoming the limitations associated with the use of this herbal product. Conclusions In this study, we established an efficient preparation method for Ephedrine alkaloids-free Ephedra Herb extract (EFE) from Ephedra Herb (EH) extract using a cation exchange resin, SK-1B. During the EFE production process, 6-methoxykynurenic acid and 6-hydroxykynurenic acid were also removed along with Ephedrine alkaloids (EAs), and the concentration of trans-cinnamic acid was slightly decreased. However, EFE showed antiproliferative effects similar to those of the EH extract, indicating that the removal of these constituents did not affect its biological activity. Furthermore, quantitative analyses of herbacetin in the EFE hydrolysate suggested that herbacetin could serve as a marker compound to control the quality of EFE for clinical use, although further studies are needed in order to clarify the pharmacological mechanisms underlying the activities of EFE. Moreover, the prepared EFE suppressed the growth of H1975 cells expressing c-Met. Therefore, EFE has the potential to become a useful carcinostatic agent against c-Met-expressing cancer cells without the adverse effects associated with EAs.

der to clarify the pharmacological mechanisms underlying the activities of EFE. Moreover, the prepared EFE suppressed the growth of H1975 cells expressing c-Met. Therefore, EFE has the potential to become a useful carcinostatic agent against c-Met-expressing cancer cells without the adverse effects associated with EAs. Electronic supplementary material Below are the links to the electronic supplementary material. Supplementary material 1 (TIFF 78 kb) Fig. 1S. LC/Orbitrap MS analysis of the resin-adsorbed fraction: a photodiode array (PDA, 254 nm) and b total ion chromatogram (TIC). Peak 1, 6-methoxykynurenic acid; peak 2, 6-hydroxykynurenic acid; peak 3, trans-cinnamic acid; EAs, ephedrine alkaloids. Supplementary material 2 (TIFF 177 kb) Fig. 2S. Extracted ion chromatograms (XICs) of: a Ephedra Herb (EH) extract and b ephedrine alkaloids-free EH extract (EFE) and authentic standard (1) at m/z 220. Supplementary material 3 (TIFF 190 kb) Fig. 3S. Extracted ion chromatograms (XICs) of: a Ephedra Herb (EH) extract and b ephedrine alkaloids-free EH extract (EFE) and synthetic compound (2) at m/z 206. Supplementary material 4 (TIFF 161 kb) Fig. 4S. Extracted ion chromatograms (XICs) of: a Ephedra Herb (EH) extract and b ephedrine alkaloids-free EH extract (EFE) and authentic standard (3) at m/z 149.

Supplementary material 3 (TIFF 190 kb) Fig. 3S. Extracted ion chromatograms (XICs) of: a Ephedra Herb (EH) extract and b ephedrine alkaloids-free EH extract (EFE) and synthetic compound (2) at m/z 206. Supplementary material 4 (TIFF 161 kb) Fig. 4S. Extracted ion chromatograms (XICs) of: a Ephedra Herb (EH) extract and b ephedrine alkaloids-free EH extract (EFE) and authentic standard (3) at m/z 149. Supplementary material 5 (TIFF 134 kb) Fig. 5S. Analyses of: A herbacetin 7-O-neohesperidoside and B herbacetin. a Extracted ion chromatogram (XIC) at m/z 609 of ephedrine alkaloids-free Ephedra herb extract (EFE), b XIC at m/z 609 of herbacetin 7-O-neohesperidoside, c XIC at m/z 301 of EFE, d XIC at m/z 301 of herbacetin. Abbreviations EHEphedra Herb EFEEphedrine alkaloids-free Ephedra Herb extract EAEphedrine alkaloid Compliance with ethical standards Funding This research is partially supported by the Research on Development of New Drugs project of the Japan Agency for Medical Research and Development (AMED). Conflict of interest We have applied for a patent under the regulations of the Patent Cooperation Treaty (PCT).

Introduction Ephedra Herb is a crude drug containing ephedrine alkaloids, and is used in Japan as a component in many Kampo formulae, including maoto, kakkonto, shoseiryuto, and eppikajutsubuto. Ephedra Herb is defined in the sixteenth edition of the Japanese Pharmacopoeia (JP) as the terrestrial stem of Ephedra sinica Staf., Ephedra intermedia Schrenk et C.A. Meyer, or Ephedra equisetina Bunge (Ephedraceae), which have stems with ephedrine alkaloids (ephedrine and pseudoephedrine) content greater than 0.7 % [1]. Ephedra Herb has anti-inflammatory [2], analgesic, anti-influenza [3], and anti-metastatic effects [4]. However, because ephedrine alkaloids stimulate both sympathetic and parasympathetic nerves, Ephedra Herb has some adverse effects, including palpitations, hypertension, insomnia, and dysuria. The Food and Drug Administration (FDA) of the United States banned the sale of dietary supplements containing ephedra plants in 2004 because of health risks [5]. Since Professor Nagayoshi Nagai reported ephedrines to be the active constituents in Ephedra Herb [6], most of the pharmacological actions of Ephedra Herb have been attributed to ephedrine alkaloids, although the plant contains other constituents, such as phenolics and tannins [7]. Therefore, the adverse effects caused by ephedrine alkaloids are thought to be an unavoidable consequence associated with the pharmacological effects of Ephedra Herb.

l actions of Ephedra Herb have been attributed to ephedrine alkaloids, although the plant contains other constituents, such as phenolics and tannins [7]. Therefore, the adverse effects caused by ephedrine alkaloids are thought to be an unavoidable consequence associated with the pharmacological effects of Ephedra Herb. Our previous research found that maoto, an Ephedra Herb-containing formulation, suppressed cancer metastasis by inhibiting cancer cell motility [8, 9] and prevented hepatocyte growth factor (HGF)-induced cancer cell motility by inhibiting phosphorylation of the c-Met receptor. Our studies confirmed that the c-Met inhibitory activity of maoto derives from Ephedra Herb, which impairs HGF-induced cancer cell motility by suppressing the HGF-c-Met signaling pathway through inhibition of c-Met tyrosine kinase activity [4]. HGF-c-Met signaling regulates several cellular processes, including cell proliferation, invasion, scattering, survival, and angiogenesis. Dysregulation of HGF-c-Met signaling promotes tumor formation, growth, progression, metastasis, and therapeutic resistance [10, 11]. Therefore, Ephedra Herb may have applications in cancer therapy as a novel c-Met inhibitor. Moreover, we have discovered that Ephedra Herb contains herbacetin 7-O-neohesperidoside and herbacetin 7-O-glucoside [12]. Herbacetin, the aglycone of these herbacetin-glycosides, inhibits HGF-induced cell migration and phosphorylation of c-Met [13]. These findings suggest that herbacetin-glycosides are bioactive constituents of Ephedra Herb that may be responsible for its pharmacological actions not mediated by ephedrine alkaloids. However, the c-Met inhibitory activity of Ephedra Herb extract cannot be explained by herbacetin-glycosides alone, because the herbacetin-glycoside content in Ephedra Herb extract is less than 0.1 % [14]. Moreover, we confirmed that ephedrine had no effect on HGF-c-Met signaling [15]. Therefore, we predicted that c-Met inhibitory activity may be produced by the non-alkaloidal fraction of Ephedra Herb extract, which contains herbacetin-glycosides and other bioactive molecules that produce synergistic effects. The non-alkaloidal fraction of Ephedra Herb is useful for cancer patients, because adverse effects caused by ephedrine alkaloids are avoided.

ivity may be produced by the non-alkaloidal fraction of Ephedra Herb extract, which contains herbacetin-glycosides and other bioactive molecules that produce synergistic effects. The non-alkaloidal fraction of Ephedra Herb is useful for cancer patients, because adverse effects caused by ephedrine alkaloids are avoided. We utilized ion-exchange column chromatography to eliminate ephedrine alkaloids from Ephedra Herb extract, resulting in ephedrine alkaloids-free Ephedra Herb extract (EFE) [14]. In this study, we report the pharmacological and toxicological properties of EFE. Materials and methods Materials Ephedrine was purchased from Dainippon Pharma Co., Ltd. (Tokyo, Japan). Methylephedrine and pseudoephedrine were purchased from Alps Pharmaceutical Ind. Co. Ltd. (Gifu, Japan). Norephedrine was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). 6-Methoxykynurenic acid was purchased from Chemicia Scientific, LLC (San Diego, CA, USA). trans-Cinnamic acid was purchased from Wako Co. (Tokyo, Japan). 6-Hydroxykynurenic acid was synthesized from 6-methoxykynurenic acid [14]. Syringin, kaempferol 3-O-rhamnoside 7-O-glucoside, and isovitexin 2″-O-rhamnoside were isolated from Ephedra Herb [12]. The methanol and water used for liquid chromatography coupled with photodiode array detection (LC-PDA) analysis were LC grade.

-Hydroxykynurenic acid was synthesized from 6-methoxykynurenic acid [14]. Syringin, kaempferol 3-O-rhamnoside 7-O-glucoside, and isovitexin 2″-O-rhamnoside were isolated from Ephedra Herb [12]. The methanol and water used for liquid chromatography coupled with photodiode array detection (LC-PDA) analysis were LC grade. Recombinant human HGF (purity ≥98 % by SDS-PAGE and HPLC analysis) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA). SU11274 (purity ≥98 % by HPLC analysis) was purchased from Sigma-Aldrich (St Louis, MO, USA). The antibodies (Abs) used were as follows: anti-p-Met (Tyr1234/1235) monoclonal Ab (mAb) (CST#3077), anti-Met mAb (CST#8198), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPD) mAb (CST#2118), and horseradish peroxidase (HRP)-labeled anti-rabbit IgG Ab (CST#7074); all were obtained from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).

ed were as follows: anti-p-Met (Tyr1234/1235) monoclonal Ab (mAb) (CST#3077), anti-Met mAb (CST#8198), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPD) mAb (CST#2118), and horseradish peroxidase (HRP)-labeled anti-rabbit IgG Ab (CST#7074); all were obtained from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). Preparation of EFE and Ephedra Herb extract Preparation of EFE and Ephedra Herb extract was carried out as described by Oshima et al. [14]. Ephedra Herb (200 g, E. sinica, Japanese pharmacopoeia grade) was added to water (2000 ml), extracted at 95 °C for 1 h, and filtered, after which the residue was washed with water (200 ml). The extract was centrifuged at 1800g for 10 min, after which half of the supernatant was concentrated under reduced pressure to obtain Ephedra Herb extract (14.1 g), while the other half was passed directly through DIAION™ SK-1B ion-exchange resin (100 ml) which was treated with 1 M HCl (30 ml) and water (100 ml) prior to use, then washed with water (100 ml). The unadsorbed fraction (1100 ml) was adjusted to pH 5 using 5 % NaHCO3 aq. (60 ml), and the solution was then evaporated under reduced pressure to obtain EFE (11.8 g). LC-PDA analysis of Ephedra Herb extract and EFE One milliliter of methanol was added to 50 mg samples of Ephedra Herb extract and EFE, which were exposed to ultrasonic waves for 30 min and centrifuged. The supernatants were filtered through 0.45-µm membrane filters, after which 20 µl of each sample was subjected to LC-PDA analysis.

is of Ephedra Herb extract and EFE One milliliter of methanol was added to 50 mg samples of Ephedra Herb extract and EFE, which were exposed to ultrasonic waves for 30 min and centrifuged. The supernatants were filtered through 0.45-µm membrane filters, after which 20 µl of each sample was subjected to LC-PDA analysis. LC-PDA analysis was performed using an LC-10A HPLC system consisting of an SCL-10Avp system controller, an LC-10ATvp pump, a DGU-12A degasser, an SIL-10A auto injector, a CTO-10Avp column oven, and an SPD-M10Avp photodiode array detector equipped with a semi-micro cell (Shimadzu Inc., Tokyo, Japan). An Inertsil ODS-3 column (4.6 × 150 mm, 5 µm) from GL Sciences (Tokyo, Japan) was used for the separation. The column was maintained at 40 °C in the column oven. The mobile phase consisted of 0.1 % formic acid in water (A) and 0.1 % formic acid in methanol (B). The flow rate was 1.0 ml/min. The mobile phase gradient was adjusted as follows: 5 % B (0–10 min), 5–75 % B (10–70 min), 75–100 % B (70–80 min), 100 % B (80–90 min). Cell lines and culture MDA-MB-231 human breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Sigma-Aldrich, St. Louis, MA, USA) containing 10 % fetal calf serum (FCS) (Invitrogen Corp., Carlsbad, CA, USA) at 37 °C in an atmosphere containing 5 % CO2.

LC-PDA analysis was performed using an LC-10A HPLC system consisting of an SCL-10Avp system controller, an LC-10ATvp pump, a DGU-12A degasser, an SIL-10A auto injector, a CTO-10Avp column oven, and an SPD-M10Avp photodiode array detector equipped with a semi-micro cell (Shimadzu Inc., Tokyo, Japan). An Inertsil ODS-3 column (4.6 × 150 mm, 5 µm) from GL Sciences (Tokyo, Japan) was used for the separation. The column was maintained at 40 °C in the column oven. The mobile phase consisted of 0.1 % formic acid in water (A) and 0.1 % formic acid in methanol (B). The flow rate was 1.0 ml/min. The mobile phase gradient was adjusted as follows: 5 % B (0–10 min), 5–75 % B (10–70 min), 75–100 % B (70–80 min), 100 % B (80–90 min). Cell lines and culture MDA-MB-231 human breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Sigma-Aldrich, St. Louis, MA, USA) containing 10 % fetal calf serum (FCS) (Invitrogen Corp., Carlsbad, CA, USA) at 37 °C in an atmosphere containing 5 % CO2. Trans-well migration assay MDA-MB-231 cells (5 × 104 cells/100 µl) were suspended in 100 μl DMEM containing EFE (10, 20, or 40 μg/ml), Ephedra Herb extract (40 μg/ml), or SU11274 (5 μM). The cells were poured into the upper well of a trans-well permeable support system (Corning Inc., Acton, MA, USA). DMEM (600 µl) containing 50 ng/ml HGF was added to the lower well of the trans-well system, which was incubated for 20 h at 37 °C. Finally, the number of cells that had migrated from the upper layer to the lower well was counted.

to the upper well of a trans-well permeable support system (Corning Inc., Acton, MA, USA). DMEM (600 µl) containing 50 ng/ml HGF was added to the lower well of the trans-well system, which was incubated for 20 h at 37 °C. Finally, the number of cells that had migrated from the upper layer to the lower well was counted. Cell viability MDA-MB-231 cells (5 × 104 cells/100 μl) were suspended in 100 μl of 10 % FCS-DMEM containing 40 μg/ml EFE, 40 μg/ml Ephedra Herb extract, or 5 μM SU11274. After 20 h, 10 μl of Cell Counting Kit-8 solution (Dojindo, Kumamoto, Japan) was added to each sample, after which the resulting mixture was incubated at 37 °C for 2 h. Formazan absorbance (450 nm) was quantified using an iMark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Herb extract, or 5 μM SU11274. After 20 h, 10 μl of Cell Counting Kit-8 solution (Dojindo, Kumamoto, Japan) was added to each sample, after which the resulting mixture was incubated at 37 °C for 2 h. Formazan absorbance (450 nm) was quantified using an iMark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Detection of phosphorylated c-Met (p-Met), c-Met, and GAPDH MDA-MB-231 cells (2 × 106 cells/4 ml) were incubated in 4 ml of 10 % FCS-DMEM for 48 h, washed three times with DMEM, and incubated in 4 ml DMEM for 24 h. After the cells were washed three times with DMEM, they were incubated for 15 min at 37 °C in 4 ml DMEM or DMEM containing 50 ng/ml of HGF with or without 0.5, 1, or 10 μg/ml EFE, 10 μg/ml Ephedra Herb extract, or 5 μM SU11274. After the cells were washed three times with cold PBS without Ca2+ and Mg2+ (PBS(-)) they were treated with 1 ml Complete Lysis-M with phosphatase inhibitor (Roche Diagnostics Co., Indianapolis, IN, USA) for 5 min on an ice bath. The lysates were collected and centrifuged, after which the supernatants were incubated with 5× sodium dodecyl sulfate (SDS) loading buffer for 5 min at 95 °C. The lysates were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and electro-transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked at room temperature for 1 h with 5 % non-fat dry milk in Tris-buffered saline (10 mM Tris–HCl, pH 7.5, 100 mM NaCl) containing 0.1 % Tween 20 (TBS-T). After the membrane was washed with TBS-T, it was incubated with the anti-p-Met (Tyr1234/1235) mAb (CST#3077), anti-Met mAb (CST #8198), or anti-GAPDH Ab (SC-25778) overnight at 4 °C and washed with TBS-T. Horseradish peroxidase-labeled anti-rabbit IgG Ab (CST#7074) was applied for 1 h at room temperature, after which the membranes were washed with TBS-T. The Abs were detected with an enhanced chemiluminescent (ECL) reaction (GE Healthcare Japan, Tokyo, Japan) and imaged using an ImageQuant Las 4000 mini system (GE Healthcare Japan).

eroxidase-labeled anti-rabbit IgG Ab (CST#7074) was applied for 1 h at room temperature, after which the membranes were washed with TBS-T. The Abs were detected with an enhanced chemiluminescent (ECL) reaction (GE Healthcare Japan, Tokyo, Japan) and imaged using an ImageQuant Las 4000 mini system (GE Healthcare Japan). Measurement of c-Met tyrosine-kinase activity and determination of IC50 values Met kinase activity was measured using the ADP-Glo kinase assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, 10 μl of a reaction mixture containing 2 μg/ml of recombinant Met kinase domain, 0.2 μg/ml poly(E4Y1), and 10 μM ATP was incubated with Ephedra Herb extract or EFE at room temperature for 60 min. The kinase reactions were terminated by the addition of 10 μl ADP-Glo reagent, after which the resulting mixture was incubated for 40 min at room temperature. Next, 20 μl of Kinase Detection Reagent was added, after which the mixture was incubated for 30 min at room temperature. Luminescence was measured with an EnSpire multi-plate reader (Perkin Elmer, Foster City, CA, USA). The experiments were repeated three times. Each IC50 was calculated using a four-parameter logistic model (Prism 5.0, GraphPad Software, San Diego, CA, USA).

r 14 days. Clinical signs and mortality were assessed several times per day. Body weight, food consumption, and water consumption were measured twice per week throughout the experiment. After 14 days, all mice were anesthetized by isoflurane inhalation, after which blood samples were collected from the abdominal aorta. Daiich Negishi Clinical Laboratory, Inc. assessed the following hematological parameters: red blood cell count (RBC), hematocrit (Ht), hemoglobin concentration (Hb), platelet count (PLT), white blood cell count (WBC), and WBC differential count (stab neutrophils, segmented neutrophils, lymphocytes, and monocytes). Serum biochemical parameters were measured by the Nagahama Institute for Biochemical Science at Oriental Yeast Co., Ltd. The selected serum biochemical parameters were albumin (ALB), aspartate aminotransferase (ALT), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), leucine aminopeptidase (LAP), gamma-glutamyl transpeptidase (γ-GT), and total bilirubin (T-BIL). After the collection of the blood samples, the organs were harvested from each mouse and weighed. Colon weight was measured after washing out the colon contents with saline solution. Statistical analysis All data are expressed as mean ± standard deviation (SD). Data were analyzed by ANOVA. Significant differences between the control and treatment groups were determined by Student’s t test, Dunnett’s test, and Tukey’s test using GraphPad Prism 5J software (MDF Co., Ltd., Tokyo, Japan). p < 0.05 was considered statistically significant.

ressed as mean ± standard deviation (SD). Data were analyzed by ANOVA. Significant differences between the control and treatment groups were determined by Student’s t test, Dunnett’s test, and Tukey’s test using GraphPad Prism 5J software (MDF Co., Ltd., Tokyo, Japan). p < 0.05 was considered statistically significant. Results 3D-HPLC analysis of Ephedra Herb extract and EFE Ephedra Herb extract and EFE displayed similar chromatographic profiles, but several peaks were absent in that of EFE (Fig. 1a, b). The retention times and UV spectra of the Ephedra Herb extract standard revealed the presence of ephedrine alkaloids (ephedrine, pseudoephedrine, norephedrine, and methylephedrine), 6-hydroxykynurenic acid, syringin, kaempferol 3-O-rhamnoside 7-O-glucoside, 6-methoxykynurenic acid, isovitexin 2″-O-rhamnoside, and cinnamic acid (Fig. 1a, c). However, ephedrine alkaloids, 6-hydroxykynurenic acid, and 6-methoxykynurenic acid were not present in the EFE chromatogram (Fig. 1a, b) [14].Fig. 1 3D-HPLC profile of Ephedra Herb extract and EFE. a Ephedra Herb extract; b EFE; c zoom at 20–45 min in Ephedra Herb extract. 1 ephedrine alkaloids; 2 syringin; 3 kaempferol 3-O-rhamnoside 7-O-glucoside; 4 isovitexin 2″-O-rhamnoside; 5 cinnamic acid; 6 6-hydroxykynurenic acid; 7 6-methoxykynurenic acid

a, b) [14].Fig. 1 3D-HPLC profile of Ephedra Herb extract and EFE. a Ephedra Herb extract; b EFE; c zoom at 20–45 min in Ephedra Herb extract. 1 ephedrine alkaloids; 2 syringin; 3 kaempferol 3-O-rhamnoside 7-O-glucoside; 4 isovitexin 2″-O-rhamnoside; 5 cinnamic acid; 6 6-hydroxykynurenic acid; 7 6-methoxykynurenic acid Inhibitory effect of EFE on HGF-induced motility of MDA-MB-231 cells We confirmed the inhibitory effect of EFE on HGF-induced motility of MDA-MB-231 cells using a trans-well permeable support system. HGF (50 ng/ml) significantly induced MDA-MB-231 cell motility; however, this effect was inhibited by 5 μM SU11274, a c-Met-specific inhibitor (Fig. 2a). We previously reported that 40 μg/ml Ephedra Herb extract suppressed the HGF-induced migration of MDA-MB-231 cells [4], so the same concentration was used in this study. The results show that both EFE and Ephedra Herb extract significantly suppressed the HGF-induced motility at a concentration of 40 μg/ml (Fig. 2a).Fig. 2 Effects of EFE, Ephedra Herb extract, and c-Met inhibitor SU11274 on HGF-induced motility and viability of MDA-MB-231 cells. a MDA-MB-231 cells (5 × 104 cells) were suspended in DMEM with or without 40 μg/ml Ephedra Herb, 40 μg/ml EFE, or 5 μM SU11274, and poured into the upper well of the trans-well system. The lower well of the trans-well system contained 600 µL of DMEM containing 50 ng/ml HGF. After 20 h, the cells that had migrated into the lower well were counted. Each assay was performed in triplicate. The error bars represent standard deviation. Statistical significance was determined by Dunnett’s test. **p < 0.001 vs. the number of cells that migrated following HGF stimulation. b 5 × 104 cells were suspended in 100 μl of DMEM containing 50 ng/ml HGF with or without 40 μg/ml Ephedra Herb, 40 μg/ml EFE, or 5 μM SU11274. After 20 h, cell viability was analyzed using the Cell Counting Kit-8 as described in the “Materials and methods” section. Viability (%) is expressed as (absorbance of cells in DMEM containing HGF and crude drug extract/absorbance of cells in DMEM containing HGF) × 100. Each assay was performed in triplicate. The error bars represent standard deviation. Statistical significance was determined by Dunnett’s test. c 5 × 104 cells were suspended in DMEM with 0, 10, 20, or 40 μg/ml EFE and poured into the upper well of the trans-well system. The lower well of the trans-well system contained DMEM with 50 ng/ml HGF.

in triplicate. The error bars represent standard deviation. Statistical significance was determined by Dunnett’s test. c 5 × 104 cells were suspended in DMEM with 0, 10, 20, or 40 μg/ml EFE and poured into the upper well of the trans-well system. The lower well of the trans-well system contained DMEM with 50 ng/ml HGF. After 20 h, the cells that had migrated to the lower well were counted. Each assay was performed in triplicate. The error bars represent standard deviation. Statistical significance was determined by Dunnett’s test. *p < 0.01, or **p < 0.001 vs. the number of cells that migrated without EFE exposure We also examined the effects of Ephedra Herb extract and EFE on the viability of MDA-MB-231 cells and found that the extracts had no effect on cell viability (Fig. 2b). This indicates that the inhibitory activities of these extracts on HGF-induced motility are independent of cytotoxicity. Subsequently, we investigated the effects of various concentrations of EFE on HGF-induced migration of MDA-MB-231 cells. We found that EFE significantly inhibited the HGF-induced motility of MDA-MB-231 cells in a concentration-dependent manner (Fig. 2c), thus confirming that EFE possesses inhibitory activity against HGF-induced cancer cell motility.

he effects of various concentrations of EFE on HGF-induced migration of MDA-MB-231 cells. We found that EFE significantly inhibited the HGF-induced motility of MDA-MB-231 cells in a concentration-dependent manner (Fig. 2c), thus confirming that EFE possesses inhibitory activity against HGF-induced cancer cell motility. Inhibitory effect of EFE on HGF-induced c-Met phosphorylation and tyrosine kinase activity HGF binding activates c-Met, which initiates receptor dimerization and auto-phosphorylation of tyrosine residues, propagating downstream signals. Accordingly, we confirmed the inhibitory effect of EFE on HGF-induced phosphorylation of c-Met in MDA-MB-231 cells. Tyrosine phosphorylation of c-Met was induced by HGF (50 ng/ml) and inhibited by 5 μM SU11274 (Fig. 3a). An Ephedra Herb extract concentration of 10 μg/ml was used in this study because we demonstrated in a previous study that 10 μg/ml Ephedra Herb extract suppressed HGF-induced phosphorylation of c-Met [4]. No phosphorylation of c-Met was observed following the addition of 50 ng/ml HGF with 10 μg/ml EFE (Fig. 3a). Moreover, we investigated the effects of various concentrations of EFE on the HGF-induced phosphorylation of c-Met. EFE prevented the HGF-induced phosphorylation of c-Met in a concentration-dependent manner (Fig. 3b). We also investigated the inhibitory activity of EFE on tyrosine kinase activity of c-Met. Ephedra Herb extract and EFE produced concentration-dependent inhibition of the tyrosine kinase activity of c-Met (Fig. 3c). The IC50 of Ephedra Herb extract and EFE were 0.887 and 0.530 μg/ml, respectively.Fig. 3 Effects of EFE, Ephedra Herb extract, and SU11274 on HGF-induced phosphorylation of c-Met, and effects of EFE and Ephedra Herb extract on the tyrosine-kinase activity of c-Met. a MDA-MB-231 cells were incubated in DMEM, DMEM containing 50 ng/ml HGF, or DMEM containing 50 ng/ml HGF with 10 μg/ml EFE, 10 μg/ml Ephedra Herb extract, or 5 μM SU11274 for 15 min at 37 °C. Tyrosine phosphorylation of c-Met was determined by immunoprecipitation and Western blot analysis. b MDA-MB-231 cells were incubated in DMEM containing 50 ng/ml of HGF with 0, 0.5, 1, 5, or 10 μg/ml of EFE for 15 min at 37 °C. The level of tyrosine phosphorylation of c-Met in the cells was determined by immunoprecipitation and Western blot analysis. c The kinase activity of c-Met was measured using the ProfilerPro Kit.

DA-MB-231 cells were incubated in DMEM containing 50 ng/ml of HGF with 0, 0.5, 1, 5, or 10 μg/ml of EFE for 15 min at 37 °C. The level of tyrosine phosphorylation of c-Met in the cells was determined by immunoprecipitation and Western blot analysis. c The kinase activity of c-Met was measured using the ProfilerPro Kit. A recombinant c-Met kinase domain was pre-incubated with and without a twofold serial dilution of 8 μg/ml EFE or Ephedra Herb extract at 28 °C for 15 min. The fluorescence-labeled peptide substrate, 1.5 μM 5-carboxyfluorescein-EAIYAAPFAKKK-NH2, and 79.5 μM ATP were added, followed by incubation at 28 °C for 90 min. The kinase reactions were terminated by the addition of 3 mM EDTA. Phosphorylated peptides were separated from substrate peptides and quantified using a LabChip 3000 These results suggest that EFE suppresses the HGF-induced motility by inhibiting the phosphorylation of c-Met though the prevention of tyrosine kinase activity. In addition, the c-Met inhibitory activity of Ephedra Herb was confirmed to be independent of ephedrine alkaloids.

A recombinant c-Met kinase domain was pre-incubated with and without a twofold serial dilution of 8 μg/ml EFE or Ephedra Herb extract at 28 °C for 15 min. The fluorescence-labeled peptide substrate, 1.5 μM 5-carboxyfluorescein-EAIYAAPFAKKK-NH2, and 79.5 μM ATP were added, followed by incubation at 28 °C for 90 min. The kinase reactions were terminated by the addition of 3 mM EDTA. Phosphorylated peptides were separated from substrate peptides and quantified using a LabChip 3000 These results suggest that EFE suppresses the HGF-induced motility by inhibiting the phosphorylation of c-Met though the prevention of tyrosine kinase activity. In addition, the c-Met inhibitory activity of Ephedra Herb was confirmed to be independent of ephedrine alkaloids. Effect of EFE on formalin-induced pain in mice The analgesic effect of Ephedra Herb has been traditionally believed to be mediated by pseudoephedrine [2, 16], but we recently found that herbacetin, a component of Ephedra Herb, suppressed the formalin-induced pain [17]. Therefore, we examined the analgesic effect of EFE using the formalin test. Ephedra Herb extract and EFE showed no effects during the first phase of the formalin test. Ephedra Herb extract and EFE reduced paw-licking time in a dose-dependent manner during the second phase of the formalin test. The paw-licking time during the second phase of the formalin test was significantly decreased by oral administration of 700 mg/kg Ephedra Herb extract, 350 mg/kg EFE, and 700 mg/kg EFE (Fig. 4). These results reveal that EFE possessed the analgesic action.Fig. 4 Effects of EFE and Ephedra Herb extract on formalin-induced pain. ICR mice were treated orally with water, 350 mg/kg EFE, 700 mg/kg EFE, or Ephedra Herb extract for 3 days. On the third day of treatment, formalin tests were performed 6 h after drug or placebo administration. The amount of time that each animal spent licking the injection paw was recorded for 30 min in two phases, the first (0–5 min) and second (15–30 min) phases. Statistical significance was determined by Dunnett’s test. *p < 0.05 or **p < 0.01 vs. control

alin tests were performed 6 h after drug or placebo administration. The amount of time that each animal spent licking the injection paw was recorded for 30 min in two phases, the first (0–5 min) and second (15–30 min) phases. Statistical significance was determined by Dunnett’s test. *p < 0.05 or **p < 0.01 vs. control Effect of EFE on influenza virus infection in MDCK cells Ephedra Herb has been reported to possess anti-influenza activity [3]. Therefore, we examined the effect of EFE on the survival rate of MDCK cells infected with influenza virus A/WSN/33(H1N1). Oseltamivir, an anti-influenza drug, suppressed influenza virus infection in MDCK cells in a concentration-dependent manner without causing cytotoxicity (See Supplemental Fig. 1). The IC50 of oseltamivir was 3.49 μM. Neither Ephedra Herb extract nor EFE affected MDCK cell viability (Fig. 5a), whereas Ephedra Herb extract and EFE prevented cell death caused by influenza virus infection in a concentration-dependent manner (Fig. 5b). The IC50 values of Ephedra Herb extract and EFE were 8.6 μg/ml and 8.3 μg/ml, respectively. These results indicate that EFE retains the anti-influenza activity of Ephedra Herb, indicating that this activity is not mediated by ephedrine alkaloids.Fig. 5 Effects of EFE and Ephedra Herb extract on influenza virus infection in MDCK cells. MDCK cells (3 × 104 cells) were incubated in 100 μl of 10 % FCS-MEM in a 96-well plate for 24 h, and then washed with MEM. They were incubated for 72 h at 37 °C in 100 μl of MEM or MEM containing a twofold serial dilution of 50 μg/ml EFE or 50 μg/ml Ephedra Herb extract with (b) or without (a) 100 TCID50 of influenza virus A/WSN/33(H1N1). Next, living cells were stained with crystal violet and the absorbance (560 nm) of each sample was quantified using a microplate reader

7 °C in 100 μl of MEM or MEM containing a twofold serial dilution of 50 μg/ml EFE or 50 μg/ml Ephedra Herb extract with (b) or without (a) 100 TCID50 of influenza virus A/WSN/33(H1N1). Next, living cells were stained with crystal violet and the absorbance (560 nm) of each sample was quantified using a microplate reader Safety assessment of EFE We evaluated the safety of EFE in comparison with that of Ephedra Herb extract and water through a repeated-dose toxicity study. Extract-related death and abnormal clinical signs were not observed in mice treated with EFE or Ephedra Herb extract during the testing period. After 2 weeks, gross abnormalities were not observed in any of the treated mice, and there were no significant differences in mice weights between the three groups (Table 1). However, the colon weights of the male mice subjected to oral administration of EFE or Ephedra Herb extract were significantly lower than those of male mice treated with water, although the reduction was moderate. Differences in the colon weights of the groups of female mice were not significant. There was no significant difference in the weight of any other tissue among the groups (Table 1). Table 1 Body and tissue weight

t were significantly lower than those of male mice treated with water, although the reduction was moderate. Differences in the colon weights of the groups of female mice were not significant. There was no significant difference in the weight of any other tissue among the groups (Table 1). Table 1 Body and tissue weight Sex Group Body weight (g) Thymus (mg) Heart (mg) Lung (mg) Liver (mg) Adrenal gland (mg) Kidney (right) (mg) Kidney (left) (mg) Spleen (mg) Stomach (mg) Small intestine (mg) Cecum (mg) Colon (mg) Male Water AVG 32.3 63.7 158.2 177.5 1939.0 7.2 262.7 244.4 97.8 256.1 1915.1 153.2 382.3 SD 1.5 14.6 13.9 34.0 186.6 1.9 37.7 35.0 16.9 28.3 279.7 14.9 14.6 EFE AVG 30.9 64.9 152.4 171.2 1777.8 7.3 237.1 239.3 90.5 231.0 1741.5 133.5 310.4** SD 2.9 22.2 14.7 17.7 169.4 1.6 27.0 24.5 13.6 28.1 318.3 16.2 35.6 Ephedra Herb AVG 30.8 43.6 151.7 195.8 1674.2 5.8 243.9 238.3 90.3 252.3 1912.2 161.7 326.9* SD 1.2 6.3 17.8 43.2 196.9 0.8 22.8 21.8 17.7 22.7 235.0 33.1 22.5 Female Water AVG 26.3 67.0 140.5 154.3 1350.3 10.0 167.3 157.2 88.1 205.7 1397.0 121.2 338.6 SD 2.3 46.4 14.9 10.2 162.3 1.2 12.3 14.0 13.0 18.2 144.3 14.3 91.3 Ephedra Herb AVG 26.1 55.6 135.9 158.8 1208.8 10.3 159.6 154.4 93.1 215.9 1202.8 109.8 282.1 SD 1.5 7.7 9.4 10.8 121.4 1.1 11.4 10.9 8.1 15.4 92.2 13.2 45.3 EFE AVG 26.5 54.2 142.5 161.6 1223.6 9.8 171.8 172.1 113.9 216.8 1324.4 140.8 274.1 SD 2.8 13.2 24.1 13.9 215.5 2.3 29.2 35.1 42.2 26.7 217.9 25.5 22.1 Sex Group Testis (mg) Vesicular gland (mg) Vas deferens (mg) Epididymis (mg) Ovary (mg) Uterus (mg) Male Water AVG 199.4 170.5 30.8 78.3 – – SD 20.9 75.5 7.0 12.3 – – EFE AVG 199.7 149.3 26.1 77.0 – – SD 24.3 37.5 2.3 6.3 – – Ephedra Herb AVG 198.6 126.0 25.7 74.4 – – SD 13.0 36.8 3.9 5.8 – – Female Water AVG – – – – 13.1 125.9 SD – – – – 1.0 43.7 Ephedra Herb AVG – – – – 12.4 167.1 SD – – – – 2.4 66.9 EFE AVG – – – – 13.8 145.4 SD – – – – 6.2 53.0 * P < 0.05 and ** P < 0.01 vs. mice administered water, by Tukey’s test

24.3 37.5 2.3 6.3 – – Ephedra Herb AVG 198.6 126.0 25.7 74.4 – – SD 13.0 36.8 3.9 5.8 – – Female Water AVG – – – – 13.1 125.9 SD – – – – 1.0 43.7 Ephedra Herb AVG – – – – 12.4 167.1 SD – – – – 2.4 66.9 EFE AVG – – – – 13.8 145.4 SD – – – – 6.2 53.0 * P < 0.05 and ** P < 0.01 vs. mice administered water, by Tukey’s test Serum biochemistry and hematological data are shown in Tables 2 and 3, respectively. The groups showed no significant differences in serum biochemistry parameters (Table 2). The PLT of male mice taking Ephedra Herb extract was significantly higher than that of the water group, but there was no significant difference between the PLT of the male EFE-treated group and that of the male water-treated group (Table 3). The WBC of male mice treated with Ephedra Herb extract was significantly lower than of the water male group, but there was no significant difference between the WBC of the male EFE-treated group and that of the male water-treated group (Table 3). The groups of female mice showed no significant differences in any hematological parameter. These results indicate that EFE may be less toxic than Ephedra Herb extract and could represent a safer alternative. Table 2 Serum biochemical values

the male EFE-treated group and that of the male water-treated group (Table 3). The groups of female mice showed no significant differences in any hematological parameter. These results indicate that EFE may be less toxic than Ephedra Herb extract and could represent a safer alternative. Table 2 Serum biochemical values Sex Parameter ALB (g/dl) AST (IU/l) ALT (IU/l) ALP (IU/l) LDH (IU/l) LAP (IU/l) γ-GT (IU/1) T-BIL (mg/dl) Group AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD Male Water 2.86 ± 0.13 51.8 ± 10.9 34.2 ± 10.8 292.6 ± 51.6 597.2 ± 447.8 58.4 ± 3.8 N.D. 0.08 ± 0.014 EFE 2.96 ± 0.17 50.2 ± 8.3 26.4 ± 2.7 280.2 ± 46.3 765.0 ± 233.2 53.8 ± 3.2 N.D. 0.10 ± 0.024 Ephedra Herb 2.90 ± 0.10 53.4 ± 4.7 29.0 ± 4.3 384.6 ± 191.2 727.0 ± 251.9 60.6 ± 2.1 N.D. 0.08 ± 0.008 Female Water 3.12 ± 0.08 53.4 ± 10.7 25.0 ± 3.9 332.8 ± 38.8 435.6 ± 214.0 54.2 ± 4.7 N.D. 0.08 ± 0.015 EFE 3.06 ± 0.18 55.0 ± 9.7 21.6 ± 2.6 361.2 ± 50.6 645.8 ± 224.5 55.0 ± 3.2 N.D. 0.10 ± 0.004 Ephedra Herb 3.24 ± 0.19 57.8 ± 6.9 21.6 ± 6.1 332.4 ± 60.1 644.2 ± 91.4 58.6 ± 7.2 N.D. 0.08 ± 0.025 N.D not detectable Table 3 Haematological values

Sex Parameter ALB (g/dl) AST (IU/l) ALT (IU/l) ALP (IU/l) LDH (IU/l) LAP (IU/l) γ-GT (IU/1) T-BIL (mg/dl) Group AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD Male Water 2.86 ± 0.13 51.8 ± 10.9 34.2 ± 10.8 292.6 ± 51.6 597.2 ± 447.8 58.4 ± 3.8 N.D. 0.08 ± 0.014 EFE 2.96 ± 0.17 50.2 ± 8.3 26.4 ± 2.7 280.2 ± 46.3 765.0 ± 233.2 53.8 ± 3.2 N.D. 0.10 ± 0.024 Ephedra Herb 2.90 ± 0.10 53.4 ± 4.7 29.0 ± 4.3 384.6 ± 191.2 727.0 ± 251.9 60.6 ± 2.1 N.D. 0.08 ± 0.008 Female Water 3.12 ± 0.08 53.4 ± 10.7 25.0 ± 3.9 332.8 ± 38.8 435.6 ± 214.0 54.2 ± 4.7 N.D. 0.08 ± 0.015 EFE 3.06 ± 0.18 55.0 ± 9.7 21.6 ± 2.6 361.2 ± 50.6 645.8 ± 224.5 55.0 ± 3.2 N.D. 0.10 ± 0.004 Ephedra Herb 3.24 ± 0.19 57.8 ± 6.9 21.6 ± 6.1 332.4 ± 60.1 644.2 ± 91.4 58.6 ± 7.2 N.D. 0.08 ± 0.025 N.D not detectable Table 3 Haematological values Sex Parameter RBC (10,000/μl) Hb (g/dl) Ht (%) PLT (10000/μl) WBC (1000/μl) Neutro (Stab) % Neutro (Seg)% Lympho (%) Mono (%) Group AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD AVG ± SD Male Water 926 ± 25 15.1 ± 0.6 50.0 ± 1.6 49.8 ± 8.4 2.27 ± 0.94 2.2 ± 0.4 33.4 ± 9.4 59.6 ± 9.0 4.8 ± 1.1 EFE 964 ± 21 15.6 ± 0.6 49.6 ± 1.6 48.2 ± 8.6 1.27 ± 0.63 2.6 ± 0.9 18.8 ± 12.9 74.2 ± 13.8 4.4 ± 2.1 Ephedra Herb 976 ± 40 15.2 ± 0.8 49.5 ± 1.8 65.2 ± 10.4* 0.90 ± 0.17* 2.4 ± 0.9 23.6 ± 12.4 70.4 ± 13.1 3.6 ± 2.2 Female Water 947 ± 48 15.5 ± 1.4 49.6 ± 3.5 44.2 ± 12.3 2.49 ± 1.4 2.0 ± 1.0 13.0 ± 11.0 80.0 ± 11.9 5.0 ± 1.2 EFE 980 ± 37 15.9 ± 0.8 49.1 ± 1.8 56.5 ± 7.4 1.6 ± 0.81 1.4 ± 0.5 9.8 ± 12.6 85.0 ± 13.8 3.8 ± 1.6 Ephedra Herb 965 ± 52 15.5 ± 0.8 49.1 ± 1.4 53.1 ± 8.1 1.99 ± 0.76 2.2 ± 0.8 12.4 ± 6.6 80.6 ± 7.4 4.8 ± 2.3 * P < 0.05 vs. mice administered water, by Dunnett’s test

.49 ± 1.4 2.0 ± 1.0 13.0 ± 11.0 80.0 ± 11.9 5.0 ± 1.2 EFE 980 ± 37 15.9 ± 0.8 49.1 ± 1.8 56.5 ± 7.4 1.6 ± 0.81 1.4 ± 0.5 9.8 ± 12.6 85.0 ± 13.8 3.8 ± 1.6 Ephedra Herb 965 ± 52 15.5 ± 0.8 49.1 ± 1.4 53.1 ± 8.1 1.99 ± 0.76 2.2 ± 0.8 12.4 ± 6.6 80.6 ± 7.4 4.8 ± 2.3 * P < 0.05 vs. mice administered water, by Dunnett’s test Discussion Administration of Kampo medicines containing Ephedra Herb is contraindicated for patients with hypertension or cardiomyopathies, while administration of these medicines to elderly patients or those with extreme sensitivity to Ephedra Herb requires special attention. The primary effects and adverse effects of Ephedra Herb have been traditionally believed to be mediated by ephedrine alkaloids, because ephedrine alkaloids are structurally similar to adrenaline and stimulate both sympathetic and parasympathetic neurons. However, recent data suggest that Ephedra Herb contains active ingredients other than ephedrine alkaloids, such as herbacetin glycosides [12], and possesses ephedrine alkaloid-independent pharmacological actions [13]. We hypothesized that several pharmacological actions from the herb would remain after removing the ephedrine alkaloids from it. In the present study, we show that EFE has a c-Met inhibitory action, analgesic effect, and anti-influenza activity without toxicity.

hedrine alkaloid-independent pharmacological actions [13]. We hypothesized that several pharmacological actions from the herb would remain after removing the ephedrine alkaloids from it. In the present study, we show that EFE has a c-Met inhibitory action, analgesic effect, and anti-influenza activity without toxicity. We have previously reported that Ephedra Herb suppresses HGF-induced cancer cell motility by prevention of c-Met phosphorylation via inhibition of its tyrosine kinase activity [4]. Our findings suggest that Ephedra Herb could be utilized as a novel type of c-Met inhibitor in c-Met-expressing cancer patients. However, Ephedra Herb is not suitable for cancer patients, because they do not have sufficient physical strength to tolerate its associated adverse effects. In this study, EFE exhibited efficacy as a c-Met inhibitor similar to that of Ephedra Herb extract, indicating that the c-Met inhibitory activity of Ephedra Herb is not derived from ephedrine alkaloids. Therefore, EFE or pseudo-Kampo medicines containing EFE instead of Ephedra herb could be utilized as treatments for c-Met-expressing cancer patients, because ephedrine alkaloid-induced side effects should not limit their use.

ng that the c-Met inhibitory activity of Ephedra Herb is not derived from ephedrine alkaloids. Therefore, EFE or pseudo-Kampo medicines containing EFE instead of Ephedra herb could be utilized as treatments for c-Met-expressing cancer patients, because ephedrine alkaloid-induced side effects should not limit their use. Kampo medicines containing Ephedra Herb, such as eppikajutsubuto, makyoyokukanto, kakkonto, and maoto, are used to treat myalgia, arthralgia, and rheumatism. The analgesic effects of these Kampo medicines containing Ephedra Herb are explained by the anti-inflammatory action of pseudoephedrine, a constituent of Ephedra Herb. Ephedra Herb has been reported to inhibit acute inflammation [2], and its main anti-inflammatory action is thought to be carried out by pseudoephedrine due to its inhibition of prostaglandin E2 biosynthesis [16]. However, we have recently found that herbacetin, a component of Ephedra Herb, suppressed formalin-induced pain via inhibition of NGF-TrkA signaling [17]. Formalin injection induces two different phases of pain. In the first phase of formalin-induced pain, neurogenic pain is caused by direct activation of type C fibers in nociceptive nerve endings, which release substance P, glutamine, and bradykinin, among other pain mediators. Non-steroidal anti-inflammatory agents (NSAIDs) such as aspirin and diclofenac are ineffective against the first phase of the formalin test [19, 20]. The second phase of formalin-induced pain occurs through ventral horn neuronal activation at the spinal cord level and is characterized as inflammatory pain related to the release of chemical mediators such as histamine, serotonin, bradykinin, prostaglandins, and excitatory amino acids [19, 21]. The pain associated with the second phase of the formalin test is suppressed by NSAIDs. Central analgesics, such as morphine, inhibit the pain associated with the first and second phases of the formalin test. EFE reduced the second phase of formalin-induced pain in the same manner as Ephedra Herb, suggesting that EFE acts on inflammatory pain, while indicating that the analgesic effect of Ephedra Herb is independent from pseudoephedrine. EFE could represent a novel analgesic drug without the adverse effects associated with ephedrine alkaloids.

nd phase of formalin-induced pain in the same manner as Ephedra Herb, suggesting that EFE acts on inflammatory pain, while indicating that the analgesic effect of Ephedra Herb is independent from pseudoephedrine. EFE could represent a novel analgesic drug without the adverse effects associated with ephedrine alkaloids. Hayashi reported that maoto, which contains Ephedra Herb, relieved bone pain associated with treatment with zoledronic acid hydrate, a therapeutic agent used to treat patients with bone lesions derived from bone cancer metastasis [22]. Therefore, EFE and pseudo-Kampo medicines containing EFE instead of Ephedra Herb may treat cancer and cancer-related pain simultaneously.

erb, relieved bone pain associated with treatment with zoledronic acid hydrate, a therapeutic agent used to treat patients with bone lesions derived from bone cancer metastasis [22]. Therefore, EFE and pseudo-Kampo medicines containing EFE instead of Ephedra Herb may treat cancer and cancer-related pain simultaneously. The maoto formula consists of four herbal substances: Apricot Kernel, Cinnamon Bark, Glycyrrhiza, and Ephedra Herb, the principal component. Maoto affects the early phase of influenza virus infection, and its anti-influenza activity is comparable with that of oseltamivir [23]. Furthermore, it has been reported that Ephedra Herb has an inhibitory effect on the acidification of intracellular compartments, such as endosomes and lysosomes, which inhibits the growth of influenza virus [3]. Our study revealed that EFE prevented influenza virus infection, in a manner independent of ephedrine alkaloids. EFE and a pseudo-maoto formula, consisting of the herbal substances mentioned above with EFE instead of Ephedra Herb, have none of the adverse effects associated with ephedrine alkaloids; therefore, they may be of use as therapeutic and prophylactic measures against influenza infection, especially in the elderly.

oids. EFE and a pseudo-maoto formula, consisting of the herbal substances mentioned above with EFE instead of Ephedra Herb, have none of the adverse effects associated with ephedrine alkaloids; therefore, they may be of use as therapeutic and prophylactic measures against influenza infection, especially in the elderly. We evaluated the safety of EFE by carrying out repeated-dose toxicity studies. After 2 weeks of oral administration of Herb Ephedra, EFE, or water, there was no significant difference in the weight of any tissue, except for the colon, among the groups. The colon weights of the male mice treated with Herb Ephedra extract or EFE were significantly lower than that of the water group. However, the reduction in colon weight was small and not associated with morphological abnormalities. Furthermore, colon weight showed no significant difference between the groups of female mice. Thus, EFE has almost no effect on the colon. Neither serum biochemistry data nor hematological data showed any significant differences between mice taking EFE or water. On the other hand, there were significant differences in PLT and WBC between male mice taking Ephedra Herb extract and water. These results suggest that EFE may be safer than Ephedra Herb extract. Ephedra Herb has an antitussive action and removes nasal obstructions by sympathomimetic effects derived from ephedrine alkaloids, but EFE is predicted to produce neither of these effects. Therefore, EFE may be unsuitable for treatment of patients with a common cold.

We evaluated the safety of EFE by carrying out repeated-dose toxicity studies. After 2 weeks of oral administration of Herb Ephedra, EFE, or water, there was no significant difference in the weight of any tissue, except for the colon, among the groups. The colon weights of the male mice treated with Herb Ephedra extract or EFE were significantly lower than that of the water group. However, the reduction in colon weight was small and not associated with morphological abnormalities. Furthermore, colon weight showed no significant difference between the groups of female mice. Thus, EFE has almost no effect on the colon. Neither serum biochemistry data nor hematological data showed any significant differences between mice taking EFE or water. On the other hand, there were significant differences in PLT and WBC between male mice taking Ephedra Herb extract and water. These results suggest that EFE may be safer than Ephedra Herb extract. Ephedra Herb has an antitussive action and removes nasal obstructions by sympathomimetic effects derived from ephedrine alkaloids, but EFE is predicted to produce neither of these effects. Therefore, EFE may be unsuitable for treatment of patients with a common cold. Until now, the pharmacological effects of Ephedra Herb were widely believed to be mediated by ephedrine alkaloids. Harada obtained alkaloid-free Ephedra Herb by selectively removing ephedrine alkaloids through ether extraction under ammonium hydroxide alkali conditions, followed by extraction with water, evaporation of the product to dryness, and addition of the product to a neutral extract, obtained by liquid–liquid partition of the ether extract. Harada reported that alkaloid-free Ephedra Herb extract did not raise blood pressure, inhibit inflammation, or reduce the severity of carrageenan-induced edema [18]; therefore, he concluded that the pharmacological actions of Ephedra Herb were due to ephedrine alkaloids. However, as noted by the author, some components in Ephedra Herb may be altered after exposure to ammonium hydroxide [18]. For example, pyran rearrangements of procyanidins have been reported at alkaline pH [24]. Moreover, Ephedra Herb has been reported to contain proanthocyanidins [7], which might be rearranged by alkaline treatment. Therefore, it is possible that alkaloid-free Ephedra Herb may lack some of the pharmacological activities of Ephedra Herb.

an rearrangements of procyanidins have been reported at alkaline pH [24]. Moreover, Ephedra Herb has been reported to contain proanthocyanidins [7], which might be rearranged by alkaline treatment. Therefore, it is possible that alkaloid-free Ephedra Herb may lack some of the pharmacological activities of Ephedra Herb. In this study, we demonstrated for the first time that Ephedra Herb extract does not lose its pharmacological activity after elimination of its ephedrine alkaloids. Our current objectives include identifying active substances present in the non-alkaloidal fraction of Ephedra Herb extract and obtaining licensing approval for therapeutic use of EFE. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary material 1 (TIFF 30 kb) Supplemental Fig. 1. Effect of oseltamivir on influenza virus infection in MDCK cells. MDCK cells (3 × 104 cells) were incubated in 100 μl of 10 % FCS-minimal essential medium (MEM) in a 96-well plate for 24 h and washed with MEM. Next, the cells were incubated for 72 h at 37 °C in 100 μl of MEM or MEM containing a twofold serial dilution of 10 μM oseltamivir with (B) or without (A) 100 TCID50 of influenza virus A/WSN/33(H1N1). Next, living cells were stained with crystal violet and the absorbance (560 nm) of each sample was quantified using a microplate reader

were incubated for 72 h at 37 °C in 100 μl of MEM or MEM containing a twofold serial dilution of 10 μM oseltamivir with (B) or without (A) 100 TCID50 of influenza virus A/WSN/33(H1N1). Next, living cells were stained with crystal violet and the absorbance (560 nm) of each sample was quantified using a microplate reader This research is supported by the Research on Development of New Drugs from Japan Agency for Medical Research and Development, AMED, and a Grant-in-Aid from the Japan Health Sciences Foundation (Public–private sector joint research on Publicly Essential Drugs). We would like to thank Editage (http://www.editage.jp) for English language editing. Compliances with ethical standards Conflict of interest We have applied for a patent under the regulations of the Patent Cooperation Treaty (PCT).

Introduction With a history dating back 1,600 years, Kampo is a traditional Japanese medicine of therapeutic strategies and diagnostics adapted from traditional Chinese medicine (TCM). Kampo medicines are prescribed and generally prepared with combinations of crude drugs derived from herbs, animals, and minerals. Curative effects are based on synergism between pharmacologically and biologically active constituents producing minimum side-effects. Currently, 254 regulated crude drugs, as well as their processed forms, and 22 Kampo medicines are listed in the Japanese Pharmacopoeia, 16th edition [1], and 294 over-the-counter prescriptions were approved in 2012. A revival of Kampo has been accompanied by scientific re-evaluation of its relevance to modern healthcare [2, 3].

254 regulated crude drugs, as well as their processed forms, and 22 Kampo medicines are listed in the Japanese Pharmacopoeia, 16th edition [1], and 294 over-the-counter prescriptions were approved in 2012. A revival of Kampo has been accompanied by scientific re-evaluation of its relevance to modern healthcare [2, 3]. Kampo is a personalized holistic treatment system for improving the disease states of a patient by returning the patient to a balanced state. The Japanese term Sho refers to the fundamental diagnosis of the patient’s conditions and symptoms and is the term given to the summarization of the diagnostic process [4–6]. A patient’s constitution is diagnosed based on the three states in Sho—Deficiency (Kyo), Middle (Kang), and Excess (Jitsu), referring to states of weakness/hypoactivity/malnutrition, neutrality, and robustness/hyperactivity/overnutrition, respectively (Fig. 1). This systemic diagnostic method has been empirically established based on clinical and pharmacological evidence accumulated over many years. The method is quite unique to Kampo and differs from the diagnostic approach in contemporary medicine, which treats abnormal patient conditions by focusing on individual conditions and symptoms. Accordingly, the prescriptions of Kampo medicines are not limited to the treatment of only targeted symptomatic disease.Fig. 1 Patient constitutions according to Kampo diagnostic criteria Sho. Relationships between patient conditions of Deficiency, Middle, and Excess

s by focusing on individual conditions and symptoms. Accordingly, the prescriptions of Kampo medicines are not limited to the treatment of only targeted symptomatic disease.Fig. 1 Patient constitutions according to Kampo diagnostic criteria Sho. Relationships between patient conditions of Deficiency, Middle, and Excess Recently we proposed that informatics based on the comprehensive and simultaneous analysis of numerous factors may be clarified to classify the complex relationships among crude drugs and formulas, chemical constituents, and pharmacological and biological effects in traditional and modern medicine (reviewed in Refs. [7–9] ). In the present study, we investigated the relationship between the diagnostic criteria Sho and the prescriptions of Kampo medicines based on statistical factorial analysis. Specifically, this study focused on the complex correlation between the combination of patterns of crude drugs in formulating Kampo medicines and a Sho diagnosis of Deficiency/Excess. To systematically understand and interpret the theory of empirical medication in Kampo, multivariate statistics including principal component analysis (PCA) and partial least squares projection to latent structures (PLS) modeling were applied to the correlation between crude drug patterns and Deficiency/Excess. Metabolome analysis, a comprehensive and global chemical analysis of metabolites contained in samples of decoctions of Kampo prescriptions actually prepared from mixtures of crude drugs was also incorporated using mass spectrometry (MS), to substantiate the relationships between Kampo formulas and Deficiency/Excess using the chemical fingerprints of Kampo prescriptions based on the similarities and differences among their chemically complex features. In this study, we begin to unveil the complex system of Kampo medication.

using mass spectrometry (MS), to substantiate the relationships between Kampo formulas and Deficiency/Excess using the chemical fingerprints of Kampo prescriptions based on the similarities and differences among their chemically complex features. In this study, we begin to unveil the complex system of Kampo medication. Materials and methods Kampo formulas Kampo formulas analyzed in this study are listed in the KAMPO section of the KNApSAcK family database [8, 10] and in several Kampo reference texts [11–25]. Medicinal resources of crude drugs used in Kampo formulas are listed in two references texts for crude drugs [1, 26] and are described along with their scientific names and medicinally active region in Table S1. Kampo formulas are introduced using the structured Romanized notation recommended by The Japan Society of Oriental Medicine (Tokyo, Japan), and are abbreviated in subsequent appearances. For metabolome analysis, 25 Kampo prescriptions containing Cinnamon bark (Cinnamomi Cortex), as well as 9 other prescriptions, were selected from Refs [11, 13].

ed using the structured Romanized notation recommended by The Japan Society of Oriental Medicine (Tokyo, Japan), and are abbreviated in subsequent appearances. For metabolome analysis, 25 Kampo prescriptions containing Cinnamon bark (Cinnamomi Cortex), as well as 9 other prescriptions, were selected from Refs [11, 13]. Preparation of decoctions of Kampo prescriptions for metabolome analysis Crude drugs for the preparation of 34 Kampo prescriptions (Table S2) were purchased from Uchida Wakanyaku (Tokyo, Japan) and Tsumura (Tokyo, Japan). The decoctions for metabolome analysis were prepared in accordance with the standard method clinically used at the Diagnosis and Treatment Department of Kampo Medicine, Chiba University Hospital (Chiba, Japan) as follows. Kampo prescriptions tested were packed in an L-size filter bag (Uchida Wakanyaku). The packed prescription was boiled with 600 mL of water for 60 min by using a decocting pot with an electric heater (HMJ3-1000 W; Uchida Wakanyaku).

tment Department of Kampo Medicine, Chiba University Hospital (Chiba, Japan) as follows. Kampo prescriptions tested were packed in an L-size filter bag (Uchida Wakanyaku). The packed prescription was boiled with 600 mL of water for 60 min by using a decocting pot with an electric heater (HMJ3-1000 W; Uchida Wakanyaku). Acquisition of chemical fingerprints by MS-based metabolome analysis of Kampo prescriptions High-accuracy quadrupole time-of-flight (Q-TOF)–MS analysis by direct infusion was performed for acquisition of the chemical fingerprints of Kampo prescriptions using a Q-TOF mass spectrometer (Q-TOF micro™ Mass Spectrometer; JASCO International, Tokyo, Japan) and the resolution was set at 5,000. Ionization of the analyzed samples was performed by positive electrospray ionization (ESI) at m/z range of 85–1,200. The flow rates of cone and desolvation N2 gases were set at 50 and 500 L h−1, respectively. In the positive ESI source, capillary and sample cone voltages were set at 2,800 and 30 V, respectively, with the desolvation and source temperatures set to 150 and 100 °C, respectively. The ion energy was set at 2.0 V in the Q setting. TOF flight tube and tube lens voltages were set at 5,630 and 90 V, respectively, and a microchannel plate detector was set at 2,400 V.

ne voltages were set at 2,800 and 30 V, respectively, with the desolvation and source temperatures set to 150 and 100 °C, respectively. The ion energy was set at 2.0 V in the Q setting. TOF flight tube and tube lens voltages were set at 5,630 and 90 V, respectively, and a microchannel plate detector was set at 2,400 V. The spectral intensity in MS analysis was acquired under the unsaturated condition of peak detection. Test samples were diluted 100-fold with water and injected by syringe pump at a constant rate. The basal conditions of Q-TOF–MS analysis were calibrated by measured values of sodium formate solution (0.1 % formic acid and 5 mM NaOH/90 % acetonitrile). MS data were collected at a scanning rate of 1.0 s per one data scan with 0.1 s of internal delay. In addition, leucine-enkephalin, of which the m/z value in positive ESI analysis was 556.277 ([M + H]+), was analyzed as a reference compound for calibration of the measured values using a syringe pump with a lock spray module, permitting one data scan every 5.0 s. Processing of metabolome data for multivariate analysis A metabolomics data matrix for multivariate analysis was constructed by m/z values and peak intensities of mass spectra (Table S3). Data points of m/z values were reduced by integration of the peak intensities per m/z range of 0.5 (m/z: 85.0,85.5,86.0…1199.0,1199.5). Accordingly, a data matrix consisting of 2,230 m/z values and the corresponding peak intensities was generated. The acquired metabolome data were applied to PCA and PLS regression analysis.

points of m/z values were reduced by integration of the peak intensities per m/z range of 0.5 (m/z: 85.0,85.5,86.0…1199.0,1199.5). Accordingly, a data matrix consisting of 2,230 m/z values and the corresponding peak intensities was generated. The acquired metabolome data were applied to PCA and PLS regression analysis. PCA Kampo medicines are blended herbal formulas composed of several crude drugs sourced from plants, animals, and minerals that are thought to possess complex interactions. The ith Kampo formula can be represented by a vector consisting of quantities of each crude drug in the composition, that is, xi [= (xi1,xi2,…,xij,…,xiM)]. Here, xij represents the quantity of jth crude drug in ith formulas. Thus, the crude drugs in all formulas examined were represented by a matrix as in Eq. (1). 1 X=x11x12…x1Mx21x21…x2M…………xN1xN2…xNM Here, total numbers of Kampo formulas and composite crude drugs are denoted by N and M, respectively. PCA was applied to the matrix to extract the independent factors [27]. The kth principal component is a linear combination of the principal coefficients bkj, that is, Zk=bk1X1+bk2X2+⋯+bkMXM. Here, bk1 was normalized as unity (∑j=1Mbkj2=1). Variables and elements are denoted with upper and lower case letters, respectively. Correlation between the principal components Zk and Zk′(k≠k′) was zero, and the first PC (Z1) has the largest variance, the second PC was that with the second-largest variance, etc. Two parameters, proportion and factor loadings, are used to interpret the distribution of PCA. Proportion Pr(Zk) is represented by the ratio of variance of the kth PC score Var(Zk) to the total variance, and factor loading r(Zk, Xj) is represented by the correlation coefficient between the kth PC score and the jth variable.

arameters, proportion and factor loadings, are used to interpret the distribution of PCA. Proportion Pr(Zk) is represented by the ratio of variance of the kth PC score Var(Zk) to the total variance, and factor loading r(Zk, Xj) is represented by the correlation coefficient between the kth PC score and the jth variable. PLS regression analysis Kampo formulas were analyzed by reciprocal attributes Deficiency and Excess in Sho based on PLS regression analysis. If the ith formula has the attribute Deficiency, then yi was set to −1, and if it has the attribute Excess, then yi was set to 1. The discrimination function is expressed by Eq. (2). 2 y=b0+b1X1+⋯+bjXj+⋯+bMXM Here, b0,b1,…,bM are regression coefficients. A multiple linear regression model is an effective method when the number of variables M is much smaller than that of samples N, but it should be noted that in the case of datasets with strong collinearity between variables, regression coefficients cannot reflect the relationship between variables X with the effect y. In other words, two variables Xu and Xv are positively correlated to y but regression coefficients bu and bv are not always positive, which leads to difficulty in interpreting the relationship between Xu/Xv and y. A multivariate linear equation expressing y with variables derived using PCA by transforming the original variables can avoid the problem of collinearity between variables, but interpretation of the model equation becomes complicated because, Zk is a combination of the original variables Xj (j=1,2,…,M). A PLS model is a method for linearly relating the data matrix X(consisting of N × M) to vector y = (consisting of N × 1), as represented by Eqs. (4) and (5). This makes it possible to overcome the two aforementioned issues, collinearity between variables and indirect interpretation [7]. 3 y=y¯+∑k=1Atkqk+e 4 X=X¯+∑k=1AtkpkT+E Here pk and qk are the loading vectors of X, and the coefficient of y for the kth component, respectively. A is the number of components and tk is a score vector for the kth component. E and e represent the residual matrix and vector, respectively. The number of components, A, is determined by maximization of Q2 by Eq. (5). 5 Q2=1-∑(yobs-ypred)2∑yobs2 A with Q2, a value closest to 1 is the best linear regression equation to explain the relation of X to effect y. The multivariate linear relation denoted by Eq. (2) can be easily derived from Eqs. (3) and (4).

vely. The number of components, A, is determined by maximization of Q2 by Eq. (5). 5 Q2=1-∑(yobs-ypred)2∑yobs2 A with Q2, a value closest to 1 is the best linear regression equation to explain the relation of X to effect y. The multivariate linear relation denoted by Eq. (2) can be easily derived from Eqs. (3) and (4). Results and discussion Factorial analysis of Kampo formulas PCA of unsupervised learning and PLS modeling of supervised learning are very popular in multivariate analysis and have been applied in previous studies to analyze ingredients and metabolome of crude drugs (reviewed in Refs. [7, 28] ). Prior to this study, a comprehensive classification of Kampo formulas (N = 826) was performed using PCA (Fig. 2). For this multivariate analysis, a matrix represented in Eq. (1) was set using the quantities of crude drugs (M = 159) composing each Kampo formula. In the present analysis, PC 1–26 axes where each PC axis accounts for >1 % of variance were selected, and their total variance was 77.9 %. We examined p values by t test in differences in Sho diagnoses of Deficiency and Excess. Of 26 PCs, PC 8 (p = 9.2 × 10−37) and PC 24 (p = 4.9 × 10−17) were the most significantly different (Fig. S1); thus, the variation of 826 Kampo formulas was analyzed in a two-dimensional space using PC 8 and PC 24.Fig. 2 PCA projections for Kampo formulas based on Sho patient constitutions. Classification of Kampo medicines prescribed for Deficiency, Middle, and Excess by PC 8 and 24

the most significantly different (Fig. S1); thus, the variation of 826 Kampo formulas was analyzed in a two-dimensional space using PC 8 and PC 24.Fig. 2 PCA projections for Kampo formulas based on Sho patient constitutions. Classification of Kampo medicines prescribed for Deficiency, Middle, and Excess by PC 8 and 24 The scores of PCA assigned to Kampo formulas prescribed for Deficiency (382 formulas), Middle (218 formulas), and Excess (226 formulas), were projected onto a two-dimensional space (Fig. 2). In this unsupervised analysis, a discriminant boundary between the formulas prescribed for Deficiency and Excess was broadly observed in both axes (dotted line in Fig. 2). The formulas prescribed for Middle also formed a cluster at a nearby discriminant boundary between the formulas for Deficiency and Excess. The contribution of crude drugs for variation of formulas was indicated by the factor loading in PCA. For example, Pueraria root (Puerariae Radix) (0.673) and apricot kernel (Armeniacae Semen) (−0.293) in PC 24, and fennel (Foeniculi Fructus) (0.041) and ass-hide glue (Asini Gelatinum) (−0.252) in PC 8 contributed to the variation of formulas in each degree. These results suggested that the prescribing theory of Kampo medicines may be considered from the point of view of the formulations and quantities of the crude drugs composing the medicines.

li Fructus) (0.041) and ass-hide glue (Asini Gelatinum) (−0.252) in PC 8 contributed to the variation of formulas in each degree. These results suggested that the prescribing theory of Kampo medicines may be considered from the point of view of the formulations and quantities of the crude drugs composing the medicines. To verify the results of PCA, the difference of quantities in the composition of crude drugs between the prescriptions for Deficiency and Excess was examined using PLS regression analysis. From the quantities of composite crude drugs (M = 126) as well as PCA, the distinctions between Kampo formulas (N = 608) assigned to Deficiency (382 formulas) and Excess (226 formulas) were predicted based on a PLS model (Fig. 3). The regression model was constructed using 12 axes, because the Q2 value, which is estimated by cross-validation in PLS modeling, indicated that the fractions of total variation among variables X or Y was maximized in this case (0.863). The distinctions between 601 of 608 Kampo formulas (98.8 %) were accurately predicted from the combination patterns of crude drugs (Fig. 3a).Fig. 3 Classification of Kampo formulas based on Sho patient constitutions by PLS regression analysis. a Y values assigned to Kampo formulas. Negative and positive Y values suggest Deficiency and Excess, respectively. Black line indicates predicted Y values of Kampo formulas. Gray line shows the actual Y values, where −1 and 1 indicate Deficiency and Excess, respectively. The 7 formulas surrounded by a dotted line were contrary to prescriptions for Deficiency and Excess. b Regression coefficients (b) of crude drugs in Kampo medicines. Negative and positive values suggest the contribution of crude drugs to Deficiency and Excess, respectively

indicate Deficiency and Excess, respectively. The 7 formulas surrounded by a dotted line were contrary to prescriptions for Deficiency and Excess. b Regression coefficients (b) of crude drugs in Kampo medicines. Negative and positive values suggest the contribution of crude drugs to Deficiency and Excess, respectively Figure 3b shows the correlation between the linear regression coefficients, which are the b values of the linear equation predicted in Eq. (2), and the use rates of 126 crude drugs used in Kampo formulas prescribed for Deficiency or Excess by PLS regression analysis. The contribution of jth crude drug prescribed for Deficiency or Excess can be easily assessed by the coefficient bj; if bj of jth crude drug is positive, it is used more frequently for Excess, while negative bj values show that the use rate of jth crude drug is greater for Deficiency. Crude drugs that were assigned coefficient b less than −0.1 and >0.1 were selected in Table 1. For example, Japanese angelica root (Angelicae Radix), which is used in 179 of the 608 formulas (29.4 %) was predicted to contribute in cases of Deficiency (b = −0.153). Japanese angelica root generally works to replenish blood via a warming action, resulting in the improvement of weak constitution. This pharmacological role well agrees with its prescription in Kampo formulas for Deficiency. Conversely, Scutellaria root (Scutellariae Radix), which is used in 148 of 608 formulas (24.3 %) was predicted to contribute in cases of Excess (b = 0.267). Scutellaria root generally removes and brings down high fever, and this pharmacological role also agrees with its application in Kampo formulas prescribed for Excess. In addition to Scutellaria root, Bupleurum root (Bupleuri Radix), used in 104 formulas (17.1 %) also has antipyretic action, and was predicted to contribute in cases of Excess (b = 0.158). In actual practice, Kampo medicines composed of both Bupleurum root and Scutellaria root are often prescribed for Excess. In this study, 58 of 608 formulas contained both Bupleurum root and Scutellaria root, and 52 of these 58 formulas are generally prescribed for Excess. Coptis rhizome (Coptidis Rhizoma) is also often used with Scutellaria root in Kampo formulas (57 of the 608 formulas in this study), and these crude drugs resolve excessive fever in the body by their joint action. Interestingly, Coptis rhizome was predicted to contribute in cases of Deficiency (b = −0.118) in contrast to the prediction for Scutellaria root.

so often used with Scutellaria root in Kampo formulas (57 of the 608 formulas in this study), and these crude drugs resolve excessive fever in the body by their joint action. Interestingly, Coptis rhizome was predicted to contribute in cases of Deficiency (b = −0.118) in contrast to the prediction for Scutellaria root. This result is consistent with the actual use of Kampo medicines containing both Scutellaria root and Coptis rhizome, as 20 and 37 formulas of the total 57 formulas are separately prescribed for Deficiency and Excess, respectively. Thus, PLS regression analysis predicted almost the same patterns of use for Deficiency and Excess as those in practice, suggesting that Deficiency and Excess can be interpreted by the composition of crude drugs.Table 1 Linear regression coefficients b of PLS regression analysis assigned to crude drugs composing Kampo formulas prescribed for Deficiency and Excess

st the same patterns of use for Deficiency and Excess as those in practice, suggesting that Deficiency and Excess can be interpreted by the composition of crude drugs.Table 1 Linear regression coefficients b of PLS regression analysis assigned to crude drugs composing Kampo formulas prescribed for Deficiency and Excess Crude drug b Value (<−0.1) No. of formulas Percentage Trichosanthes root −0.397 6 0.99 Oyster shell −0.276 17 2.80 Bamboo shavings −0.202 20 3.29 Processed ginger −0.200 86 14.14 Japanese angelica root −0.153 179 29.44 Hemp fruit −0.149 20 3.29 Rehmannia root (steamed) −0.134 19 3.13 Lycium bark −0.130 6 0.99 Immature orange −0.127 66 10.86 Orange −0.123 12 1.97 Coptis rhizome −0.118 66 10.86 Achyranthes root −0.113 14 2.30 Glycyrrhiza −0.109 429 70.56 Euodia fruit −0.106 21 3.45 Crude drug b Value (>0.1) No. of formulas Percentage Scutellaria root 0.267 148 24.34 Akebia stem 0.265 19 3.13 Rhubarb 0.249 97 15.95 Polyporus sclerotium 0.243 17 2.80 Common rush 0.197 13 2.14 Trichosanthes seed 0.171 8 1.32 Ephedra herb 0.170 47 7.73 Areca peel 0.169 13 2.14 Bupleurum root 0.158 104 17.11 Saussurea root 0.155 37 6.09 Saposhnikovia root and rhizome 0.148 41 6.74 Aralia rhizome 0.138 10 1.64 Japanese cherry bark 0.134 8 1.32 Plantago seed 0.121 15 2.47 Pueraria root 0.119 17 2.80 Cyperus rhizome 0.107 37 6.09 Schisandra fruit 0.106 26 4.28 Magnolia bark 0.105 59 9.70 The b values less than −0.1 and >0.1 were listed

root 0.155 37 6.09 Saposhnikovia root and rhizome 0.148 41 6.74 Aralia rhizome 0.138 10 1.64 Japanese cherry bark 0.134 8 1.32 Plantago seed 0.121 15 2.47 Pueraria root 0.119 17 2.80 Cyperus rhizome 0.107 37 6.09 Schisandra fruit 0.106 26 4.28 Magnolia bark 0.105 59 9.70 The b values less than −0.1 and >0.1 were listed As shown by the dotted line in Fig. 3a, Y values −0.221 to 0.479 predicted for 3 of 5 formulas—Keishi-ka-shakuyaku-daio-To (KSTSD; Keishikashakuyakudaioto) variants, Daio-bushi-To (DBST; Daiobushito) and Mao-bushi-saishin-To (MBST; Maobushisaishinto)—were contrary to the prescriptions for Deficiency and Excess. KSTSD was predicted for Deficiency but is generally prescribed for Excess. The formula is prepared from Keishi-ka-shakuyaku-To (KSTS; Keishikashakuyakuto), usually prescribed for Deficiency, by adding rhubarb (Rhei Rhizoma) (b = 0.249 in Fig. 3b), which is often added to Excess prescriptions (see nos. 6 and 7 in Tables 2 and S2). This difference of use from Excess to Deficiency may be due to its use as a combination of crude drugs in KSTS, such that the addition of rhubarb to KSTS did not affect the predicted prescription of KSTSD for Deficiency or Excess. DBST containing rhubarb is also generally prescribed for Excess, but was predicted for Deficiency, probably due to the presence of the two other crude drugs in DBST, Asiasarum root (Asiasari Radix) (b = −0.053) and processed aconite root (Processi Aconiti Radix) (b = −0.099), because they are used to treat Deficiency. MBST was predicted for Excess but prescribed for Deficiency, and also contains Asiasarum root and processed aconite root, which play important roles in Deficiency; however, the effect of Ephedra herb (Ephedrae Herba) (b = 0.170 in Fig. 3b) for Excess may have contributed to this difference.Table 2 34 Kampo prescriptions analyzed by direct infusion Q-TOF–MS for metabolomic analysis

d also contains Asiasarum root and processed aconite root, which play important roles in Deficiency; however, the effect of Ephedra herb (Ephedrae Herba) (b = 0.170 in Fig. 3b) for Excess may have contributed to this difference.Table 2 34 Kampo prescriptions analyzed by direct infusion Q-TOF–MS for metabolomic analysis Prescription no. Kampo prescription Patient constitution according to Sho

d also contains Asiasarum root and processed aconite root, which play important roles in Deficiency; however, the effect of Ephedra herb (Ephedrae Herba) (b = 0.170 in Fig. 3b) for Excess may have contributed to this difference.Table 2 34 Kampo prescriptions analyzed by direct infusion Q-TOF–MS for metabolomic analysis Prescription no. Kampo prescription Patient constitution according to Sho 1 Keishi-To Deficiency 2 Keishi-ka-ogi-To Deficiency 3 Keishi-ka-kakkon-To Deficiency 4 Keishi-ka-kei-To Deficiency 5 Keishi-ka-koboku-kyonin-To Deficiency 6 Keishi-ka-shakuyaku-To (KSTS) Deficiency 7 Keishi-ka-shakuyaku-daio-To (KSTSD) Excess 8 Keishi-ka-shakyaku-shokyo-ninjin-To Deficiency 9 Keishi-ka-ryukotsu-borei-To Deficiency 10 Keishi-ni-eppi-Itto (Keishinieppiitto) Excess 11 Keishi-ni-mao-Itto (Keishinimaoitto) Deficiency 12 Keishi-mao-kakuhan-To Middle 13 I-rei-To Middle 14 Kakkon-To Excess 15 Kakkon-To-ka-senkyu-shin’i Excess 16 Kakkon-ka-hange-To Excess 17 Goshaku-San Deficiency 18 Saiko-keishi-To Deficiency 19 Keishi-kanzo-To (Keishikanzoto) Deficiency 20 Keishi-kyo-kei-ka-bukuryo-To (Keishikyokeikabukuryoto) Deficiency 21 Keishi-kyo-shakuyaku-To (Keishikyoshakuyakuto) Deficiency 22 Keishi-ninjin-To Deficiency 23 Keishi-bukuryo-Gan (KBG) Excess 24 Ogi-keishi-gomotsu-To Deficiency 25 Ogon-To Excess 26 Ogon-ka-hange-shokyo-To Excess 27 Kikyo-To Middle 28 Sai-kan-To Excess 29 Sho-saiko-To Excess 30 Sho-saiko-To-ka-kikyo-sekko Excess 31 Shokyo-shashin-To Deficiency 32 Dai-saiko-To Excess 33 Toki-shigyaku-To Deficiency 34 Toki-shigyaku-ka-goshuyu-shokyo-To Deficiency Kampo prescriptions analyzed are listed alongside the appropriate patient Sho diagnoses

Kikyo-To Middle 28 Sai-kan-To Excess 29 Sho-saiko-To Excess 30 Sho-saiko-To-ka-kikyo-sekko Excess 31 Shokyo-shashin-To Deficiency 32 Dai-saiko-To Excess 33 Toki-shigyaku-To Deficiency 34 Toki-shigyaku-ka-goshuyu-shokyo-To Deficiency Kampo prescriptions analyzed are listed alongside the appropriate patient Sho diagnoses Thus, PLS regression analysis distinguished between Kampo formulas for Deficiency and Excess as well as did PCA (Fig. 2), and enabled an assessment of the contributions of crude drugs to those groups.

Kikyo-To Middle 28 Sai-kan-To Excess 29 Sho-saiko-To Excess 30 Sho-saiko-To-ka-kikyo-sekko Excess 31 Shokyo-shashin-To Deficiency 32 Dai-saiko-To Excess 33 Toki-shigyaku-To Deficiency 34 Toki-shigyaku-ka-goshuyu-shokyo-To Deficiency Kampo prescriptions analyzed are listed alongside the appropriate patient Sho diagnoses Thus, PLS regression analysis distinguished between Kampo formulas for Deficiency and Excess as well as did PCA (Fig. 2), and enabled an assessment of the contributions of crude drugs to those groups. Metabolome analysis of Kampo prescriptions Metabolomic analysis revealed the chemical fingerprints of prescriptions to verify correlations between Kampo concepts and crude drugs. The principal crude drug, Cinnamon bark, as well as other drugs in 34 prescriptions were analyzed by Q-TOF–MS with positive ESI by direct infusion (Tables 2, S2, S3), and factor analysis was applied. Most metabolomes could be classified for Sho prescription by PCA (Fig. 4). Variations in prescriptions were represented by PCs 1 and 2 at 60.2 % (Z1–Z2) of variance, because PCs 1–5 contributed 89.4 % (Z1–Z5) [32.5 % (Z1) and 27.7 % (Z2), 12.2 % (Z3), 10.6 % (Z4) and 6.4 % (Z5)]. Variations in 34 prescriptions were assessed in terms of prescription for patient conditions Deficiency/Middle/Excess. All except 2 Deficiency/Excess prescriptions were classified, and Middle prescriptions were plotted around the boundary line. Compared with the results of PCA for Kampo formulas (Fig. 2), these results were perhaps due to differences in the data properties of factor analysis. Variations in prescriptions for Deficiency/Excess based on metabolomes were interpreted by lower dimensions consisting of PCs 1 and 2 (Fig. 4) rather than by combinations of PCs 8 and 24 (Fig. 2). Thus, the chemical fingerprints of Kampo prescriptions acquired by metabolomic analysis showed greater diversity than the formulation of crude drugs in Kampo medicines.Fig. 4 Variation of Kampo prescriptions indicated by metabolome analysis. Total variance contributions of PCs 1 and 2 were 32.5 % (Z 1) and 27.7 % (Z 2), respectively. The PCA result displays variation from the point of view of Sho diagnoses of Deficiency, Middle, and Excess

ormulation of crude drugs in Kampo medicines.Fig. 4 Variation of Kampo prescriptions indicated by metabolome analysis. Total variance contributions of PCs 1 and 2 were 32.5 % (Z 1) and 27.7 % (Z 2), respectively. The PCA result displays variation from the point of view of Sho diagnoses of Deficiency, Middle, and Excess Metabolomic data were acquired by chemical analysis and contained latent information about crude drugs in the formulas; thus, multivariate analysis was useful in considering individual substances. KSTSD (prescription no. 7) was plotted as a Deficiency group outlier, although it is generally prescribed for Excess (Fig. 4); this may suggest that it was plotted near KSTS (prescription no. 6) on the PCA score plot because of the addition of crude drug rhubarb to KSTS. PLS regression analysis was also applied to the metabolomic data of 31 of 34 Kampo prescriptions excluding prescriptions for Middle (Fig. 5). Thus, KSTSD was predicted for Deficiency at a recognition rate of 87.1 %, but if KSTSD was removed from the 31 prescriptions, the recognition rate of the remaining 30 prescriptions for Deficiency/Excess increased to 93.3 %. The results suggest that the pharmacological and biological effects of rhubarb on Deficiency prescriptions are exceedingly high compared with other crude drugs in KSTS. Keishi-bukuryo-Gan (KBG; Keishibukuryogan) (prescription no. 23), which is usually prescribed for Excess and Middle, was plotted in the Deficiency group (Fig. 4); this outlying plot may be a result of differences in dosage form, because all samples were dissolved in water for metabolomic analysis. KBG is given in the form of a solid tablet (-Gan in Japanese), while the other 33 Kampo prescriptions are in the form of liquid and powder (-To and -San in Japanese, respectively). Thus, the efficacy of Kampo prescriptions appeared to differ by dosage form.Fig. 5 PLS regression analysis of the chemical fingerprints obtained from metabolomic analysis of Kampo prescriptions. The original Y values were set to ‘−1’ in Deficiency or ‘1’ in Excess. The predicted Y values for Deficiency or Excess were calculated from the metabolomic data of 31 Kampo prescriptions (a) and 30 Kampo prescriptions (when KSTSD was removed) (prescription no. 7) (b). Arrow indicates KSTSD

lysis of Kampo prescriptions. The original Y values were set to ‘−1’ in Deficiency or ‘1’ in Excess. The predicted Y values for Deficiency or Excess were calculated from the metabolomic data of 31 Kampo prescriptions (a) and 30 Kampo prescriptions (when KSTSD was removed) (prescription no. 7) (b). Arrow indicates KSTSD Conclusions In conclusion, systematic and comprehensive interpretation of Kampo medication was achieved by integration of the accumulated informatics. The factor analyses simultaneously processed a number of Kampo medicines, enabling comprehensive classification and correlation analysis of multiple factors; namely, Sho diagnosis of Deficiency/Excess, Kampo formulas, and the crude drugs. This finding and suggestion could be chemically substantiated by the metabolome analysis of Kampo prescriptions. The approaches in this study could lead to systems medicine, a comprehensive analysis of correlations between ingredients and practices in traditional (empirically verified) and modern medicines [7]. Factor and correlation analyses may lead to generalization of the diagnostic criteria and prescribing methods of Kampo, and be applied to other traditional medicines such as TCM and Jamu (an Indonesian traditional medicine). Moreover, Kampo medicines more suitable for the health of people in the modern age may be proposed from the new information assembled by systems medicine. Practically, TCM tends to create new combinations of crude drugs for therapeutic use, while Kampo medicine is often dependent on traditional prescriptions [29]. We believe that factor analysis of a complex medical system by informatics will have implications for systems medicine.

ormation assembled by systems medicine. Practically, TCM tends to create new combinations of crude drugs for therapeutic use, while Kampo medicine is often dependent on traditional prescriptions [29]. We believe that factor analysis of a complex medical system by informatics will have implications for systems medicine. Electronic supplementary material Supplementary Table S1 (XLSX 16 kb) Supplementary Table S2 (XLSX 14 kb) Supplementary Table S3 (XLSX 560 kb) Supplementary Fig. S1 (PDF 341 kb) Abbreviations of Kampo formulas DBSTDaio-bushi-To (Daiobushito) KBGKeishi-bukuryo-Gan (Keishibukuryogan) KSTSKeishi-ka-shakuyaku-To (Keishikashakuyakuto) KSTSDKeishi-ka-shakuyaku-daio-To (Keishikashakuyakudaioto) MBSTMao-bushi-saishin-To (Maobushisaishinto) T. Okada, F. M. Afendi and M. Yamazaki contributed equally to this work. This study was partially funded by a research grant from the Japanese Ministry of Health, Labour and Welfare (Grant Number 24202001) and a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (Grant Number 25860085). Conflict of interest The authors declare that they have no conflict of interest.

Introduction Ephedra Herb (EH) is defined as the terrestrial stem of Ephedra sinica Staf, E. intermedia Schrenk et C. A. Meyer, or E. equisetina Bunge (Ephedraceae) [1]. It is one of the most important crude drugs used in Japan and is a component of many Kampo formulae such as maoto, kakkonto, eppikajutsubuto, makyoyokukanto, and maobushisaishinto (http://kconsort.umin.jp/framepage.html) that have been used to treat rheumatism, myalgia, and arthralgia [2–4]. The analgesic actions of these Kampo medicines are thought to result from the anti-inflammatory properties of EH [3, 5, 6]. Recently, we demonstrated that EH extract (EHE) suppresses the late phase of formalin-induced pain [7], which is characterized as an inflammatory pain related to the release of chemical mediators such as histamine, serotonin, bradykinin (BK), and prostaglandins (PGs), and is suppressed by nonsteroidal anti-inflammatory drugs [8]. Thus, EH is thought to be involved in the regulation of inflammatory pain. However, the molecular mechanism of its analgesic effect remains to be clarified.

lease of chemical mediators such as histamine, serotonin, bradykinin (BK), and prostaglandins (PGs), and is suppressed by nonsteroidal anti-inflammatory drugs [8]. Thus, EH is thought to be involved in the regulation of inflammatory pain. However, the molecular mechanism of its analgesic effect remains to be clarified. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective ligand-gated cation channel expressed in primary sensory nerves [9]. It is an integrator of many noxious physical and chemical stimuli such as heat (>43 °C), proton, and capsaicin [9, 10], as well as endogenous lipids such as anandamide and 12-(S)-hydroperoxyeicosatetraenoic acid [11–13]. Upon the activation of TRPV1 by these stimuli, a variety of pro-algesic neuropeptides such as substance P and calcitonin gene-related peptide are released from the peripheral nerve terminals, and subsequently, a neurogenic inflammation is induced [14]. Furthermore, TRPV1 is sensitized by the stimulation of certain inflammatory mediators such as BK and PGs, and is transported to the plasma membrane by nerve growth factor stimulation, which indirectly modulates TRPV1 through its phosphorylation [14–16]. These inflammatory mediators reduce the temperature threshold for activation of TRPV1 from 43 to 35 °C, thus inducing inflammatory pain [10]. In this study, we assessed the effects of EHE on mouse TRPV1-expressing Flp-In293 cells and capsaicin-induced pain in vivo, to investigate whether EHE elicits a direct nociceptive action.

Transient receptor potential vanilloid 1 (TRPV1) is a nonselective ligand-gated cation channel expressed in primary sensory nerves [9]. It is an integrator of many noxious physical and chemical stimuli such as heat (>43 °C), proton, and capsaicin [9, 10], as well as endogenous lipids such as anandamide and 12-(S)-hydroperoxyeicosatetraenoic acid [11–13]. Upon the activation of TRPV1 by these stimuli, a variety of pro-algesic neuropeptides such as substance P and calcitonin gene-related peptide are released from the peripheral nerve terminals, and subsequently, a neurogenic inflammation is induced [14]. Furthermore, TRPV1 is sensitized by the stimulation of certain inflammatory mediators such as BK and PGs, and is transported to the plasma membrane by nerve growth factor stimulation, which indirectly modulates TRPV1 through its phosphorylation [14–16]. These inflammatory mediators reduce the temperature threshold for activation of TRPV1 from 43 to 35 °C, thus inducing inflammatory pain [10]. In this study, we assessed the effects of EHE on mouse TRPV1-expressing Flp-In293 cells and capsaicin-induced pain in vivo, to investigate whether EHE elicits a direct nociceptive action. Materials and methods Materials EHE (Lot. 2091037010) was purchased from Tsumura & Co. (Tokyo, Japan). The ephedrine content of EHE was quantified by HPLC, and was approximately 2 % (Fig. S1). Capsaicin and N-(4-tert-butylphenyl)-1,2-dihydro-4-(3-chloropyridine-2-yl) tetrahydropyrazine-1-carboxamide (BCTC) were purchased from Funakoshi Co., Ltd. (Tokyo, Japan).

091037010) was purchased from Tsumura & Co. (Tokyo, Japan). The ephedrine content of EHE was quantified by HPLC, and was approximately 2 % (Fig. S1). Capsaicin and N-(4-tert-butylphenyl)-1,2-dihydro-4-(3-chloropyridine-2-yl) tetrahydropyrazine-1-carboxamide (BCTC) were purchased from Funakoshi Co., Ltd. (Tokyo, Japan). Animals Specific pathogen-free ddY mice (5-week-old, male) were purchased from Japan SLC, Inc. (Shizuoka, Japan). Prior to experimentation, the mice were acclimatized for 1 week at a temperature of 25 ± 2 °C, humidity of 50 ± 10 %, and a 12-h light/12-h dark cycle. All animal experiments were performed between 10:00 a.m. and 5:00 p.m. The protocol for animal experiments was approved by the Institutional Animal Care and Use Committee of Kitasato University, and was performed in accordance with the Kitasato University guidelines for animal care, handling, and termination, which are in line with the international and Japanese guidelines for animal care and welfare.

for animal experiments was approved by the Institutional Animal Care and Use Committee of Kitasato University, and was performed in accordance with the Kitasato University guidelines for animal care, handling, and termination, which are in line with the international and Japanese guidelines for animal care and welfare. Transfectant Flp-In293 cells Flp-In293 cells, derived from the HEK293 cell line containing a stably integrated FRT site, were transfected using Lipofectamine (Thermo Fisher Scientific Inc., Waltham, MA, USA) with pOG44 vector (Thermo Fisher Scientific Inc.) and pEF5/FRT/V5-DEST vector (Thermo Fisher Scientific Inc.) harboring full-length mouse TRPV1 cDNA (GeneCopoeia Inc., Rockville, MD, USA). The cells were cultivated in hygromycin B (200 μg/ml) for 4 weeks, and stable mouse TRPV1-expressing transfectants (mTRPV1/Flp-In293 cells) were established. The expression levels of TRPV1 protein in mTRPV1/Flp-In293 and Flp-In293 cells were determined by Western blotting using anti-TRPV1 antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Cell culture The mTRPV1/Flp-In293 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 2 mM GlutaMAX, 0.1 mM MEM non-essential amino acid solution (MEM NEAA), 200 µg/ml hygromycin B, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 5 % CO2. Flp-In293 cells were cultured under the same conditions without hygromycin B. The reagents for cell culture were purchased from Thermo Fisher Scientific Inc.

mM GlutaMAX, 0.1 mM MEM non-essential amino acid solution (MEM NEAA), 200 µg/ml hygromycin B, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 5 % CO2. Flp-In293 cells were cultured under the same conditions without hygromycin B. The reagents for cell culture were purchased from Thermo Fisher Scientific Inc. Measurement of intracellular Ca2+ concentration in mTRPV1/Flp-In293 and Flp-In293 cells Measurement of intracellular Ca2+ concentration was performed as previously described [17, 18]. Mouse TRPV1/Flp-In293 and Flp-In293 cells (4 × 104 cells/well) were cultured in 100 µl of DMEM with 10 % FBS, 2 mM GlutaMAX, and 0.1 mM MEM NEAA in 96-well, poly-d-lysine black-walled, clear-bottomed plates (Greiner Bio-One, Frickenhausen, Germany) for 24 h. The medium was exchanged and the cells incubated in Hank’s balanced salt solution (HBSS) buffer and 20 mM HEPES buffer (pH 7.4) containing FLIPR® calcium 5 assay reagent (Molecular Devices, Sunnyvale, CA, USA) for 1 h at 37 °C. The fluorescence was immediately measured using a FlexStation 3 microplate reader (Molecular Devices) (excitation at 485 nm and emission at 525 nm, using a 515-nm cut-off) for 20 s. Subsequently, HBSS buffer containing 0–1000 µg/ml of EHE or 0–0.2 µM capsaicin was added, and the fluorescence was immediately measured.

. The fluorescence was immediately measured using a FlexStation 3 microplate reader (Molecular Devices) (excitation at 485 nm and emission at 525 nm, using a 515-nm cut-off) for 20 s. Subsequently, HBSS buffer containing 0–1000 µg/ml of EHE or 0–0.2 µM capsaicin was added, and the fluorescence was immediately measured. To examine the effect of BCTC, a TRPV1 antagonist, on EHE- or capsaicin-induced increase in intracellular Ca2+ concentration, HBSS buffer containing 0–10 nM BCTC was added after the initial fluorescence measurement was made. After 60 s, HBSS buffer containing 1000 µg/ml EHE or 0.00625 µM capsaicin was added, and the fluorescence was immediately measured. In these experiments, capsaicin and BCTC were dissolved in dimethyl sulfoxide (DMSO) and diluted with HBSS buffer. The final DMSO concentration was adjusted to within 0.1–0.2 %. The data were analyzed by Soft Max Pro 5.4 software (Molecular Devices).

M capsaicin was added, and the fluorescence was immediately measured. In these experiments, capsaicin and BCTC were dissolved in dimethyl sulfoxide (DMSO) and diluted with HBSS buffer. The final DMSO concentration was adjusted to within 0.1–0.2 %. The data were analyzed by Soft Max Pro 5.4 software (Molecular Devices). EHE- or capsaicin-induced paw licking test The capsaicin-induced paw licking test was performed as previously described [19]. Mice were grouped into 4 or 5 groups treated with different doses of capsaicin or EHE, with 5–8 mice in each group. The mice were placed individually for adaptation in transparent acrylic cylinder cages with a height of 200 mm and a diameter of 100 mm. After 20 min, the mice were injected with 10 μl of vehicle (DMSO:Tween-80:saline = 1:1:8) containing 0.031–3.1 μg/paw capsaicin, or 0.3–10 mg/paw EHE, into the plantar surface of the left hind paw using a microsyringe (MS-NG50; Ito Microsyringe Co., Ltd, Tokyo, Japan) with a sharp-edged needle (28 G; Ito Microsyringe, Co., Ltd). The licking behavior was recorded using a digital video camera for a period of 5 min. To investigate the effect of BCTC on EHE- or capsaicin-induced pain, the mice were grouped into 3 or 4 groups treated with three different doses of BCTC, with 3–7 mice in each group. The experiment was performed as described above except by injecting the mice with 10 μl of the vehicle containing 5 mg/paw EHE, together with 0.037–0.37 μg/paw BCTC.

BCTC on EHE- or capsaicin-induced pain, the mice were grouped into 3 or 4 groups treated with three different doses of BCTC, with 3–7 mice in each group. The experiment was performed as described above except by injecting the mice with 10 μl of the vehicle containing 5 mg/paw EHE, together with 0.037–0.37 μg/paw BCTC. To compare the time course of EHE-induced paw licking with that induced by capsaicin, the mice were grouped into 3 groups treated with vehicle, capsaicin, and EHE, with 3 mice in each group. The mice were injected with 10 µl of the vehicle, with or without 3 mg/paw EHE or 0.92 μg/paw capsaicin, into the plantar surface of the left hind paw. The licking behavior was recorded using the digital video camera for a period of 60 min. Analgesic effect of i.d. administration of EHE The mice were grouped into 6 groups treated with vehicle, EHE or capsaicin, with 6 mice in each group. The mice were injected with 10 µl of the vehicle, with or without 3 mg/paw EHE or 0.92 μg/paw capsaicin into the plantar surface of the left hind paw. After 30 or 60 min, 0.18 μg/paw capsaicin (10 µl) was injected into the same area, and the licking time was measured.

hicle, EHE or capsaicin, with 6 mice in each group. The mice were injected with 10 µl of the vehicle, with or without 3 mg/paw EHE or 0.92 μg/paw capsaicin into the plantar surface of the left hind paw. After 30 or 60 min, 0.18 μg/paw capsaicin (10 µl) was injected into the same area, and the licking time was measured. Analgesic effect of oral (p.o.) administration of EHE The mice were grouped into 14 groups treated with either EHE or water, with 4–8 mice in each group. The mice were administered p.o. with 700 mg/kg of EHE. After a period of 0, 0.25, 0.5, 1, 2, 6, and 24 h, 0.18 μg/paw capsaicin (10 µl) was injected into the plantar surface of the left hind paw, and the licking time was measured for 5 min. To analyze the effects of different doses of EHE, the mice were administered p.o. with 87.5–700 mg/kg of EHE. After 30 min, 0.18 μg/paw capsaicin (10 µl) was injected as described above. Rotarod test The rotarod test was performed as previously reported [20]. A rotarod treadmill (MK-600; Muromachi Kikai Co., Ltd, Tokyo, Japan) was used in this study. To adapt the mice to the rotarod, they were placed on the rod rotating at 28 rpm for 5 min each hour six times, 1 day before the test. On the day of the test, another episode of training was performed, and the mice that fell off the rotating shaft were excluded from the experiment. The mice were administered water or 175–700 mg/kg of EHE orally. After 30 min, we measured the endurance time the mice could remain on the rotarod.

, 1 day before the test. On the day of the test, another episode of training was performed, and the mice that fell off the rotating shaft were excluded from the experiment. The mice were administered water or 175–700 mg/kg of EHE orally. After 30 min, we measured the endurance time the mice could remain on the rotarod. Statistical analysis All data are expressed as mean ± standard error of the mean (SEM) and analyzed by one-way analysis of variance (ANOVA). Significant differences between the control and treatment groups were determined by Dunnett’s multiple comparison test or Student’s t test. All statistical analyses were performed using Prism 5 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was determined based on values of p < 0.05, 0.01, and 0.001.

ficant differences between the control and treatment groups were determined by Dunnett’s multiple comparison test or Student’s t test. All statistical analyses were performed using Prism 5 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was determined based on values of p < 0.05, 0.01, and 0.001. Results Confirmation of functional expression of mTRPV1 in mTRPV1/Flp-In293 cells Expression levels of mTRPV1 were determined by Western blotting (Fig. 1a). Mouse TRPV1 was detected in mTRPV1/Flp-In293 cells, but not in Flp-In293 cells. The intracellular Ca2+ concentration in mTRPV1/Flp-In293 cells was increased by capsaicin, but that in Flp-In293 cells was unaffected (Fig. 1b). In addition, capsaicin produced a dose-dependent increase in intracellular Ca2+ concentration in mTRPV1/Flp-In293 cells (Fig. 1c). A capsaicin concentration of 0.00625 μM, which induced approximately 80 % of the maximum response produced by 0.2 μM capsaicin, was used to investigate the inhibitory effects of the TRPV1 antagonist, BCTC. As shown in Fig. 1d, BCTC dose-dependently inhibited the 0.00625 μM capsaicin-induced increase in intracellular Ca2+ concentration. These results indicated that mTRPV1 was functionally expressed in mTRPV1/Flp-In293 cells.Fig. 1 Confirmation of functional expression of mTRPV1 in mTRPV1/Flp-In293 cells. a Expression levels of mTRPV1 in mTRPV1/Flp-In293 and Flp-In293 cells were determined by Western blot analysis. b The effect of 0.2 μM capsaicin (Cap) on the uptake of Ca2+ in mTRPV1/Flp-In293 and Flp-In293 cells. c The ratio of fluorescence intensity induced by different concentrations of capsaicin over that induced by 0.2 µM capsaicin. d The ratio of fluorescence intensity induced by 0.00625 µM capsaicin in the presence of 0−10 nM BCTC over that induced in its absence. Each assay was performed in triplicate. The error bar represents the standard error. Statistical significance was determined with Tukey’s test; *p < 0.001 vs Flp-In293 cells

capsaicin. d The ratio of fluorescence intensity induced by 0.00625 µM capsaicin in the presence of 0−10 nM BCTC over that induced in its absence. Each assay was performed in triplicate. The error bar represents the standard error. Statistical significance was determined with Tukey’s test; *p < 0.001 vs Flp-In293 cells Effect of EHE on TRPV1 studied using mTRPV1/Flp-In293 cells The presence of EHE (1000 μg/ml) significantly increased the intracellular Ca2+ concentration in mTRPV1/Flp-In293 cells, but not in Flp-In293 cells (Fig. 2a). In addition, the increase in intracellular Ca2+ concentration by EHE was dose-dependent, with an EC50 value of 271.6 µg/ml (Fig. 2b), and was inhibited by BCTC in a similar manner (Fig. 2c). These results suggested that EHE directly activates mTRPV1. The time course of increasing intracellular Ca2+ concentration in mTRPV1/Flp-In293 cells induced by 1000 µg/ml EHE and 0.2 μM capsaicin is shown in Fig. 2d. The rate of increase of intracellular Ca2+ concentration in the presence of EHE was much slower than in the presence of capsaicin.Fig. 2 Effect of EHE on the uptake of Ca2+ into mTRPV1/Flp-In293 cells. a The effects of EHE on mTRPV1/Flp-In293 and Flp-In293 cells. b The ratio of fluorescence intensity induced by 0–1000 µg/ml EHE over that induced by 0.2 µM capsaicin. c The ratio of fluorescence intensity induced by 1000 µg/ml EHE in the presence of 0–10 nM BCTC over that induced in its absence. d Fluorescence kinetics measuring Ca2+ uptake by mTRPV1/Flp-In293 cells induced by 1000 µg/ml EHE (closed circle) and 0.2 μM capsaicin (open circle). Each assay was performed in triplicate. The error bar represents the standard error. Statistical significance was determined with Tukey’s test; *p < 0.001 vs Flp-In293 cells

luorescence kinetics measuring Ca2+ uptake by mTRPV1/Flp-In293 cells induced by 1000 µg/ml EHE (closed circle) and 0.2 μM capsaicin (open circle). Each assay was performed in triplicate. The error bar represents the standard error. Statistical significance was determined with Tukey’s test; *p < 0.001 vs Flp-In293 cells EHE-induced nociceptive pain through the stimulation of TRPV1 An i.d. injection of 0.3–10 mg/paw EHE (10 μl) into the hind paw of mice induced paw licking, a pain-related behavior, and the paw licking times increased in a dose-dependent manner (Fig. 3a). Similarly, the paw licking times decreased in a dose-dependent manner upon co-injection with BCTC (Fig. 3b), suggesting that EHE induces nociceptive pain through the activation of TRPV1. It is well known that capsaicin induces transient paw licking. The time course of EHE-induced paw licking was similar to that induced by capsaicin. The paw licking behavior induced by capsaicin and EHE was not observed starting from 25 min after the injection (Fig. 3c).Fig. 3 EHE-induced nociceptive pain. Mice were injected with 10 µl of vehicle (DMSO:Tween-80:physiological saline = 1:1:8) containing a 0.3–10 mg/paw EHE, b 5 mg/paw EHE with 0.037–0.37 μg/paw BCTC, and c 3 mg/paw EHE or 0.92 μg/paw capsaicin into the plantar surface of the left hind paw. Licking behaviors were observed for a, b 5 min, and c 60 min. Data represent the mean ± standard error of a 7–8, b 3–4, and c 3 mice

ical saline = 1:1:8) containing a 0.3–10 mg/paw EHE, b 5 mg/paw EHE with 0.037–0.37 μg/paw BCTC, and c 3 mg/paw EHE or 0.92 μg/paw capsaicin into the plantar surface of the left hind paw. Licking behaviors were observed for a, b 5 min, and c 60 min. Data represent the mean ± standard error of a 7–8, b 3–4, and c 3 mice Desensitization of TRPV1 in peripheral sensory nerves by EHE It is well known that a large dose of capsaicin desensitizes TRPV1, relieving nociceptive pain. In this experiment, the transient desensitization of capsaicin-induced pain was triggered by i.d. administration of a single large dose of EHE. Capsaicin-induced paw licking time significantly decreased 30 min after the injection of 3 mg/paw EHE or 0.92 μg/paw capsaicin into the hind paw (Fig. 4a); the desensitizing effects were abolished 60 min after their injection (Fig. 4b).Fig. 4 Suppression of capsaicin-induced pain by i.d. administration of EHE. Mice were injected with 10 µl of vehicle (DMSO:Tween-80:physiological saline = 1:1:8) containing 3 mg/paw EHE or 0.92 μg/paw capsaicin (Cap) into the plantar surface of left hind paw. After 30 min (a) or 60 min (b), 0.18 μg/paw capsaicin was injected into the same area. Licking behaviors were observed for 5 min. Data represent the mean ± standard error of 6 mice. Statistical significance was determined with Dunnett’s test; *p < 0.01, and **p < 0.001 vs vehicle group (Veh)

r surface of left hind paw. After 30 min (a) or 60 min (b), 0.18 μg/paw capsaicin was injected into the same area. Licking behaviors were observed for 5 min. Data represent the mean ± standard error of 6 mice. Statistical significance was determined with Dunnett’s test; *p < 0.01, and **p < 0.001 vs vehicle group (Veh) Analgesic effect of oral administration of EHE on capsaicin-induced nociceptive pain Capsaicin-induced paw licking times were recorded between 0 and 24 h after p.o. administration of EHE (700 mg/kg). Paw licking times significantly decreased for up to 2 h after the administration of EHE, with the maximal effect observed between 15 and 30 min after EHE administration (Fig. 5a). The antinociceptive effect of p.o. administration of EHE reduced the capsaicin-induced paw licking times in a dose-dependent manner (Fig. 5b). Furthermore, the physical performance of mice was evaluated 30 min after p.o. administration of EHE (175–700 mg/kg) and compared with mice from the vehicle group using a rotarod treadmill. There were no significant differences in physical performance between the two groups (Fig. 6). Thus, these results indicated that p.o. administration of EHE has an analgesic effect on capsaicin-induced pain.Fig. 5 Suppression of capsaicin-induced pain by p.o. administration of EHE. a Mice were orally administered 700 mg/kg of EHE (closed circle) or water (closed square). After 0, 0.25, 0.5, 1, 2, 6, and 24 h, the mice were injected with 10 µl of solution (DMSO:Tween-80:physiological saline = 1:1:8) containing 0.06 mM capsaicin into the plantar surface of left hind paw. b Mice were orally administered 87.5–700 mg/kg of EHE. After 30 min, the mice were injected with 10 µl of solution (DMSO:Tween-80:physiological saline = 1:1:8) containing 0.18 μg/paw capsaicin into the plantar surface of left hind paw. Licking behaviors were observed for 5 min. Data represent the mean ± standard error of a 4–8 and b 3–4 mice. Statistical significance was determined with a Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs the water group and b Dunnett’s test; *p < 0.05 vs vehicle group (Veh)

f left hind paw. Licking behaviors were observed for 5 min. Data represent the mean ± standard error of a 4–8 and b 3–4 mice. Statistical significance was determined with a Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs the water group and b Dunnett’s test; *p < 0.05 vs vehicle group (Veh) Fig. 6 Effect of EHE on rotarod performance of mice. Mice were orally administered 175–700 mg/kg of EHE. After 30 min, the mice were placed on the rod rotating at 28 rpm, and their performance was observed for 5 min. Data represent the mean ± standard error of 4 mice. Values above the columns represent the number of mice that did not fall during a period of 5min. Statistical significance was determined with Dunnett’s test; *p < 0.05 vs vehicle group (Veh) Discussion The analgesic effect of EH has previously been thought to be an indirect effect elicited through anti-inflammatory action [5, 6]. In this study, we demonstrated that EHE exhibits a direct antinociceptive effect by affecting the TRPV1-mediated nociceptive pathway. TRPV1 is a polymodal receptor that converts multiple noxious stimulation into electric signals [10]. It is extensively modulated by neurotransmitters, inflammatory cytokines, growth factors, local hormones, and oxidative chemicals, thereby serving as an integrator for processing nociceptive information [21].

e pathway. TRPV1 is a polymodal receptor that converts multiple noxious stimulation into electric signals [10]. It is extensively modulated by neurotransmitters, inflammatory cytokines, growth factors, local hormones, and oxidative chemicals, thereby serving as an integrator for processing nociceptive information [21]. We investigated the effect of EHE on TRPV1 using stable mouse TRPV1-expressing transfected mTRPV1/Flp-In293 cells (Fig. 2), and showed that EHE increased the intracellular Ca2+ concentration in these cells, and that this increase was inhibited by BCTC, a TRPV1 antagonist (Fig. 2a–c) [22–25]. These results indicate the presence of components in EHE that directly activate mTRPV1. The kinetics of the increase in the Ca2+ concentration in mTRPV1/Flp-In293 cells induced by EHE was different from that produced by capsaicin (Fig. 2d), suggesting that the binding mode of the compounds in EHE to TRPV1 might be different from that of capsaicin. A number of TRPV1 activators have been found in natural products, including capsaicin, 6-gingerol, piperine, and evodiamine found in hot pepper, ginger, black pepper, and evodia fruits, respectively [26, 27]. However, there are currently no reports describing the effect of the compounds contained in EHE on TRPV1. Investigations are now underway to identify the active molecules in EHE.

ncluding capsaicin, 6-gingerol, piperine, and evodiamine found in hot pepper, ginger, black pepper, and evodia fruits, respectively [26, 27]. However, there are currently no reports describing the effect of the compounds contained in EHE on TRPV1. Investigations are now underway to identify the active molecules in EHE. Capsaicin, a TRPV1 ligand, is thought to stimulate nociceptive pain by the activation of TRPV1 on sensory neurons, since the nociceptive pain that it induces is specifically inhibited by BCTC (Fig. S2) [23]. An i.d. injection of EHE induced paw licking in mice that was suppressed by BCTC administration (Fig. 3), suggesting that EHE similarly induces pain through the activation of TRPV1 on sensory neurons in vivo. Moreover, pretreatment with a high dose of EHE suppressed the capsaicin-induced paw licking, suggesting that EHE elicits an analgesic action by affecting TRPV1 function on peripheral sensory nerves. Although capsaicin is known to induce pain, it also exhibits analgesic effects [28]. Therefore, capsaicin has been used to treat neuropathic pain, and there are many clinical trials testing its topical application, such as the use of capsaicin patches [28–30]. The analgesic effects of capsaicin are considered to be derived from both the desensitization of TRPV1 and defunctionalization of TRPV1-positive nerves [16]. The desensitization of TRPV1 by capsaicin depends on an increase in intracellular Ca2+ concentration [31–33], and the analgesic effects induced by this process are thought to be transient. Several mechanisms of desensitization of TRPV1 have been suggested—(1) the phosphatase, calcineurin, is activated by an increase in intracellular Ca2+ concentration through the activation of TRPV1, and the dephosphorylation of TRPV1 by calcineurin is followed by the desensitization of TRPV1 while it recovers from desensitization through the inhibition of calcineurin [34]; (2) the interaction between the Ca2+/calmodulin complex and TRPV1 induces a reduction in TRPV1 responsiveness [35–37]; and (3) TRPV1 agonists that rapidly downregulate the membrane expression of TRPV1 through endocytosis and lysosomal degradation. This process is modulated by an increase in intracellular Ca2+ concentration and protein kinase A-dependent phosphorylation of TRPV1 [38]. In contrast, the long-lasting analgesic effect of capsaicin is considered to arise from the defunctionalization of TRPV1-positive nerves [28–30].

ocytosis and lysosomal degradation. This process is modulated by an increase in intracellular Ca2+ concentration and protein kinase A-dependent phosphorylation of TRPV1 [38]. In contrast, the long-lasting analgesic effect of capsaicin is considered to arise from the defunctionalization of TRPV1-positive nerves [28–30]. Prolonged activation of TRPV1 by repeated or high-concentration capsaicin treatment causes long-lasting loss of sensitivity of sensory neurons, as the overload of Ca2+ by prolonged activation of TRPV1 induces degeneration of sensory nerve terminals [39]. In this study, the analgesic effect of an i.d. injection of a high dose of EHE (3 mg/paw) or capsaicin (0.92 μg/paw) was transient, and was abolished 60 min after the injection of EHE or capsaicin (Fig. 4). Therefore, this analgesic effect possibly originated from the desensitization of TRPV1. Oral administration of EHE also relieved capsaicin-induced paw licking without a drop in physical performance. Surprisingly, the analgesic effect of EHE was evident immediately after its p.o. administration, and was highest between 15 and 30 min after administration (Fig. 5a). These results suggest that EHE contains components that are rapidly absorbed from the digestive system. Wei et al. [40] recently reported that ephedrine alkaloids, the major alkaloid components of EH, are rapidly absorbed in rats. However, ephedrine did not activate or inhibit TRPV1 (Fig. S3A and B). Efforts are underway in our laboratory to identify the active compounds of EHE.

apidly absorbed from the digestive system. Wei et al. [40] recently reported that ephedrine alkaloids, the major alkaloid components of EH, are rapidly absorbed in rats. However, ephedrine did not activate or inhibit TRPV1 (Fig. S3A and B). Efforts are underway in our laboratory to identify the active compounds of EHE. Desensitization of TRPV1 is likely to be one of the molecular mechanisms of EHE action following its i.d. administration. However, it is unclear if the analgesic effect of EHE by oral administration (Fig. 5a) is similarly mediated by the TRPV1 pathway. Thus, further investigation of the mechanism of the analgesic effect of oral administration of EHE is required. Antagonists of TRPV1 have been reported to alleviate various types of pain, such as neuropathic pain, inflammatory pain, and allodynia [41]. However, these agents also induce undesirable side-effects such as hyperthermia and an increase in the threshold of noxious heat in humans [42, 43]. On the other hand, EH has not been reported to induce the above adverse effects. Therefore, it is possible that EHE is more effective than other TRPV1 antagonists in addressing acute and chronic pain. We report for the first time that EHE activates TRPV1 in vitro. Topical administration of EHE activates and desensitizes TRPV1, and alleviates capsaicin-induced pain in vivo. These results suggest that the TRPV1 pathway may be integral to the molecular mechanism of EHE action. Although oral administration of EHE also reduced the capsaicin-induced pain, the mechanism of action remains to be elucidated.

on of EHE activates and desensitizes TRPV1, and alleviates capsaicin-induced pain in vivo. These results suggest that the TRPV1 pathway may be integral to the molecular mechanism of EHE action. Although oral administration of EHE also reduced the capsaicin-induced pain, the mechanism of action remains to be elucidated. Electronic supplementary material Below is the link to the electronic supplementary material. Fig. S1 Chromatogram of EHE obtained using an HPLC system (Shimadzu, Kyoto, Japan) consisting of an SIL-20A auto-injector, SPD-M20A photodiode array detector, LC-20AD pump, DGU-20A3 degasser, and CBM-20A communications bus module. Separations were carried out with an YMC-Triart C18 plus column (5 µm particle size, 4.6 mm (inner diameter) × 150 mm; YMC Co., Ltd, Kyoto, Japan). The mobile phase was a mixture of water, acetonitrile, and phosphoric acid (650:350:1, v/v/v) containing 0.5 % SDS delivered at a flow rate of 1 ml/min. The column temperature was maintained at 40 °C with a CTO-20A column oven (Shimadzu). The detection wavelength was set at 210 nm for quantitative determination. The test samples were resolved by methanol and injected (10 µl injection volume) by an auto-injector. (TIFF 112 kb) Fig. S2 a Capsaicin-induced nociceptive pain. Mice were injected with 10 μl of solution (DMSO:Tween-80:physiological saline = 1:1:8) containing 0.031–3.1 μg/paw capsaicin into the plantar surface of the left hind paw using a microsyringe. Licking behaviors were observed for 5 min. Each data represents the mean ± standard error of 5–7 mice. (TIFF 37 kb)

ptive pain. Mice were injected with 10 μl of solution (DMSO:Tween-80:physiological saline = 1:1:8) containing 0.031–3.1 μg/paw capsaicin into the plantar surface of the left hind paw using a microsyringe. Licking behaviors were observed for 5 min. Each data represents the mean ± standard error of 5–7 mice. (TIFF 37 kb) b Inhibition of the capsaicin-induced pain by BCTC. Mice were injected with 10 μl of solution (DMSO:Tween-80:physiological saline = 1:1:8) containing 0.18 μg/paw capsaicin with 0.0011–0.037 μg/paw BCTC into the plantar surface of the left hind paw using a microsyringe. Licking behaviors were observed for 5 min. Each data represents the mean ± standard error of 6 mice. (TIFF 34 kb) Fig. S3 a Effect of ephedrine on the uptake of Ca2+ into mTRPV1/Flp-In293 cells through the activation of TRPV1. The ratio of fluorescence intensity induced by HBSS buffer alone (Con) and buffer containing 0.2 µM capsaicin (Cap), 1000 µg/ml EHE, or 40 µg/ml ephedrine (Eph), in the absence and presence of 1 µM BCTC, over that induced by 0.2 µM capsaicin. (TIFF 61 kb) b Effects of ephedrine on the uptake of Ca2+ into mTRPV1/Flp-In293 cells through TRPV1 activation by capsaicin. The ratio of fluorescence intensity induced by 0−0.2 µM capsaicin in the absence (closed circle) and presence of 40 μg/ml ephedrine (closed square) or 1 nM BCTC (closed triangle), over that induced by 0.2 μM capsaicin. Each assay was performed in triplicate. The error bar represents the standard error. Statistical significance was determined with Tukey’s test; *p < 0.001 vs BCTC-treated group. (TIFF 60 kb)

circle) and presence of 40 μg/ml ephedrine (closed square) or 1 nM BCTC (closed triangle), over that induced by 0.2 μM capsaicin. Each assay was performed in triplicate. The error bar represents the standard error. Statistical significance was determined with Tukey’s test; *p < 0.001 vs BCTC-treated group. (TIFF 60 kb) Electronic supplementary material The online version of this article (doi:10.1007/s11418-016-1034-9) contains supplementary material, which is available to authorized users. A correction to this article is available online at https://doi.org/10.1007/s11418-018-1196-8. This research is supported by a Grant-in-Aid from the Japan Health Sciences Foundation (public–private sector joint research on publicly essential drugs), the Research on Development of New Drugs from the Japan Agency for Medical Research and Development (AMED), and the All Kitasato Project Study (AKPS) Collaborative Research. We would like to thank Editage (http://www.editage.jp) for English language editing.

Introduction Constipation affects multiple aspects of a person’s health, including health-related quality of life. It is one of the most frequently reported functional gastrointestinal disorders. The prevalence of constipation varies from 2.6 to 26.9%, being most frequent in females and in advanced age [1, 2]. Constipation is caused by decreased internal organ function, lack of water fluid, etc. [3]. In general, improvement in dietary habits, water intake, exercise and other activities in daily life is prioritized for relieving constipation, but more active interventions become indispensable for severe constipation. In recent years, a novel chloride-channel activator, lubiprostone, which is not classified as a prokinetic, has been developed [4] and is attracting attention. The mechanism of lubiprostone’s laxative actions is accounted for by the activation of Chloride Channel-2 (ClC-2) channels that results in chloride efflux across the apical membrane and subsequent paracellular passive movement of sodium and water into the intestinal lumen. The luminal distension caused by increased intestinal fluid then promotes gut motility, thereby enhancing intestinal and colonic transits [4]. However, there is contrasting evidence that the molecular target of lubiprostone may rather be the cystic fibrosis transmembrane conductance regulator (CFTR) channel than ClC-2. In the Xenopus oocyte expression system, CFTR but not ClC-2 has been found to be activated via the prostaglandin receptor sub-type 4 (EP-4) [5]. In the intestinal epithelia of both mice and human, endogenous expression of CFTR is restricted to the apical membrane while that of ClC-2 is localized largely in the basolateral membrane, and, moreover, only the former can be activated by lubiprostone [6]. Thus, it still remains controversial what type of ion channels/transporters are involved in lubiprostone’s laxative actions.

expression of CFTR is restricted to the apical membrane while that of ClC-2 is localized largely in the basolateral membrane, and, moreover, only the former can be activated by lubiprostone [6]. Thus, it still remains controversial what type of ion channels/transporters are involved in lubiprostone’s laxative actions. It is also reported that guanylate cyclase-C (GC-C) receptor activators, linaclotide and plecanatide, exert similar gastrokinetic actions, through enhanced intracellular cGMP synthesis and subsequent phosphorylation of CFTR protein by cGMP-dependent protein kinase II (PKG II), which facilitates luminal chloride secretion and paracellular movement of sodium and water [3, 7].

ptor activators, linaclotide and plecanatide, exert similar gastrokinetic actions, through enhanced intracellular cGMP synthesis and subsequent phosphorylation of CFTR protein by cGMP-dependent protein kinase II (PKG II), which facilitates luminal chloride secretion and paracellular movement of sodium and water [3, 7]. Kampo medicines are composed of various medicinal herbs. Two classes of Kampo medicines, Rhei Rhizoma-based (class 1) and Kenchuto-based ones (class 2) are frequently used for the treatment of constipation [8]. In Rhei Rhizoma-based medicines, Junchoto (JCT) and Mashiningan (MNG) constitute a unique subgroup that contains Cannabis Fructus, as well as a small amount of Rhei Rhizoma. JCT and MNG are prescribed exclusively for elderly patients suffering from spastic constipation, which results mostly in softened stool. Recently, it was suggested that such laxative actions of JCT and MNG may involve CFTR activation [9, 10]. However, this speculation relies entirely on the presumptive specificity of an organic CFTR inhibitor used (CFTRinh-172) which also inhibits other types of Cl− channels including volume-sensitive anion channels [11] and ClC-2 [12] at micromolar concentrations, thus lacking rigorous proof at the molecular level.

9, 10]. However, this speculation relies entirely on the presumptive specificity of an organic CFTR inhibitor used (CFTRinh-172) which also inhibits other types of Cl− channels including volume-sensitive anion channels [11] and ClC-2 [12] at micromolar concentrations, thus lacking rigorous proof at the molecular level. In the present study, we therefore adopted more direct gene-based approaches to manipulate CFTR expression, in order to unequivocally determine the molecular target of JCT’s actions. Furthermore, to confirm whether JCT can actually promote water secretion as the consequence of CFTR activation (or induction of Cl− efflux), we compared the time courses of and causal relationship between JCT-induced cell volume decrease and CFTR activation. Additionally, the cellular mechanism by which JCT induces CFTR-mediated Cl− conductance was investigated in some detail. Methods Reagents DMSO was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Forskolin, CFTR inhibitor-172 and SQ22536 were obtained from Sigma-Aldrich (St. Louis, MO, USA). KT5823 was obtained from Cayman (Cayman Chemical Co, Ann Arbor, MI, USA). Junchoto compound was obtained from Tsumura (Tsumura Co., Ltd, Tokyo, Japan: http://www.tsumura.co.jp/english/products/pi/JPR_T051.pdf). Junchoto powder was dissolved in DMSO at concentrations from 400 to 800 mg/mL and used on the same day. All other chemical reagents were purchased from commercial suppliers.

MI, USA). Junchoto compound was obtained from Tsumura (Tsumura Co., Ltd, Tokyo, Japan: http://www.tsumura.co.jp/english/products/pi/JPR_T051.pdf). Junchoto powder was dissolved in DMSO at concentrations from 400 to 800 mg/mL and used on the same day. All other chemical reagents were purchased from commercial suppliers. Cell cultures and cDNA expression HEK293T cells and Caco-2 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 30 units/ml penicillin and 30 μg/ml streptomycin (in the case of Caco-2 cells, 1% non-essential amino acids were further added), under a 95% air–5% CO2 atmosphere at 37 °C. Twenty-four hours after plating, HEK293T cells were transfected with either pCIneo-IRES-GFP vector or human CFTR-pCIneo-IRES-GFP vector (a generous gift from Dr. RZ Sabirov [13]). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used as a transfection reagent following the manufacturer’s instructions. Electrophysiological measurements and Western blot analysis were performed 36–72 h after transfection.

vector or human CFTR-pCIneo-IRES-GFP vector (a generous gift from Dr. RZ Sabirov [13]). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used as a transfection reagent following the manufacturer’s instructions. Electrophysiological measurements and Western blot analysis were performed 36–72 h after transfection. Mean cell volume measurements Mean cell volume was measured at room temperature by electronic sizing with a Coulter-type cell size analyzer (CDA-500; Sysmex, Hyogo, Japan). The mean volume of the cell population was calculated from the cell volume distribution measured after the machine was calibrated with latex beads of known volume. Isotonic “Tyrode solution” (300 mosmol/kg H2O adjusted by d-mannitol) contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 d-glucose and 10 HEPES (pH 7.4 adjusted by NaOH). Relative cell volumes in Fig. 6a–d are defined by the following equation: relative cell volume = VA/VCtl, where VCtl and VA are the mean cell volumes before and after DMSO (control) or JCT application, respectively.

ontained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 d-glucose and 10 HEPES (pH 7.4 adjusted by NaOH). Relative cell volumes in Fig. 6a–d are defined by the following equation: relative cell volume = VA/VCtl, where VCtl and VA are the mean cell volumes before and after DMSO (control) or JCT application, respectively. Single-cell size measurements Single-cell size was measured at room temperature in cells adhering to a non-coated cover glass in Tyrode solution. The experiments were performed in a 1-ml recording chamber in which the cover glass was placed. The cells were visualized through a charge-coupled device camera (XC-ST70, Sony, Tokyo, Japan) and images were recorded with the mAgicTV software (I-O DATA, Ishikawa, Japan). The cross-sectional area (CSA) of the cell of interest was measured as an indicator of cell size by ImageJ software [14]. Relative CSAs in Fig. 6e and h are defined by the following equation: relative CSA = AA/ACtl, where ACtl and AA are the CSA values before and after JCT application, respectively.

, Ishikawa, Japan). The cross-sectional area (CSA) of the cell of interest was measured as an indicator of cell size by ImageJ software [14]. Relative CSAs in Fig. 6e and h are defined by the following equation: relative CSA = AA/ACtl, where ACtl and AA are the CSA values before and after JCT application, respectively. Electrophysiology After transfection with human CFTR-pCIneo-IRES-GFP or pCIneo-IRES-GFP plasmid, cells were dissociated by mechanical agitation and lodged onto coverslips placed in tissue culture dishes. Membrane currents of these cells were recorded at room temperature (22–27 °C) using the whole-cell mode of the patch-clamp technique, with an Axopatch 200B (Axon Instruments/Molecular Devices, Union City, CA, USA) patch-clamp amplifier. For whole-cell recordings, patch electrodes prepared from borosilicate glass capillaries had an input resistance of 3–5 MΩ. Current signals were filtered at 5 kHz with a four-pole Bessel filter and digitized at 20 kHz. pCLAMP (version 10.5.1.0; Axon Instruments/Molecular Devices) software was used for command pulse control, data acquisition and analysis. Data were also analyzed using Origin (OriginLab Corp., Northampton, MA, USA) software. For whole-cell recordings, the series resistance was compensated (to 70–80%) to minimize voltage errors. The external solution contained (in mM) 110 CsCl, 2 CaCl2, 1 MgCl2, 5 glucose and 10 HEPES (pH 7.4 adjusted with CsOH, and osmolality adjusted to 310 mmol/kg with d-mannitol). The pipette solution contained (in mM) 110 CsCl, 2 MgSO4, 1 EGTA, 10 HEPES, 1 Na2ATP and 15 Na-HEPES (pH 7.4 adjusted with CsOH, and osmolality adjusted to 300 mmol/kg with d-mannitol). To test the ion selectivity of the macroscopic channel currents, 110 mM Cs-aspartate in the bath solution was replaced with 55 mM CsCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose and 10 mM HEPES (pH 7.4 adjusted with CsOH, and osmolality adjusted to 310 mmol/kg with d-mannitol). To test the nystatin-perforated whole-cell currents with single-cell size measurements, the Na+-based bath solution contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES and 10 d-glucose (pH adjusted to 7.4 with NaOH, and osmolality adjusted to 320 mosmol/kgH2O with d-mannitol). The pipette solution contained (in mM) 55 K2SO4, 20 KCl, 5 MgCl2, 0.2 EGTA and 5 HEPES (pH adjusted to 7.4 with KOH, and osmolality adjusted to 300 mosmol/kgH2O with d-mannitol).

KCl, 2 CaCl2, 1 MgCl2, 10 HEPES and 10 d-glucose (pH adjusted to 7.4 with NaOH, and osmolality adjusted to 320 mosmol/kgH2O with d-mannitol). The pipette solution contained (in mM) 55 K2SO4, 20 KCl, 5 MgCl2, 0.2 EGTA and 5 HEPES (pH adjusted to 7.4 with KOH, and osmolality adjusted to 300 mosmol/kgH2O with d-mannitol). Western blot analysis After 36 h of transfection, Caco-2 cells were solubilized in the radioimmunoprecipitation assay (RIPA) buffer (pH 8.0) containing 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P40, 150 mM NaCl, 50 mM Tris–HCl, 1 mM PMSF and 10 μg/μl leupeptin, then centrifuged at 17,400g for 20 min. Whole-cell lysates were fractionated by 7.5% SDS-PAGE and electro-transferred onto a poly-vinylidene fluoride (PVDF) membrane. The blots were incubated with anti-CFTR antibody (1:1000 dilution, CUSABIO and CUSAb, MD, USA: CSB-PA001608) or monoclonal anti-α-tubulin (as an internal standard, 1:2000 dilution; Sigma-Aldrich: T6074), and stained using the enhanced chemiluminescence system (Thermo Scientific, Rockford, IL, USA).

ide (PVDF) membrane. The blots were incubated with anti-CFTR antibody (1:1000 dilution, CUSABIO and CUSAb, MD, USA: CSB-PA001608) or monoclonal anti-α-tubulin (as an internal standard, 1:2000 dilution; Sigma-Aldrich: T6074), and stained using the enhanced chemiluminescence system (Thermo Scientific, Rockford, IL, USA). RNA isolation and RT-PCR Total cellular RNA was extracted from Caco-2 cells by using NucleoSpin® RNA Plus (Takara-Bio, Shiga, Japan) according to the protocol supplied by the manufacturer. The concentration and purity of RNA were determined using a Nanodrop-ND1000 (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA samples were reverse-transcribed at 42 °C for 30 min with Prime Script RTase using the PrimeScript™ II High Fidelity RT-PCR Kit (Takara-Bio, Shiga, Japan), according to the manufacturer’s protocols. Expression levels of CFTR in the cDNA from Caco-2 were determined by PCR. As a positive control, we amplified the partial sequence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Suppression of RNA expression was confirmed by RT-PCR analysis. PCR was done using KOD-Plus-Ver.2 (Toyobo, Osaka, Japan) under the following conditions: pre-denaturation at 94 °C for 2 min, followed by 32–35 cycles of denaturation at 98 °C for 10 s and annealing at 55–63 °C for 30 s, and final extension at 68 °C for 30 s. The sequences of gene-specific primers (synthesized by Sigma-Aldrich) and the predicted lengths of PCR products are as follows: hGAPDH (496 bp) forward and reverse primers: 5′-GGTGAAGGTCGGAGTCAACG-3′ and 5′-CAAAGTTGTCATGGATGACC-3′ respectively; hCFTR (327 bp) forward and reverse primers: 5′-AGGAGGAACGCTCTATCG-3′ and 5′-GCAGACGCCTGTAACAAC-3′, respectively.

s (synthesized by Sigma-Aldrich) and the predicted lengths of PCR products are as follows: hGAPDH (496 bp) forward and reverse primers: 5′-GGTGAAGGTCGGAGTCAACG-3′ and 5′-CAAAGTTGTCATGGATGACC-3′ respectively; hCFTR (327 bp) forward and reverse primers: 5′-AGGAGGAACGCTCTATCG-3′ and 5′-GCAGACGCCTGTAACAAC-3′, respectively. siRNA transfection Caco-2 cells were transfected with 1 µg small interfering RNA (siRNA) using the RNAiMAX Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions, and used for experiments 48–72 h later. To determine transfection efficacy, we used the pEGFP-N1 vector (Takara-Bio). As a negative control, we used a non-silencing siRNA (or mock siRNA). The mock siRNA and the siRNA against CFTR were purchased from Bioneer (Bioneer Daejon, S. Korea). Statistical evaluation All data are expressed as mean ± SEM. We accumulated the data for each condition from at least three independent experiments. Statistical analyses were performed using Student’s t test. P < 0.05 was considered significant.

siRNA transfection Caco-2 cells were transfected with 1 µg small interfering RNA (siRNA) using the RNAiMAX Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions, and used for experiments 48–72 h later. To determine transfection efficacy, we used the pEGFP-N1 vector (Takara-Bio). As a negative control, we used a non-silencing siRNA (or mock siRNA). The mock siRNA and the siRNA against CFTR were purchased from Bioneer (Bioneer Daejon, S. Korea). Statistical evaluation All data are expressed as mean ± SEM. We accumulated the data for each condition from at least three independent experiments. Statistical analyses were performed using Student’s t test. P < 0.05 was considered significant. Results Concentration-dependent activation of CFTR-mediated Cl− current by Junchoto Previous studies with short-circuit measurements using the Ussing chamber reported that Junchoto (JCT) increased the net membrane current across a polarized human bronchial epithelial cell layer [9]. Since this trans-epithelial current was effectively reduced by the CFTR inhibitor-172 [9], it was simply concluded that the current arose from CFTR activation. However, the electrophysiological details of this current remain entirely undetermined. To more unequivocally investigate the identification of JCT’s target, we first performed patch-clamp measurements in HEK293T cells over-expressing human CFTR channels (Fig. 1a, inset). As shown in Fig. 1a (left panel), application of JCT resulted in slow development of a large whole-cell current. No such current was induced in empty vector-transfected cells (Fig. 1a, right trace: Fig. 1b, bottom traces). The JCT-induced current responded to step voltage pulses with almost instantaneous activation and deactivation time courses (Fig. 1b, upper traces), and showed a linear current–voltage (I–V) relationship (Fig. 1c, open circles). These electrophysiological properties are characteristic of heterologously expressed CFTR-mediated Cl− channels, suggesting that JCT is a robust activator of the CFTR channel.Fig. 1 Junchoto (JCT)-induced CFTR currents in HEK293T cells transiently transfected with CFTR. a Representative records of whole-cell current activation in CFTR- (left) and vector-transfected (right) cells before and after application of 400 μg/mL JCT (filled bars), taken during the application of alternating pulses from 0 to ± 40 mV every 10 s. The asterisks denote times when step pulses were applied. The inset shows a membrane displaying immunoblot of CFTR protein from control (vector-transfected) and CFTR transfected HEK293T cells in the upper lane. Note that the lower band is only detected in CFTR-transfected HEK293T cells. Alpha-tubulin bands with molecular mass 50 kDa were detected at equal levels in the lower lane. b The current response to step pulses from −100 to +100 mV for CFTR (top traces) and the vector (bottom trace).

ed HEK293T cells in the upper lane. Note that the lower band is only detected in CFTR-transfected HEK293T cells. Alpha-tubulin bands with molecular mass 50 kDa were detected at equal levels in the lower lane. b The current response to step pulses from −100 to +100 mV for CFTR (top traces) and the vector (bottom trace). c I–V relationships for the mean JCT-activated current densities for cells expressing CFTR (open circles; n = 6) and vector (filled triangles; n = 6) Figure 2a demonstrates the concentration-dependent effects of JCT on inducing Cl− current assessed by a cumulative application protocol. To attain maximal activation, forskolin (10 μM) was applied at the end of the protocol. The degree of activation of Cl− current by JCT (expressed as the fraction of forskolin-induced Cl− current) was increased in a concentration-dependent manner, and half maximal activation (at +100 mV) occurred at 279 µg/ml (EC50) with a cooperativity coefficient of 2.14 (Fig. 2c).Fig. 2 JCT dose–response of CFTR channel in HEK293T cells transiently transfected with CFTR. a Representative time courses of the JCT evoked whole-cell currents recorded at +100 and −100 mV under ramp clamp. b Corresponding I–V relationships at time points a, b, c, d and e. c Peak current densities induced by JCT normalized to that of 10 μM FSK (n = 5–8). Data points show the mean ± S.E.M

ransfected with CFTR. a Representative time courses of the JCT evoked whole-cell currents recorded at +100 and −100 mV under ramp clamp. b Corresponding I–V relationships at time points a, b, c, d and e. c Peak current densities induced by JCT normalized to that of 10 μM FSK (n = 5–8). Data points show the mean ± S.E.M Junchoto activates endogenous CFTR channels in Caco-2 cells In the next step, to test the effects of JCT on endogenous CFTR channels, we repeated similar experiments in Caco-2 cells. As in CFTR-expressing HEK293T cells, JCT induced whole-cell Cl− currents that developed slowly and had a linear I–V relationship with a reversal potential near 0 mV under symmetric Cl− conditions (Fig. 3a, b). When the extracellular Cl− concentration was reduced from 116 to 61 mM (from 110 to 55 mM CsCl bath solution), the reversal potential of JCT-activated current shifted to the right (Fig. 3b: inset) by 14.1 ± 0.8 mV (n = 6). This value is in good agreement with that predicted for an ideal anion electrode (15.1 mV). JCT-induced currents were blocked by extracellular application of the so-called CFTR specific inhibitor, CFTR-inhibitor-172 (CFTR-inh.: Fig. 3c–e). These properties are essentially the same as those of recombinant CFTR channels observed elsewhere.Fig. 3 Whole-cell currents evoked by JCT in human colonic Caco-2 cells. a, c Representative time courses of the JCT-evoked whole-cell currents recorded at +100 and −100 mV under ramp clamp. Gray bar and solid bar show application of 400 μg/mL of JCT and 20 μM of CFTR inhibitor-172 (CFTR-inh.), respectively. b, d Corresponding I–V relationships at time points a, b and c. e Averages of JCT-induced whole-cell current in control and CFTR inh. (n = 5–6). The inset shows JCT-induced whole-cell current when the extracellular Cl− concentration is reduced from control to 55 mM CsCl (55 Cs-Cl) bath solution. Data points show the mean ± SEM. ***P < 0.001 compared to control at +100 mV

, b and c. e Averages of JCT-induced whole-cell current in control and CFTR inh. (n = 5–6). The inset shows JCT-induced whole-cell current when the extracellular Cl− concentration is reduced from control to 55 mM CsCl (55 Cs-Cl) bath solution. Data points show the mean ± SEM. ***P < 0.001 compared to control at +100 mV Suppression of JCT-induced currents by siRNA of CFTR To confirm that the JCT-induced current in Caco-2 cells indeed reflects the activation of the CFTR channel, we next conducted an RT-PCR assay to detect the cftr mRNA expression. As shown in Fig. 4a (top left), robust amplification of the cftr transcript of an expected size (327 bp) occurred from the reverse-transcribed RNA of Caco-2 cells. Moreover, 24-h treatment of Caco-2 cells with CFTR-specific siRNA almost completely eliminated the cftr transcript, whereas mock siRNA had no effect. Neither siRNA affected the mRNA expression of a housekeeping enzyme GAPDH (Fig. 4a, top right).Fig. 4 Effects of small interfering RNA (siRNA) silencing of CFTR on JCT-induced anion currents in Caco-2 cells. a RT-PCR analysis of CFTR mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in control, mock-treated and CFTR siRNA-treated Caco-2 cells. The data represent 3 similar experiments. No PCR product was amplified when reverse transcriptase was omitted from the reaction in the RT(−) group. The nucleotide sequences of the PCR products obtained with CFTR-specific primers were completely identical to the corresponding sequences in human CFTR (4302-4722 Sequence ID: NM_000492.3). b Immunoblot of CFTR protein from control, mock-treated and CFTR siRNA-treated Caco-2 cells. Alpha-tubulin bands with molecular mass 50 kDa were detected at equal levels. c–h Representative time courses of the JCT-evoked whole-cell currents recorded at +100 and −100 mV under ramp clamp in mock siRNA-treated cells (c) and in CFTR siRNA-treated cells (e). Gray bar and solid bar show application of 400 μg/mL of JCT and 10 μM of FSK, respectively. Corresponding I–V relationships at time points a–c in d and a–c in f. g, h Averages of JCT-induced whole-cell current at +100 mV in mock and CFTR siRNA (g), and FSK-induced whole-cell current (h) (n = 5–6). Data points show the mean ± SEM. *P < 0.05 compared to mock at +100 mV

/mL of JCT and 10 μM of FSK, respectively. Corresponding I–V relationships at time points a–c in d and a–c in f. g, h Averages of JCT-induced whole-cell current at +100 mV in mock and CFTR siRNA (g), and FSK-induced whole-cell current (h) (n = 5–6). Data points show the mean ± SEM. *P < 0.05 compared to mock at +100 mV Western blot analyses were performed for whole-cell lysate extracted from Caco-2 cells treated with none (control), mock siRNA or CFTR-siRNA with a polyclonal anti-CFTR antibody (Fig. 4b). The immunoreactive bands detected in control and mock-transfection lanes had a molecular mass of 168 kDa, and this band was diminished by CFTR-siRNA treatment. The functional impact of siRNA was confirmed by patch-clamp experiments. While JCT failed to induce noticeable membrane currents after CFTR-specific siRNA transfection (Fig. 4e–g), it still induced robust currents in mock siRNA-transfected Caco-2 cells (Fig. 4c, d, g). Essentially the same results were obtained when FSK, instead of JCT, was used as a stimulant (Fig. 4c–h). These results collectively suggest that endogenous CFTR protein of Caco-2 cells is functionally and effectively activated by JCT.

The functional impact of siRNA was confirmed by patch-clamp experiments. While JCT failed to induce noticeable membrane currents after CFTR-specific siRNA transfection (Fig. 4e–g), it still induced robust currents in mock siRNA-transfected Caco-2 cells (Fig. 4c, d, g). Essentially the same results were obtained when FSK, instead of JCT, was used as a stimulant (Fig. 4c–h). These results collectively suggest that endogenous CFTR protein of Caco-2 cells is functionally and effectively activated by JCT. Mechanisms of CFTR channel activation It is well known that the CFTR channel is regulated by cyclic nucleotides such as cAMP and cGMP. Because of its apical localization in intestinal epithelia, this channel serves as the main route for Cl− and HCO3− effluxes to the lumen [15]. Therefore, to investigate the cellular mechanism underlying CFTR activation by JCT in Caco-2 cells, we employed KT5823 and SQ22536, relatively specific inhibitors for protein kinase G (PKG) and adenylate cyclase (AC), respectively.

lia, this channel serves as the main route for Cl− and HCO3− effluxes to the lumen [15]. Therefore, to investigate the cellular mechanism underlying CFTR activation by JCT in Caco-2 cells, we employed KT5823 and SQ22536, relatively specific inhibitors for protein kinase G (PKG) and adenylate cyclase (AC), respectively. As demonstrated and summarized in Fig. 5, the magnitude of JCT-induced current was not significantly affected by KT5823 treatment (Fig. 5a–d, g). In contrast, SQ22536 almost completely eliminated JCT-induced currents (Fig. 5e–g), suggesting that cAMP rather than cGMP mediates CFTR channel activation by JCT. Essentially the same results were obtained with FSK (Fig. 5a–f, h). To reinforce these observations further, we next assessed the ability of JCT to stimulate intracellular cAMP synthesis using a luciferase-based chemiluminescence method. As anticipated, JCT elicited an increase in chemiluminescence, which was antagonized by pretreatment with SQ22536 at a concentration which effectively inhibited FSK-induced cAMP increase (Fig. 5i).Fig. 5 JCT-induced CFTR activation is mediated by adenylyl cyclase and cAMP signaling pathway. Effects of KT5823 (1 μM) and SQ22536 (100 μM) were measured on whole-cell currents activated by JCT (400 μg/ml) in Caco-2 cells. Representative time courses and corresponding I–V relationships in control (a, b), KT5823 (c, d) and SQ22536 (e, f). Current densities evoked by JCT (g) and FSK (10 μM) (h) (n = 5–6). Averages of JCT-induced whole-cell current densities recorded at +100 mV (g), and FSK-induced whole-cell current densities (h) (n = 5–6). Data points are the mean ± SEM. ***P < 0.001. i Biochemical determination of cAMP levels. Caco-2 cells were pretreated with or without SQ22536 (100 μM), and then treated with JCT (400 μg/ml) or FSK (10 μM). Data are expressed as a percentage of the cAMP levels (n = 6 independent experiments). ***P < 0.001 compared with control, †P < 0.001 cAMP levels in cells pretreated with JCT or FSK. Error bars indicate the SEM

cells were pretreated with or without SQ22536 (100 μM), and then treated with JCT (400 μg/ml) or FSK (10 μM). Data are expressed as a percentage of the cAMP levels (n = 6 independent experiments). ***P < 0.001 compared with control, †P < 0.001 cAMP levels in cells pretreated with JCT or FSK. Error bars indicate the SEM Evaluation of epithelial fluid secretion by cell volume measurements Solute secretion from the cell inevitably accompanies osmotic water movement to cause cell volume decrease. We therefore estimated the extent of water secretion caused by JCT-induced Cl− efflux via CFTR in Caco-2 cells by using a Coulter-type volume measurement and video-microscopic measurement of secretory volume decrease (SVD) [16].

from the cell inevitably accompanies osmotic water movement to cause cell volume decrease. We therefore estimated the extent of water secretion caused by JCT-induced Cl− efflux via CFTR in Caco-2 cells by using a Coulter-type volume measurement and video-microscopic measurement of secretory volume decrease (SVD) [16]. As shown in Fig. 6a and b, JCT applied to non-treated Caco-2 cells (control) caused a ~15% SVD at 30 min after its administration. The extent of SVD was significantly diminished when CFTR channel activity was blocked by CFTR inhibitor-172 (CFTR-inh.: 20 μM) or SQ22536 (SQ: 100 μM). More prominent SVD (~24%) occurred when CFTR-mediated Cl− efflux was maximally activated by FSK (10 μM) (data not shown). The extent of SVD of Caco-2 cells was also greatly reduced by CFTR-specific siRNA treatment, while mock siRNA had little effect (Fig. 6c, d). Simultaneous measurement of whole-cell currents and cross-sectional area (CSA) was performed by combining the patch-clamp technique and video-microscopy. Under nystatin-perforated whole-cell recording, administration of 400 µg/mL JCT into the bath evoked an inward current at a holding potential of −60 mV (Fig. 6e). The whole-cell current reached a peak within a few minutes and persisted. The JCT-induced current was almost completely blocked by CFTR-specific siRNA (Fig. 6e, f). Administration of JCT reduced the size of Caco-2 cells and its time course paralleled the development of the inward current (Fig. 6e). This cell volume decrease was greatly attenuated by CFTR-siRNA treatment (Fig. 6g, h).Fig. 6 Involvement of the CFTR channel in JCT-induced cell-volume decrease of Caco-2 cells. a Time course of changes in mean cell volume. At time 0, JCT (400 μg/ml) was applied except in control. The conditions denote JCT, JCT with SQ22536 (100 μM), and JCT with CFTR-inh. (20 μM) on secretory volume decrease (SVD) monitored by an electronic sizing technique. b Percentage of initial cell volume (SVD) at 30 min (n = 5–10). c Effects of treatment with CFTR siRNA or mock siRNA on SVD. d Percentage of initial cell volume (SVD) at 30 min after application with or without JCT (n = 5). Data points are the mean ± SEM. *P < 0.05 compared to control or mock. §P < 0.05 compared to JCT. e–f Time course of the JCT-induced inward current and cell volume decrease.

treatment with CFTR siRNA or mock siRNA on SVD. d Percentage of initial cell volume (SVD) at 30 min after application with or without JCT (n = 5). Data points are the mean ± SEM. *P < 0.05 compared to control or mock. §P < 0.05 compared to JCT. e–f Time course of the JCT-induced inward current and cell volume decrease. e Averaged time courses of the JCT-evoked whole-cell currents recorded at −100 mV under ramp clamp in mock siRNA-treated cells and in CFTR siRNA-treated cells with nystatin-perforated recording. The holding potential was at −60 mV. f Averages of JCT-induced whole cell current at −100 mV (n = 6–7). Data points are the mean ± SEM. *P < 0.05. g Time-dependent profile of JCT-induced cell volume change in mock siRNA-treated cells and in CFTR siRNA-treated cells. JCT (400 μg/ml) was applied at 3 min. h Percentage of initial CSA at 22 min after application with JCT (n = 6–7). Data points are the mean ± SEM. *P < 0.05 compared to mock It is thus highly likely that cAMP-dependent activation of the CFTR channel significantly contributes to the JCT-induced SVD of Caco-2 cells, which involves facilitated Cl− efflux and accompanying water movement.

e Averaged time courses of the JCT-evoked whole-cell currents recorded at −100 mV under ramp clamp in mock siRNA-treated cells and in CFTR siRNA-treated cells with nystatin-perforated recording. The holding potential was at −60 mV. f Averages of JCT-induced whole cell current at −100 mV (n = 6–7). Data points are the mean ± SEM. *P < 0.05. g Time-dependent profile of JCT-induced cell volume change in mock siRNA-treated cells and in CFTR siRNA-treated cells. JCT (400 μg/ml) was applied at 3 min. h Percentage of initial CSA at 22 min after application with JCT (n = 6–7). Data points are the mean ± SEM. *P < 0.05 compared to mock It is thus highly likely that cAMP-dependent activation of the CFTR channel significantly contributes to the JCT-induced SVD of Caco-2 cells, which involves facilitated Cl− efflux and accompanying water movement. Discussion and conclusions The results of the present study make it apparent that JCT can facilitate Cl− and water secretion via cAMP-dependent activation of the CFTR channel. Several lines of evidence strongly support this view. First, JCT dose-dependently induced Cl− currents showing the hallmark properties of CFTR channels in both a heterologous expression system (HEK293T cells) and a cultured intestinal epithelial cell line (Caco-2 cells). Second, the JCT-induced current in Caco-2 cells was not only suppressed pharmacologically (i.e. by CFTR inhibitor-172) but also effectively eliminated by specific siRNA knockdown of CFTR. Third, the activation of CFTR-mediated Cl− current in Caco-2 cells by JCT was eliminated by the adenylate cyclase inhibitor SQ22536 (but not the PKG inhibitor KT5823), thus likely involving a cAMP-dependent mechanism. Finally, the JCT-induced secretory volume decrease of Caco-2 cells was greatly attenuated by CFTR inhibitor-172, CFTR-specific siRNA and SQ22536 with similar efficacies to inhibit CFTR-mediated Cl− currents. This observation is interpreted as indicating that facilitated Cl− efflux through the CFTR channel by JCT promoted water secretion from Caco-2 cells and caused their volume size reduction.

ly attenuated by CFTR inhibitor-172, CFTR-specific siRNA and SQ22536 with similar efficacies to inhibit CFTR-mediated Cl− currents. This observation is interpreted as indicating that facilitated Cl− efflux through the CFTR channel by JCT promoted water secretion from Caco-2 cells and caused their volume size reduction. The possibility that JCT may activate epithelial CFTR was previously suggested by short-circuit current measurements in human bronchial epithelia [9]. However, this was based merely on the inhibitory effect of a relatively specific CFTR inhibitor, CFTR inhibitor-172, at a concentration (20 μM) which non-specifically inhibited other types of Cl− channels [11, 12]. In this regard, the main progress of the present study lies in the unequivocal demonstration that JCT activates CFTR and facilitates subsequent water secretion, as well as clarification of the mechanism for this drug’s actions (i.e. via stimulated cAMP synthesis).

fically inhibited other types of Cl− channels [11, 12]. In this regard, the main progress of the present study lies in the unequivocal demonstration that JCT activates CFTR and facilitates subsequent water secretion, as well as clarification of the mechanism for this drug’s actions (i.e. via stimulated cAMP synthesis). The functional significance of CFTR-mediated Cl− secretion has been implicated in various epithelia, including the proximal small intestine, by the use of CFTR knockout mice. For instance, the ablation of the cftr gene is found to cause intestinal obstruction due to decreased epithelial fluid secretion [17, 18]. Mutations of this gene in human cystic fibrosis patients are also well known to accompany severe secretory defects in various organs such as exocrine glands, airways and the gastrointestinal tract [19], which frequently culminates in luminal obstruction. Thus, there is little doubt that targeting CFTR, which resides predominantly in the apical surface of epithelia, would be rational and beneficial for alleviating the symptoms associated with defective epithelial secretion such as constipation.

rointestinal tract [19], which frequently culminates in luminal obstruction. Thus, there is little doubt that targeting CFTR, which resides predominantly in the apical surface of epithelia, would be rational and beneficial for alleviating the symptoms associated with defective epithelial secretion such as constipation. Forskolin is an established adenylate cyclase activator that is made from the root of a plant in the mint family (Coleus forskohlii) and is capable of inducing the maximal activity of CFTR at 10 μM [20]. In our experiments as well, this concentration of forskolin maximally activated the CFTR channel (Fig. 2) as well as markedly increasing the intracellular cAMP level (Fig. 5). Importantly, the estimated efficacy of JCT (at 800 μg/ml) in the present study is comparable to that of forskolin (10 μM) in both activating the recombinant CFTR channel and stimulating cAMP synthesis in Caco-2 cells (Figs. 2 and 5). The potency of JCT to activate CFTR (half-maximal activation occurs at 279 μg/ml) is also similar to that to induce significant cell volume decrease or Cl−/water secretion in an intestinal epithelial cell line Caco-2 (Fig. 6). These pharmacological profiles strongly point to the clinical utility of this Kampo compound for re-hydrating dry contents in the intestine. Indeed, the evidence supportive of these observations has been briefly described: oral administration of JCT (300 or 1000 mg/kg) greatly improves opioid-induced severe constipation in a rat model with increased fecal count and dried fecal weight [9]. A similar CFTR-targeted improvement of constipation has been investigated in detail in a rat model treated with another Kampo medicine, Mashiningan, although in this case stimulation of cGMP-mediated signaling was presumed to be responsible for CFTR activation [10]. However, again, whether CFTR is involved here was not unequivocally proved because of the considerably higher concentration of the drug used to selectively inhibit CFTR (CFTR-inhbitor-172, 20 μM).

ngan, although in this case stimulation of cGMP-mediated signaling was presumed to be responsible for CFTR activation [10]. However, again, whether CFTR is involved here was not unequivocally proved because of the considerably higher concentration of the drug used to selectively inhibit CFTR (CFTR-inhbitor-172, 20 μM). The active ingredient(s) of JCT which stimulate cAMP production and thereby cause CFTR activation/water movement remains to be determined. Junchoto consists of 10 crude plant-extract herbs (viz. Cannabis Fructus, Aurantii Fructus Immaturus, Rhei Rhizoma, Magnoliae Cortex, Paeoniae Radixm, Glycyrrhizae radix, Rehmanniae Radix, Angelicae Radix, Scutellariae Radix, Persicae Semen) (http://wakankensaku.inm.u-toyama.ac.jp/wiki/Main_Page), each of which contains many active ingredients. Consultation with the literature shows that, of the 10 herbs, only three (Paeoniae Radixm, Glycyrrhizae radix, Scutellariae Radix) may have cAMP-mediated actions. Paeoniflorin, an active ingredient from Paeoniae Radixm, was shown to stimulate noradrenaline release from nerve terminals in a Ca2+- and cAMP-dependent manner, probably through an as-yet-unelucidated mechanism involving tetrodotoxin-sensitive presynaptic depolarization [21]. GU-7, a 3-arylcoumarin derivative extracted from Glycyrrhizae radix, was found capable of increasing the intraplatelet cAMP concentration to inhibit platelet aggregation through phosphodiesterase (PDE) inhibition [22]. A more recent study using isoform-specific inhibitors has revealed that this compound (GU-7 or glycycoumarin) can dose-dependently accumulate intracellular cAMP (but not cGMP) via specific inhibition of type-3 PDE activity [23]. Finally, the most striking finding is that biacalein, a major flavonoid extracted from Scutellariae Radix, likely stimulates Cl− secretion across rat colon epithelia. More detailed investigations using human colonal epithelial T84 cells suggested that baicalein causes a dose-dependent increase in a short-circuit current representing the apical Cl− efflux via enhanced cAMP production without affecting the intracellular Ca2+ level [24]. These multiple actions through both stimulating the synthesis and inhibiting the degradation of cAMP would render Junchoto a potent cAMP-producing agent and thus an effective CFTR activator whose efficacy is comparable to that of forskolin (Figs. 2, 5).

MP production without affecting the intracellular Ca2+ level [24]. These multiple actions through both stimulating the synthesis and inhibiting the degradation of cAMP would render Junchoto a potent cAMP-producing agent and thus an effective CFTR activator whose efficacy is comparable to that of forskolin (Figs. 2, 5). Interestingly, another mechanistically similar laxative, Mashiningan, does not contain two of the above three active ingredients (viz. glycycoumarin, baicalein). This difference may distinguish the laxative efficacies of Junchoto and Mashiningan. Indeed, the former is generally believed to be superior to the latter in softening stools. This powerful re-hydrating action of Junchoto may also be beneficial for secretory disorders in other organs such as airways, pancreas, salivary glands [25], eyes [26] (e.g. dry eyes) and uterus/oviduct [27] (e.g. infertility). In addition, it is becoming widely recognized that beside their well-known anti-oxidant effects, flavonoids modulate many biological functions by opening K+ channels, blocking voltage-dependent Ca2+ channels, decreasing inflammatory signals, modulating apoptotic processes [28] and activating/inhibiting epithelial Cl− transports via, e.g., CFTR channels (stimulation; biacalein, tangeretin; inhibition: quercetin, lutelin) [29, 30]. These last effects potentially are expected to underlie the development of new anti-diarrheals and laxatives based on the ‘flavonoid’ pharmacophore [31].

esses [28] and activating/inhibiting epithelial Cl− transports via, e.g., CFTR channels (stimulation; biacalein, tangeretin; inhibition: quercetin, lutelin) [29, 30]. These last effects potentially are expected to underlie the development of new anti-diarrheals and laxatives based on the ‘flavonoid’ pharmacophore [31]. Acknowledgements We acknowledge Tsumura Co., Ltd for a generous gift of Junchoto powder. This work was supported in part by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science (No. 15K08197). Caco-2 (RCB0988) was purchased from RIKEN BRC through the National Bio-Resource Project of the MEXT (Tsukuba, Japan). Author contributions T.N. conducted all experiments and analysis. K.S.-N. conducted the siRNA experiments. Y.O. helped design the work and commented on the draft. T.N and R.I. conceived and designed the work and wrote the manuscript. Compliance with ethical standards Conflicts of interest The authors declare no conflicts of interest. Declaration of transparency and scientific rigour This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Introduction Cell adhesion molecules (CAMs) such as intercellular adhesion molecule 1 (ICAM-1, CD54 and immunoglobulin), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin (CD62E) are critical in the regulation of immune response and inflammation. The extracellular interactions between specific CAMs expressing on the endothelium and leukocytes mediate leukocyte entry into tissues, T cell proliferation, and antigen presentation [1–4]. The key event in autoimmune disease is the migration of leukocytes to the disease site. Agents that inhibit leukocyte adhesion, transmigration and expression of related CAMs represent therapeutic potential as immunosuppressives and anti-inflammatory drugs. The major adhesive force for lymphocyte extravasation from the blood stream into tissue site is the protein–protein interaction of the adhesion molecules lymphocyte function-associated molecule 1 (LFA-1, CD11a/CD18 and β2 integrin) and its endothelial counter-receptor ICAM-1 [5, 6]. Monoclonal antibodies to ICAM-1 have been shown to inhibit lymphocyte transendothelial migration and have yielded very promising results in clinical trials for rheumatoid arthritis and organ transplantation [7, 8]. Therefore, the search for specific inhibitors of integrin-mediated cell adhesion with a small molecule in expectation of anti-inflammatory and anti-metastatic drugs started in the 1990s.

elial migration and have yielded very promising results in clinical trials for rheumatoid arthritis and organ transplantation [7, 8]. Therefore, the search for specific inhibitors of integrin-mediated cell adhesion with a small molecule in expectation of anti-inflammatory and anti-metastatic drugs started in the 1990s. Generally, a cell adhesion inhibitor is categorized as target for cell–cell adhesion and for expression of cell adhesion molecules. Though certain small molecules such as flavonoids [9, 10] and others [11, 12] affecting expression of cell adhesion molecules are known, specific inhibitors for cell–cell contact are limited here in the review, as possible. So far, some cell adhesion inhibitors based on synthetic methods and computer-aided drug design have been developed [13, 14]; however, natural products can still make unexpected structural discovery possible and they are believed to be a reservoir of resources for new types of drugs. This review introduces cell adhesion inhibitors focusing on those of naturally occurring plant, microbial and marine origin.

design have been developed [13, 14]; however, natural products can still make unexpected structural discovery possible and they are believed to be a reservoir of resources for new types of drugs. This review introduces cell adhesion inhibitors focusing on those of naturally occurring plant, microbial and marine origin. Cell adhesion inhibitors of plant origin Terpenoid-sesquiterpene Chloranthus japonicas Sieb. (Chloranthaceae) is a perennial herb that grows in the southern part of Korea, Japan and China. It has been used for boils, dermatological disorder, and enteric fever in Korea as a folk remedy. Three active dimeric sesquiterpenoids of shizukaol B (1), cycloshizukaol A (2) and shizukaol F (3) were isolated from the MeOH extract of roots of C. japonicus [15]. These compounds inhibited phorbol 12-myristate-13-acetate (PMA)-induced homotypic aggregation of human promyelocytic leukemia (HL-60) cells without cytotoxicity with MIC values of 34.1 nM (1), 0.9 µM (2) and 27.3 nM (3), respectively. Although 1–3 did not affect the direct binding of LFA-1 to ICAM-1, these compounds markedly inhibited ICAM-1 expression in HL-60 cells in a dose-dependent manner. On the other hand, when human umbilical vein endothelial cells (HUVECs) were pretreated with 1–3 and stimulated with tumor necrosis factor α (TNF-α), adhesion of THP-1 cells to HUVECs decreased in a dose-dependent manner with IC50 values of 54.6 nM, 1.2 µM and 34.1 nM, respectively. In fact, 1 inhibited TNF-α-induced surface expression of the ICAM-1, VCAM-1 and E-selectin in HUVECs with IC50 values of 5.4 nM, 13.6 µM and 95.6 nM, respectively.

osis factor α (TNF-α), adhesion of THP-1 cells to HUVECs decreased in a dose-dependent manner with IC50 values of 54.6 nM, 1.2 µM and 34.1 nM, respectively. In fact, 1 inhibited TNF-α-induced surface expression of the ICAM-1, VCAM-1 and E-selectin in HUVECs with IC50 values of 5.4 nM, 13.6 µM and 95.6 nM, respectively. α-Iso-cubebene (4), a novel cubebene sesquiterpene from Schisandra chinensis (Schisandraceae), attenuated the activities of adhesion molecules in TNF-α-stimulated HUVECs [16]. α-Iso-cubebene (4) significantly suppressed the TNF-α-induced cell surface expression of VCAM-1 and E-selectin (43.8 and 29.6% inhibition, respectively) at 25 μg/mL, but not ICAM-1 expression. α-Iso-cubebene (4) attenuates TNF-α-stimulated endothelial adhesion to monocytes by inhibiting intracellular reactive oxygen species (ROS) production, the activation of redox-sensitive nuclear factor κB (NF-κB) transcription factor and expression of VCAM-1 and E-selectin. Terpenoid-diterpene Four clerodane diterpenes, 18,19-diacetoxyclerodane 18,19-oxide acetals, casearinols A (5) and B (6), and casearinones A (7) and B (8), were isolated from the leaves of Casearia guianensis (Flacourtiaceae) [17]. Compounds 5–8 inhibited the binding of LFA-1 to ICAM-1. Quantitative data were obtained for casearinol A (5), which inhibited the binding of LFA-1 to ICAM-1 in a dose-responsive manner, yielding an IC50 of 50 µM. This is the first report of immunomodulatory activity for this class of diterpenes.

(Flacourtiaceae) [17]. Compounds 5–8 inhibited the binding of LFA-1 to ICAM-1. Quantitative data were obtained for casearinol A (5), which inhibited the binding of LFA-1 to ICAM-1 in a dose-responsive manner, yielding an IC50 of 50 µM. This is the first report of immunomodulatory activity for this class of diterpenes. Andrographolide (9), an ent-labdane diterpenoid lactone isolated from the Chinese official herbal Andrographis paniculata (Acanthaceae), has been reported to have anticancer activity [18–20]. Jiang and co-workers reported that 9 inhibited the adhesion of gastric cancer cells with a highly expressing level of sialyl LewisX (SLeX) to the TNF-α-stimulated human endothelial cells by blocking E-selectin expression in a dose-dependent manner, in a concentration range of 1–10 µM [21].

anticancer activity [18–20]. Jiang and co-workers reported that 9 inhibited the adhesion of gastric cancer cells with a highly expressing level of sialyl LewisX (SLeX) to the TNF-α-stimulated human endothelial cells by blocking E-selectin expression in a dose-dependent manner, in a concentration range of 1–10 µM [21]. Terpenoid-triterpene, steroid and related compound Cucurbitacin E (10) was isolated from CH2C12 extract of the stem and leaves of Conobea scoparioides Benth. (Scrophulariaceae) as an antagonist of CD18-mediated cell adhesion. Cucurbitacin E (10) is a tetracyclic triterpenoid with an unsaturated side chain present in various plant families such as the Cucurbitaceae, Scrophulariaceae, Euphorbiaceae, Liliaceae and Elaeocarpaceae. Cucurbitacin E (10) and five related analogues, cucurbitacins B (11), I (12), D (13), L (14) and R (15) obtained separately, were tested in the cell adhesion assay. Compounds 10–13 showed inhibition of JY/HeLa cell binding through LFA-1/ICAM-1-mediated adhesion, with IC50 values of 0.18, 0.30, 0.95 and 1.36 µM, respectively. Cucurbitacin E (10) was demonstrated to inhibit cell adhesion to HeLa cells by interfering with LFA-1 and not ICAM-1 [22]. Touihri-Barakati and co-workers reported that cucurbitacin B (10) from the leaves of Tunisian Ecballium elaterium (Cucurbitaceae) showed anti-integrin activity on human glioblastoma U87 cells, without being cytotoxic at concentrations up to 500 nM [23].

Terpenoid-triterpene, steroid and related compound Cucurbitacin E (10) was isolated from CH2C12 extract of the stem and leaves of Conobea scoparioides Benth. (Scrophulariaceae) as an antagonist of CD18-mediated cell adhesion. Cucurbitacin E (10) is a tetracyclic triterpenoid with an unsaturated side chain present in various plant families such as the Cucurbitaceae, Scrophulariaceae, Euphorbiaceae, Liliaceae and Elaeocarpaceae. Cucurbitacin E (10) and five related analogues, cucurbitacins B (11), I (12), D (13), L (14) and R (15) obtained separately, were tested in the cell adhesion assay. Compounds 10–13 showed inhibition of JY/HeLa cell binding through LFA-1/ICAM-1-mediated adhesion, with IC50 values of 0.18, 0.30, 0.95 and 1.36 µM, respectively. Cucurbitacin E (10) was demonstrated to inhibit cell adhesion to HeLa cells by interfering with LFA-1 and not ICAM-1 [22]. Touihri-Barakati and co-workers reported that cucurbitacin B (10) from the leaves of Tunisian Ecballium elaterium (Cucurbitaceae) showed anti-integrin activity on human glioblastoma U87 cells, without being cytotoxic at concentrations up to 500 nM [23]. The extract from the root of Trichilia rubra (Meliaceae) was identified as having potent inhibitory activity in a bioassay for LFA-l/ICAM-I-mediated adhesion of JY and HeLa cells [24]. A series of seco-limonoids (16–22) with uncommon hemi ortho ester A-rings, was isolated. Compounds 16–22 exhibited potent inhibitory activity in the LFA-l/ICAM-1-mediated cell adhesion assay with IC50 values in the range of 10–25 nM. None of the compounds showed cytotoxicity at concentrations up to 20 µM.

a cells [24]. A series of seco-limonoids (16–22) with uncommon hemi ortho ester A-rings, was isolated. Compounds 16–22 exhibited potent inhibitory activity in the LFA-l/ICAM-1-mediated cell adhesion assay with IC50 values in the range of 10–25 nM. None of the compounds showed cytotoxicity at concentrations up to 20 µM. The tetracyclic triterpene euphol (23) is the main constituent found in the sap of Euphorbia tirucalli (Euphorbiaceae), widely known in Brazilian traditional medicine for its use in the treatment of several kinds of cancer. The effect of euphol (23) on experimental models of colitis and the underlying mechanisms involved in its action has been reported [25]. The euphol (23) decreased lipopolysaccharide (LPS)-induced monocyte chemotactic protein 1 (MCP-1), TNF-α, interleukin 6 (IL-6) and interferon γ (IFN-γ), but increased IL-10 secretion from bone marrow-derived macrophages in vitro, and markedly inhibited both selectin (P- and E-selectin) and integrin (ICAM-1, VCAM-1 and LFA-1) expression in colonic tissue. Moreover, euphol (23) treatment markedly inhibited the activation of NF-κB in mouse colon tissue. α-Tomatine (24), a glycoalkaloid isolated from Lycopersicon esculentum Linn, was reported to inhibit the PMA-induced abilities of adhesion, morphology/actin cytoskeleton arrangement, invasion and migration by cell–matrix adhesion assay, through blocking protein kinase Cα (PKC-α), extracellular signal-regulated kinase (ERK) and NF-κB activation. [26]

d isolated from Lycopersicon esculentum Linn, was reported to inhibit the PMA-induced abilities of adhesion, morphology/actin cytoskeleton arrangement, invasion and migration by cell–matrix adhesion assay, through blocking protein kinase Cα (PKC-α), extracellular signal-regulated kinase (ERK) and NF-κB activation. [26] Lignane Manassantin A (25) and B (26), dineolignans isolated from Saururus chinensis, inhibited PMA-induced ICAM-1/LFA-1-mediated homotypic aggregation of HL-60 cells without cytotoxicity and with MIC values of 1.0 and 5.5 nM, respectively. After pretreating HUVECs with 25 and 26 followed by stimulation with TNF-α, adhesion of human acute monocytic leukemia cell line THP-1 to HUVECs decreased in a dose-dependent manner with IC50 values of 5 and 7 ng/mL, respectively, without cytotoxicity [27]. Both 25 and 26 also inhibited TNF-α-induced up-regulation of ICAM-1, VCAM-1 and E-selectin. Flavonoide Astilbin [3,3′,4′,5,7-pentahydroxyflavanone 3-(6-deoxy-(l-mannopyranoside)] (27) from the rhizome of Smilax glabra (Liliaceae) was demonstrated to show a selective immunosuppressive feature [28]. The effect of 27 on concanavalin A (Con A)-induced liver injury by focusing on the TNF-α production and T lymphocyte adhesion was investigated. Inhibitory effect of 27 on the adhesion of Con A-activated human Jurkat T cells to endothelial cell line ECV-304 was reported.

show a selective immunosuppressive feature [28]. The effect of 27 on concanavalin A (Con A)-induced liver injury by focusing on the TNF-α production and T lymphocyte adhesion was investigated. Inhibitory effect of 27 on the adhesion of Con A-activated human Jurkat T cells to endothelial cell line ECV-304 was reported. Alkaloid Piperine (28) and ethyl 3′,4′,5′-trimethoxycinnamate (34; the structure is shown in a section of other compounds described below) from the combined hexane and chloroform extracts of Piper longum (Piperaceae) were isolated as potent inhibitors of cell adhesion molecules on HUVECs [29]. Both 28 and 34 inhibited the TNF-α-induced expression of ICAM-1 at IC50 values of 45 and 25 µg/mL, respectively. In further study, 34 significantly blocked the adhesion of neutrophils to endothelium in a concentration-dependent manner. Compound 34 also significantly inhibited TNF-α-induced expression of VCAM-1 and E-selectin at 50 µg/mL. To elucidate its structure–activity relationship, effects of synthesized analogues of 34 and its thio, thiono analogues, and synthesized 7-hydroxy-4-methylcoumarin derivatives on cell adhesion molecules were studied [30, 31]. Lee and co-workers reported four quinolone alkaloids (29–32) isolated from the methanol extracts of Evodiae fructus, as the specific inhibitor on the binding of LFA-1 and ICAM-1 [32]. Evodiae fructus is natural medicine originated from Evodia rutaecarpa (Juss.) Benth. (Rutaceae), which has been used for treatment of gastrointestinal disorders and headaches, as an analgesic and antiemetic, and for amenorrhea in Korea.

Evodiae fructus, as the specific inhibitor on the binding of LFA-1 and ICAM-1 [32]. Evodiae fructus is natural medicine originated from Evodia rutaecarpa (Juss.) Benth. (Rutaceae), which has been used for treatment of gastrointestinal disorders and headaches, as an analgesic and antiemetic, and for amenorrhea in Korea. The four quinolone alkaloids inhibited the interaction of sICAM-1 and LFA-1 in THP-1 cells at IC50 values of > 150 (29), 109.8 (30), > 150 (31) and 40.9 μM (32), respectively [vs. lovastatin (66) as a positive control, IC50 33 µM]. On the other hand, they had no effect on direct binding assay using sVCAM-1 and E-selectin. They did not show cytotoxicity at the concentrations employed in the study (ca. 70–80% of THP-1 cell viability at 150 µM). Among four quinolone alkaloids (29–32), cell adhesion inhibitory activity was suggested to be positively influenced by the presence of a double bond and an increase in aliphatic side chain length.

The four quinolone alkaloids inhibited the interaction of sICAM-1 and LFA-1 in THP-1 cells at IC50 values of > 150 (29), 109.8 (30), > 150 (31) and 40.9 μM (32), respectively [vs. lovastatin (66) as a positive control, IC50 33 µM]. On the other hand, they had no effect on direct binding assay using sVCAM-1 and E-selectin. They did not show cytotoxicity at the concentrations employed in the study (ca. 70–80% of THP-1 cell viability at 150 µM). Among four quinolone alkaloids (29–32), cell adhesion inhibitory activity was suggested to be positively influenced by the presence of a double bond and an increase in aliphatic side chain length. Castanospermine (33) is an indolizidine alkaloid originally isolated from the seeds of Castanospermum austral (the Australian Moreton Bay Chestnut, Fabaceae). Effects of 33 in a range of concentrations from 16,384 to 0.25 μM on mononuclear/endothelial cell binding and expression of their cell adhesion molecules were reported [33]. Upon HUVECs, 33 reduced expression of E-selectin, ICAM-1, ICAM-2 and platelet endothelial cell adhesion molecule (PECAM)-1, but increased it for P-selectin. Upon peripheral blood mononuclear cells, 33 reduced expression of L-selectin, LFA-1α, very-late antigen 4 (VLA-4; integrin α4β1), macrophage adhesion ligand 1 (Mac-1) and complement receptor 4 (CR-4; CD11c/CD18), but increased expression of P-selectin glycoprotein ligand 1 (PSGL-1) and PECAM-1. Similar changes of expression were found in the subset of lymphocytes and monocytes, but the reductions in LFA-1α and VLA-4 on lymphocytes and Mac-1 (CD11b/CD18) and CR-4 on monocytes were greater.

mplement receptor 4 (CR-4; CD11c/CD18), but increased expression of P-selectin glycoprotein ligand 1 (PSGL-1) and PECAM-1. Similar changes of expression were found in the subset of lymphocytes and monocytes, but the reductions in LFA-1α and VLA-4 on lymphocytes and Mac-1 (CD11b/CD18) and CR-4 on monocytes were greater. Other compounds Lee and co-workers found an inhibitory effect of methanol extract of Rheum undulatum (Polygonaceae) rhizomes on cell adhesion in search for anti-inflammatory or anti-metastasis agents, and isolated six stilbenes from the by bioactivity-guided fractionation. Six stilbenes were identified as desoxyrhapontigenin (35), rhapontigenin (36), trans-resveratrol (37), piceatannol (38), piceatannol-3′-O-β-D-glucopyranoside and isorhapontin. Among them, 35–38 inhibited the direct binding between sICAM-1 and LFA-1 of the THP-1 cells in a dose-dependent manner with IC50 values of 50.1, 25.4, 33.4 and 45.9 μM, respectively (Table 1). Compounds 36, 37 and 38 also had an inhibitory effect on direct binding between sVCAM-1 and VLA-4 of THP-1 cells [34].Table 1 Inhibitory effect of 35–38 on cell adhesion-mediated LFA-1/sICAM-1 and E-selectin/VCAM-1 LFA-1/sICAM-1 (µM) E-selectin/sVCAM-1 (µM) 35 a 50.1 Inactive 36 25.4 > 100 37 33.4 > 100 38 45.9 44.1 Lovastatin (66) 57.2 – aCompound 35 showed cytotoxicity at 100 µM In addition, compound 38 can interfere with the binding between integrin (LFA-1 and VLA-4) and immunoglobulins (ICAM-1 and VCAM-1). A lovastatin (66) was used as a positive control for the binding between sICAM-1 and LFA-1 of the THP-1 cells (IC50 57.2 µM; Table 1).

38 45.9 44.1 Lovastatin (66) 57.2 – aCompound 35 showed cytotoxicity at 100 µM In addition, compound 38 can interfere with the binding between integrin (LFA-1 and VLA-4) and immunoglobulins (ICAM-1 and VCAM-1). A lovastatin (66) was used as a positive control for the binding between sICAM-1 and LFA-1 of the THP-1 cells (IC50 57.2 µM; Table 1). Sparstolonin B (39) is an isocoumarin compound isolated from the tubers of both Sparganium stoloniferum and Scirpus yagara. Sparstolonin B (39) inhibited LPS-induced expression of ICAM-1 and VCAM-1 in HUVECs at 10 and 100 µM, respectively [35]. Sparstolonin B (39) significantly suppressed the adhesion of THP-1 cells to LPS-activated HUVECs at a concentration of 100 µM. The inhibitory effect of 39 on LPS-induced phosphorylation of extracellular signal-regulated kinase (Erk1/2) and serine/threonine kinase (Akt, protein kinase B) was also reported. Plant extract, etc. Effects of crude plant extract, snake venom and other naturally occurring fatty acid derivatives on cell adhesion molecules are also reported. As they are not isolated as pure components, only references are shown [36–45].

Sparstolonin B (39) significantly suppressed the adhesion of THP-1 cells to LPS-activated HUVECs at a concentration of 100 µM. The inhibitory effect of 39 on LPS-induced phosphorylation of extracellular signal-regulated kinase (Erk1/2) and serine/threonine kinase (Akt, protein kinase B) was also reported. Plant extract, etc. Effects of crude plant extract, snake venom and other naturally occurring fatty acid derivatives on cell adhesion molecules are also reported. As they are not isolated as pure components, only references are shown [36–45]. Cell adhesion inhibitors of microbial origin Macrosphelides (MSs) are 16-membered macrolides, embodying 3 ester linkages produced by several fungal strains. MSs A–D (40–43), J (49) and K (50) were originally isolated from the culture broth of Microsphaeropsis sp. FO-5050 as cell adhesion inhibitors [46–49]. At almost the same time, MSs A (40), C (42), E–I (44–48), L (51), seco-MS E (52) and MS-M (53) were isolated from a fungal strain of Periconia byssoides originally separated from the sea hare Aplysia kurodai [50–54]. MSs A–D (40–43) inhibited the adhesion of SLex-expressing HL-60 cells to endothelial cell leukocyte adhesion molecule 1 (ELAM-1)-expressing HUVECs in a dose-dependent fashion, with IC50 values of 3.5, 36, 67.5 and 25 µM, respectively. Among MSs, MS-A (40) showed the most potent inhibitory activity. On the one hand, MSs J (49) and K (50) were inactive (IC50 value: > 100 µg/mL).

L-60 cells to endothelial cell leukocyte adhesion molecule 1 (ELAM-1)-expressing HUVECs in a dose-dependent fashion, with IC50 values of 3.5, 36, 67.5 and 25 µM, respectively. Among MSs, MS-A (40) showed the most potent inhibitory activity. On the one hand, MSs J (49) and K (50) were inactive (IC50 value: > 100 µg/mL). It was suggested that they prevent the cell–cell adhesion by blocking the binding of SLex to ELAM-1. However, MSs showed no effect on the adhesion of sialyl Lewis A (SLea)-expressing HL-60 cells to HUVECs. Furthermore, pretreatment of HL-60 cells, not HUVECs, with MSs caused inhibition of the adhesion of HL-60 to HUVECs. These findings indicated that MSs specifically bound to SLex on HL-60 cells to block the cell–cell adhesion [55]. MSs proved to be effective in several in vivo models. In the mouse model of B16/BL6 melanoma lung metastasis, MS-B (41) caused a dose-dependent decrease in lung metastatic nodules without any toxic effect including body weight loss in the range of 5–20 mg/kg. Furthermore, its efficacy in combination therapy with anti-cancer drugs was demonstrated. Combined therapy of MS-B (41) and cisplatin (CDDP) induced remarkable lung metastasis inhibition without adverse effects of CDDP to the host [56, 57].

without any toxic effect including body weight loss in the range of 5–20 mg/kg. Furthermore, its efficacy in combination therapy with anti-cancer drugs was demonstrated. Combined therapy of MS-B (41) and cisplatin (CDDP) induced remarkable lung metastasis inhibition without adverse effects of CDDP to the host [56, 57]. The total synthesis of MS-A (40) has been reported by several groups. A novel total synthesis have been accomplished by the group of the Kitasato Institute [58, 59]. The combinatorial synthesis of a 122-member MS library including MSs A (40), C (42), E (44) and F (45) has been achieved based on a unique strategy for a three-component coupling utilizing a palladium-catalyzed chemoselective carbonylation and an unprecedented macrolactonization on a polymer support [60]. Synthetic approaches to MS derivatives, based on medicinal chemistry, were reviewed [61]. At present, MS-A (40) is commercially available as a reagent. HUN-7293 (54) is a fungal cyclodepsipeptide that was first identified as an inhibitor of VCAM expression [62]. HUN-7293 (54) inhibited expression of VCAM-1 and ICAM-1 on TNF-α-stimulated human microvascular endothelial cells (HMEC-1), with IC50 values of 2 and 50 nM, respectively. HUN-7293 (54) almost inhibited cell adhesion between the human Burkitt’s lymphoma B (BL 2) cell and TNF-α-stimulated HMEC-1 at a concentration of 20 nM. Total synthesis of 54 had done and evaluation of synthetic analogues as inhibitors of VCAM-1 expression was further reported [63, 64].

HUN-7293 (54) is a fungal cyclodepsipeptide that was first identified as an inhibitor of VCAM expression [62]. HUN-7293 (54) inhibited expression of VCAM-1 and ICAM-1 on TNF-α-stimulated human microvascular endothelial cells (HMEC-1), with IC50 values of 2 and 50 nM, respectively. HUN-7293 (54) almost inhibited cell adhesion between the human Burkitt’s lymphoma B (BL 2) cell and TNF-α-stimulated HMEC-1 at a concentration of 20 nM. Total synthesis of 54 had done and evaluation of synthetic analogues as inhibitors of VCAM-1 expression was further reported [63, 64]. Nakagawa and co-workers found two inhibitors adxanthromycins A (55) and B (56) of ICAM-1/LFA-1-mediated cell adhesion molecule produced by Streptomyces sp. NA-148 [65–67]. The structure of 55 and 56 were characterized as dimeric anthrone peroxide skeleton containing an α-d-galactose for 55 and two α-d-galactose for 56. Both 55 and 56 inhibited homotypic aggregation of Epstein–Barr virus (EBV)-immortalised B cell lymphoblastoid line (JY cell) from 1.5 µg/mL in a dose dependent manner. A complete inhibition was observed at 6.25 µg/mL.

Nakagawa and co-workers found two inhibitors adxanthromycins A (55) and B (56) of ICAM-1/LFA-1-mediated cell adhesion molecule produced by Streptomyces sp. NA-148 [65–67]. The structure of 55 and 56 were characterized as dimeric anthrone peroxide skeleton containing an α-d-galactose for 55 and two α-d-galactose for 56. Both 55 and 56 inhibited homotypic aggregation of Epstein–Barr virus (EBV)-immortalised B cell lymphoblastoid line (JY cell) from 1.5 µg/mL in a dose dependent manner. A complete inhibition was observed at 6.25 µg/mL. The toxicity (IC50) of 55 and 56 against JY cell was 15.2 µg/mL. Compounds 55 and 56 also inhibited SKW-3 adhesion to soluble ICAM-1 in a dose-dependent manner with an IC50 of 18.8 and 25.0 µg/mL, respectively. The cell toxicity (IC50) of adxanthromycins against SKW-3 was 110.0 µg/mL. In the cell-free receptor binding assay, both 55 and 56 showed weak inhibition with an IC50 of 760 µg/mL. They were reported as the first example of inhibitors of ICAM-l/LFA-1-mediated adhesion molecule isolated from microbial sources.

ectively. The cell toxicity (IC50) of adxanthromycins against SKW-3 was 110.0 µg/mL. In the cell-free receptor binding assay, both 55 and 56 showed weak inhibition with an IC50 of 760 µg/mL. They were reported as the first example of inhibitors of ICAM-l/LFA-1-mediated adhesion molecule isolated from microbial sources. The benzopyran derivative (57) was found in the culture of Streptomyces sp. Mer-88 as ICAM-1/LFA-1 binding inhibitors, ICAM-1 inhibitors and LFA-1 inhibitors [68]. Compound 57 inhibited binding between ICAM-1 and LFA-1 in the range of 31–2500 µg/mL dose dependently, without cytotoxicity up to a concentration of 1000 µg/mL. Then, Xu and co-workers reported isolation of the same compound, N-[[3,4-dihydro-3S-hydroxy-2S-methyl-2-(4′R-methyl-3′S-pentenyl)-2H-1-benzopyran-6-yl]carbonyl]-threonine (57), produced by Streptomyces xiamenensis, its structure, including the absolute configuration, and its anti-fibrotic properties [69]. Three derivatives of cytochalasin were isolated from the cultured broth of the fungal strain Mycotypha sp. UMF-006, as inhibitors of cell adhesion based on LFA-1/ICAM-1 [70]. These compounds were identified to be cytochalasin E (58), 5,6-dehydro-7-hydroxy derivative of cytochalasin E (59) and Δ6,12-isomer of 59 (60). All these components inhibited adhesion of HL-60 cells to CHO-ICAM-1 cells at IC50 values of 30 µg/mL for 58, 75 µg/mL for 59 and 90 µg/mL for 60.

n based on LFA-1/ICAM-1 [70]. These compounds were identified to be cytochalasin E (58), 5,6-dehydro-7-hydroxy derivative of cytochalasin E (59) and Δ6,12-isomer of 59 (60). All these components inhibited adhesion of HL-60 cells to CHO-ICAM-1 cells at IC50 values of 30 µg/mL for 58, 75 µg/mL for 59 and 90 µg/mL for 60. Members of the efomycine family from Streptomyces BS1261 were found to inhibit leukocyte adhesion, from a screening library of 20,000 natural compounds [71]. Finally, efomycines A B, E and G (61) were isolated as active substances [only structures of G (61) and M (62) were shown]. Members of the efomycine family inhibited the binding of human or porcine neutrophils by 50–60% at 10−5 M, whereas efomycine M (62) did not have a significant effect. Efomycine M (62) showed the most selective inhibitory effects on selectin-mediated leukocyte-endothelial adhesion in vitro, significantly diminishing rolling in mouse ear venules in vivo. In addition, efomycine M (70) alleviated cutaneous inflammation in two complementary mouse models of psoriasis, one of the most common chronic inflammatory skin disorders. Molecular modeling demonstrated a spatial conformation of efomycines mimicking naturally occurring selectin ligands.

g in mouse ear venules in vivo. In addition, efomycine M (70) alleviated cutaneous inflammation in two complementary mouse models of psoriasis, one of the most common chronic inflammatory skin disorders. Molecular modeling demonstrated a spatial conformation of efomycines mimicking naturally occurring selectin ligands. Three compounds, NP25301 (63), NP25302 (deoxybohemamine 64) and bohemamine (65), inhibitors of cell adhesion based on LFA-1/ICAM-1, were isolated from the cultured broth of the strain Streptomyces sp. UMA-044 [72]. Compounds 63–65 inhibited adhesion of HL-60 cells to CHO-ICAM-1 cells at IC50 values of 29.5 µg/ml for 63, 24.3 µg/ml for 64 and 27.2 µg/ml for 65. Random screening of chemical libraries identified the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin (66), a drug clinically used for lowering cholesterol levels, as an inhibitor of the LFA-1/ICAM-1 interaction [73]. Lovastatin (66) showed binding inhibition of recombinant ICAM-1 to purified LFA-1 with an IC50 value of 2.1 µM. Inhibitory effects of statin-derived compounds on the binding were also shown with a range of IC50 0.04–14 µM. The biological relevance of LFA-1 inhibition by statins with respect to the overall benefit of this drug class was reviewed [74].

nhibition of recombinant ICAM-1 to purified LFA-1 with an IC50 value of 2.1 µM. Inhibitory effects of statin-derived compounds on the binding were also shown with a range of IC50 0.04–14 µM. The biological relevance of LFA-1 inhibition by statins with respect to the overall benefit of this drug class was reviewed [74]. Structurally related to known naturally occurring cyclic heptadepsipeptides, HUN-7293 (54), named heptadepsin (67), was isolated from the culture broth of Paenibacillus sp. [75]. Compound 67 inhibited LPS-stimulated adhesion between HUVECs and HL-60 cells with an IC50 value of 0.92 µg/mL, without showing any cytotoxicity up to 30 µg/mL. Compound 67 also inhibited cellular adhesion induced by lipid A, the active component of LPS, but it did not inhibit TNF-α- or IL-1β-induced cell adhesion. Heptadepsin (67) was shown to inactivate LPS by direct interaction with LPS and lipid A from the results of surface plasmon resonance analysis.

city up to 30 µg/mL. Compound 67 also inhibited cellular adhesion induced by lipid A, the active component of LPS, but it did not inhibit TNF-α- or IL-1β-induced cell adhesion. Heptadepsin (67) was shown to inactivate LPS by direct interaction with LPS and lipid A from the results of surface plasmon resonance analysis. Peribysins A–J (68–77) including a furanofuran, were isolated from the culture of a strain Periconia byssoides OUPS-N133 separated from the sea hare Aplysia kurodai. They inhibited the adhesion of HL-60 cells to LPS-stimulated HUVECs with an IC50 range of 0.1–20 µM. Among them, compounds A (68) and D (71) showed the most potent cell adhesion inhibitory activity with IC50 values 0.3 and 0.1 µM, respectively, as compared to that of herbimycin A (standard, IC50 38 µM) [54, 76–78]. Interestingly, the producing strain OUPS-N133 of peribysins was the same as that of MSs A (40), C (42), E (44), F–I (45–48), L (51) and M (53). The total synthesis necessitated revision of the assignment of the absolute configuration of 72 [79]. Cell adhesion inhibitors derived from a marine organism In the screening for P-selectin inhibitors, sulfonoquinovosyl dipalmitoyl glyceride (78) and phosphatidylglycerol (79) were isolated from the 85% EtOH extract of the marine alga Dictyochloris fragrans. Both 78 and 79 inhibited P-selectin binding to sulfatides in the P-selectin–IgG ELISA assay, with IC50 values of 5 and 1 µM, respectively [80].

lectin inhibitors, sulfonoquinovosyl dipalmitoyl glyceride (78) and phosphatidylglycerol (79) were isolated from the 85% EtOH extract of the marine alga Dictyochloris fragrans. Both 78 and 79 inhibited P-selectin binding to sulfatides in the P-selectin–IgG ELISA assay, with IC50 values of 5 and 1 µM, respectively [80]. The inhibitory effect of 78 on HL-60 cell adhesion to immobilized P-selectin receptor globulin (Rg) was only shown with an IC50 value of 40 µM. Compound 78 was further shown for its ability to inhibit (24%) the P-selectin-dependent binding of activated platelets to HL-60 cells. From a panel of 60 unusual marine natural products, 17 compounds inhibited LFA-1/ICAM-1-based cell aggregation without showing significant cytotoxicity in the primary assay. Six compounds inhibited the cell–cell adhesion of HL-60 cells to CHO-ICAM-1 cells. The unusual oxylipin Cymathere aldehyde methyl ester (80; IC50 3.5 µM), cyanobacterial lipopeptide microcolins B (81; IC50 0.15 µM) and D (82; IC50 0.9 µM), bromophenol avrainvilleol (83; IC50 2.2 µM), sesquiterpene cymopol (84; IC50 2.7 µM) and cryptophyte-derived compound styrylchromone hormothamnione diacetate (85; IC50 1.5 µM) significantly inhibited LFA-1/ICAM-1-mediated cell adhesion (Table 2). The pharmacological activity and structure–activity relationships of selected marine algal metabolites are described [81].Table 2 Effects of marine natural products (80–85) on cell adhesion of HL-60 cells to CHO-ICAM-1 cells, and on proliferation of CHO-ICAM-1 cells

ibited LFA-1/ICAM-1-mediated cell adhesion (Table 2). The pharmacological activity and structure–activity relationships of selected marine algal metabolites are described [81].Table 2 Effects of marine natural products (80–85) on cell adhesion of HL-60 cells to CHO-ICAM-1 cells, and on proliferation of CHO-ICAM-1 cells Compound Sources HL-60 HL-60/CHO-ICAM-1 CHO-ICAM-1 (A) C. A.a (B) C. P.b S.I.c (C) Cell adhesion (D) C. P.b MICd (µM) IC50 (µM) B/A IC50 (µM) IC50 (µM) 80 Cymathere aldehyde methyl ester Cymathere triplicate 5.9 > 5.9 > 1.0 3.5 ± 0.3 2.7 ± 0.5 81 Microcolin B Lyngbya majuscula 1.7 1.3 ± 0.2 0.7 0.15 ± 0.09 > 1.4 82 Microcolin D Lyngbya majuscula 1.8 > 1.8 > 1.0 0.9 ± 0.1 1.1 ± 0.2 83 Avrainvilleol Avrainvillea longicaulis 3.1 > 3.1 > 1.0 2.2 ± 0.2 0.8 ± 0.3 84 Cymopol Cymopolia barbata 1.3 > 3.8 > 3.0 2.7 ± 0.2 > 3.1 85 Hormothamnione diacetate Chrysophaeum taylori 2.6 > 2.6 > 1.0 1.5 ± 0.2 0.6 ± 0.2 Cytochalasin Be 0.3 > 2.6 8.9 1.2 ± 0.2 0.2 ± 0.01 Values are means of three independent determinations ± SE aCell aggregation; bCell proliferation; cSpecific index; dMinimum inhibitory concentration; eUsed as a standard Conclusions As listed above, natural cell adhesion inhibitors are still proved essential to drug development due to their great variety of unexpected structures, such as plant-derived terpenoid (1–24), lignans (25–26), flavonoid (27) and alkaloid (28–33), and macrolide (40–53, 58–60), cyclodepsipeptide (54, 67), dimeric anthrone (55, 56), and furanofuran (68–77) of microbial origin, and marine-derived compounds (78–85).

pment due to their great variety of unexpected structures, such as plant-derived terpenoid (1–24), lignans (25–26), flavonoid (27) and alkaloid (28–33), and macrolide (40–53, 58–60), cyclodepsipeptide (54, 67), dimeric anthrone (55, 56), and furanofuran (68–77) of microbial origin, and marine-derived compounds (78–85). Among those from different origin, it is interesting to note that casearinols (5, 6), casearinones (7, 8), andrographolide (9), lovastatin (66) and peribysins A–J (68–77) were structurally similar based on a decalin (decahydronaphthalene) skeleton with a side chain or condensed furanofuran. Basically, all inhibitors covered this time were found using a cell-based assay for cell–cell adhesion or cell-soluble cell adhesion molecule. In a different way from the cell-based assay, the pharmacophore of sLex, recognized by a family of selectin, was used to search a three-dimensional database of chemical structures. As result of a search for a binding inhibitor between selectins and sLex, glycyrrhizin (structure was not shown), a saponin and sweet-tasting constituent of Glycyrrhiza glabra (liquorice, Fabaceae) root, was matched as a pharmacophore [82].

lectin, was used to search a three-dimensional database of chemical structures. As result of a search for a binding inhibitor between selectins and sLex, glycyrrhizin (structure was not shown), a saponin and sweet-tasting constituent of Glycyrrhiza glabra (liquorice, Fabaceae) root, was matched as a pharmacophore [82]. Commercially available cell adhesion inhibitors are almost all developed based on peptide [83, 84]. But naturally occurring inhibitors such as MSs A–D (40–43) were not only first isolated in the culture of Microsphaeropsis sp. FO-5050 as cell adhesion inhibitors, but also possess a novel macrocyclic skeleton with tri-ester groups. Furthermore, MSs (40–53), nontoxic against HL-60 cells and HUVECs, have recently become an active area of research for anticancer drugs [61]. It is of great interest that MSs (40–53) were found in the culture of Periconia byssoides OUPS-N133, a strain producing peribysins A–J (68–77) [54, 76–78]. Application of a diversity of microorganisms displays an infinite number of possibilities and unexploited production capacity. Therefore, natural products are still of particular interest as seeds for drug discovery. The original version of this article was revised due to a retrospective Open Access order. This review is dedicated to the Japanese Society of Pharmacognosy (JSP) Award for Scientific Contributions 2015. Change history 8/3/2018 The article Naturally occurring cell adhesion inhibitors, written by Satoshi Takamatsu, was originally published electronically on the publisher’s internet portal.

The original version of this article was revised due to a retrospective Open Access order. This review is dedicated to the Japanese Society of Pharmacognosy (JSP) Award for Scientific Contributions 2015. Change history 8/3/2018 The article Naturally occurring cell adhesion inhibitors, written by Satoshi Takamatsu, was originally published electronically on the publisher’s internet portal. Acknowledgements I am indebted particularly to Prof. Kazuki Saito of the Graduate School of Pharmaceutical Sciences, Chiba University. I am deeply indebted to the late Prof. Kazuo Toriizuka of the School of Pharmacy, Showa University. I deeply grateful to Dr. Satoshi Ōmura, Distinguished Emeritus Professor of Kitasato University. I also sincerely thank Dr. Kanki Komiyama of Kitasato Research Center for Environmental Science, Prof. Yong-pil Kim and Prof. Masahiko Hayashi of the Department of Pharmacy, Iwaki Meisei University, for collaboration at the Kitasato Institute. This research was supported in part by a Grant-in-Aid for Scientific Research (C; no. 15K08002).

Introduction Many reports have demonstrated that various metabolizing enzymes and transporters have important roles in the pharmacokinetics of xenobiotics [1–4]. P-glycoprotein (P-gp) is one of the major drug transporters expressed in different organs, including the small intestine, kidney, liver, lungs, colon, and blood–brain barrier [5, 6]. In the small intestine, P-gp is localized in the apical surface of intestinal epithelial mucosa and has the primary function of promoting removal of toxic compounds by active efflux back to the intestinal lumen [7]. Recently, venetoclax, which is indicated for the treatment of patients with chronic lymphocytic leukemia, enhanced the plasma concentration of co-administrated digoxin, a P-gp probe substrate, by inhibition of P-gp transporter [8]. Similarly, the levels of expression and functionality of P-gp can be modulated by inhibition or induction, which can affect the pharmacokinetics, efficacy, safety, or tissue levels of P-gp substrates, i.e., the safety and efficacy of drugs can be dramatically altered by drug–drug interactions (DDIs) which are influenced by the increase and decrease in P-gp activity.

ality of P-gp can be modulated by inhibition or induction, which can affect the pharmacokinetics, efficacy, safety, or tissue levels of P-gp substrates, i.e., the safety and efficacy of drugs can be dramatically altered by drug–drug interactions (DDIs) which are influenced by the increase and decrease in P-gp activity. Caco-2 cells derived from human colon carcinoma cells, which express P-gp and metabolizing enzymes such as peptidase, are widely used as an in vitro model of human small intestinal mucosa to predict the absorption of orally administered drugs [9] because of their morphological and functional similarity with human enterocytes [9, 10]. Digoxin is often used as a model P-gp substrate to assess transporter-mediated transport and inhibition in the Caco-2 permeability assay [11].

human small intestinal mucosa to predict the absorption of orally administered drugs [9] because of their morphological and functional similarity with human enterocytes [9, 10]. Digoxin is often used as a model P-gp substrate to assess transporter-mediated transport and inhibition in the Caco-2 permeability assay [11]. Many traditional Japanese Kampo medicines approved by the Japanese Ministry of Health, Labor, and Welfare (MHLW) have been used for the treatment of various diseases in Japan for many years. In modern medical care in which Kampo and Western drugs are often combined, it is important to clarify DDI to ensure the safety and efficacy of their combined use. However, there is little evidence of DDI in Kampo medicines. Yokukansan (YKS), rikkunshito (RKT), hangeshashinto (HST), goshajinkigan (GJG), and shakuyakukanzoto (SKT) are Kampo medicines which have been used for treating the behavioral and psychological symptoms of dementia, including aggression and insomnia [12], functional dyspepsia [13], stomatitis [14], and peripheral neuropathy [15], in patients with dementia and cancer or with muscle cramps [16]. Because the frequency of clinical use for these five prescriptions has recently increased rapidly, and opportunities to be used concomitantly with Western medicines have increased, safety information including drug interactions is required in medical practice. In order to promote the fostering and evolution of these Kampo medicines, we are actively accumulating scientific evidence to ensure their effectiveness and safety. Several studies on the DDIs or the side-effects of these five Kampo medicines have been reported previously, which include information on side-effects frequency surveys [17, 18] for YKS and SKT, cytochrome P-450 (CYP) [19] for YKS and RKT, and P-gp function using ATPase assay [20] or other drug transporter polypeptide 2B1 to organic anion-transporting polypeptide 2B1 [21] for various Kampo medicines. The results suggest that the possibility of causing severe pharmacokinetic DDIs through some types of drug-metabolizing enzyme and transporter or side-effects is low. However, to our knowledge, there is no evidence examining the effects of the five Kampo medicines on P-gp function using a Caco-2 permeability assay recommended by the Japanese Ministry of Health, Labor, and Welfare (MHLW) [22] and the US Food and Drug Administration (FDA) [23].

and transporter or side-effects is low. However, to our knowledge, there is no evidence examining the effects of the five Kampo medicines on P-gp function using a Caco-2 permeability assay recommended by the Japanese Ministry of Health, Labor, and Welfare (MHLW) [22] and the US Food and Drug Administration (FDA) [23]. Therefore, in this study, the effects of five Kampo medicines (YKS, RKT, HST, GJG, and SKT) on P-gp drug transport were first examined. Then, the effects of the constituent crude drugs and some ingredients in YKS, which showed the strongest P-gp inhibitory effect, were further examined to clarify the contribution of YKS activity. Finally, the Igut/IC50 values for the five Kampo medicines were calculated, and the DDI risk was objectively evaluated according to the criteria in the DDI guidance of the MHLW [22] and FDA [23].

Introduction Structure of carotenoids Carotenoids are tetraterpene pigments, which exhibit yellow, orange, red and purple colors. Carotenoids are the most widely distributed pigments in nature and are present in photosynthetic bacteria, some species of archaea and fungi, algae, plants, and animals. Most carotenoids consist of eight isoprene units with a 40-carbon skeleton. Their general structures commonly consist of a polyene chain with nine conjugated double bonds and an end group at both ends of the polyene chain. The structures of the polyene chain and end groups of carotenoids are shown in Fig. 1a [1]. Carotenoids are divided into two groups: carotenes and xanthophylls. Carotenes, such as α-carotene, β-carotene, β,ψ-carotene (γ-carotene), and lycopene, are hydrocarbons. About 50 kinds of carotenes are present in nature [1]. On the other hand, xanthophylls, such as β-cryptoxanthin, lutein, zeaxanthin, astaxanthin, fucoxanthin, and peridinin, are carotenoids containing oxygen atoms as hydroxy, carbonyl, aldehyde, carboxylic, epoxide, and furanoxide groups in these molecules. Some xanthophylls are present as fatty acid esters, glycosides, sulfates, and protein complexes. Structures of xanthophylls show marked diversity. About 800 kinds of xanthophylls have been reported in nature up until 2018 [1, 2]. Figure 1b shows structures of typical carotenes and xanthophylls. Most carotenoids have 40-carbon skeleton (C40 carotenoid). Some carotenoids have a 45- or 50-carbon skeleton, which are called higher carotenoids. About 40 kinds of higher carotenoids are present in some species of archaea. On the other hand, carotenoids composed of carbon skeletons with fewer than 40 carbons are called apocarotenoids. About 120 kinds of apocarotenoids are present in some species of plants and animals as degradation products of C40 carotenoids [1, 2].Fig. 1 a Basic structures of carotenoids and end groups. b Structures of typical carotenes and xanthophylls

n YKS, which showed the strongest P-gp inhibitory effect, were further examined to clarify the contribution of YKS activity. Finally, the Igut/IC50 values for the five Kampo medicines were calculated, and the DDI risk was objectively evaluated according to the criteria in the DDI guidance of the MHLW [22] and FDA [23]. Materials and methods Test substances and reagents The biological activities of the five Kampo medicines and the composition of the constituent crude drugs are shown in Table 1. In the present study, the dry powdered extracts of YKS (Lot No. 321017700), RKT (Lot No. 332003900), HST (Lot No. 302149300), GJG (Lot No. 2120107020), and SKT (Lot No. 322098500), and seven crude drugs, constituting YKS {i.e., Uncariae Uncis Cum Ramulus (UUCR; Lot No. 2071089010), GR (Lot No. 281013010), Atractylodis Lanceae Rhizoma (ALR; Lot No. 2031005010), Poria (Lot No. 2031007010), Angelicae Acutilobae Radix (Lot No. 2031002010), Cnidii Rhizoma (Lot No. 2031004010), and Bupleuri Radix (Lot No. 2081020010)} were supplied by Tsumura & Co. (Tokyo, Japan). They were prepared by spray-drying a hot water extract from one crude drug or a mixture of several crude drugs. UUCR alkaloids geissoschizine methyl ether (GM) and rhynchophylline (RP) were obtained from AvaChem Scientific (San Antonio, TX, USA) and Tsumura & Co., respectively.Table 1 Biological activity of the five Kampo medicines used in the present study and the composition of the constituent crude drugs

several crude drugs. UUCR alkaloids geissoschizine methyl ether (GM) and rhynchophylline (RP) were obtained from AvaChem Scientific (San Antonio, TX, USA) and Tsumura & Co., respectively.Table 1 Biological activity of the five Kampo medicines used in the present study and the composition of the constituent crude drugs Kampo medicine Known pharmacological activities Constituent crude drugs (% composition) Yokukansan Amelioration of behavioral and psychological symptoms of dementia [12] Glycyrrhizae radix (7.4), Atractylodis lanceae rhizoma (19.5), poria (19.5), cnidii rhizoma (14.6), Uncariae uncis cum ramulus (14.6), Angelicae acutilobae radix (14.6), bupleuri radix (9.8) Rikkunshito Improvement of upper gastrointestinal disorders [13] Atractylodis lanceae rhizoma (18.6), Ginseng radix (18.6), Pinelliae tuber (18.6), poria (18.6), zizyphi fructus (9.3), citri unshiu pericarpium (9.3), Glycyrrhizae radix (4.7), Zingiberis rhizoma (2.3) Shakuyakukanzoto Amelioration of painful muscle cramps [16] Glycyrrhizae radix (50), Paeoniae radix (50) Hangeshashinto Reducing chemotherapy-induced oral mucositis [14] Pinelliae tuber (27.0), Scutellariae radix (13.5), Zingiberis rhizoma processum (13.5), Glycyrrhizae radix (13.5), zizyphi fructus (13.5), Ginseng radix (13.5), coptidis rhizoma (5.5) Goshajinkigan Reducing chemotherapy-induced peripheral neuropathy [15] Rehmanniae radix (17.9), Achyranthis radix (10.7), corni fructus (10.7), Dioscoreae rhizoma (10.7), plantaginis semen (10.7), alismatis tuber (10.7), poria (10.7), moutan cortex (10.7), Cinnamomi cortex (3.6), Aconiti radix processa et pulverata (3.6)

shajinkigan Reducing chemotherapy-induced peripheral neuropathy [15] Rehmanniae radix (17.9), Achyranthis radix (10.7), corni fructus (10.7), Dioscoreae rhizoma (10.7), plantaginis semen (10.7), alismatis tuber (10.7), poria (10.7), moutan cortex (10.7), Cinnamomi cortex (3.6), Aconiti radix processa et pulverata (3.6) [3H]-Digoxin (26.3–39.8 Ci/mmol), [3H]-mannitol (17.2 Ci/mmol), and ULTIMA Gold scintillation fluid were obtained from PerkinElmer Life and Analytical Sciences (Waltham, MA, USA). Digoxin was obtained from Alfa Aesar (Ward Hill, MA, USA). Mannitol was obtained from Wako Pure Chemical Industries (Osaka, Japan). Verapamil hydrochloride was obtained from Enzo Life Sciences (Farmingdale, NY, USA). Fetal bovine serum, nonessential amino acids, penicillin, streptomycin, and l-glutamine used for Caco-2 cell culture were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Other chemicals were purchased from commercial sources. Test substance solutions Various concentrations of the five Kampo medicines, seven crude drugs, and two ingredients (GM and RP) were prepared by dissolving in dimethyl sulfoxide (DMSO) or a mixture of DMSO and HEPES-buffered HBSS (HBSS-HEPES, pH 7.4) and then diluting with HBSS-HEPES buffer. The final concentration of DMSO was adjusted to ≤1% (v/v) in each study.

ns of the five Kampo medicines, seven crude drugs, and two ingredients (GM and RP) were prepared by dissolving in dimethyl sulfoxide (DMSO) or a mixture of DMSO and HEPES-buffered HBSS (HBSS-HEPES, pH 7.4) and then diluting with HBSS-HEPES buffer. The final concentration of DMSO was adjusted to ≤1% (v/v) in each study. Caco-2 cell culture Caco-2 cells obtained from the American Type Culture Collection (Manassas, VA, USA) were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific), including 10% fetal bovine serum, 1% nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 292 µg/ml l-glutamine, and incubated at 37 °C in an atmosphere with 5% CO2. They were split three times per week at a ratio of 1:4 upon reaching 80–90% confluence to use in the following cell viability and P-gp permeability assays. The cells used in this study were between 25 and 52 passages.

00 µg/ml streptomycin, and 292 µg/ml l-glutamine, and incubated at 37 °C in an atmosphere with 5% CO2. They were split three times per week at a ratio of 1:4 upon reaching 80–90% confluence to use in the following cell viability and P-gp permeability assays. The cells used in this study were between 25 and 52 passages. Cell viability assay The confluent Caco-2 cells (1.0 × 104 cells/well) were seeded into 96-well plates and incubated for 2 days in the same constituent DMEM as described above. The media were then replaced with DMEM that included various concentrations of test substance or vehicle. Three or more wells were used for evaluation of each concentration. After the test substance-added wells were incubated for 2 h, the supernatant was removed, and 90 μl of HBSS-HEPES buffer and 10 μl of Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) were then added to the well. The cells were incubated for another 2–4 h at 37 °C in a CO2 incubator and measured at 450 nm using a Tecan Infinite M200 plate reader (Mannedorf, Switzerland). Cell viability was evaluated as a percentage of the absorbance of drug-treated cells relative to that of the vehicle-treated control (100% viability).

he well. The cells were incubated for another 2–4 h at 37 °C in a CO2 incubator and measured at 450 nm using a Tecan Infinite M200 plate reader (Mannedorf, Switzerland). Cell viability was evaluated as a percentage of the absorbance of drug-treated cells relative to that of the vehicle-treated control (100% viability). Digoxin transport assay via P-gp Caco-2 cells (2.7 × 104 cells/insert) were seeded into a polyethylene terephthalate insert (0.4 mm pore size; Corning Life Sciences, Acton, MA, USA) in 24-well tissue culture plates. After culturing for approximately 21 days, the cell monolayer insert side was washed with warm phosphate-buffered saline (PBS), and the PBS in the apical chamber and the medium in the basolateral chamber were replaced with HBSS-HEPES buffer. The transepithelial electrical resistance (TEER) to evaluate the integrity of the cell monolayer was measured using the Millicell ER-2 (Millipore, Billerica, MA, USA). For further verification of the tight junctions in the monolayer, warm HBSS-HEPES buffer containing mannitol (10 µmol/l) and [3H]-mannitol (0.058 µmol/l) was loaded into apical chambers, and the monolayer permeability of mannitol was measured in the same procedure as that of the following test substances. The integrity of a cell monolayer was confirmed by measuring the TEER, and mannitol permeability was used for evaluation of the test substance.

mannitol (0.058 µmol/l) was loaded into apical chambers, and the monolayer permeability of mannitol was measured in the same procedure as that of the following test substances. The integrity of a cell monolayer was confirmed by measuring the TEER, and mannitol permeability was used for evaluation of the test substance. To evaluate the test substance effect on the permeability of digoxin via the cell monolayer, warm HBSS-HEPES buffer containing digoxin (10 µmol/l) and [3H]-digoxin (0.025–0.038 µmol/l) was added into either the apical or basolateral chamber in the absence or presence of various concentrations of test substances or verapamil (50 µmol/l). Aliquot solution in the apical or basolateral chamber was collected after incubation at 37 °C for 120 min with slow rotation in the horizontal direction and addition of an ULTIMA GOLD scintillation cocktail. The mixture was immediately injected into a liquid scintillation counter (AccuFLEX LSC-7200; Hitachi Aloka Medical, Tokyo, Japan) to measure the radioactivity. The apparent permeability (Papp) of digoxin was calculated as follows: Papp=dQdt×VA×C0 where dQ/dt is the slope of the linear portion of the permeated amount versus time curve (µmol/l/s), A is the effective surface area of the Transwell insert (0.3 cm2), C0 is the initial concentration of the digoxin applied at t = 0 (µmol/l), and V is the volume of the receiver chamber (ml).

as follows: Papp=dQdt×VA×C0 where dQ/dt is the slope of the linear portion of the permeated amount versus time curve (µmol/l/s), A is the effective surface area of the Transwell insert (0.3 cm2), C0 is the initial concentration of the digoxin applied at t = 0 (µmol/l), and V is the volume of the receiver chamber (ml). The efflux ratio to evaluate the P-gp inhibitory effect of the test substance was calculated as follows: Efflux ratio=Papp,B→APapp,A→B where Papp, A→B is the apparent permeability of digoxin from the apical-to-basolateral direction and Papp, B→A is that from the basolateral-to-apical direction. The inhibition ratios of P-gp by the test substances were determined as follows: Inhibition ratio(%)=(efflux ratiotest substance/efflux ratiocontrol)×100. Data analysis The efflux ratios in each experiment were expressed as the mean ± standard deviation (SD). The statistical significance was evaluated by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test. A P value of < 0.05 indicated significance. IC50 values for P-gp inhibition of the five Kampo medicines were calculated by non-linear regression analysis using SAS 9.2 software (SAS Institute, Inc., Cary, NC, USA) or from the least square regression line. The concentration–response correlative formula and correlation coefficient (R2) for each crude drug constituting YKS were determined by regression analysis using Excel (Microsoft, Redmond, WA, USA).

regression analysis using SAS 9.2 software (SAS Institute, Inc., Cary, NC, USA) or from the least square regression line. The concentration–response correlative formula and correlation coefficient (R2) for each crude drug constituting YKS were determined by regression analysis using Excel (Microsoft, Redmond, WA, USA). Results Cell viability The viabilities of Caco-2 cells treated with various concentrations of test substances are summarized in Supplementary Table S1. From the results, the concentrations of test substances that showed little cell toxicity (i.e., cell viabilities >80%) were used in the digoxin transport assay using a Caco-2 cell monolayer. Effects of test substances on digoxin transport across a Caco-2 cell monolayer The TEER value of the Caco-2 cell monolayer guarantees tight junction formation, which depends on the cell source, passage number, number of seeded cells, and culture conditions [24]. Based on this information, the TEER value corresponding to our experimental condition was judged to be ≥250 Ω cm2. Therefore, Caco-2 cell monolayers showing a TEER value of ≥250 Ω cm2 were first selected for this experiment. Next, the flux of mannitol across the cell membrane was examined to verify the integrity of the monolayer. The permeability of mannitol was < 1.5 × 10−6 cm/s, which indicated that the tight junctions of the cell monolayer were completed.

layers showing a TEER value of ≥250 Ω cm2 were first selected for this experiment. Next, the flux of mannitol across the cell membrane was examined to verify the integrity of the monolayer. The permeability of mannitol was < 1.5 × 10−6 cm/s, which indicated that the tight junctions of the cell monolayer were completed. Furthermore, to confirm the expression and function of P-gp in the completed cell monolayer, transport of digoxin via the cell monolayer was investigated. The efflux ratio, an index for permeability of digoxin (6.98), was inhibited (0.832) by co-treating a P-gp inhibitor, verapamil (50 µmol/l). The inhibition rate was 88.1%, which shows that P-gp was expressed in the cell monolayer and functioned.

l monolayer, transport of digoxin via the cell monolayer was investigated. The efflux ratio, an index for permeability of digoxin (6.98), was inhibited (0.832) by co-treating a P-gp inhibitor, verapamil (50 µmol/l). The inhibition rate was 88.1%, which shows that P-gp was expressed in the cell monolayer and functioned. Figure 1 shows the effects of the five Kampo medicines (YKS, RKT, SKT, HST, and GJG) on digoxin transport across the cell monolayer. All the Kampo medicines significantly inhibited the efflux ratio in a dose-dependent manner (YKS, F5,18 = 69.3, P < 0.001; RKT, F5,12 = 129.0, P < 0.001; SKT, F5,12 = 12.0, P < 0.001; HST, F6,14 = 35.2, P < 0.001; GJG, F5,12 = 32.0, P < 0.001). The IC50 values of YKS, RKT, SKT, HST, and GJG calculated by non-linear regression analysis were 1.94, 3.36, 10.80, 6.37, and 6.34 mg/ml, and the Igut/IC50 values as P-gp inhibition index were 3.4, 2.4, 0.5, 1.4, and 1.4, respectively. However, the IC50 values of SKT and HST were calculated as the estimated values, because both medicines did not show 50% inhibition in the examined concentration range.Fig. 1 Inhibitory effects of five Kampo medicines on the efflux ratio across the Caco-2 cell monolayer of the P-gp substrate digoxin. Data represent the mean ± SD (n = 3–4). One-way ANOVA showed that each Kampo medicine significantly inhibited the efflux ratio in a concentration-dependent manner. The IC50 values for P-gp inhibition were calculated by non-linear regression analysis using SAS 9.2 software. The Igut/IC50 values were calculated as follows: Igut/IC50 = Inhibitordose in 250 ml/IC50

-way ANOVA showed that each Kampo medicine significantly inhibited the efflux ratio in a concentration-dependent manner. The IC50 values for P-gp inhibition were calculated by non-linear regression analysis using SAS 9.2 software. The Igut/IC50 values were calculated as follows: Igut/IC50 = Inhibitordose in 250 ml/IC50 Next, the P-gp inhibition rates of seven crude drugs constituting YKS showing the strongest suppression of P-gp were examined. As shown in Fig. 2, four crude drugs (ALR, Poria, GR, and UUCR) among the seven constituents significantly inhibited the efflux ratio in a concentration-dependent manner (ALR, F3,12 = 229.0, P < 0.001; Poria, F3,12 = 58.4, P < 0.001; GR, F3,12 = 471.0, P < 0.001; UUCR, F2,9 = 577.0, P < 0.001). The correlativity was observed as y = − 0.031x + 95.264 and the correlation coefficient as (R2) = 0.9537 for ALR, y = − 0.033x + 96.634 and R2 = 0.9379 for Poria, y = − 0.046x + 104.790 and R2 = 0.9757 for GR, and y = − 0.191x + 97.706 and R2 = 0.9926 for UUCR, where y is the efflux ratio and x is the drug concentration. From these formulas, the IC50 values were calculated to be 1,470 µg/ml for ALR, 1,430 µg/ml for Poria, 1,190 µg/ml for GR, and 249 µg/ml for UUCR. Other crude drugs (Angelicae Acutilobae Radix, Cnidii Rhizoma, and Bupleuri Radix) had low correlation, and the IC50 values could not be determined in the concentration range examined.Fig. 2 Inhibitory effect of four among seven crude drugs constituting YKS on the efflux ratio across a Caco-2 cell monolayer of the P-gp substrate digoxin. The dose–response correlative formula and correction coefficient (R2) of each crude drug were determined by regression analysis. Each data point represents the mean ± SD (n = 4). Concentration reactivity was statistically evaluated by one-way ANOVA

lux ratio across a Caco-2 cell monolayer of the P-gp substrate digoxin. The dose–response correlative formula and correction coefficient (R2) of each crude drug were determined by regression analysis. Each data point represents the mean ± SD (n = 4). Concentration reactivity was statistically evaluated by one-way ANOVA Table 2 shows the P-gp inhibition rate for each concentration of each constituent crude drug contained in the IC50 concentration (1.94 mg/ml, Fig. 1) of YKS to determine the contribution of each crude drug to the inhibitory effect of YKS. The concentration of each crude drug contained in the IC50 concentration of YKS was obtained from the composition ratio of the seven crude drugs constituting YKS. The inhibition rate at the concentration of each crude drug contained in the IC50 concentration of the YKS extract was calculated using the correlative formulas in Fig. 2. UUCR inhibited the majority (53.1%) of the total inhibitory activity of the seven crude drugs.Table 2 The P-gp inhibition rate of seven crude drugs constituting YKS Crude drug Composition ratio of crude drug in YKS (%) Concentration of crude drug in YKS (μg/ml)a Contribution rate (%)b Uncariae uncis cum ramulus 14.6 284 53.1 Glycyrrhizae radix 7.4 143 1.7 Atractylodis lanceae rhizoma 19.5 378 15.3 Poria 19.5 378 14.7 Angelicae acutilobae radix 14.6 284 3.0 Cnidii rhizoma 14.6 284 7.4 Bupleuri radix 9.8 189 4.8 Total 100.0 1,940 100.0 aConcentration of each crude drug in the IC50 concentration (1.94 mg/ml) of YKS extract was calculated from the composition ratio of seven crude drugs constituting YKS

19.5 378 15.3 Poria 19.5 378 14.7 Angelicae acutilobae radix 14.6 284 3.0 Cnidii rhizoma 14.6 284 7.4 Bupleuri radix 9.8 189 4.8 Total 100.0 1,940 100.0 aConcentration of each crude drug in the IC50 concentration (1.94 mg/ml) of YKS extract was calculated from the composition ratio of seven crude drugs constituting YKS bThe contribution rate of each crude drug was calculated as a percentage of the total inhibition at each concentration included in the YKS (IC50 concentration) by using the correlative formulas described in result section and Fig. 2 Next, the effects of GM and RP, which are UUCR alkaloids in YKS, were evaluated using a Caco-2 permeability assay (Fig. 3). GM (>10 μmol/l) and RP (100 μmol/l) significantly inhibited the efflux ratio (P < 0.001) compared to those in the control, and the IC50 values were 16.5 and 56.0 µmol/l, respectively.Fig. 3 Inhibitory effects of GM and RP on the efflux ratio across the Caco-2 cell monolayer of the P-gp substrate digoxin. Each data point represents the mean ± SD (n = 3). ***P < 0.001 vs vehicle control: concentration reactivity was statistically evaluated by Dunnett’s test following one-way ANOVA The concentrations of GM and RP were 0.238 nmol/ml (0.238 µmol/l) and 0.179 nmol/ml (0.179 µmol/l) in the 1.94 mg/ml of YKS, respectively. To clarify the contributions of both ingredients to the inhibitory effect of YKS, although the inhibition rate at the concentration of each ingredient contained in the YKS extract were examined in Fig. 3, both ingredients were found to hardly inhibit P-gp at those concentrations (≤1.5% inhibitions).

YKS, respectively. To clarify the contributions of both ingredients to the inhibitory effect of YKS, although the inhibition rate at the concentration of each ingredient contained in the YKS extract were examined in Fig. 3, both ingredients were found to hardly inhibit P-gp at those concentrations (≤1.5% inhibitions). Discussion We examined the inhibitory effect of the five Kampo medicines (YKS, RKT, SKT, HST, and GJG) widely used in clinical treatments for various diseases in Japan on P-gp transport of digoxin across a Caco-2 cell monolayer. Djuv and Nilsen [25] suggested the importance of measuring the cellular TEER, mannitol transport, and inhibition of verapamil on digoxin transport in a cultured cell monolayer to ensure the integrity of cell morphology and function in the Caco-2 permeability assay. In the present study, the monolayer, of which the integrity was confirmed by these indices, was used for evaluation of the test substance.

ol transport, and inhibition of verapamil on digoxin transport in a cultured cell monolayer to ensure the integrity of cell morphology and function in the Caco-2 permeability assay. In the present study, the monolayer, of which the integrity was confirmed by these indices, was used for evaluation of the test substance. The P-gp transporter assay using a completed Caco-2 cell monolayer showed that all Kampo medicines examined inhibited the P-gp transport of digoxin, with YKS showing the strongest inhibitory action. Subsequent examination to clarify the contribution of the seven crude drugs constituting YKS showed that P-gp inhibitory action was observed in UUCR, GR, ALR, and Poria (Fig. 2). Although GR has been reported to suppress P-gp activity by ATPase assay [20], its contribution to the inhibitory activity of YKS was low as shown in Table 2. This result was also supported by other results, i.e., GR is included in four Kampo medicines (SKT, YKS, RKT, and HST) other than GJG. Of these GR-containing Kampo medicines, the GR content contained in SKT is 2–7 times greater than those of the other Kampo medicines. However, the P-gp inhibitory action of SKT was the weakest among the five Kampo medicines (IC50: 10.80 mg/ml). This result suggests that the GR level contained in each Kampo medicine may not contribute much to the P-gp inhibitory action of the five Kampo medicines, including YKS. On the other hand, UUCR is a crude drug that was contained only in YKS among the Kampo medicines examined in the present study, among which the inhibitory action of UUCR was stronger than the others (Fig. 2), and its contribution to the inhibitory activity of YKS was 53.1% (Table 2). Therefore, the potent P-gp inhibitory effect of YKS may be mainly due to the inhibitory action of UUCR, although the possibility that the effect may be due to additive or synergistic action with other crude drugs, such as GR, cannot be ruled out from this experiment.

the inhibitory activity of YKS was 53.1% (Table 2). Therefore, the potent P-gp inhibitory effect of YKS may be mainly due to the inhibitory action of UUCR, although the possibility that the effect may be due to additive or synergistic action with other crude drugs, such as GR, cannot be ruled out from this experiment. We further investigated the P-gp inhibitory effect of the two major UUCR alkaloids (GM and RP) that were available for this study in order to to clarify the components contributing to the inhibitory activity of UCCR, and found that both ingredients had P-gp inhibitory activity. However, the P-gp inhibition rate at the GM and RP concentrations in the YKS extract powder was slight (≤1.5%), suggesting that other ingredients are involved in the inhibitory activity of UCCR. Because many other active alkaloids have been identified in YKS in addition to GM and RP [26], it is necessary to examine the P-gp inhibitory actions and additive/synergistic actions of other active alkaloids in the future. In addition, P-gp is also expressed in the luminal membrane of the blood–brain barrier, in the apical membranes of excretory cells, such as hepatocytes and kidney proximal tubule epithelia, and has an important role in the pharmacokinetics, efficacy, safety, or tissue levels of P-gp substrates [6]. We previously demonstrated that several UUCR alkaloids, including GM and PR, were detected in the plasma of rats treated with oral YKS [27]. It will be necessary to clarify the effect of these alkaloids on P-gp localized in other organs.

n the pharmacokinetics, efficacy, safety, or tissue levels of P-gp substrates [6]. We previously demonstrated that several UUCR alkaloids, including GM and PR, were detected in the plasma of rats treated with oral YKS [27]. It will be necessary to clarify the effect of these alkaloids on P-gp localized in other organs. Both the Japanese MHLW [22] and US FDA [23] draft DDI guidance state that test drugs having an Igut/IC50 ≥10 are likely to be P-gp inhibitors. For test substances evaluated as P-gp inhibitors it is stated that more detailed DDI tests should be conducted in humans. Therefore, the Igut/IC50 values for the five Kampo medicines were calculated, and the DDI risk was objectively evaluated according to the criteria in the DDI guidance. The rates of YKS, RKT, SKT, HST, and GJG examined in the present study were 3.4, 2.4, 0.5, 1.4, and 1.4, respectively. These values were <10, which was evaluated as a weak P-gp inhibitory effect that does not require further verification in humans, suggesting that the DDI risk due to P-gp inhibition for these Kampo medicines may be low. Furthermore, it has been reported that the inhibitory potential of YKS and RKT against P-gp was low in an ATPase assay [28] and did not affect the plasma pharmacokinetics of digoxin in mice [19]. Ito et al. [19] and Soraoka et al. [29] also evaluated the effects of several Kampo medicines on CYP3A, which is known to have considerable overlap in substrate specificity and tissue localization of P-gp [30], and it has been reported that the inhibition rate of YKS on metabolic activity was low in mice and humans. Combining the above results, the P-gp inhibitory effect appears to be low in all five Kampo medicines examined in this study. In other words, in the present five Kampo medicines, it is thought that the possibility of drug interaction due to the inhibition of P-gp is extremely low in combination therapy with Western medicine.

ing the above results, the P-gp inhibitory effect appears to be low in all five Kampo medicines examined in this study. In other words, in the present five Kampo medicines, it is thought that the possibility of drug interaction due to the inhibition of P-gp is extremely low in combination therapy with Western medicine. Conclusion All Kampo medicines examined (YKS, RKT, SKT, HST, and GJG) inhibited the P-gp transport of digoxin in a Caco-2 permeability assay but to a low degree according to the criteria in the DDI draft guidance of the MHLW and FDA. These results suggest that the risk of drug interaction due to inhibition of P-gp in combination therapy with Western medicines is extremely low for these Kampo medicines. This finding should provide useful clinical information on the safety and efficacy of combinations of Kampo medicines and Western medicines. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary material 1 (DOCX 23 kb) Competing financial interests The authors of this manuscript have the following competing interests: TM, NK, YM, and JW are employees of Tsumura & Co. EW received a research grant from Tsumura & Co. Acknowledgements The authors are grateful to Dr. Yasushi Ikarashi (Tsumura Kampo Research Laboratory, Tsumura & Co.) for his valuable advice and support related to this manuscript. The authors would like to thank Enago (www.enago.jp) for the English language review.

Competing financial interests The authors of this manuscript have the following competing interests: TM, NK, YM, and JW are employees of Tsumura & Co. EW received a research grant from Tsumura & Co. Acknowledgements The authors are grateful to Dr. Yasushi Ikarashi (Tsumura Kampo Research Laboratory, Tsumura & Co.) for his valuable advice and support related to this manuscript. The authors would like to thank Enago (www.enago.jp) for the English language review. Author contributions TM and NK conceived and designed the experiments. TM, NK, and YM performed the experiments. TM, NK, and YM analyzed the data. TM and NK contributed reagents/materials/analysis tools. TM wrote the paper. EW and JW provided some useful suggestions. All authors read and approved the final manuscript.

osed of carbon skeletons with fewer than 40 carbons are called apocarotenoids. About 120 kinds of apocarotenoids are present in some species of plants and animals as degradation products of C40 carotenoids [1, 2].Fig. 1 a Basic structures of carotenoids and end groups. b Structures of typical carotenes and xanthophylls History of carotenoid research in natural product chemistry In the early part of the nineteenth century, carotenoids were found in paprika (1817), saffron (1818), annatto (1825), carrots (1831), and autumn leaves (1837). In 1906, Zwet succeeded in the separation of carotene, xanthophyll and chlorophyll from green leaves using column chromatography. In the 1930s, Karrer and Khun elucidated the structures of β-carotene and lycopene. Furthermore, they found that β-carotene was a precursor of vitamin A. They won the Nobel Prize in chemistry for this work. Subsequently, structures of lutein, zeaxanthin, and astaxanthin were revealed by their groups. These structural studies were based on the oxidative degradation of carotenoids with KMnO4, and structures were analyzed using elemental analysis. In the 1950s, the Zechmeister group studied E/Z (cis–trans) isomerization of carotenoids. In the 1960s, the Weedon group and Liaaen-Jensen group elucidated the structure of fucoxanthin and peridinin, respectively, using NMR and MS spectrometry [3]. Since the first structural elucidation of β-carotene by Kuhn and Karrer in 1928–1930, about 750 naturally occurring carotenoids had been reported up until 2004 [1]. Improvements of analytical instruments such as NMR, MS, and HPLC have made it possible to perform the structural elucidation of very minor carotenoids in nature [2]. Annually, several new structures of carotenoids are being reported. Our research group has performed the structural elucidation and analysis of naturally occurring carotenoids using NMR, MS, MS/MS, and LC/MS [2] over the last decade. About 100 kinds of natural carotenoids were reported from 2004 to 2018 [2]. Synthetic studies of carotenoids revealed the stereochemistry of several complex structures of natural carotenoids such as peridinin, fucoxanthin, crassostreaxanthin B, and cucurbitaxanthin A [4].

MS, MS/MS, and LC/MS [2] over the last decade. About 100 kinds of natural carotenoids were reported from 2004 to 2018 [2]. Synthetic studies of carotenoids revealed the stereochemistry of several complex structures of natural carotenoids such as peridinin, fucoxanthin, crassostreaxanthin B, and cucurbitaxanthin A [4]. Carotenoids in photosynthetic bacteria, some species of fungi, algae, and plants Carotenoid biosynthesis Basic carotenoid biosynthetic pathways are indicated in Fig. 2a and b. The first step, dimethylallyl pyrophosphate is formed from acetyl CoA or pyruvic acid through mevalonate pathway or non-mevanolate pathway, respectively. Then, phytoene, with a C40 carotenoid skeleton, is formed from dimethylallyl pyrophosphate through geranyl pyrophosphate and geranylgeranyl pyrophosphate (Fig. 2a). Phytoene is a colorless carotenoid with three conjugated double bonds. Phytoene is stepwisely desaturated to form lycopene via phytofluene, ζ-carotene, and neurosporene by phytoene desaturase. Lycopene cyclases produce carotenoids with cyclic terminal end groups such as α-carotene and β-carotene, as shown in Fig. 2b. Several xanthophylls are produced by carotene hydroxylases, ketolases, and epoxidase. These carotenoid biosynthetic pathways have been comprehensively revealed by enzymatic and genetic studies [5].Fig. 2 a Carotenoid biosynthetic pathways (formation of phytoene). b Desaturation of polyene chain and cyclization of end groups

al xanthophylls are produced by carotene hydroxylases, ketolases, and epoxidase. These carotenoid biosynthetic pathways have been comprehensively revealed by enzymatic and genetic studies [5].Fig. 2 a Carotenoid biosynthetic pathways (formation of phytoene). b Desaturation of polyene chain and cyclization of end groups Carotenoids in photosynthetic organs of plants Carotenoids are essential compounds along with chlorophylls in photosynthetic bacteria, algae, and plants and are involved in photosynthesis and photo-protection. Carotenoids harvest light energy and transfer this energy to chlorophylls through singlet–singlet excitation transfer. This singlet–singlet transfer is a lower energy state transfer used during photosynthesis. Carotenoids absorb excessive energy from chlorophylls through triplet–triplet transfer and release excessive energy by polyene vibration. The triplet–triplet transfer is a higher energy state essential in photo-protection. Reactive oxygen species such as singlet oxygen, hydroxy radicals, and superoxide anion radicals are produced from oxygen and light during photosynthesis. Carotenoids with more than eleven conjugated double bonds show a marked capacity to quench singlet oxygen. The mechanism for quenching singlet oxygen is a physical reaction. Carotenoids take up thermal energy from singlet oxygen and release this energy by polyene vibration.

from oxygen and light during photosynthesis. Carotenoids with more than eleven conjugated double bonds show a marked capacity to quench singlet oxygen. The mechanism for quenching singlet oxygen is a physical reaction. Carotenoids take up thermal energy from singlet oxygen and release this energy by polyene vibration. The xanthophyll cycle involves the enzymatic removal of epoxy groups from xanthophylls (violaxanthin, antheraxanthin, and lutein epoxide) to create the so-called de-epoxy xanthophylls (zeaxanthin and lutein). These enzymatic cycles were found to play a key role in stimulating energy dissipation within light-harvesting antenna proteins by non-photochemical quenching, a mechanism to reduce the amount of energy that reaches the photosynthetic reaction centers. Non-photochemical quenching is one of the main ways to protect against photoinhibition. In higher plants, xanthophyll cycles consist of violaxanthin–antheraxanthin–zeaxanthin. During light stress, violaxanthin is converted to zeaxanthin via the intermediate antheraxanthin, which plays a direct photo-protective role acting as a lipid-protective antioxidant and by stimulating non-photochemical quenching within light-harvesting proteins. This conversion of violaxanthin to zeaxanthin is medicated by the enzyme violaxanthin de-epoxidase, while the reverse reaction is performed by zeaxanthin epoxidase (Fig. 3) [6]. Lutein epoxide and lutein are members of xanthophylls cycles in higher plants. In diatoms, the xanthophyll cycle consists of diadinoxanthin and diatoxanthin (Fig. 3) [6].Fig. 3 Xanthophyll cycles

cated by the enzyme violaxanthin de-epoxidase, while the reverse reaction is performed by zeaxanthin epoxidase (Fig. 3) [6]. Lutein epoxide and lutein are members of xanthophylls cycles in higher plants. In diatoms, the xanthophyll cycle consists of diadinoxanthin and diatoxanthin (Fig. 3) [6].Fig. 3 Xanthophyll cycles Carotenoids in non-photosynthetic organs of plants Carotenoids are also present in non-photosynthetic organs of plants such as fruits, pericarps, seeds, roots, and flowers. Carotenoids in these none-photosynthetic organs show structural diversity and are formed by secondary metabolic reactions, such as oxidation, the cleavage of polyene chains, and (Z/E) (cis–trans) isomerization [5, 6]. Carotenoids in non-photosynthetic organs act as photo-protectors, antioxidants, color attractants, and precursors of plant hormones. Many fruits and seeds turn red or purple during the ripening stage. This color change is due to the formation of carotenoids and/or anthocyanins. For example, the color of the pericarp of tomato turns from greenish-yellow to deep red during the ripening stage. This color change is due to the conversion of phytoene to lycopene in the pericap of the tomato. Phytoene (colorless), which is the major carotenoid in greenish-yellow tomato, is converted to phytofluene (pale yellow), ζ-carotene (yellow), neurosporene (orange), and lycopene (red) by phytoene desaturase, as shown in Fig. 2B [5, 6]. Lycopene is a carotenoid with some of the strongest singlet oxygen quenching and photo-protection activities [5, 6].

e major carotenoid in greenish-yellow tomato, is converted to phytofluene (pale yellow), ζ-carotene (yellow), neurosporene (orange), and lycopene (red) by phytoene desaturase, as shown in Fig. 2B [5, 6]. Lycopene is a carotenoid with some of the strongest singlet oxygen quenching and photo-protection activities [5, 6]. Pittosporum tobira (Tobera in Japanese) is a small, slender, evergreen tree growing in southern Japan. In summer, the seeds have a pale yellow color and are covered with a capsule. In autumn, the seeds are exposed to sunlight and change color from yellow to red. The major carotenoid in the yellow seeds is violaxanthin, with a pale yellow color, and related epoxy carotenoids. On the other hand, the major carotenoid in the red seeds is a series of red seco-carotenoids named as tobiraxanthin A, B, and C. During the autumn season, violaxanthin is converted to tobiraxanthin A by oxidative cleavage of C5–C6 and C5′–C6′ bonds, as shown in Fig. 4a. Tobiraxanthin A shows an approximately 30-nm longer wavelength shift than violaxanthin because of the introduction of two conjugated carbonyl groups at C6 and C6′ in the polyene chain of violaxanthin. Therefore, tobiraxanthin A shows strong activity to quench singlet oxygen induced by sunlight. Furthermore, the red color of the seed acts as an attractant for birds to eat seeds to disperse them. Therefore, red seco-carotenoids act as antioxidants, photo-protectors, and color attractants in P. tobira (Fig. 4a) [2, 7].Fig. 4 a Formation of tobiraxanthin and b formation of pittosporumxanthins in the seeds of Pittosporum tobira

he red color of the seed acts as an attractant for birds to eat seeds to disperse them. Therefore, red seco-carotenoids act as antioxidants, photo-protectors, and color attractants in P. tobira (Fig. 4a) [2, 7].Fig. 4 a Formation of tobiraxanthin and b formation of pittosporumxanthins in the seeds of Pittosporum tobira Novel carotenoid and α-tocopherol complexes named pittosporumxanthins were also isolated from the red-colored seeds of P. tobira [2, 8, 9] by our research group. Pittosporumxanthin A1 and A2 are diastereomeric pairs of the cycloaddition product of violaxanthin at the C-11′ and C-12′ positions, with α-tocopherol (Fig. 4b). The mechanism of carotenoid and α-tocopherol complex formation in the seeds of P. tobira may be considered as follows: an α-tocopherol radical is formed from α-tocopherol by the quenching or scavenging of reactive oxygen species in the seed. The α-tocopherol radical itself becomes a pro-oxidant and causes oxidative damage. Therefore, carotenoids such as violaxanthin take up α-tocopherol radicals through the formation of adduct products. This suggests that carotenoids can quench phenoxy radicals by reacting with these polyene chains [2, 8, 9]. Carotenoids also participate in different types of cell signaling. They are able to signal the production of abscisic acid, which regulate plant growth, seed dormancy, embryo maturation and germination, cell division and elongation, floral growth, and stress responses. Carotenoids are also precursors of some aroma compounds [6].

s also participate in different types of cell signaling. They are able to signal the production of abscisic acid, which regulate plant growth, seed dormancy, embryo maturation and germination, cell division and elongation, floral growth, and stress responses. Carotenoids are also precursors of some aroma compounds [6]. Carotenoids in animals In general, animals do not synthesize carotenoids de novo, and so those found in animals are either directly obtained from food or partly modified through metabolic reactions. The major metabolic conversions of carotenoids found in these animals are oxidation, reduction, translation of double bonds, oxidative cleavage of double bonds, and cleavage of epoxy bonds [5, 6, 10]. It is well-known that carotenoids that contain unsubstituted β-ionone rings such as β-carotene, α-carotene, β-cryptoxanthin. and β,ψ-carotene (γ-carotene) are precursor of retinoids and are called pro-vitamin A. Furthermore, carotenoids in animals play important roles such as photo-protectors, antioxidants, enhancers of immunity, and contributors to reproduction. Several animals use carotenoids as signals for intra-species (sexual signaling, social status signaling, and parent–offspring signaling) and inter-species (species recognition, warning coloration, mimicry, and crypsis) communication [5, 6, 10].

ors, antioxidants, enhancers of immunity, and contributors to reproduction. Several animals use carotenoids as signals for intra-species (sexual signaling, social status signaling, and parent–offspring signaling) and inter-species (species recognition, warning coloration, mimicry, and crypsis) communication [5, 6, 10]. Food chain and metabolism of carotenoids in aquatic animals Aquatic animals contain various carotenoids that show structural diversity. Aquatic animals obtain carotenoids from foods such as algae and other animals and modify them through metabolic reactions. Many of the carotenoids present in aquatic animals are metabolites of β-carotene, fucoxanthin, peridinin, diatoxanthin, alloxanthin, and astaxanthin [2, 10, 11].

ids that show structural diversity. Aquatic animals obtain carotenoids from foods such as algae and other animals and modify them through metabolic reactions. Many of the carotenoids present in aquatic animals are metabolites of β-carotene, fucoxanthin, peridinin, diatoxanthin, alloxanthin, and astaxanthin [2, 10, 11]. Bivalves (oysters, clams, scallops, mussels, and ark shells) and tunicates (sea squirts) are filter feeders. They feed on micro-algae such as diatoms, dinoflagellates, blue-green algae, and green algae and obtain carotenoids from these dietary sources. The major carotenoid in diatoms is fucoxanthin. Fucoxanthin has several functional groups, such as allenic bond, epoxide, carbonyl, and acetyl groups. Therefore, metabolites of fucoxanthin in bivalves and tunicates show structural diversity, as shown in Fig. 5a. The major metabolic conversions of fucoxanthin found in these animals are the conversion of allenic bond to acetylenic bond, hydrolysis cleavage of epoxy group, and oxidative cleavage of epoxy group, as shown in Fig. 5b [2, 10].Fig. 5 a Metabolic pathways of fucoxanthin in bivalves and tunicates. b Metabolic conversion mechanisms of end groups of fucoxanthin in bivalves and tunicates Peridinin, with its C37 carbon skeleton, is a major red carotenoid in dinoflagellates. Peridinin also has several functional groups, such as an allenic bond, epoxide, and a lactone ring. As well as fucoxanthin, peridinin is also converted to several metabolites in bivalves and tunicates, as shown in Fig. 6 [2, 10].Fig. 6 Metabolic pathways of peridinin in bivalves and tunicates

tenoid in dinoflagellates. Peridinin also has several functional groups, such as an allenic bond, epoxide, and a lactone ring. As well as fucoxanthin, peridinin is also converted to several metabolites in bivalves and tunicates, as shown in Fig. 6 [2, 10].Fig. 6 Metabolic pathways of peridinin in bivalves and tunicates Astaxanthin is a characteristic marine carotenoid in crustaceans (shrimps and crabs). Many crustaceans can synthesize astaxanthin from β-carotene, ingested in dietary algae, via echinenone, 3-hydroxyechinenone, canthaxanthin, and adonirubin, as shown in Fig. 7 [5, 11]. In many crustaceans, hydroxylation at C-3 (C-3′) in the 4-oxo-β-end group is non-stereo-selective. Therefore, astaxanthin and related carotenoids with a 3-hydroxy-4-oxo-β-end group, present in crustaceans, are comprised of a mixture of these optical isomers [5, 11].Fig. 7 Oxidative metabolism of β-carotene in crustaceans Carp, crucian carp, and goldfish belonging to Cyprinidae can convert zeaxanthin to (3S,3′S)-astaxanthin via adonixanthin and idoxanthin (Fig. 8). Therefore, spirulina, which contains zeaxanthin as the major carotenoid, is used for pigmentation in red carp and goldfish [10, 11].Fig. 8 Metabolic conversion of zeaxanthin to (3S,3′S)-astaxanthin in Cyprinidae fish

to Cyprinidae can convert zeaxanthin to (3S,3′S)-astaxanthin via adonixanthin and idoxanthin (Fig. 8). Therefore, spirulina, which contains zeaxanthin as the major carotenoid, is used for pigmentation in red carp and goldfish [10, 11].Fig. 8 Metabolic conversion of zeaxanthin to (3S,3′S)-astaxanthin in Cyprinidae fish On the other hand, several marine fish (red sea bream, cod, tuna, and yellow-tail) and Salmonidae fish (salmon and trout) cannot synthesize astaxanthin from other carotenoids such as β-carotene and zeaxanthin [5, 10, 11]. Therefore, astaxanthin present in these fish originates from dietary zooplankton belonging to Crustacea such as krill. So, astaxanthin is used for pigmentation in red sea bream and salmon. The bright yellow color of the fin and skin of several marine fish is due to the presence of tunaxanthin (ε,ε-carotene-3,3′-diol). Tunaxanthin is metabolized from astaxanthin via zeaxanthin, as shown in Fig. 9 [5, 10, 11]. Carotenoids with a 3-oxo-ε-end group such as 3-hydroxy-β,ε-caroten-3′-one and ε,ε-carotene-3,3′-dione are key intermediates in this metabolic conversion.Fig. 9 Reductive metabolic pathway of astaxanthin in marine fish Biological function of carotenoids in marine animals As described above, marine animals convert dietary carotenoids and accumulate them in these organs. Through these metabolic conversions, antioxidative and photo-protective activities of carotenoids are increased.

On the other hand, several marine fish (red sea bream, cod, tuna, and yellow-tail) and Salmonidae fish (salmon and trout) cannot synthesize astaxanthin from other carotenoids such as β-carotene and zeaxanthin [5, 10, 11]. Therefore, astaxanthin present in these fish originates from dietary zooplankton belonging to Crustacea such as krill. So, astaxanthin is used for pigmentation in red sea bream and salmon. The bright yellow color of the fin and skin of several marine fish is due to the presence of tunaxanthin (ε,ε-carotene-3,3′-diol). Tunaxanthin is metabolized from astaxanthin via zeaxanthin, as shown in Fig. 9 [5, 10, 11]. Carotenoids with a 3-oxo-ε-end group such as 3-hydroxy-β,ε-caroten-3′-one and ε,ε-carotene-3,3′-dione are key intermediates in this metabolic conversion.Fig. 9 Reductive metabolic pathway of astaxanthin in marine fish Biological function of carotenoids in marine animals As described above, marine animals convert dietary carotenoids and accumulate them in these organs. Through these metabolic conversions, antioxidative and photo-protective activities of carotenoids are increased. For example, many marine invertebrates such as crustaceans convert β-carotene to astaxanthin and accumulate it in integuments, carapaces, eggs, and ovaries. Through the metabolic conversion, the carotenoid changes its color from yellow (β-carotene) to red (astaxanthin). Astaxanthin in marine invertebrates sometimes forms a carotenoid protein complex and is a red, blue, or purple color. These colors may serve to camouflage the animals in the prevailing undersea light conditions, serve as general photoreceptors, or provide protection against possible harmful effects of light. Furthermore, through this metabolic conversion, the antioxidant effects of carotenoids such as the quenching of singlet oxygen, inhibiting lipid peroxidation, and protection against photo-oxidation are enhanced. Therefore, astaxanthin in these animals acts as an antioxidant and prevents oxidative stress [6].

of light. Furthermore, through this metabolic conversion, the antioxidant effects of carotenoids such as the quenching of singlet oxygen, inhibiting lipid peroxidation, and protection against photo-oxidation are enhanced. Therefore, astaxanthin in these animals acts as an antioxidant and prevents oxidative stress [6]. The next example involves carotenoids in the gonads of the sea angel Clione limacine [12]. The sea angel is a small, floating sea slug belonging to the class Gastropoda. It inhabits under the drift-ice in the sea of Okhotsk and is exposed to strong sunlight. Its body is gelatinous and transparent. On the other hand, its gonads and viscera are a bright orange-red color due to the presence of carotenoids. The sea angel is carnivorous and feeds exclusively on a small sea snail Limacina helicina, which is herbivorous and feeds on micro-algae such as diatoms and dinoflagellates. Therefore, carotenoids produced by micro-algae are made available to the sea angel through L. helicina in the food chain. L. helicina directly absorbs carotenoids such as diatoxanthin from dietary algae and accumulates them without metabolic modification. On the other hand, the sea angel oxidatively metabolizes ingested diatoxanthin from L. helicina to pectenolone, as shown in Fig. 10 [12]. By introducing a carbonyl group at C-4′ in diatoxanthin, the carotenoid changes color from yellow to red and shows enhanced antioxidative and photo-protective activities. Therefore, the sea angel accumulates pectenolone in the gonads as an anti-oxidant and a photo-protector [12].Fig. 10 Food chain and metabolism of carotenoids in sea angel

onyl group at C-4′ in diatoxanthin, the carotenoid changes color from yellow to red and shows enhanced antioxidative and photo-protective activities. Therefore, the sea angel accumulates pectenolone in the gonads as an anti-oxidant and a photo-protector [12].Fig. 10 Food chain and metabolism of carotenoids in sea angel The red carotenoid mytiloxanthin is a metabolite of fucoxanthin present in shellfish and tunicates. Dietal fucoxanthin from diatoms is metabolized to mytiloxanthin via fucoxanthinol and halocynthiaxanthin in shellfish and tunicates, as shown in Fig. 5a. Through this metabolic conversion, antioxidative activities such as singlet oxygen quenching, scavenging of hydroxy radicals, and inhibition of lipid peroxidation of carotenoids are increased. Mytiloxanthin showed almost the same excellent antioxidative activity as that of astaxanthin [13]. Therefore, it was concluded that marine animals metabolize dietary carotenoids to a more active antioxidative form and accumulate them in their bodies and reproductive organs.

id peroxidation of carotenoids are increased. Mytiloxanthin showed almost the same excellent antioxidative activity as that of astaxanthin [13]. Therefore, it was concluded that marine animals metabolize dietary carotenoids to a more active antioxidative form and accumulate them in their bodies and reproductive organs. A novel fucoxanthin pyropheophorbide A ester (Fig. 11) was isolated from the viscera of the abalone Haliotis diversicolor aquatilis. The major food sources of abalone are macro-algae such as brown algae, which contain fucoxanthin as the major carotenoid. Pyropheophorbide A is a metabolite of chlorophyll A in the viscera of abalone. This carotenoid pyropheophorbide A ester might be formed from fucoxanthin and pyropheophorbide A by esterase in the abalone viscera. It is well-known that pyropheophorbide A is a photosensitizer that generates singlet oxygen from ground-state molecular oxygen in the presence of light. On the other hand, carotenoids are excellent quenchers of singlet oxygen and they prevent photo-oxidation. Therefore, it is interesting that compounds acting as singlet oxygen generators and quenchers are linked with esterified bonds. Indeed, fucoxanthin pyropheophorbide A ester shows weaker singlet oxygen generation than pyropheophorbide A [14].Fig. 11 Novel carotenoid pyropheophorbide A ester from abalone

photo-oxidation. Therefore, it is interesting that compounds acting as singlet oxygen generators and quenchers are linked with esterified bonds. Indeed, fucoxanthin pyropheophorbide A ester shows weaker singlet oxygen generation than pyropheophorbide A [14].Fig. 11 Novel carotenoid pyropheophorbide A ester from abalone Carotenoids in terrestrial animals As with aquatic animals, most terrestrial animals cannot synthesize carotenoids de novo, and so must obtain them from their diet. Therefore, carotenoids in terrestrial animals mainly originate from plants that they feed on. Many of the carotenoids present in terrestrial animals are β-carotene, β-cryptoxanthin, lutein, zeaxanthin, and their metabolites [5, 6].

animals cannot synthesize carotenoids de novo, and so must obtain them from their diet. Therefore, carotenoids in terrestrial animals mainly originate from plants that they feed on. Many of the carotenoids present in terrestrial animals are β-carotene, β-cryptoxanthin, lutein, zeaxanthin, and their metabolites [5, 6]. Carotenoids in insects and spiders Insects are the most diverse group of animals. Therefore, carotenoids in insects show structural diversity. Many of the carotenoids present in insect are β-carotene, β-cryptoxanthin, lutein, and zeaxanthin, which originate from their food, and their metabolites. On the other hand, the aphid and whitefly can synthesize carotenoids de novo by carotenoid biosynthesis genes that are acquired via horizontal gene transfer from fungi or endosymbiotic bacteria. These insect synthesize β-zeacarotene, β,ψ-carotene (γ-carotene), torulene, β,γ-carotene, and γ,γ-carotene by carotenoid biosynthesis genes transferred from endosymbiotic bacteria (Fig. 12) [15]. Furthermore, blue-green aphids synthesize polycyclic quinines using genes of the endosymbiotic bacterium Rickettsiella [16]. Therefore, aphids make their own carotenoids and quinines by horizontal transfer genes from fungi or symbiotic bacteria for coloration, depending on the environmental context. These aphid carotenoids are also accumulated in beetles and dragonflies through the food chain.Fig. 12 Carotenoid biosynthetic pathways by horizontal transfer genes from fungi and symbiotic bacteria in aphid and whitefly

sfer genes from fungi or symbiotic bacteria for coloration, depending on the environmental context. These aphid carotenoids are also accumulated in beetles and dragonflies through the food chain.Fig. 12 Carotenoid biosynthetic pathways by horizontal transfer genes from fungi and symbiotic bacteria in aphid and whitefly Stick insects change their body color from green to red for camouflage in autumn. In summer, stick insects accumulate chlorophyll and β-carotene from dietary green leaves and show a green body color. In autumn, they convert β-carotene to 3,4,3′,4′-tetradehydro-β,β-carotene-2,2′-dione, with 15 conjugated double bonds system, which exhibits deep red color. This carotenoid is also accumulated in eggs for reproduction [17]. A series of ketocarotenoids, 3-hydroxyechinenone, adonirubin, and astaxanthin, was identified in the two-spotted spider mite Tetranychus urticae. In response to long nights and lower temperatures, female spider mites enter a facultative diapause characterized by the cessation of reproduction and a marked change in body color from faint yellow to bright red–orange. This body color change results from the accumulation of ketocarotenoids, like astaxanthin, which has been suggested to protect against the physical stresses of overwintering.

r a facultative diapause characterized by the cessation of reproduction and a marked change in body color from faint yellow to bright red–orange. This body color change results from the accumulation of ketocarotenoids, like astaxanthin, which has been suggested to protect against the physical stresses of overwintering. A recent investigation revealed that carotenoid cyclase/synthase and carotenoid desaturase genes, which may be responsible for the conversion of phytoene to β-carotene, were present in the two-spotted spider mite T. urticae [18, 19]. Phylogenetic analyses suggest that these carotenoid biosynthetic genes were transferred from fungi into the spider mite genome.

tenoid cyclase/synthase and carotenoid desaturase genes, which may be responsible for the conversion of phytoene to β-carotene, were present in the two-spotted spider mite T. urticae [18, 19]. Phylogenetic analyses suggest that these carotenoid biosynthetic genes were transferred from fungi into the spider mite genome. Carotenoids in birds Most of the bright red, orange, and yellow pigments of plumage (feathers) are due to the presence of carotenoids. In birds, carotenoids are an important signal of a good nutritional condition and they are used in ornamental displays as a sign of fitness and to increase sexual attractiveness. Carotenoid-based colors of feathers (plumage) catch the attention of the opposite sex to promote mating. For example, manipulation of the dietary carotenoid supply invokes parallel changes in cell-mediated immune function and sexual attractiveness in male zebra finches [6, 20]. At least ten kinds of carotenoids have been documented in red feathers. Most of these are produced through metabolic modification of dietary precursor compounds. A series of yellow carotenoids with the 3-hydoxy- and/or 3-oxo-ε-end group were also reported in colored feathers of the goldfinch Carduelis. They are also metabolized from lutein and zeaxanthin [21].

n documented in red feathers. Most of these are produced through metabolic modification of dietary precursor compounds. A series of yellow carotenoids with the 3-hydoxy- and/or 3-oxo-ε-end group were also reported in colored feathers of the goldfinch Carduelis. They are also metabolized from lutein and zeaxanthin [21]. Recently, Mundy et al. identified genes required for the bright-red coloration that birds use for communication, such as attracting mates. They revealed a genetic link between red coloration and color vision in the zebra finch, and proposed that redness may be an honest signal of mate quality by indicating a bird’s ability to detoxify harmful substances [22]. Carotenoids are also present in frogs, snails, and lizards. These yellow and red colors are due to the presence of carotenoids such as β-carotene, β-cryptoxanthin, lutein, and astaxanthin. Carotenoids in mammals It has been reported that mammals are categorized into three groups in terms of their ability to absorb carotenoids. White-fat animals such as pigs, sheep, goats, cats, and rodents do not absorb carotenoids at all or in very small amounts. Yellow-fat animals, such as ruminant cattle and horses exclusively accumulate carotenes and not xanthophylls. The third group, humans and monkeys, accumulate both carotenes and xanthophylls equally well [6].

animals such as pigs, sheep, goats, cats, and rodents do not absorb carotenoids at all or in very small amounts. Yellow-fat animals, such as ruminant cattle and horses exclusively accumulate carotenes and not xanthophylls. The third group, humans and monkeys, accumulate both carotenes and xanthophylls equally well [6]. A feeding experiment revealed that monkeys effectively absorbed not only β-carotene but also β-cryptoxanthin, lutein, and zeaxanthin into plasma. In the liver, both β-carotene and xanthophylls were well-deposited. In the lung, heart, muscle, fat, skin, and brain, less polar carotenoids such as β-carotene and β-cryptoxanthin, were well-deposited rather than polar xanthophylls such as lutein and zeaxanthin. Namely, the plasma carotenoid profile in monkeys reflected the dietary carotenoid composition, as in humans. Monkeys effectively accumulated not only β-carotene but also β-cryptoxanthin, lutein, and zeaxanthin in plasma. Interestingly, monkeys were similar with regard to the preferential accumulation of β-cryptoxanthin in the blood and brain [23].

profile in monkeys reflected the dietary carotenoid composition, as in humans. Monkeys effectively accumulated not only β-carotene but also β-cryptoxanthin, lutein, and zeaxanthin in plasma. Interestingly, monkeys were similar with regard to the preferential accumulation of β-cryptoxanthin in the blood and brain [23]. Xanthophylls with the 3-oxo-ε-end group, such as β,ε-caroten-3′-one, 3-hydroxy-β,ε-caroten-3′-one, 3′-hydroxy-ε,ε-caroten-3′-one, and ε,ε-carotene-3,3′-dione, are present in several mammals. Recently, Nagao et al. revealed that carotenoids with a 3-hydroxy-β-end group in xanthophylls were oxidized to carotenoids with a 3-oxo-ε-end group via an unstable intermediate with a 3-oxo-β-end group by a NAD+-dependent dehydrogenase of the mouse liver, as shown in Fig. 13 [24].Fig. 13 Oxidation pathway of xanthophylls with 3-hydroxy-β-end in mammals

at carotenoids with a 3-hydroxy-β-end group in xanthophylls were oxidized to carotenoids with a 3-oxo-ε-end group via an unstable intermediate with a 3-oxo-β-end group by a NAD+-dependent dehydrogenase of the mouse liver, as shown in Fig. 13 [24].Fig. 13 Oxidation pathway of xanthophylls with 3-hydroxy-β-end in mammals Carotenoids in humans About 50 kinds of carotenoids are found in common human foods, and among them, about 20 types ingested from food are found in the blood (plasma or serum). Of these, β-carotene, α-carotene, lycopene, β-cryptoxanthin, lutein, and zeaxanthin have been found to be the major components and make up more than 90% of the total carotenoids [25, 26]. Carotenoids also accumulate in human erythrocytes [23]. Oxidative metabolites of lycopene, lutein, and zeaxanthin are also found in human plasma [23, 25, 26]. Capsanthin, a major carotenoid in paprika, is also absorbed in humans and part of it is metabolized to capsanthone [23]. However, epoxy carotenoids, such as antheraxanthin, violaxanthin, neoxanthin, and lutein epoxide, which are present in vegetables, are not found in human blood. These epoxy carotenoids might be degraded by to the acidic conditions in the stomach [27–29].

rbed in humans and part of it is metabolized to capsanthone [23]. However, epoxy carotenoids, such as antheraxanthin, violaxanthin, neoxanthin, and lutein epoxide, which are present in vegetables, are not found in human blood. These epoxy carotenoids might be degraded by to the acidic conditions in the stomach [27–29]. Carotenoids ingested from the diet are absorbed by the small intestine. Xanthophyll esters are hydrolyzed by lipase or esterase and absorbed. A part of provitamin A carotenoids are converted into retinal in the mucous of the small intestine by β-carotene-15,15′-dioxygenase. Absorbed carotenoids are incorporated into chylomicrons and then transported to the liver and various organs through the blood. All three major lipoproteins: very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), are involved in the transport of carotenoids [28]. Carotenoids can be found in several human organs, such as the liver, adrenal gland, ovaries, skin, lung, testes, prostate, and blood serum. The distribution of carotenoids in human organs shows specificity. Lutein and zeaxanthin are found in the surface of the skin and subcutaneous tissue in an esterified form and act as UV absorbers and quenchers of singlet oxygen [30]. Xanthophylls such as β-cryptoxanthin, lutein, and zeaxanthin are found in the brain [31]. In the eye, lutein (meso)-zeaxanthin, and zeaxanthin are present as macular pigments [6]. Lycopene accumulates in the prostate [28, 29].

us tissue in an esterified form and act as UV absorbers and quenchers of singlet oxygen [30]. Xanthophylls such as β-cryptoxanthin, lutein, and zeaxanthin are found in the brain [31]. In the eye, lutein (meso)-zeaxanthin, and zeaxanthin are present as macular pigments [6]. Lycopene accumulates in the prostate [28, 29]. Several investigations have revealed that dietary carotenoids are associated with reduced risks of some cancers and other serious conditions, stimulation of the immune systems and benefits for skin health of humans [6, 28, 29]. In 1981, Peto et al. reported that dietary β-carotene reduced human cancer rates [32]. Since then, several epidemiological studies have demonstrated that intake of green-yellow vegetables and fruits, which contain various carotenoids, is associated with a reduced risk of cancer [6, 28, 29]. For example, β-cryptoxanthin, which is rich in Satuma mandarin (Citrus unshiu), could be associated with a reduced risk of cancer. Intake of lycopene also reduced the risk of prostate cancer [6, 28, 29]. Furthermore, clinical trials have also revealed that the administration of natural multi carotenoids (mixture of α-carotene, β-carotene, lutein, and lycopene) and α-tocopherol resulted in significant suppression of hepatoma development in a hepatitis virus-induced cirrhosis patient [33, 34]. Carotenoids were also reported to help prevent cardiovascular disease, diabetes, obesity, and several lifestyle-related diseases and to enhance immunity. Furthermore, carotenoids improve endurance and skin health [6, 28, 29].

t suppression of hepatoma development in a hepatitis virus-induced cirrhosis patient [33, 34]. Carotenoids were also reported to help prevent cardiovascular disease, diabetes, obesity, and several lifestyle-related diseases and to enhance immunity. Furthermore, carotenoids improve endurance and skin health [6, 28, 29]. Chemical mechanism of scavenging of active oxygen species by carotenoids It was reported that the mechanism whereby carotenoids scavenge singlet oxygen was a physical reaction. Namely, carotenoids take up thermal energy from singlet oxygen and release this energy by polyene vibration [6]. However, recent investigations including our research group studies revealed that carotenoids could scavenge reactive oxygen species through chemical reactions.

avenge singlet oxygen was a physical reaction. Namely, carotenoids take up thermal energy from singlet oxygen and release this energy by polyene vibration [6]. However, recent investigations including our research group studies revealed that carotenoids could scavenge reactive oxygen species through chemical reactions. A series of apo-astaxanthin was obtained as out-oxidation products of astaxanthin. These compounds were considered to take up oxygen by polyene in astaxanthin (Fig. 14) [35]. Astaxanthin also forms nito-astaxanthins by the reaction of peroxynitrite and inhibits the nitration of tyrosine in an in vitro model (Fig. 14). These results indicate that astaxanthin is able to capture peroxynitrite and nitrogen dioxide radicals from their molecules to form nitro-astaxanthin and protects against the nitration of tyrosine. Similar results were obtained in cases of β-carotene, lutein, zeaxanthin, capsanthin, and fucoxanthin. These results suggest that carotenoids might have higher reactivity with peroxynitrite and/or nitrogen dioxide radicals than phenoic compounds such as tyrosine. Therefore, carotenoids could inhibit the nitration of tyrosine [36, 37].Fig. 14 Auto oxidation products of astaxanthin and reaction products of astaxanthin with peroxynitrite

uggest that carotenoids might have higher reactivity with peroxynitrite and/or nitrogen dioxide radicals than phenoic compounds such as tyrosine. Therefore, carotenoids could inhibit the nitration of tyrosine [36, 37].Fig. 14 Auto oxidation products of astaxanthin and reaction products of astaxanthin with peroxynitrite Recently we investigated the reaction products of astaxanthin with hydroxy radicals, superoxide anion radicals, and singlet oxygen by LC/PDA ESI–MS and ESR spectrometry. The ESR study revealed that astaxanthin could quench not only singlet oxygen but also superoxide anion radicals and hydroxy radicals. The LC/PDA ESI–MS study revealed that astaxanthin epoxides were major reaction products of astaxanthin with superoxide anion radicals and hydroxyl radicals. Astaxanthin endoperoxides were identified as major reaction products of astaxanthin with singlet oxygen (Fig. 15) [38]. Similar results were also obtained in the case of β-carotene, zeaxanthin, and capsanthin [39, 40]. These results suggest that carotenoids could take up singlet oxygen, superoxide anion radicals and hydroxyl radicals by the formation of endoperoxide or epoxide.Fig. 15 Reaction products of ataxanthin with hydroxy radicals, superoxide anion radicals, and singlet oxygen

f β-carotene, zeaxanthin, and capsanthin [39, 40]. These results suggest that carotenoids could take up singlet oxygen, superoxide anion radicals and hydroxyl radicals by the formation of endoperoxide or epoxide.Fig. 15 Reaction products of ataxanthin with hydroxy radicals, superoxide anion radicals, and singlet oxygen Conclusion Until 1980, interest in carotenoid research was exclusively focused in the field of natural product chemistry and fisheries science in Japan. Many interesting structural carotenoids were identified in aquatic animals. Since then, several biological functions of natural carotenoids have been established through investigation of natural product chemistry, aquaculture, human-health science, and photosynthesis. Japanese researchers have been contributing to the progress of carotenoids science, technology, and commercial application. Now, carotenoids are well-known to be important for human health, playing roles in the prevention of cancer and lifestyle-related diseases and contributing to the beauty industry. Carotenoids, such as astaxanthin, are therefore industrially produced and used for supplements and cosmetics. The 19th International Symposium on Carotenoids will held in Toyama from July 12 to 17, 2020. I hope for the success of this symposium and further progress in carotenoid science and business in Japan. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Compliance with ethical standards Conflict of interest No conflict of interests for the author.

Correction to: Journal of Natural Medicines 10.1007/s11418-019-01372-x In the original publication of the article, Figures 2, 3, 5, 11 and 13 were published incorrectly. The correct version of Fig. 2, 3, 5, 11 and 13 are given below,Fig. 2 Representative H&E staining in area postrema along with ileum of rats and minks (100 × magnification). The figures show the H&E staining in area postrema along with ileum of rats and minks (rats: n = 5, minks: n = 6). Bar indicates 100 µm. C normal control group, CG simple gingerol control group, V cisplatin control group, M cisplatin + metoclopramide group, GL cisplatin + low-dose gingerol group, GM cisplatin + middle-dose gingerol group, GH cisplatin + high-dose gingerol group. The red arrow shows the nerve cell, black arrow shows the epithelial cell, and blue arrow shows the inflammatory cell Fig. 3 5-HT immunostaining expression in area postrema in addition to ileum of rats and minks. a Immunohistochemistry manifestation of 5-HT in area postrema in addition to ileum of rats and minks (rats: n = 5, minks: n = 6). Bar indicates 100 µm. b Mean optical density values of 5-HT. The images were quantified by Image-Pro Plus. C normal control group, CG simple gingerol control group, V cisplatin control group, M cisplatin + metoclopramide group, GL cisplatin + low-dose gingerol group, GM cisplatin + middle-dose gingerol group, GH cisplatin + high-dose gingerol group. *P < 0.05 vs. Group C, ‡P < 0.05 vs. Group CG, †P < 0.05 vs. Group V, §P < 0.05 vs. Group M, ||P < 0.05 vs. Group GL, ¶P < 0.05 vs. Group GM. 5-HT 5-tyrosine hydroxylase

in + metoclopramide group, GL cisplatin + low-dose gingerol group, GM cisplatin + middle-dose gingerol group, GH cisplatin + high-dose gingerol group. *P < 0.05 vs. Group C, ‡P < 0.05 vs. Group CG, †P < 0.05 vs. Group V, §P < 0.05 vs. Group M, ||P < 0.05 vs. Group GL, ¶P < 0.05 vs. Group GM. 5-HT 5-tyrosine hydroxylase Fig. 5 TPH immunostaining manifestation in area postrema as well as ileum of rats and minks. a Immunohistochemistry manifestation of TPH2 in area postrema of rats plus minks, and TPH1 in ileum of rats and minks (rats: n = 5, minks: n = 6). Bar indicates 100 µm. b Mean optical density values of TPH2 and TPH1. The images were quantified by Image-Pro Plus. C normal control group, CG simple gingerol control group, V cisplatin control group, M cisplatin + metoclopramide group, GL cisplatin + low-dose gingerol group, GM cisplatin + middle-dose gingerol group, GH cisplatin + high-dose gingerol group. *P < 0.05 vs. Group C, ‡P < 0.05 vs. Group CG, †P < 0.05 vs. Group V, §P < 0.05 vs. Group M, ||P < 0.05 vs. Group GL, ¶P < 0.05 vs. Group GM. TPH1 tryptophan hydroxylase 1, TPH2 tryptophan hydroxylase 2

platin + low-dose gingerol group, GM cisplatin + middle-dose gingerol group, GH cisplatin + high-dose gingerol group. *P < 0.05 vs. Group C, ‡P < 0.05 vs. Group CG, †P < 0.05 vs. Group V, §P < 0.05 vs. Group M, ||P < 0.05 vs. Group GL, ¶P < 0.05 vs. Group GM. TPH1 tryptophan hydroxylase 1, TPH2 tryptophan hydroxylase 2 Fig. 11 DA immunostaining expression in area postrema in addition to ileum of rats and minks. a Immunohistochemistry manifestation of DA in area postrema plus ileum of rats and minks (rats: n = 5, minks: n = 6). Bar indicates 100 µm. b Mean optical density values of DA. The images were quantified by Image-Pro Plus. C normal control group, CG simple gingerol control group, V cisplatin control group, M cisplatin + metoclopramide group, GL cisplatin + low-dose gingerol group, GM cisplatin + middle-dose gingerol group, GH cisplatin + high-dose gingerol group. *P < 0.05 vs. Group C, ‡P < 0.05 vs. Group CG, †P < 0.05 vs. Group V, §P < 0.05 vs. Group M, ||P < 0.05 vs. Group GL, ¶P < 0.05 vs. Group GM. DA dopamine

group, M cisplatin + metoclopramide group, GL cisplatin + low-dose gingerol group, GM cisplatin + middle-dose gingerol group, GH cisplatin + high-dose gingerol group. *P < 0.05 vs. Group C, ‡P < 0.05 vs. Group CG, †P < 0.05 vs. Group V, §P < 0.05 vs. Group M, ||P < 0.05 vs. Group GL, ¶P < 0.05 vs. Group GM. DA dopamine Fig. 13 TH immunostaining manifestation in area postrema in addition to ileum of rats and minks. a Immunohistochemistry manifestation of TH in area postrema plus ileum of rats and minks (rats: n = 5, minks: n = 6). Bar indicates 100 µm. b Mean optical density values of TH. The images were quantified by Image-Pro Plus. C normal control group, CG simple gingerol control group, V cisplatin control group, M cisplatin + metoclopramide group, GL cisplatin + low-dose gingerol group, GM cisplatin + middle-dose gingerol group, GH cisplatin + high-dose gingerol group. *P < 0.05 vs. Group C, ‡P < 0.05 vs. Group CG, †P < 0.05 vs. Group V, §P < 0.05 vs. Group M, ||P < 0.05 vs. Group GL, ¶P < 0.05 vs. Group GM. TH tyrosine hydroxylase Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Introduction Alzheimer’s disease (AD), a type of progressive dementia, is a neurodegenerative disorder characterized by a progressive cognitive decline resulting from selective neuronal dysfunction, synaptic loss, and neuronal cell death [1–5]. Numerous medications are undergoing testing for the treatment of the dementia associated with AD, however, there is no known cure for AD. Studies of patients with AD revealed the depleted levels of acetylcholine in an AD patient’s brain. Centrally acting cholinergic drugs have been reported to increase the regional cerebral blood flow of acetylcholine in the brain regions affected by AD [6]. Thus, the maintaining of the acetylcholine level in the brain is important for the cure of AD patients. One method for this is blocking the activity of acetylcholinesterase (AChE), the enzyme-degrading acetylcholine. The cholinesterase inhibitors (AChEI) donepezil hydrochloride, galantamine hydrobromide, and rivastigmine tartrate are the current approved drugs for the treatment of AD patients [7]. However, AChEI presents some limitations, such as their short half-lives and excessive side effects caused by the activation of peripheral cholinergic systems, as well as hepatotoxicity, which is the most frequent and important side effect of these drug therapies [8–11]. For this reason, alternative and complementary therapies need to be developed. Thus, novel AChE inhibitors from plant sources could be valuable alternatives in the context of the treatment of AD. Several studies have shown the neuroprotective and/or cognition-enhancing properties of natural products and their components using different animal models [12–18].

entary therapies need to be developed. Thus, novel AChE inhibitors from plant sources could be valuable alternatives in the context of the treatment of AD. Several studies have shown the neuroprotective and/or cognition-enhancing properties of natural products and their components using different animal models [12–18]. Magnolol, honokiol, and obovatol are well-known bioactive constituents of the bark of Magnolia officinalis. Magnolol and honokiol have been known to have various pharmacological activities, such as anti-inflammatory, anti-bacterial, and anti-allergic activities, and have also been used as traditional Chinese medicines for the treatment of neurosis, anxiety, stroke, fever, and headache [19]. Honokiol was known to promote a potassium-induced release of acetylcholine in a rat hippocampal slice [20] and to enhance neurite sprouting [21]. In addition, magnolol and honokiol exhibited an AChE inhibitory property in rat spleen microsomes and human polymorphonuclear leukocytes [22]. Moreover, we recently found that obovatol has high anti-anxiety activity [23] and anti-AChE activity (unpublished data). Among various constituents of the ethanol extract of Magnolia officinalis, we have isolated a major compound (40–50%) identified as 4-O-methylhonokiol that has not demonstrated any pharmacological activities. Therefore, in this study, we investigated whether the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol could inhibit the memory impairment induced by scopolamine through the inhibition of AChE.

jor compound (40–50%) identified as 4-O-methylhonokiol that has not demonstrated any pharmacological activities. Therefore, in this study, we investigated whether the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol could inhibit the memory impairment induced by scopolamine through the inhibition of AChE. Materials and methods Animals Male ICR mice weighing 25–30 g (Samtako, Gyeonggi-do, Korea) were maintained in accordance with the guidelines of the National Institute of Toxicological Research of the Korea Food and Drug Administration for the care and use of laboratory animals. Animals were housed three per cage, allowed access to water and food ad libitum, and maintained on a 12-h light/dark cycle regulated at 23°C room temperature. The experiments were performed at least 1 week after their arrival in individual home cages. Sixty mice were used for the water maze test and another 60 mice were used for the step-through test. Ten mice per group were used for each of the memory tests; Group 1: control, Group 2: scopolamine-treated, Group 3: scopolamine with 5 mg/kg ethanol extract, Group 4: scopolamine with 10 mg/kg ethanol extract, Group 5: scopolamine with 0.75 mg/kg 4-O-methylhonokiol, Group 6: scopolamine with 1.5 mg/kg 4-O-methylhonokiol. The mice used for the water maze test were used for the assay of AChE activity.

2: scopolamine-treated, Group 3: scopolamine with 5 mg/kg ethanol extract, Group 4: scopolamine with 10 mg/kg ethanol extract, Group 5: scopolamine with 0.75 mg/kg 4-O-methylhonokiol, Group 6: scopolamine with 1.5 mg/kg 4-O-methylhonokiol. The mice used for the water maze test were used for the assay of AChE activity. Materials The bark of Magnolia officinalis Rehd. et Wils. was dried in the shade at room temperature and stored in a dark, cold room until use. The air-dried bark of Magnolia officinalis Rehd. et Wils. (3 kg) was cut into pieces and extracted twice with 95% (v/v) ethanol (four times as much as the weight of the dried plants) for 3 days at room temperature. After filtration through the 400-mesh filter cloth, the filtrate was filtered again through filter paper (Whatman Grade No. 5) and concentrated under reduced pressure. The combined extract (450 g) was suspended in H2O and the aqueous suspension was extracted with n-hexane, ethyl acetate, and n-BuOH, respectively. The n-hexane layer was evaporated to dryness to give a residue (70 g), which was chromatographed on silica gel with n-hexane:ethyl acetate (9:1) gradient to yield a crude fraction that included 4-O-methylhonokiol. This fraction was repeatedly purified by silica gel chromatography using n-hexane:ethyl acetate as the eluent to obtain pure 4-O-methylhonokiol (Fig. 1a). 4-O-methylhonokiol was identified by 1H-NMR and 13C-NMR. The results of the NMR data are as follows and are in agreement with previously published data [24]. 1H-NMR (400 MHz, CDCl3): δ 3.36 (2H, d, J = 7 Hz, H-7), 3.44 (2H, d, J = 7 Hz, 7′-H), 3.89 (3H, s, OMe), 5.05–5.14 (5H, m, H-9, H-9′, OH), 5.93–6.07 (2H, m, H-8, H-8′), 6.92 (1H, d, J = 7 Hz, Ar-H), 6.97 (1H, d, J = 8 Hz, Ar-H), 7.04–7.08 (2H, m, Ar-H), 7.24–7.31 (2H, m, Ar-H). 13C-NMR (100 MHz, CDCl3): δ 34.5 (C-7), 39.6 (C-7′), 55.8 (OMe), 111.2 (C-3′), 115.7 (C-4′), 115.8 (C-9), 116.1 (C-9′), 128.0 (C-1′), 128.1 (C-6), 129.0 (C-3), 129.2 (C-1), 130.0 (C-5), 130.4 (C-6′), 130.7 (C-2), 132.4 (C-5′), 136.7 (C-8), 138.0 (C-8′), 151.0 (C-2′), 157.2 (C-4). The ethanol extract of Magnolia officinalis contained 16.6% 4-O-methylhonokiol, followed by 16.5% honokiol and 12.9% magnolol, and 42–45% others.Fig. 1 Chemical structure of 4-O-methylhonokiol (a) and experimental scheme (b)

130.4 (C-6′), 130.7 (C-2), 132.4 (C-5′), 136.7 (C-8), 138.0 (C-8′), 151.0 (C-2′), 157.2 (C-4). The ethanol extract of Magnolia officinalis contained 16.6% 4-O-methylhonokiol, followed by 16.5% honokiol and 12.9% magnolol, and 42–45% others.Fig. 1 Chemical structure of 4-O-methylhonokiol (a) and experimental scheme (b) Passive avoidance performance test The passive avoidance performance test is widely accepted as a simple and rapid method for memory testing [24]. The passive avoidance response was determined using a ‘step-through’ apparatus (Med Associates Inc., Vermont, USA) that consisted of an illuminated and a dark compartment (each of dimensions 20.3 × 15.9 × 21.3 cm) adjoining each other through a small gate with a grid floor, 3.175-mm stainless steel rod set 8 mm apart. On a training trial, the ICR mice were placed in the illuminated compartment facing away from the dark compartment. When the mice moved completely into the dark compartment, it received an electric shock (1 mA, 3 s duration). Then, the mice were returned to their home cage. Twenty-four hours later after the training trial, the mice were placed in the illuminated compartment and the latency period to enter the dark compartment defined as ‘retention.’ The time when the mice entered the dark compartment was recorded and described as step-through latency. Scopolamine (1 mg/kg) was injected intraperitoneally (i.p.) into mice 30 min before the retention trial. The retention trials were set at a limit of 180 s of cut-off time.

the dark compartment defined as ‘retention.’ The time when the mice entered the dark compartment was recorded and described as step-through latency. Scopolamine (1 mg/kg) was injected intraperitoneally (i.p.) into mice 30 min before the retention trial. The retention trials were set at a limit of 180 s of cut-off time. Water maze test The water maze test is a widely accepted method for memory testing, and we performed this test following the method described by Morris et al. [25]. Maze testing was performed by the SMART-CS (Panlab, Barcelona, Spain) program and equipment. A circular plastic pool (height 35 cm, diameter 100 cm) was filled with milky water and kept at 22–25°C. An escape platform (height 14.5 cm, diameter 4.5 cm) was submerged 0.5–1 cm below the surface of the water in position. On training trials, the mice were placed in a pool of water and allowed to remain on the platform for 10 s, and then returned to the home cage. The mice that did not find the platform within 60 s were placed on the platform for 10 s. Twenty-four hours after the last training trial (six training trials, 2 times/day for 3 days), the mice were given the test trial. Scopolamine (1 mg/kg, i.p.) was injected into mice 30 min before the test trial. They were allowed to swim until they discovered the escape platform. The escape latency, escape distance, swimming speed, and swimming pattern of each mouse was monitored by a camera above the center of the pool connected to the SMART-LD program (Panlab, Barcelona, Spain).

jected into mice 30 min before the test trial. They were allowed to swim until they discovered the escape platform. The escape latency, escape distance, swimming speed, and swimming pattern of each mouse was monitored by a camera above the center of the pool connected to the SMART-LD program (Panlab, Barcelona, Spain). AChE activity assay After behavior testing, the animals were perfused with PBS under inhaled chloroform anesthetization. The brains were immediately collected in the same manner and frozen stored at −20°C, and separated into cortical and hippocampal regions. Brain tissues were homogenized with PBS and lyzed by 60 min of incubation on ice. The lysate was centrifuged at 15,000 rpm for 15 min and the supernatant was used for the assays. AChE activity was determined by Ellman’s method [26]. Briefly, 5 μl of sample was mixed with 200 μl of reaction buffer (0.5 mM PBS pH 7.4) containing 0.02% DTNB (5,5′-dithio-bis-2-nitrobenzoic acid), 0.02% acetylcholine, and 0.1 mM isoOMPA (tetraisopropyl pyrophosphoramide). The activity of the enzyme was determined after 5 min of incubation at 37°C and stopped with 1 mM BW284c51, a potent selective inhibitor of AChE. The reaction mixture was converted to yellow color. The optical density was measured at 405 nm and then expressed as units of the quantity (μM) of the acetylcholine that were hydrolyzed to thiocholine per 1 min per mg. Specific activity was standardized by the amount (mg) of protein of the sample (μM/min/mg protein).

. The reaction mixture was converted to yellow color. The optical density was measured at 405 nm and then expressed as units of the quantity (μM) of the acetylcholine that were hydrolyzed to thiocholine per 1 min per mg. Specific activity was standardized by the amount (mg) of protein of the sample (μM/min/mg protein). To test the in vitro AChE activity, the whole mouse brain was homogenized in a glass Teflon homogenizer containing ten volumes of homogenization buffer (12.5 mM sodium phosphate buffer, pH 7.0, 400 mM NaCl) and then centrifuged at 1,000×g for 10 min at 4°C. The supernatant was used as an enzyme source for the assay. Aliquots of diluted 4-O-methylhonokiol were then mixed with reaction buffer and reacted at room temperature for 5 min and the activity was determined by the same method as described above. The concentrations of 4-O-methylhonokiol required to inhibit AChE activity by 50% (IC50) were calculated using enzyme inhibition dose response curves. Tacrine was used as a positive control.

reaction buffer and reacted at room temperature for 5 min and the activity was determined by the same method as described above. The concentrations of 4-O-methylhonokiol required to inhibit AChE activity by 50% (IC50) were calculated using enzyme inhibition dose response curves. Tacrine was used as a positive control. Statistics The data were analyzed using GraphPad Prism 4 software (Version 4.03, GraphPad Software, Inc.). The data are presented as mean ± standard error (SE). The homogeneity of variances was assessed using a Bartlett test. If variances were homogeneous, differences between groups and treatment were assessed by one-way or two-way analysis of variance (ANOVA). If the P-value in the ANOVA test was significant, the differences between pairs of means were assessed by Dunnet’s test. One-way ANOVA was used to analyze data for AChE, while data obtained from the water Morris maze (swimming distance, escape latency, and average speed) were analyzed using two-way ANOVA. When variances were not homogeneous, the Kruskal–Wallis test was used to assess differences between groups for nonparametric analyses. Data from the step-through avoidance test (latency) were analyzed using the nonparametric tests mentioned above, followed by Dunnet’s test. A value of P < 0.05 was considered to be statistically significant.

t homogeneous, the Kruskal–Wallis test was used to assess differences between groups for nonparametric analyses. Data from the step-through avoidance test (latency) were analyzed using the nonparametric tests mentioned above, followed by Dunnet’s test. A value of P < 0.05 was considered to be statistically significant. Results Effect of the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol on the memory impairment induced by scopolamine Escape latency (s) of animals treated with scopolamine (1 mg/kg, i.p.) was decreased (12 ± 5.5 s, P < 0.01) in comparison with the control group (28 ± 7.3 s), indicating that scopolamine induced memory impairment. On the other hand, the escape latency of animals pretreated with 5 or 10 mg/kg of ethanol extract of Magnolia officinalis for 1 week showed significant preventive effect on memory impairment compared to that of scopolamine-treated animals (24 ± 7.1 s, P > 0.05 or 19 ± 6.1 s, P < 0.05) (Fig. 2a). Tacrine (3 mg/kg) also showed a significant preventive effect against scopolamine-induced memory impairment (25 ± 1.9 s, P > 0.05). The escape latency of animals administered with 0.75 or 1.5 mg/kg 4-O-methylhonokiol was approximately 2–3 times longer than that of scopolamine-treated animals, and showed significant preventive effect on memory impairment (28 ± 1.8 s, P < 0.01 or 33 ± 8.7 s, P < 0.01) (Fig. 2b).Fig. 2 Inhibitory effect of the ethanol extract of Magnolia officinalis (a) and 4-O-methylhonokiol (b) on memory impairment induced by scopolamine in the passive avoidance test. Mice were administered the ethanol extract of Magnolia officinalis at 5.0 and 10.0 mg/kg and 4-O-methylhonokiol at 0.75 and 1.5 mg/kg for 1 week into drinking water, and then the training trial was given on the last day. After 24 h, the passive avoidance test was performed. Scopolamine (i.p., 1 mg/kg) was treated 30 min before the test trial as a positive control. Each value is mean ± standard error (SE) from ten animals. #P < 0.05 showed a significant difference compared with the controls. *P < 0.05 showed a significant difference compared with the scopolamine-treated controls

performed. Scopolamine (i.p., 1 mg/kg) was treated 30 min before the test trial as a positive control. Each value is mean ± standard error (SE) from ten animals. #P < 0.05 showed a significant difference compared with the controls. *P < 0.05 showed a significant difference compared with the scopolamine-treated controls To further examine the memory-enhancing activity of the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol, we determined the improvement of the spatial memory function of these compounds using the Morris water maze. The mice were pretreated continuously with the ethanol extract of Magnolia officinalis (5 or 10 mg/kg into drinking water) and 4-O-methylhonokiol (0.75 or 1.5 mg/kg into drinking water) for 1 week, and then the animals were given the training trial. After finishing the training trial for 3 days (2 times/day, six times training), scopolamine (1 mg/kg, i.p.) was then injected into the mice to induce memory impairment. Two-way ANOVA revealed an effect of the number of days (F3, 144 = 6.981, P = 0.0002) and the treated groups (F3, 144 = 6.956, P = 0.0002) on the escape latency, and an effect of the number of days (F3, 144 = 6.254, P < 0.0001) and the treated groups (F3, 144 = 9.423, P = 0.0005) on the escape distance.

mory impairment. Two-way ANOVA revealed an effect of the number of days (F3, 144 = 6.981, P = 0.0002) and the treated groups (F3, 144 = 6.956, P = 0.0002) on the escape latency, and an effect of the number of days (F3, 144 = 6.254, P < 0.0001) and the treated groups (F3, 144 = 9.423, P = 0.0005) on the escape distance. The escape latency on the next day of training to the platform was about 450 ± 127 cm and 19 ± 3 s. Scopolamine-treated mice arrived slower at the location of the platform compared to the controls (885 ± 47 cm, P < 0.05; 42 ± 2 s, P < 0.05). The escape latency of animals pretreated with 5 or 10 mg/kg ethanol extract of Magnolia officinalis for 1 week showed a memory-enhancing effect. The escape latency of animals pretreated with 5 or 10 mg/kg ethanol extract of Magnolia officinalis for 1 week and subsequently treated with scopolamine was shown to be approximately 2.5 or 2.4 times shorter (354 ± 40 cm, P < 0.05; 16 ± 3 s, P < 0.05 or 361 ± 50 cm, P < 0.05; 17 ± 2 s, P < 0.05) than that of scopolamine-treated animals determined at 4 day after the training trial (Fig. 3a).Fig. 3 Inhibitory effect of the ethanol extract of Magnolia officinalis (a) and 4-O-methylhonokiol (b) on memory impairment induced by scopolamine in the water maze test. Mice were administered the ethanol extract of Magnolia officinalis at 5.0 and 10.0 mg/kg and 4-O-methylhonokiol at 0.75 and 1.5 mg/kg for 1 week into drinking water. The mice were then given training trials six times (2 times/day), and the test was performed 24 h after the last training trial. Scopolamine (i.p., 1 mg/kg), as a positive control, was treated 10 min before the first test trial, as shown in the Fig. 1b. Each value is the mean ± SE from ten animals. #P < 0.05 showed a significant difference compared with the controls. *P < 0.05 showed a significant difference compared with the scopolamine-treated controls

ne (i.p., 1 mg/kg), as a positive control, was treated 10 min before the first test trial, as shown in the Fig. 1b. Each value is the mean ± SE from ten animals. #P < 0.05 showed a significant difference compared with the controls. *P < 0.05 showed a significant difference compared with the scopolamine-treated controls There was also shown to be a significant memory-improving effect by the treatment with 4-O-methylhonokiol. Elevated escape latency and escape distance by scopolamine was inhibited by the time (third and fourth day) and treatment (1.5 mg/kg). An amount of 1.5 mg/kg of 4-O-methylhonokiol significantly reduced the escape latency (s) (F3, 39 = 5.983, P < 0.05 and P < 0.01 at the third and fourth days after training, respectively) and distance (cm) (F3, 39 = 4.93, P < 0.05 and P < 0.01 at the third and fourth days after training, respectively) (Fig. 3b). The average speed was not affected by any of the treatments during all days of the evaluation (data not shown).

, P < 0.05 and P < 0.01 at the third and fourth days after training, respectively) and distance (cm) (F3, 39 = 4.93, P < 0.05 and P < 0.01 at the third and fourth days after training, respectively) (Fig. 3b). The average speed was not affected by any of the treatments during all days of the evaluation (data not shown). Effect of the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol on the activities of AChE induced by scopolamine In animals treated with scopolamine (1 mg/kg, i.p.), the AChE activity was significantly increased in comparison with the control group, and in animals pretreated with the ethanol extract of Magnolia officinalis (5 or 10 mg/kg) for 1 week and subsequently treated with scopolamine (1 mg/kg, i.p.), the AChE activity was significantly (P < 0.05) suppressed in both the hippocampus and cortex in a dose-dependent manner (Fig. 4a). In animals pretreated with 4-O-methylhonokiol (0.75 or 1.5 mg/kg), the AChE activity was also significantly (P < 0.05) suppressed in the hippocampus and cortex in a dose-dependent manner (Fig. 4b). By in vitro testing, 4-O-methylhonokiol also inhibited AChE activity in a concentration (1 nM–100 μM)-dependent manner, with IC50 values of 12 nM. The IC50 value of tacrine (positive control) was 135.4 nM.Fig. 4 Inhibitory effect of the ethanol extract of Magnolia officinalis (a) and 4-O-methylhonokiol (b) on the AChE activity in the cortex and hippocampus of scopolamine-induced mice. Mice were treated with the ethanol extract of Magnolia officinalis (5 and 10 mg/kg) and 4-O-methylhonokiol (0.75 and 1.5 mg/kg) for 1 week into drinking water, and then the AChE activity was measured. Each value is the mean ± SE from ten animals. #P < 0.05 showed a significant difference compared with the controls. *P < 0.05 showed a significant difference compared with the scopolamine-treated controls

nd 4-O-methylhonokiol (0.75 and 1.5 mg/kg) for 1 week into drinking water, and then the AChE activity was measured. Each value is the mean ± SE from ten animals. #P < 0.05 showed a significant difference compared with the controls. *P < 0.05 showed a significant difference compared with the scopolamine-treated controls Discussion Alzheimer’s disease (AD) is a progressive and eventually fatal disease of the brain, and it is the cause of more than 50% of dementia patients developing in the population older than 60 years of age [27–30]. A deficiency in acetylcholine is probably the single most common cause of declining memory [20]. AChE modulates acetylcholine to proper levels by degradation, thus, excessive AChE activity leads to constant acetylcholine deficiency, causing memory and cognitive impairments in AD [16]. Currently, AChE inhibitors (AChEI) are the first group of drugs for AD treatment. Several AChEI have been approved by the FDA for the treatment of AD, such as tacrine (Cognex®), donepezil (Aricept®), rivastigmine (Exelon®), and galantamine (Reminyl®) [10]. However, these drugs have limitations in their usage, such as their short half-lives and excessive side effects caused by the activation of peripheral cholinergic systems, as well as hepatotoxicity [8, 9, 11]. Thus, a complementary therapeutic strategy is required. Neuroprotective and/or cognition-enhancing properties of natural products and their components using different animal models have been suggested as alternative therapies in several studies [12–18].

ipheral cholinergic systems, as well as hepatotoxicity [8, 9, 11]. Thus, a complementary therapeutic strategy is required. Neuroprotective and/or cognition-enhancing properties of natural products and their components using different animal models have been suggested as alternative therapies in several studies [12–18]. In this study, the ethanol extract of Magnolia officinalis and its major ingredient, 4-O-methylhonokiol, recovered the memory impairment induced by scopolamine, and its effect may be related to the ability of AChE inhibition. The step-through latency, which was reduced by scopolamine treatment, was recovered to 60–70% of the vehicle-treated control group. The Morris water maze learning task was used to assess hippocampal-dependent spatial learning ability [25, 31] and long-term memory [25]. Impairment in the long-term memory was observed in the scopolamine-treated group. However, the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol also significantly shortened the escape latencies prolonged by the scopolamine treatment. At the probe trial session, the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol improved the swimming time and distance within the zone of the platform to the control level. Collectively, these behavioral studies suggest that the ethanol extract of Magnolia officinalis improves the long-term memory in an amnestic mouse model induced by scopolamine treatment, and 4-O-methylhonokiol may be a major active ingredient.

ming time and distance within the zone of the platform to the control level. Collectively, these behavioral studies suggest that the ethanol extract of Magnolia officinalis improves the long-term memory in an amnestic mouse model induced by scopolamine treatment, and 4-O-methylhonokiol may be a major active ingredient. To confirm the mechanism of the action of the ethanol extract of Magnolia officinalis and its major ingredient, 4-O-methylhonokiol, their inhibitory activity on AChE was assessed in vivo. It is well known that the memory-improving effect of the anti-amnestic effect of tacrine and donepezil is due to the inhibition of AChE in the brain. The present data showed that the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol inhibited AChE activity in a dose-dependent manner in both the cortex and hippocampus. Thus, it is likely that the memory-improving effect of the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol may be, at least in part, mediated by the AChE inhibition. AChEI, such as tacrine and donepezil, causes severe hepatic toxicity, nausea, vomiting, etc., so these drugs must be used carefully. The efficacy of the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol is superior compared with conventional drugs such as tacrine (Table 1). In the present study, we found that 5 or 10 mg/kg of ethanol extract of Magnolia officinalis or 0.75 or 1.5 mg/kg of 4-O-methylhonokiol has a similar inhibitory effect to 3 mg/kg tacrine on scopolamine-induced memory impairment as well as the inhibition of AChE activity in vivo. The inhibitory effect on the AChE activity may be related to the inhibitory effect of 4-O-methylhonokiol on the expression of AChE. We observed that the elevated expression of AChE by scopolamine in both the cortex and hippocampus was decreased by the treatment with 4-O-methylhonokiol (data not shown). In the in vivo study, however, we did not observe clear dose-dependent prevention on the memory impairment. These unexpected data (similar dose effects) may be caused by the non differential effect of 4-O-methylhonokiol by these two doses with only two times the difference, thus, additional increased or decreased doses could be tested in further studies.

d not observe clear dose-dependent prevention on the memory impairment. These unexpected data (similar dose effects) may be caused by the non differential effect of 4-O-methylhonokiol by these two doses with only two times the difference, thus, additional increased or decreased doses could be tested in further studies. We are currently undertaking another investigation using AD transgenic mice with three different doses with three times the difference among the dosing, since the present memory prevention effects were likely to be saturated, and the effect was similar to the effect of tacrine. In in vitro acetylcholine esterase, 4-O-methylhonokiol (IC50: 12 nM) showed more than ten times stronger inhibitory effect than tacrine (IC50: 135.4 nM). Moreover, the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol originate from a natural plant, and, thus, side effects could be relatively smaller. In fact, 4-week oral treatment (up to 300 mg/kg) of the ethanol extract of Magnolia officinalis to mice and rats did not show any toxicities in the liver, kidney, intestine, or cardiovascular and central nervous systems, and the animals did not have any high cholinergic symptoms (data not shown). Similar effects of other ingredients isolated from Magnolia officinalis, such as honokiol and magnolol, were reported to increase acetylcholine release in freely moving rats, and these effects may be related to the inhibitory effects on the AChE activity [20, 32]. Therefore, from the above behavioral and biochemical data, it can be concluded that the ethanol extract of Magnolia officinalis has an ability to improve or ameliorate spatial long-term memory impairment, in part, via enhancement of the cholinergic nervous system, and 4-O-methylhonokiol may be one of major components of the memory-improving effect of Magnolia officinalis. It is also noteworthy that cognition-enhancing agents activate cholinergic transmission via an agonistic or antagonistic effect on GABAA/benzodiazepine receptor [33–35], and GABAA/benzodiazepine receptor complex controls acetylcholine release [36]. We previously found that obovatol, a similar compound isolated from Magnolia officinalis, has high affinity of GABAA/benzodiazepine receptor [23]. Therefore, it can also be speculated that the GABAA/benzodiazepine receptor ligand properties of 4-O-methylhonokiol may also be related to the ameliorating effects on scopolamine-induced memory impairment. Further studies are needed to clarify these points.

alis, has high affinity of GABAA/benzodiazepine receptor [23]. Therefore, it can also be speculated that the GABAA/benzodiazepine receptor ligand properties of 4-O-methylhonokiol may also be related to the ameliorating effects on scopolamine-induced memory impairment. Further studies are needed to clarify these points. We are currently attempting to identify the mechanisms of the interactions between 4-O-methylhonokiol and GABAA/benzodiazepine receptors. Taken together, these data suggest that the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol may be useful leading compounds in anti-AD drug development.Table 1 Inhibitory activities of acetylcholinesterase (AChE) Compound Concentration Inhibition (%) 4-O-methylhonokiol 0.1 nM 0.4 ± 0.1 1 nM 10.43 ± 1.43 10 nM 47.4 ± 2.54 100 nM 68.11 ± 7.64 1 μM 78.11 ± 4.34 10 μM 84.15 ± 2.60 Tacrine 1 nM 1.14 ± 1.24 10 nM 9.62 ± 2.32 100 nM 46.54 ± 3.86 1 μM 54.71 ± 7.74 10 μM 61.41 ± 2.94 100 μM 59.97 ± 5.31 Efficacy of inhibition was expressed as percent inhibition versus the control values (100%). Values represent mean ± SD (n = 5) This work was supported by a grant (KRF-2005-005-J15001) from the Korea Research Foundation (MOEHRD, Basic Research Promotion Fund) and by the Research Grant of Bioland Ltd. (2007–2008). Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Introduction Psidium guajava (Myrtaceae) is a small medicinal tree that is native to South America. All parts of this tree, including fruits, leaves, bark, and roots, have been used for treating stomachache and diarrhea in many countries. Previous studies of this plant have led to the isolation of tannins [1, 2], tannins and other phenolic compounds [3], flavonol glycosides [4, 5], triterpenoids [6], terpenoids [7], and carotenoids [8]. It is well known that an extract of the leaves of P. guajava improves symptoms of allergic disease, and we are interested in small molecules from the leaves of P. guajava, other than tannins, and their potential anti-inflammatory activities. In this investigation, two new benzophenone galloyl glycosides, guavinosides A (1) and B (2), and a quercetin galloyl glycoside, guavinoside C (3), were isolated from the leaves of P. guajava together with known quercetin glycosides (4–8). These structures of the novel glycosides were established through detailed analysis of their spectroscopic data and chemical evidence, and their inhibitory activities against histamine release from rat mast cells and nitric oxide (NO) from RAW 264.7 were also determined.

together with known quercetin glycosides (4–8). These structures of the novel glycosides were established through detailed analysis of their spectroscopic data and chemical evidence, and their inhibitory activities against histamine release from rat mast cells and nitric oxide (NO) from RAW 264.7 were also determined. Results and discussion Guavinoside A (1) was obtained as a yellow powder. The positive ion mode FAB-MS of 1 showed a quasimolecular ion peak at m/z 545 [M + H]+, and the molecular formula was determined to be C26H24O13 based on its high-resolution (HR)-FAB-MS (found 545.8418, calcd. 545.8413 for C26H25O13). In the IR spectrum, the strong absorbance at 1,700 cm−1 indicated the presence of conjugated carbonyl groups in 1. Acid hydrolysis of 1 furnished d-glucose which was identical with HPLC analysis, using an optical rotation (OR) detector. In the 1H-NMR spectrum of 1, two aromatic signals at δH 6.11 (2H, s) and 6.98, (2H, s) indicated the presence of two 1,3,4,5-tetrasubstituted phenyl groups, and the spin system of δH 7.68 (2H, dd, J = 7.0, 1.0 Hz), 7.46 (2H, t, J = 7.0 Hz), and 7.57 (1H, t, J = 7.0 Hz) showed the presence of a phenyl group. Additionally, an anomeric proton signal at δH 4.86 (1H, d, J = 7.6 Hz) indicated that the glucose residue was in the β-form. Correlations in the 1H–1H-COSY spectrum were observed from the anomeric proton to δH 3.15 (1H, m), 3.20 (1H, m), 3.50 (1H, m), 3.30 (1H, m), and 4.39 (2H, br s) indicating the presence of β-glucose. The signal pattern in the aromatic region of the 13C-NMR spectrum indicated the presence of three aromatic rings. In addition, the 13C-NMR and DEPT spectra showed an anomeric carbon (δC 100.5), four oxymethines (δC 73.0, 76.1, 68.9, and 73.6), an oxymethylene (δC 62.5), and two carbonyl carbons (δC 165.7 and 195.3). All proton–carbon connectivities assigned by using HMQC experiments are summarized in Table 1. The HMBC correlations from 2″′, 6″′-H (δH 6.98, 2H, s) to C-1″′ (δC 119.2), C-3″′, 5″′ (δC 145.4), C-4″′ (δC 138.4), and a carbonyl carbon (δC 165.7) revealed the presence of a galloyl moiety. A phenyl proton signal at δH 7.68 (2H, dd, J = 7.0, 1.0 Hz, 2′, 6′-H) correlated with a carbonyl carbon signal at δC 195.3, and an aromatic proton at δH 6.11 (2H, s, 3, 5-H) correlated with C-4 (δC 159.8) and the carbonyl carbon by weak 4J correlation. Furthermore, the anomeric proton signal correlated with C-4. NOE enhancement was observed between the anomeric proton signal and the signal of 3, 5-H.

th a carbonyl carbon signal at δC 195.3, and an aromatic proton at δH 6.11 (2H, s, 3, 5-H) correlated with C-4 (δC 159.8) and the carbonyl carbon by weak 4J correlation. Furthermore, the anomeric proton signal correlated with C-4. NOE enhancement was observed between the anomeric proton signal and the signal of 3, 5-H. These data suggested that the aglycone of 1 was 2,4,6-trihydroxybenzophenone, and a sugar was attached at C-4. The signal of 6′′-H, however, was downfield shifted at δH 4.39 (2H, br s), and correlated with the galloyl carbonyl carbon signal at δC 165.7 in the HMBC experiment. From these data, the structure of 1 was established to be 2,4,6-dihydroxybenzophenone 4-O-(6″-O-galloyl)-β-d-glucopyranoside (Figs. 1, 2).Table 1 13C- and 1H-NMR spectral data of guavinosides A (1) and B (2)

t δH 4.39 (2H, br s), and correlated with the galloyl carbonyl carbon signal at δC 165.7 in the HMBC experiment. From these data, the structure of 1 was established to be 2,4,6-dihydroxybenzophenone 4-O-(6″-O-galloyl)-β-d-glucopyranoside (Figs. 1, 2).Table 1 13C- and 1H-NMR spectral data of guavinosides A (1) and B (2) 1 2 δC δH δC δH Aglycone 1 108.9 s 113.0 s 2, 6 157.3 s 151.8 s 3, 5 94.9 d 6.11 (2H, s) 110.8 s 4 159.8 s 155.6 s 1′ 138.4 s 138.7 s 2′, 6′ 128.7 d 7.68 (2H, dd, J = 7.0, 1.0 Hz) 128.6 d 7.65 (2H, dd, J = 7.0, 1.0 Hz) 3′, 5′ 128.2 d 7.46 (2H, t, J = 7.0 Hz) 128.1 d 7.45 (2H, t, J = 7.0 Hz) 4′ 132.5 d 7.57 (1H, t, J = 7.0 Hz) 132.3 d 7.54 (1H, t, J = 7.0 Hz) C=O 195.3 s 196.7s 3, 5-CH3 9.9 q 2.00 (6H, s) Glucosyl 1″ 100.5 d 4.86 (1H, d, J = 7.6 Hz) 104.2 d 4.63 (1H, d, J = 8.5 Hz) 2″ 73.0 d 3.15 (1H, m) 74.2 d 3.36 (1H, m) 3″ 76.1 d 3.20 (1H, m) 76.1 d 3.30 (1H, m) 4″ 68.9 d 3.50 (1H, m) 69.3 d 3.44 (1H, m) 5″ 73.6 d 3.30 (1H, m) 73.5 d 3.42 (1H, m) 6″ 62.5 t 4.39 (2H, br s) 62.7 t 4.23 (1H, d, J = 11.0, 3.0 Hz) 4.32 (1H, d, J = 11.0 Hz) Galloyl 1″′ 119.2 s 119.6 s 2″′, 6″′ 108.6 d 6.98 (2H, s) 108.5 d 6.96 (2H, s) 3″′, 5″′ 145.4 s 145.3 s 4″′ 138.4 s 138.3 s C=O 165.7 s 165.6 s 1H-NMR (600 MHz) and 13C-NMR (150 MHz) spectra were measured in DMSO-d6 with TMS as internal standard Fig. 1 Structures of guavinosides A (1), B (2), and C (3) Fig. 2 Key HMBC and NOE correlations of guavinosides A (1) and B (2)

1 2 δC δH δC δH Aglycone 1 108.9 s 113.0 s 2, 6 157.3 s 151.8 s 3, 5 94.9 d 6.11 (2H, s) 110.8 s 4 159.8 s 155.6 s 1′ 138.4 s 138.7 s 2′, 6′ 128.7 d 7.68 (2H, dd, J = 7.0, 1.0 Hz) 128.6 d 7.65 (2H, dd, J = 7.0, 1.0 Hz) 3′, 5′ 128.2 d 7.46 (2H, t, J = 7.0 Hz) 128.1 d 7.45 (2H, t, J = 7.0 Hz) 4′ 132.5 d 7.57 (1H, t, J = 7.0 Hz) 132.3 d 7.54 (1H, t, J = 7.0 Hz) C=O 195.3 s 196.7s 3, 5-CH3 9.9 q 2.00 (6H, s) Glucosyl 1″ 100.5 d 4.86 (1H, d, J = 7.6 Hz) 104.2 d 4.63 (1H, d, J = 8.5 Hz) 2″ 73.0 d 3.15 (1H, m) 74.2 d 3.36 (1H, m) 3″ 76.1 d 3.20 (1H, m) 76.1 d 3.30 (1H, m) 4″ 68.9 d 3.50 (1H, m) 69.3 d 3.44 (1H, m) 5″ 73.6 d 3.30 (1H, m) 73.5 d 3.42 (1H, m) 6″ 62.5 t 4.39 (2H, br s) 62.7 t 4.23 (1H, d, J = 11.0, 3.0 Hz) 4.32 (1H, d, J = 11.0 Hz) Galloyl 1″′ 119.2 s 119.6 s 2″′, 6″′ 108.6 d 6.98 (2H, s) 108.5 d 6.96 (2H, s) 3″′, 5″′ 145.4 s 145.3 s 4″′ 138.4 s 138.3 s C=O 165.7 s 165.6 s 1H-NMR (600 MHz) and 13C-NMR (150 MHz) spectra were measured in DMSO-d6 with TMS as internal standard Fig. 1 Structures of guavinosides A (1), B (2), and C (3) Fig. 2 Key HMBC and NOE correlations of guavinosides A (1) and B (2) Guavinoside B (2) was obtained as a brownish solid. The negative ion mode FAB-MS of 2 showed quasimolecular ion peak at m/z 571 [M − H]−, and the molecular formula was determined to be C28H28O13 based on its HR-FAB-MS (found 571.1474, calcd. 571.1451 for C28H27O13). The molecular weight was 28 mass units (C2H4) greater than that of 1. The 1H- and 13C-NMR spectra of 2 were very similar to those of 1 except for absence of the aromatic methine signal [δH 6.11 (s); δC 94.9] in 1, and a new aryl methyl signal [δH 2.00 (6H, s); δC 9.9] and a quaternary carbon (δC 110.8) were observed. In the 13C-NMR spectrum, the appearance of a high-field region shifted methyl signal suggested that the methyl is linked to a benzene ring in the ortho-position and attached via an oxygen atom [9]. HMBC correlations were observed from the methyl proton signal to δC 110.8 (C-3, 5), 151.8 (C-2, 6), and 155.6 (C-4). NOE enhancement was also observed between the methyls and an anomeric proton at δH 4.63. These data suggested that the aglycone of 2 was 2,4,6-trihydroxy-3,5-dimethylbenzophenone. Absolute configuration of the glucose moiety was determined to be d by using HPLC analysis with an OR detector. From the above data, the structure of 2 was identified to be 2,4,6-trihydroxy-3,5-dimethylbenzophenone 4-O-(6″-O-galloyl)-β-d-glucopyranoside.

the aglycone of 2 was 2,4,6-trihydroxy-3,5-dimethylbenzophenone. Absolute configuration of the glucose moiety was determined to be d by using HPLC analysis with an OR detector. From the above data, the structure of 2 was identified to be 2,4,6-trihydroxy-3,5-dimethylbenzophenone 4-O-(6″-O-galloyl)-β-d-glucopyranoside. Guavinoside C (3) was obtained as a yellow powder. Its HR-FAB-MS showed a quasimolecular ion peak at m/z 585.0868, corresponding to the molecular formula C27H22O15. The UV absorbances at 211, 265, and 355 nm were characteristic of flavonol. The IR spectrum indicated the presence of hydroxyl (3,400 cm−1), ester (1,710 cm−1), and conjugated carbonyl group (1,690 cm−1). In the 1H-NMR spectrum, meta-coupled signals at δH 6.20 and δH 6.41 and a hydrogen-bonded hydroxyl signal at δH 12.62 indicated the presence of a 5,7-dihydroxy A ring system in flavonol. A spin system of three aromatic signals at δH 7.46 (1H, d, J = 2.2 Hz), δH 6.85 (1H, d, J = 8.8 Hz), and δH 7.49 (1H, dd, J = 8.8, 2.2 Hz) indicated the presence of a 3′,4′-dihydroxy B ring system in flavonol. In the 13C-NMR spectrum, significant flavonol signals at δC 157.3 (C-2), 133.0 (C-3), and 177.4 (C-4) were observed. In the HMBC experiment, the correlations from 2′-H to C-2, δC 144.9 (C-3′), 148.3 (C-4′), and 121.3 (C-6′), from 6′-H to C-2, C-4′ and δC 115.6 (C-2′), and from 5′-H to C-3′, C-4′, and δC 120.8 (C-1′) were observed. From these data, the aglycone of 3 was determined to be quercetin. In addition, an aromatic methine (δH 6.89, 2H, s) correlated with δC 118.9 (C-1″′), 108.5 (C-2″′, 6″′), 145.3 (C-3″′, 5″′), 138.4 (C-4″′), and 165.4 (carbonyl), indicating the presence of a galloyl moiety the same as 1 and 2. Acid hydrolysis of 3 with 2 M HCl afforded (+)-l-arabinose that was identical by HPLC analysis using OR detector comparison to an authentic sample of l-arabinose. The small coupling constant of the anomeric proton (δH 5.56, d, J = 1.4 Hz) indicated the presence of the α-form of arabinose. Correlations in the 1H–1H COSY spectrum were observed for a spin system from the anomeric signal to three oxymethine signals at δH 4.18 (br s, 2″-H), 3.81 (m, 3″-H), and 3.74 (m, 4″-H), and methylene signals (δH 4.11 and 4.02, 5″-H2) was observed. Furthermore, two hydroxy signals at δH 5.72 (br s) and 5.48 (br s) both coupled with 2″-H and 3″-H, respectively.

ere observed for a spin system from the anomeric signal to three oxymethine signals at δH 4.18 (br s, 2″-H), 3.81 (m, 3″-H), and 3.74 (m, 4″-H), and methylene signals (δH 4.11 and 4.02, 5″-H2) was observed. Furthermore, two hydroxy signals at δH 5.72 (br s) and 5.48 (br s) both coupled with 2″-H and 3″-H, respectively. Additionally, the signals of 5″-H2 of 3 were shifted downfield compared with those of 4 (δH 3.36 and 3.32), and correlated with a galloyl carbonyl carbon signal in the HMBC experiment. From these observations, the sugar moiety was determined to be l-α-arabinofuranose. Thus, the structure of 3 was determined to be quercetin 3-O-(5″-O-galloyl)-α-l-arabinofuranoside. The structures of 4–8 were elucidated to be quercetin 3-O-α-l-arabinofuranoside (4), quercetin 3-O-α-l-arabinopyranoside (5), quercetin 3-O-β-d-xylopyranoside (6), quercetin 3-O-β-d-galactopyranoside (7), and quercetin 3-O-β-d-glucopyranoside (8) by comparison with spectroscopic data [10, 11] and chemical degradation methods. Benzophenone glycosides have been isolated from many kind of plants, but this is first reported isolation of a dimethylbenzophene glycoside from a natural source. The substitution pattern is the well-known A ring of flavonoid; a possible biosynthesis pathway to the aglycone moiety of 2 would be methylation of the benzophenone skeleton.

osides have been isolated from many kind of plants, but this is first reported isolation of a dimethylbenzophene glycoside from a natural source. The substitution pattern is the well-known A ring of flavonoid; a possible biosynthesis pathway to the aglycone moiety of 2 would be methylation of the benzophenone skeleton. Isolated compounds were evaluated for inhibitory activities against histamine release from rat peripheral mast cells [12] and nitric oxide (NO) production from a murine macrophage-like cell line, RAW264.7 cells [13]. Compounds 3–8 (at 100 μg/ml) inhibited histamine release from mast cells with inhibition ratios of 94.4, 21.9, 30.5, 23.9, 100, and 93.5%, respectively. But 1 and 2 did not show inhibitory activity against histamine release at this concentration. Compounds 3–8 (at 100 μg/ml) inhibited NO production by RAW 264.7 cells stimulated with lipopolysaccharide and interferon gamma with inhibition ratios of 50.0, 33.2, 32.4, 65.1, 55.3, and 52.1%, respectively. The isolated compounds therefore inhibited chemical mediators, such as histamine and NO, and increased IL-12 release from RAW 264.7 cells. In conclusion, phenolic compounds isolated from P. guajava might be valuable candidates for treating various inflammatory diseases.

Isolated compounds were evaluated for inhibitory activities against histamine release from rat peripheral mast cells [12] and nitric oxide (NO) production from a murine macrophage-like cell line, RAW264.7 cells [13]. Compounds 3–8 (at 100 μg/ml) inhibited histamine release from mast cells with inhibition ratios of 94.4, 21.9, 30.5, 23.9, 100, and 93.5%, respectively. But 1 and 2 did not show inhibitory activity against histamine release at this concentration. Compounds 3–8 (at 100 μg/ml) inhibited NO production by RAW 264.7 cells stimulated with lipopolysaccharide and interferon gamma with inhibition ratios of 50.0, 33.2, 32.4, 65.1, 55.3, and 52.1%, respectively. The isolated compounds therefore inhibited chemical mediators, such as histamine and NO, and increased IL-12 release from RAW 264.7 cells. In conclusion, phenolic compounds isolated from P. guajava might be valuable candidates for treating various inflammatory diseases. Experimental General The UV spectra were recorded on a Shimadzu model UV-160 spectrophotometer. IR spectra were recorded on a Horiba FT-210 diffraction infrared spectrometer. FAB-MS were obtained with a JEOL model JMS-AX505 HA spectrometer. 1H-NMR (600 MHz) and 13C-NMR (150 MHz) spectra were obtained on a Varian Inova™ 600 spectrometer and a JEOL Delta 600 spectrometer. NMR spectra were measured in DMSO-d6 with TMS as internal standard. Optical rotation was measured with a Jasco DIP-370 polarimeter. The inhibitory activities against histamine release from rat mast cells and NO production were carried out as described in the literature method [14].

er and a JEOL Delta 600 spectrometer. NMR spectra were measured in DMSO-d6 with TMS as internal standard. Optical rotation was measured with a Jasco DIP-370 polarimeter. The inhibitory activities against histamine release from rat mast cells and NO production were carried out as described in the literature method [14]. Plant material Leaves of P. guajava were donated by OS Industrial Co. Ltd. (Tokyo, Japan).

er and a JEOL Delta 600 spectrometer. NMR spectra were measured in DMSO-d6 with TMS as internal standard. Optical rotation was measured with a Jasco DIP-370 polarimeter. The inhibitory activities against histamine release from rat mast cells and NO production were carried out as described in the literature method [14]. Plant material Leaves of P. guajava were donated by OS Industrial Co. Ltd. (Tokyo, Japan). Extraction and isolation The dried leaves of P. guajava (5 kg) were extracted with 15 l of 80% MeOH at room temperature for 7 days. The solution was filtered and concentrated under reduced pressure to give a crude extract. The extract was dissolved in water and passed through a Diaion HP-20 column (Mitsubishi Kasei, Tokyo, Japan), and eluted stepwise with 50, 70, and 100% MeOH. The 70% MeOH eluate was dissolved in EtOH and passed through a Sephadex LH-20 column (Pharmacia, Uppsala, Sweden). The effluent was chromatographed on a Diaion CHP-20P column (Mitsubishi Kasei, Tokyo, Japan), and eluted with 50 and 70% MeOH. All fractions were monitored by TLC, and the fractions containing the same compound(s) (as evidenced by TLC) were combined to give four fractions. Fraction 3 (900 mg) was chromatographed on a Sephadex LH 20 column developed with CHCl3–MeOH (1:1) to give four fractions. Fraction 2 (770 mg) was further applied to a reversed-phase column (SSC ODS, Senshu Scientific Co. Ltd., Tokyo, Japan) eluted stepwise with 0–25% MeOH, and recrystallized from MeOH to give 1 (47 mg). Fraction 4 (124 mg) was purified by medium pressure liquid chromatography (Yamazen Baker-bond ODS Yamazen, Kyoto, Japan) column eluted with MeCN–MeOH–H2O (5:35:60) to give 2 (44 mg). Fraction 3 (130 mg) was purified by reversed-phase HPLC [column: Shiseido Capcell pak C18 UG120 (10-mm i.d. × 250 mm, Shiseido, Tokyo, Japan); mobile phase: MeCN–MeOH–H2O (5:30:65); flow rate: 3.0 ml/min; detection: UV at 254 nm] to give 3 (14 mg). The 100% MeOH eluate of Diaion HP-20 was dissolved in MeOH, and chromatographed on a Sephadex LH-20 column (2.5 × 100 cm) to give ten fractions. Fraction 3 (1.9 g) was chromatographed on a silica gel column developed with CHCl3–MeOH to give eight fractions. Fraction 6 (515 mg) was purified by reversed-phase HPLC [column: Shiseido Capcell pak C18 UG120 (10-mm i.d. × 250 mm); mobile phase: MeCN–H2O (18:82); flow rate: 3.0 ml/min.; detection: UV at 250 nm] to give 4 (20 mg), 5 (50 mg), 6 (62 mg), 7 (62 mg), and 8 (30 mg), respectively.

ith CHCl3–MeOH to give eight fractions. Fraction 6 (515 mg) was purified by reversed-phase HPLC [column: Shiseido Capcell pak C18 UG120 (10-mm i.d. × 250 mm); mobile phase: MeCN–H2O (18:82); flow rate: 3.0 ml/min.; detection: UV at 250 nm] to give 4 (20 mg), 5 (50 mg), 6 (62 mg), 7 (62 mg), and 8 (30 mg), respectively. Guavinoside A (1) was obtained as a yellow powder. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ [\alpha ]_{D}^{26} \; - 114^{ \circ } $$\end{document} (c = 0.1, MeOH); FAB-MS m/z 545 (M + H)+; HR-FAB-MS (found 545.8418, calcd for C26H25O13: 545.8413); UV \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \lambda_{\max}^{\rm MeOH} $$\end{document} nm (ε): 218 (26,800), 288 (16,200); IR \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \upsilon_{\max}^{\rm KBr} $$\end{document} cm−1: 3,390 (OH), 1,700 (ester). 1H- and 13C-NMR data, see Table 1.

minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \upsilon_{\max}^{\rm KBr} $$\end{document} cm−1: 3,390 (OH), 1,700 (ester). 1H- and 13C-NMR data, see Table 1. Guavinoside B (2) was obtained as a yellow powder; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ [\alpha ]_{D}^{26} \; - 83^{ \circ } $$\end{document} (c = 0.5, MeOH); FAB-MS m/z 571 (M − H)−; HR-FAB-MS (found 571.1474, calcd for C28H27O13: 571.1451); UV \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \lambda_{\max}^{\rm MeOH} $$\end{document} nm (ε): 218 (26,400), 283 (15,100); IR \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \upsilon_{\max}^{\rm KBr} $$\end{document} cm−1: 3,410 (OH), 1,710 (ester), 1,690 (C=O). 1H- and 13C-NMR data, see Table 1.

package{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \upsilon_{\max}^{\rm KBr} $$\end{document} cm−1: 3,410 (OH), 1,710 (ester), 1,690 (C=O). 1H- and 13C-NMR data, see Table 1. Guavinoside C (3) was obtained as a yellow powder; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ [\alpha ]_{D}^{26} \; - 73^{ \circ } $$\end{document} (c = 1.0, MeOH); FAB-MS m/z 585 (M − H)−; HR-FAB-MS (found 585.0868, calcd for C27H21O15: 585.0880); \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \lambda_{\max}^{\rm MeOH} $$\end{document} nm (ε): 211 (32,000), 265 (16,200), 355 (10,000); IR \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \upsilon_{\max}^{\rm KBr} $$\end{document} cm−1: 3,400 (OH), 1,710 (ester), 1,690 (C=O). 1H- and 13C-NMR data, see Table 2.Table 2 13C- and 1H-NMR spectral data of guavinoside C (3)

} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \upsilon_{\max}^{\rm KBr} $$\end{document} cm−1: 3,400 (OH), 1,710 (ester), 1,690 (C=O). 1H- and 13C-NMR data, see Table 2.Table 2 13C- and 1H-NMR spectral data of guavinoside C (3) No. δC δH 2 157.3 s 3 133.0 s 4 177.4 s 5 161.9 s 6 98.5 d 6.20 (1H, d, J = 1.5 Hz) 7 164.1 s 8 93.5 d 6.41 (1H, d, J = 1.5 Hz) 9 156.3 s 10 103.9 s 1′ 120.8 s 2′ 115.6 d 7.46 (1H, d, J = 2.2 Hz) 3′ 144.9 s 4′ 148.3 s 5′ 115.4 d 6.85 (1H, d, J = 8.8 Hz) 6′ 121.3 d 7.49 (1H, d, J = 8.8, 2.2 Hz) 5-OH 12.62 (1H, s) Arabinosyl 1″ 107.7 d 5.56 (1H, J = 1.4 Hz) 2″ 81.7 d 4.18 (1H, br s) 3″ 76.9 d 3.81 (1H, m) 4″ 81.9 d 3.74 (1H, m) 5″ 62.5 t 4.11 (1H, dd, J = 12.0, 3.3 Hz) 4.02 (1H, dd, J = 12.0, 5.8 Hz) 2″-OH 5.72 (1H, br s) 3″-OH 5.48 (1H, br s) Galloyl 1″′ 118.9 s 2″′, 6″′ 108.5 d 6.89 (2H, s) 3″′, 5″′ 145.3 s 4″′ 138.4 s C=O 165.4 s 1H-NMR (600 MHz) and 13C-NMR (150 MHz) spectra were measured in DMSO-d6 with TMS as internal standard

4 (1H, m) 5″ 62.5 t 4.11 (1H, dd, J = 12.0, 3.3 Hz) 4.02 (1H, dd, J = 12.0, 5.8 Hz) 2″-OH 5.72 (1H, br s) 3″-OH 5.48 (1H, br s) Galloyl 1″′ 118.9 s 2″′, 6″′ 108.5 d 6.89 (2H, s) 3″′, 5″′ 145.3 s 4″′ 138.4 s C=O 165.4 s 1H-NMR (600 MHz) and 13C-NMR (150 MHz) spectra were measured in DMSO-d6 with TMS as internal standard Acid hydrolysis of 1–3 Compound 1, 2, or 3 (3.0 mg, each) was treated with 0.5 ml of 2 M HCl for 2 h at 110°C in a sealed tube. The reaction mixture was diluted with 1 ml of H2O, and extracted with an equal volume of EtOAc, and the water layer was evaporated to dryness. The residue was dissolved in H2O (200 μl) and subjected to HPLC analysis [column: Asahi pak NH2P-50, (4.6-mm i.d. × 250 mm, Showa Denko, Tokyo, Japan); mobile phase: MeCN–H2O (75:25); flow rate: 1.0 ml/min.; detection: OR detector (Shodex OR-2, Showa Denko, Tokyo, Japan) and RI (Shodex RI-72, Showa Denko, Tokyo, Japan), column temperature: 40°C]. Retention time and optical rotation of these samples (1, 2, and 3) were found to be 8.5 min (positive), 8.5 min (positive), and 6.0 min (positive), respectively. Retention time of standard samples of (+)-d-glucose, (+)-l-arabinose, (−)-d-arabinose, and (+)-d-xylose were found to be 8.5 min (positive), 6.0 min (positive), 6.0 min (negative), and 6.3 min (positive), respectively.

and 3) were found to be 8.5 min (positive), 8.5 min (positive), and 6.0 min (positive), respectively. Retention time of standard samples of (+)-d-glucose, (+)-l-arabinose, (−)-d-arabinose, and (+)-d-xylose were found to be 8.5 min (positive), 6.0 min (positive), 6.0 min (negative), and 6.3 min (positive), respectively. This work was supported in part by a Grant-in-Aids from the Ministry of Health and Welfare of Japan (No. 19980070), by a Special Research Grant-in Aid for the development of characteristic education and High-Tech Research Centers from the Ministry of Education, Science, Sports and Culture of Japan to Nihon University, and by the Japan–China Medical Association, and “Academic Frontier” Project for Private Universities matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology) 2002–2007 and 2007–2010 of Japan. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Introduction One of the main causes of stroke is hypertension. In patients with long-term hypertension, functional modifications in the cerebrovascular system have also been found [1]. Therefore, it is important to avoid high blood pressure as a preventative measure. Hypertensive drug therapy is an easy recently developed approach that can reduce blood pressure by the administration of an antihypertensive agent. However, there is a risk of side effects and the treatment lasts for a long time. This explains the utility of blood pressure control by selection of appropriate food for which there are few side effects. The medical and pharmacological effects of mushrooms have been shown to vary greatly in terms of antihypertension, antitumor, and antidiabetic properties as well as those against various other diseases [2]. Some mushrooms have been used in Chinese medicine and folk medicine for a long time. Recently published reports have demonstrated that various kinds of mushrooms have beneficial effects on blood pressure [3–6]. Sparassis crispa (S. crispa) is an edible mushroom that has recently been cultivated in Japan. It contains a remarkably high concentration of beta-glucan, which exhibits antitumor [7] and antidiabetic [8] activities. However, there have not yet been any detailed studies of the effects of S. crispa on hypertension and stroke.

sis crispa (S. crispa) is an edible mushroom that has recently been cultivated in Japan. It contains a remarkably high concentration of beta-glucan, which exhibits antitumor [7] and antidiabetic [8] activities. However, there have not yet been any detailed studies of the effects of S. crispa on hypertension and stroke. Stroke-prone spontaneously hypertensive rats (SHRSP), a substrain of SHR created by selective breeding, have become a model for basic research on hypertension-associated cerebrovascular injury, because this model (SHRSP) presents with marked elevation of blood pressure and almost all of these animals develop stroke. Numerous nutritional and pathological studies using SHRSP have revealed that various foods are protective against hypertension and stroke [9, 10]. The present study investigated whether S. crispa exhibits preventive effects against hypertension and stroke and the mechanisms behind such effects by using SHRSP.

Stroke-prone spontaneously hypertensive rats (SHRSP), a substrain of SHR created by selective breeding, have become a model for basic research on hypertension-associated cerebrovascular injury, because this model (SHRSP) presents with marked elevation of blood pressure and almost all of these animals develop stroke. Numerous nutritional and pathological studies using SHRSP have revealed that various foods are protective against hypertension and stroke [9, 10]. The present study investigated whether S. crispa exhibits preventive effects against hypertension and stroke and the mechanisms behind such effects by using SHRSP. Methods Animals Study 1 Six-week-old male SPF (specific pathogen free) stroke-prone spontaneously hypertensive rats (SHRSP) were purchased from Japan SLC (Shizuoka, Japan). All rats were housed in a climate-controlled (temperature 22–24°C, humidity 40–60%) light-regulated room with a 12-h light and dark cycle. SHRSP were separated into 2 groups (n = 8–9): a control group (not treated) and an S. crispa group (administered 1.5% S. crispa, which was supplied by Katsuragi Industry Co., Ltd., Japan). All groups consumed an SP diet (Funahashi Co., Tokyo, Japan) and had free access to rat chow and water throughout the experiment. The survival rate of the rats was determined. Autopsies were conducted on the day of death and all rats were checked for stroke at the brain. All procedures were carried out in accordance with the guiding principles for the care and use of animals in the field of physiological sciences established by the Physiological Society of Japan and the guidelines for the care and use of animals set by Mukogawa University.

all rats were checked for stroke at the brain. All procedures were carried out in accordance with the guiding principles for the care and use of animals in the field of physiological sciences established by the Physiological Society of Japan and the guidelines for the care and use of animals set by Mukogawa University. Study 2 Male SHRSP at 6 weeks of age were divided into 2 groups: a control group and an S. crispa group administered 1.5% S. crispa (n = 8 each). All rats were housed as in study 1. We measured daily water and food intakes of the rats. Body weight and blood pressure were measured every 2 weeks. Blood flow of venous microcirculation in the tail was measured before and after 4 weeks of treatment. At the conclusion of the 4-week treatment period, 24-h urine samples were collected by placing the rats in metabolic cages. Food was withdrawn, but water was provided. Then, the animals were anesthetized with intraperitoneal injections of pentobarbital sodium (Nembutal, 100 mg/kg). The tissues were immediately harvested and cleaned for measurement of tissue weight, and brain was promptly frozen in liquid nitrogen for analysis. The samples were then stored at −80°C. Blood pressure measurements Systolic blood pressure (SBP) was measured by using a sphygmomanometer (UR-1000, Ueda Co., Chiba) using the tail-cuff method. An average of 3 measurements were taken as the initial mean SBP.

Study 2 Male SHRSP at 6 weeks of age were divided into 2 groups: a control group and an S. crispa group administered 1.5% S. crispa (n = 8 each). All rats were housed as in study 1. We measured daily water and food intakes of the rats. Body weight and blood pressure were measured every 2 weeks. Blood flow of venous microcirculation in the tail was measured before and after 4 weeks of treatment. At the conclusion of the 4-week treatment period, 24-h urine samples were collected by placing the rats in metabolic cages. Food was withdrawn, but water was provided. Then, the animals were anesthetized with intraperitoneal injections of pentobarbital sodium (Nembutal, 100 mg/kg). The tissues were immediately harvested and cleaned for measurement of tissue weight, and brain was promptly frozen in liquid nitrogen for analysis. The samples were then stored at −80°C. Blood pressure measurements Systolic blood pressure (SBP) was measured by using a sphygmomanometer (UR-1000, Ueda Co., Chiba) using the tail-cuff method. An average of 3 measurements were taken as the initial mean SBP. Blood flow measurements The blood flow through the venous microcirculation in the tail was monitored by using a laser Doppler blood flow meter of the contact type (FLO-C1, Muromachi, Tokyo). Briefly, all rats were pre-warmed at 36°C and blood flow was measured while keeping the animal at 36°C on a holder. The rate of change in blood flow was calculated as follows: Rate of change = (blood flow before treatment/blood flow after treatment) × 100.

lood flow meter of the contact type (FLO-C1, Muromachi, Tokyo). Briefly, all rats were pre-warmed at 36°C and blood flow was measured while keeping the animal at 36°C on a holder. The rate of change in blood flow was calculated as follows: Rate of change = (blood flow before treatment/blood flow after treatment) × 100. Urinary and cerebral nitrate/nitrite assay Samples of cerebral cortex were obtained by homogenization with twofold methanol followed by centrifugation at 3,000 rpm for 5 min; urinary samples were also centrifuged under the same conditions, and supernatants were then analyzed. Nitrate and nitrite (NOx) concentrations in the urine and cerebral cortex were measured by the Griess method using an automated NO detector high-performance liquid chromatography system (ENO-20, Eicom, Kyoto) as described previously [11]. Primary antibodies used Immunoblotting was performed with the following commercially available antibodies: anti-rabbit eNOS from Affinity BioReagents Inc. (golden); anti-rabbit phospho-eNOS (Ser1177), anti-rabbit Akt, anti-rabbit phospho-Akt (Ser473, Thr308), anti-rabbit mTOR, anti-rabbit phospho-mTOR (Ser2448), and anti-rabbit nNOS from Cell Signaling Technology (Beverly, MA). Anti-mouse β-actin and anti-rabbit iNOS were obtained from Sigma (St. Louis, MO, USA).

ts Inc. (golden); anti-rabbit phospho-eNOS (Ser1177), anti-rabbit Akt, anti-rabbit phospho-Akt (Ser473, Thr308), anti-rabbit mTOR, anti-rabbit phospho-mTOR (Ser2448), and anti-rabbit nNOS from Cell Signaling Technology (Beverly, MA). Anti-mouse β-actin and anti-rabbit iNOS were obtained from Sigma (St. Louis, MO, USA). Western blot analysis The cerebral cortex was homogenized with ice-cold homogenized buffer containing 50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 1% Nonidet-P40, 0.25% Na deoxycholate, 0.1% SDS, 1 mM EDTA, 50 mM NaF, 2 mM Na3VO4, 30 mM Na pyrophosphate, 2 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine, 0.02 g/mL trypsin inhibitor, 0.02 g/mL leupeptin, and 0.02 g/mL aprotinin. After incubation for 2 h, lysates were centrifuged at 15,000 rpm for 20 min and supernatants were isolated. Proteins were extracted by boiling the tissues in 0.5 mmol/l Tris/HCl, pH 6.8, glycerol, 10% SDS, 0.1% bromophenol blue, and 2-mercaptoethanol. The proteins (25 µg) were electrophoresed by using 7.5–12.5% SDS-PAGE gel at 100 V for 2 h. After fractionating, the proteins were transferred onto a poly(vinylidene difluoride) (PVDF) membrane (Amersham Inc. Buckinghamshire) at 100 mA for 2 h. The membrane was blocked in Blocking One (Nacalai Tesque, Kyoto) for 20 min. After appropriate blocking, the blot was incubated with anti-iNOS (1:5,000), anti-nNOS, anti-eNOS, anti-phospho-eNOS (Ser1177), anti-Akt, anti-phospho-Akt (Ser473 and Thr308), anti-mTOR, and anti-phosphor mTOR (1:1,000) in antibody solution 1 (Toyobo, Osaka) overnight. It was then washed with Tris-buffered saline (TTBS) and finally incubated for 1 h with a 1:5,000 dilution of anti-rabbit and mouse IgG–horseradish peroxidase. Detection was achieved by using an ECL kit (Amersham Inc. Buckinghamshire). β-actin was used as an internal control. The density of the bands was measured by using NIH Image.

ashed with Tris-buffered saline (TTBS) and finally incubated for 1 h with a 1:5,000 dilution of anti-rabbit and mouse IgG–horseradish peroxidase. Detection was achieved by using an ECL kit (Amersham Inc. Buckinghamshire). β-actin was used as an internal control. The density of the bands was measured by using NIH Image. Statistical analysis Data are expressed as the mean ± SEM. Cumulative survival was analyzed for differences according to Kaplan–Meier followed by Wilcoxon test. Statistical analysis of the data was performed by Student’s t test to determine the significance of differences. A p value of less than 0.05 was considered significant. Results Intakes of water and food, and volume of urine Water and food intakes over the 4-week period of the study did not differ between the control group and the S. crispa group. In addition, no difference was observed in terms of the volume of urine between the two groups (Table 1).Table 1 Water, food intake and urinary extract volume at 4 weeks after administration Water intake (mL) Food intake (g) Urinary volume (mL) Control 21.50 ± 4.54 18.60 ± 0.19 19.08 ± 3.67 S. crispa 25.67 ± 4.25 18.47 ± 0.53 19.00 ± 1.98 Data are means ± SEM n = 6

Results Intakes of water and food, and volume of urine Water and food intakes over the 4-week period of the study did not differ between the control group and the S. crispa group. In addition, no difference was observed in terms of the volume of urine between the two groups (Table 1).Table 1 Water, food intake and urinary extract volume at 4 weeks after administration Water intake (mL) Food intake (g) Urinary volume (mL) Control 21.50 ± 4.54 18.60 ± 0.19 19.08 ± 3.67 S. crispa 25.67 ± 4.25 18.47 ± 0.53 19.00 ± 1.98 Data are means ± SEM n = 6 Survival rate, blood pressure, and body weight Kaplan–Meier survival curves revealed that rats in the S. crispa group had a significantly higher survival rate than rats in the control group (Fig. 1). Moreover, SHRSP rats treated with S. crispa showed significant inhibition of the rise in blood pressure after 2 and 4 weeks of oral administration (Fig. 2a). However, body weight did not differ between the SHRSP group and the S. crispa group (Fig. 2b).Fig. 1 Kaplan–Meier survival curves showing that SHRSP administered S. crispa had a significantly higher survival rate than the control group (Wilcoxon test) Fig. 2 Effect of S. crispa on systolic blood pressure and body weight: control group (n = 8) and S. crispa group (n = 8). All values are the means ± SEM; *p < 0.01 versus control group (Student’s t test)

Survival rate, blood pressure, and body weight Kaplan–Meier survival curves revealed that rats in the S. crispa group had a significantly higher survival rate than rats in the control group (Fig. 1). Moreover, SHRSP rats treated with S. crispa showed significant inhibition of the rise in blood pressure after 2 and 4 weeks of oral administration (Fig. 2a). However, body weight did not differ between the SHRSP group and the S. crispa group (Fig. 2b).Fig. 1 Kaplan–Meier survival curves showing that SHRSP administered S. crispa had a significantly higher survival rate than the control group (Wilcoxon test) Fig. 2 Effect of S. crispa on systolic blood pressure and body weight: control group (n = 8) and S. crispa group (n = 8). All values are the means ± SEM; *p < 0.01 versus control group (Student’s t test) Change in blood flow and NO production The change in the amount of blood flow in venous microcirculation in the tail after treatment is shown in Fig. 3a. The administration of S. crispa significantly increased blood flow compared with that in the control group. In addition, 24-h urinary NO metabolite (nitrate and nitrite) excretion and concentration in the cerebral cortex in the S. crispa group was 2–3-fold higher than that in the control group (Fig. 3b, c). None of these changes reached significance, but these experimental data correspond to the blood pressure and blood flow data.Fig. 3 a Measurement of blood flow in the tail of rats in the control group (n = 8) and the S. crispa group (n = 8) before and after administration. The rate of change in blood flow is expressed as a percentage relative to the value before treatment. b Urinary nitrate/nitrite (NOx) excretion and c cerebral nitrate/nitrite (NOx) concentration at 4 weeks after administration. Control group (n = 4) and S. crispa group (n = 4). Data are means ± SEM; *p < 0.01 versus control group (Student’s t test)

ow is expressed as a percentage relative to the value before treatment. b Urinary nitrate/nitrite (NOx) excretion and c cerebral nitrate/nitrite (NOx) concentration at 4 weeks after administration. Control group (n = 4) and S. crispa group (n = 4). Data are means ± SEM; *p < 0.01 versus control group (Student’s t test) Measurement of tissue weight Table 2 shows the ratio of tissue weight to body weight. There were no significant differences in this ratio between the control group and the S. crispa group at 4 weeks after administration.Table 2 Effects of S. crispa on tissue weight (g/100 g body weight) Tissues Control (n = 6) S. crispa (n = 6) Brain 0.60 ± 0.04 0.57 ± 0.02 Heart 0.47 ± 0.04 0.44 ± 0.03 Spleen 0.22 ± 0.01 0.22 ± 0.01 Liver 3.39 ± 0.12 3.18 ± 0.10 Kidney 0.85 ± 0.03 0.82 ± 0.03 Adrenal gland 0.02 ± 0.00 0.02 ± 0.00 Testis 1.20 ± 0.07 1.14 ± 0.04 Values are means ± SEM Various types of NOS protein expression To investigate whether expressions of synthases for NO, which are vasodilators, showed any differences between the control group and the S. crispa group, we used antibodies to measure iNOS, nNOS, and eNOS. We determined that SHRSP receiving S. crispa did not show any changes in these protein levels (Fig. 4).Fig. 4 Western blot analysis of the protein expression of iNOS (a), nNOS (b), and eNOS (c) in the cerebral cortex at 4 weeks after administration. β-actin served as protein quantity control in each experiment. Control group (n = 4) and S. crispa group (n = 4). Data are means ± SEM (Student’s t test)

ein levels (Fig. 4).Fig. 4 Western blot analysis of the protein expression of iNOS (a), nNOS (b), and eNOS (c) in the cerebral cortex at 4 weeks after administration. β-actin served as protein quantity control in each experiment. Control group (n = 4) and S. crispa group (n = 4). Data are means ± SEM (Student’s t test) Effects of phosphorylation of eNOS via PI3 K/Akt pathway The level of phosphorylation of eNOS at Ser1177 in the cerebral cortex of the S. crispa group was significantly higher than that in the control group, as determined by western blotting with phosphospecific antibody (Fig. 5d). Because activation of the serine/threonine kinase Akt phosphorylates and activates eNOS, which in turn induces NO production and vasorelaxation, the phosphorylation of Akt was also studied. The results of western blot analysis demonstrated that cerebral cortex expression of Akt phosphorylated at Ser473 was significantly induced in the S. crispa group compared with that in the control group (Fig. 5b). Immunoblot analysis with antibodies against total Akt showed equal protein levels in all groups, thus demonstrating that the induced amount of phosphor-Akt (Ser473) was not due to increased expression of Akt (Fig. 5a). In addition, there was no difference in Thr308 between the two groups (Fig. 5c).Fig. 5 Analysis of Akt/eNOS signaling by western blotting in cerebral cortex in SHRSP (control and S. crispa treatment). a The total protein expression of Akt. Densitometric analysis of the phosphorylation level on Akt Ser473 (b), Akt Thr308 (c), eNOS Ser1177 (d), and mTOR Ser2448 (e). Control group (n = 4) and S. crispa group (n = 4). Data are means ± SEM; *p < 0.01 versus control group (Student’s t test)

ntrol and S. crispa treatment). a The total protein expression of Akt. Densitometric analysis of the phosphorylation level on Akt Ser473 (b), Akt Thr308 (c), eNOS Ser1177 (d), and mTOR Ser2448 (e). Control group (n = 4) and S. crispa group (n = 4). Data are means ± SEM; *p < 0.01 versus control group (Student’s t test) Effects of phosphorylation of mTOR Although mTOR is located downstream of Akt, the ratio of phosphor-mTOR (Ser2448) to total mTOR did not differ between the control group and the S. crispa group (Fig. 5e). Discussion The major findings of the present study are as follows: (1) The administration of Sparassis crispa (S. crispa) to SHRSP from a young age improves survival and reduces the elevation of blood pressure. (2) The mechanism for this action was improvement of endothelial dysfunction via increases in NO production with activation of the Akt/eNOS signaling pathway on vessel cerebral cortex.

administration of Sparassis crispa (S. crispa) to SHRSP from a young age improves survival and reduces the elevation of blood pressure. (2) The mechanism for this action was improvement of endothelial dysfunction via increases in NO production with activation of the Akt/eNOS signaling pathway on vessel cerebral cortex. The SHRSP is a model of essential hypertension in humans, and was developed from SHR demonstrating abnormalities of the renin-angiotensin system, catecholamines, vasopressin, and vasoactive intestinal peptide [12]. It is well known that blood pressure in SHRSP is already significantly higher than that in WKY, a normal blood pressure rat, at a young age and that almost all SHRSP develop stroke. Moreover, a significant decrease in regional cerebral blood flow in SHRSP frontal cortex at 6 weeks of age compared with that in WKY was observed [13]. The data presented in our survival studies clearly showed that S. crispa protected SHRSP against stroke death. Furthermore, the administration of S. crispa was associated with an inhibition of elevated blood pressure and a restoration of impaired peripheral blood flow, which indicates that S. crispa may improve vascular function including that in brain blood vessels.

y showed that S. crispa protected SHRSP against stroke death. Furthermore, the administration of S. crispa was associated with an inhibition of elevated blood pressure and a restoration of impaired peripheral blood flow, which indicates that S. crispa may improve vascular function including that in brain blood vessels. Nitric oxide (NO) plays an important role in the modulation of vascular tone and structure. In animal models of hypertension and patients with hypertension, nitric oxide (NO) production is abnormal, leading to hypertensive vascular lesion formation [14]. NO in the brain inhibits sympathetic nerve activity [15, 16] or directly affects vasodilatation via a cGMP mechanism, thereby reducing blood pressure [17–19]. The present study showed that S. crispa induced a higher level of NOx in the cerebral cortex compared with that in the control group.

ular lesion formation [14]. NO in the brain inhibits sympathetic nerve activity [15, 16] or directly affects vasodilatation via a cGMP mechanism, thereby reducing blood pressure [17–19]. The present study showed that S. crispa induced a higher level of NOx in the cerebral cortex compared with that in the control group. It is well known that NO is generated by nitric oxide synthases (NOSs), which have three distinct isoforms. NO from endothelial NOS (eNOS) when present in vessel endothelial cells is known to regulate vascular tone, while neuronal NOS (nNOS) is involved in neural signaling, and inducible NOS (iNOS) modulates immune function [20–22]. Our study suggests that none of the types of NOS showed any differences in expression between the control group and the S. crispa group at 4 weeks after administration. In particular, of all NOS types, NO generation from eNOS would initially limit stroke and brain injury by inducing vasodilation [23]. We previously reported that eNOS activity plays an important role in vasodilation, rather than eNOS expression, at a younger age in the SHRSP strain. The enzymatic activity of eNOS is regulated by phosphorylation at Ser1177, and one of the kinases that mediates phosphorylation of eNOS is Akt, which has an activation process involving two phosphorylation sites, Ser473 and Thr308, by phosphoinositide-dependent protein kinases [24]. Our previous study also demonstrated that phosphorylation of eNOS and Akt was decreased in the cerebral cortex of SHRSP compared with that in WKY. Interestingly, S. crispa restored eNOS phosphorylation, which was markedly impaired in the cerebral cortex of SHRSP, and this improvement was associated with the recovery of Akt, especially at the site of Ser473 phosphorylation. Enhanced Akt/eNOS signaling upon the administration of S. crispa was associated with an inhibition of elevated blood pressure, restoration of impaired blood flow, and increased NOx in the cerebral cortex.

SP, and this improvement was associated with the recovery of Akt, especially at the site of Ser473 phosphorylation. Enhanced Akt/eNOS signaling upon the administration of S. crispa was associated with an inhibition of elevated blood pressure, restoration of impaired blood flow, and increased NOx in the cerebral cortex. In contrast to eNOS, the mammalian target of rapamycin (mTOR), which is also downstream of Akt, is an important molecule in the regulation of protein synthesis and cell growth, and mTOR activity also requires phosphorylation at Ser2448 by Akt. Many neurodegenerative diseases are characterized by neuronal death via apoptosis, and it is possible that modulation of mTOR activity may offer some protection against these effects [24]. In particular, diseases involving oxygen and nutrient deprivation, such as stroke, would be expected to be associated with inhibition of the mTOR pathway [25]. Moreover, previous studies have demonstrated that focal ischemic brain injury significantly decreases the levels of phosphorylated Akt and mTOR, and suppresses protein synthesis [26]. We expected activation of mTOR with the increase in phosphor-Akt due to the administration of S. crispa; however, no such effect was observed. In brief, upon the administration of S. crispa, there is only improvement of Akt/eNOS signaling without an effect on mTOR in SHRSP brain.

and suppresses protein synthesis [26]. We expected activation of mTOR with the increase in phosphor-Akt due to the administration of S. crispa; however, no such effect was observed. In brief, upon the administration of S. crispa, there is only improvement of Akt/eNOS signaling without an effect on mTOR in SHRSP brain. Many mushrooms have been reported to exhibit antihypertension effects. It has been reported that mushroom species such as Lentinula edodes, Ganoderma lucidum, Grifola frondosa, Pleurotuscornucopiae, and Pleurotus nebrodensis have antihypertension activities; moreover, their mechanisms of action have been suggested to involve the decrease blood pressure by improvement in blood levels of triglyceride and cholesterol as well as kidney function and inhibition of angiotensin-converting enzymes [3, 4, 27, 28]. However, this is the first report to indicate an antihypertensive mechanism involving an improvement in endothelial signaling due to mushrooms. In conclusion, long-term hypertension has been implicated in functional modifications in the cerebrovascular system and causes of stroke. Therefore, it is important to avoid high blood pressure as a preventative measure. S. crispa can delay stroke through restoration of cerebrovascular endothelial dysfunction via enhancement of the Akt/eNOS/NO system in the cerebral cortex of SHRSP. This suggests that S. crispa may have utility as a functional food for the prevention of hypertension and stroke; moreover, novel components from this mushroom may be discovered in the future.

storation of cerebrovascular endothelial dysfunction via enhancement of the Akt/eNOS/NO system in the cerebral cortex of SHRSP. This suggests that S. crispa may have utility as a functional food for the prevention of hypertension and stroke; moreover, novel components from this mushroom may be discovered in the future. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Introduction Etiology of dengue fever Dengue fever is caused by the arthropode-borne flavivirus named dengue virus (DENV), transmitted by the Aedes aegypti mosquito [1]. To date, four antigenically related but distinct virus serotypes (DENV-1, 2, 3 and 4) have been identified as belonging to the genus Flavivirus in the Flaviviridae family [2–4]. Infection with one DENV serotype produces only specific antibody against that serotype. When antibody from the first infection is neutralized, secondary infections by other serotypes can cause more serious infection [5]. Although DENV-2 is known to be more lethal than other serotypes [6], some studies have revealed that primary infection with DENV-1 or DENV-3 always results in more dangerous disease than infection with DENV-2 or DENV-4 [3, 7]. In recent years, the current dengue epidemic has become a focus of international public health awareness. Unlike malaria, which is more prevalent in remote areas, cases of dengue are distributed mostly in urban and sub-urban areas [8, 9]. This has made the epidemic more lethal as an outbreak is difficult to control due to highly populated areas in cities.

pidemic has become a focus of international public health awareness. Unlike malaria, which is more prevalent in remote areas, cases of dengue are distributed mostly in urban and sub-urban areas [8, 9]. This has made the epidemic more lethal as an outbreak is difficult to control due to highly populated areas in cities. Types of DENV infection include mild fever known as dengue fever (DF), which constitutes about 95 % of cases, and a more serious type known as dengue hemorrhagic fever and/or dengue shock syndrome (DHF/DSS, 5 % of cases) [10, 11]. Recovery from first type of infection provides lifelong immunity; however, it affords only half protection from subsequent viral infection that ultimately results in the risk of DHF. Most dengue infections are characterized by non-specific symptoms including frontal headache, retro-orbital pain, body aches, nausea and vomiting, joint pains, weakness and rash [12, 13].

elong immunity; however, it affords only half protection from subsequent viral infection that ultimately results in the risk of DHF. Most dengue infections are characterized by non-specific symptoms including frontal headache, retro-orbital pain, body aches, nausea and vomiting, joint pains, weakness and rash [12, 13]. Epidemiology of dengue fever International travel, increasing human population [14, 15] and urbanisation create suitable conditions for the mosquito vector Ae. aegyti, and thus spread the virus to new areas, causing major epidemics [13, 16, 17]. Dengue epidemics are endemic in over 100 countries in Africa, America, Eastern Mediterranean, Southeast Asia and Western Pacific, with Southeast Asia and the Western Pacific being the regions most affected (Fig. 1) [13, 18–20]. The first case of DHF was discovered in the 1950s in Thailand and the Philippines [4], where the first two DENV serotypes were identified, followed by the third and fourth serotypes in 1954 [14]. Since then, DHF has recorded major cases resulting in hospitalization and death among children in regions stretching from Asia to Africa and the Pacific [4]. Approximately 2.5 billion people, or half the world’s population [14], are now at risk of Dengue, and 50 million infections globally occur annually [4]. Over 100 million cases of DF and at least 500,000 cases of DHF [21] and approximately 18,000 deaths may occur each year [22]. Despite its lethal consequences, the staggering numbers of those affected are increased by the fact that, at present, there is no specific antiviral treatment or vaccine for DF [3]. Early diagnosis and strict hospitalization often save the life of patients with DHF [3, 4, 10]. Efforts to combat the vector have been undertaken by regulatory bodies in an attempt to tackle this problem by awareness campaigns and vector control [16]. Others strategies include the use of plants with bioactive substances that have toxic properties to the vector or insecticidal properties [20]. Clearly, development of antiviral drugs and vaccines is needed in order to support these programs. Moreover, a safe, low-cost, and effective vaccine to control DENV woudl be needed, especially in the most affected countries, which are poor [2, 16]. Therefore, the search of highly selective but non-toxic antiviral compounds is urgently needed in view of the spread of dengue disease throughout the world [23].Fig. 1 Green Countries or areas at risk of dengue, 2012.

ne to control DENV woudl be needed, especially in the most affected countries, which are poor [2, 16]. Therefore, the search of highly selective but non-toxic antiviral compounds is urgently needed in view of the spread of dengue disease throughout the world [23].Fig. 1 Green Countries or areas at risk of dengue, 2012. The contour lines of the January and July isotherms indicate the potential geographical limits of the northern and southern hemispheres for year-round survival of Aedes aegypti, the principal mosquito vector of dengue viruses. This copyrighted map is reproduced with acknowledgment to the World Health Organization (WHO) Global distribution of dengue fever Guangdong province in China has become a major area with reported cases of dengue [24]. From 2000 to 2005, a total of 2,496 cases of dengue were recorded. The epidemic peaked in 2002. In Northern Thailand there were 13,915, 11,092, 6,147, 6,992 and 6,914 DF cases reported during the period 2002–2006 [25]. Outbreaks of DF and DHF have been reported in India over the past four decades [26]. From 2001 to 2002, Delhi recorded a decline in cases of DF/DHF, with a total of 1,380 cases, but deaths decreasing from 53 cases (2001) to 35 cases (2002). However, outbreaks of DF cases rose sharply in 2003, with a total of 12,754 cases and 215 deaths.

d DHF have been reported in India over the past four decades [26]. From 2001 to 2002, Delhi recorded a decline in cases of DF/DHF, with a total of 1,380 cases, but deaths decreasing from 53 cases (2001) to 35 cases (2002). However, outbreaks of DF cases rose sharply in 2003, with a total of 12,754 cases and 215 deaths. Dengue fever in Malaysia In Malaysia, with a population of 27.7 million and a population density of 84 per km2 [27], outbreaks of dengue cases are endemic, with increasing cases of dengue over the past two decades. The first case was documented in 1902 [16, 28, 29]. During the period 1973–1982, 12,077 dengue cases were reported, with a fatality rate of 3.38 %. The number of cases rose in following decade of 1983–1992 with 26,361 cases; however, the fatality rate was down to 0.55 % [28]. In 2004 and 2005, dengue was reported with 13,558 and 15,862 incidence rate, respectively, per 100,000 population. With an increase of 16.99 % of cases, a total of 107 deaths were recorded in 2005 compared to 102 cases in 2004 [29]. According to Health Facts 2006 (Ministry of Health Malaysia), the incidence rates of DF and DHF were 64.37 and 4.10 per 100,000 population, respectively, with mortality rate of 0.01 (DF) and 0.25 (DHF) [30]. In a press statement, the Director General of Health Malaysia, reported a total of 545 cases and four deaths in 5 weeks in 2012 as the highest increase of dengue cases and deaths, with an increase of 57 cases (12 %) compared to 488 cases with two deaths the previous week [31]. In the period 2009–2011, the number of dengue cases decreased to 21,602 cases with the peak appearing in 2010 (Fig. 2) [32–36].Fig. 2 Reported dengue cases in the years 2009–2012 in Malaysia [32–36]. *Data up to 24 March 2012

h an increase of 57 cases (12 %) compared to 488 cases with two deaths the previous week [31]. In the period 2009–2011, the number of dengue cases decreased to 21,602 cases with the peak appearing in 2010 (Fig. 2) [32–36].Fig. 2 Reported dengue cases in the years 2009–2012 in Malaysia [32–36]. *Data up to 24 March 2012 Since early human civilization, plants have been a source of traditional medicine, and demands for herbal and natural product have recently increased. About 70–95 % people worldwide now rely on traditional herbs as the primary treatment for various diseases [37]. It is estimated that about 25 % of modern drugs, including antiviral agent, originate from natural products [38] with over 60 % of anti-cancer compounds and 75 % of infectious disease drugs being derived from natural ingredients, which are more acceptable, less toxic and less expensive than synthetic drugs [39, 40]. Several studies have reported potential antiviral agents from plants in the form of crude extracts, essential oils or purified compounds [41, 42]. Recent studies have reported the potential of some flavonoid compounds as antivirals against DENV-2 [40, 43].

e, less toxic and less expensive than synthetic drugs [39, 40]. Several studies have reported potential antiviral agents from plants in the form of crude extracts, essential oils or purified compounds [41, 42]. Recent studies have reported the potential of some flavonoid compounds as antivirals against DENV-2 [40, 43]. Pathophysiology of dengue fever Dengue infection is caused by bites of the female Ae. aegypti mosquito carrying Flavivirus. After a person is bitten, the virus incubation period varies between 3 and 14 days [3, 18], after which the person may experience early symptoms such as fever, headache, rash, nausea, and joint and musculoskeletal pain [3, 13]. This classic DF records temperatures between 39 and 40 °C and usually lasts 5–7 days [6]. During this period, the virus may get into the peripheral bloodstream and, if left untreated, can damage blood vessels and lymph nodes resulting in DHF with symptoms such as bleeding from the nose, gums or under the skin [18]. DHF patients also have difficulty in breathing and severe development can lead to DSS. DSS can result in death if proper treatment is not provided.

ral bloodstream and, if left untreated, can damage blood vessels and lymph nodes resulting in DHF with symptoms such as bleeding from the nose, gums or under the skin [18]. DHF patients also have difficulty in breathing and severe development can lead to DSS. DSS can result in death if proper treatment is not provided. Aedes mosquitoes are small and black with white markings on the body and legs. Female mosquitoes need blood from biting humans or animals to produce live eggs. It takes 2–3 days for egg development. The principal vector of dengue (Ae. aegypti) has adapted well to the urban environment [14, 17] and always breeds in stagnant containers. Eggs need moist conditions, and mature in 24–72 h [44]. Mosquito bites are the only route of DENV spread. The transmission of DENV is often from human to human through domestic mosquitoes [6]. An outbreak starts after a mosquito sucks the blood of a patient with DF/DHF (Fig. 3) [44]. After being transmitted to a new human host by infected mosquitoes, the virus replicates in the lymph nodes and spreads through the lymph and blood to other tissues [6]. To identify a potential antiviral treatment for DENV, it is necessary to understand the life cycle of the virus. The dengue virion is a small particle with a lipoprotein envelope and an icosahedral nucleocapsid containing a positive single-stranded RNA genome [6, 12, 23]. Virus infection of the cell begins with binding to the host cell surface. It enters the cell by receptor-mediated endocytosis [15], with the cell membrane forming a sac-like structure known as an endosome. In the endosome, the virus penetrates deep into the cell until the endosome membrane acquires a negative charge, which allows it to fuse with the endosomal membrane to open a port for release of genetic material. At this point, the virus in the cell fluid starts to reproduce. Changes in the acidity of the secretory pathway during this viral journey travel play an important role in its maturation (Fig. 4).Fig. 3 Dengue virus transmission cycle

it to fuse with the endosomal membrane to open a port for release of genetic material. At this point, the virus in the cell fluid starts to reproduce. Changes in the acidity of the secretory pathway during this viral journey travel play an important role in its maturation (Fig. 4).Fig. 3 Dengue virus transmission cycle Fig. 4 Dengue virus infection cycle in cells

it to fuse with the endosomal membrane to open a port for release of genetic material. At this point, the virus in the cell fluid starts to reproduce. Changes in the acidity of the secretory pathway during this viral journey travel play an important role in its maturation (Fig. 4).Fig. 3 Dengue virus transmission cycle Fig. 4 Dengue virus infection cycle in cells Possible mechanisms and pathways in the treatment of dengue There are currently no specific treatments for dengue fever [22]. Only standard treatment for management of fever is given, i.e., nursing care, fluid balance, electrolytes and blood clotting parameters [18]. Patients with dengue fever will be treated symptomatically, for example, sponging, acetaminophen [9], bed rest and oral rehydration therapy, and if signs of dehydration or bleeding occur the patients are usually hospitalized [6]. Aspirin should be avoided because it may cause bleeding [9]. Platelet count and Hematocrit should be measured daily from the suspected day of illness until 1–2 days after defervescence [9]. Current prevention of dengue by potential dengue vaccine and vector control is highly cost effective [22, 45]. In addition, mosquito control programs are the most important preventive method [6]. However, these are difficult to implement and maintain [39]. Development of a vaccine for dengue is difficult since there are four closely related, but antigenically distinct, serotypes of the virus that can cause disease [6, 46]. Infection by one serotype does not ensure protection of the patient from infection by the other three serotypes [15]. Therefore, if vaccine were produced for only one or two serotypes, the other serotypes would increase the risk of more serious illness [47]. Ribavirin has shown significant in vivo activity against RNA viruses; however, it exhibited only very weak activity against Flaviviruses [21]. A possible strategy in the treatment of dengue is to use chimeric tetravalent vaccines that show high neutralizing antibody against all dengue serotypes [9, 15]. Studies on the development of tetravalent vaccines are ongoing in Thailand and these should be available in the near future [6]. In addition, recombinant vaccines against capsid, premembrane and envelope genes of DENV-1, -2 and -3 inserted into a copy of a DNA infectious clone of DENV-2 are being developed and are currently undergoing clinical trials [48].

ent vaccines are ongoing in Thailand and these should be available in the near future [6]. In addition, recombinant vaccines against capsid, premembrane and envelope genes of DENV-1, -2 and -3 inserted into a copy of a DNA infectious clone of DENV-2 are being developed and are currently undergoing clinical trials [48]. Plants traditionally used to treat dengue According to a World Health Organization (WHO) fact sheet dated December 2008, 80 % of the population in some Asian and African countries depends on traditional medicine as their primary health care due to economic and geographical constraints [49]. Natural products have become the main source of test material in the development of antiviral drugs based on traditional medical practices [50]. Traditional medicines are based on knowledge, experience and practices based on indigenous cultural beliefs and knowledge, and are used to maintain health, prevent, treat and diagnose physical or mental illness [49]. Traditional medicinal plants have been reported to have antiviral activity [49, 51, 52] and some have been used to treat viral infections in animals and humans.

and practices based on indigenous cultural beliefs and knowledge, and are used to maintain health, prevent, treat and diagnose physical or mental illness [49]. Traditional medicinal plants have been reported to have antiviral activity [49, 51, 52] and some have been used to treat viral infections in animals and humans. To date, 31 different species have been found to have the potential to treat dengue; some of these have not yet been investigated scientifically (as indicated in Table 1). In the Philippines, Euphorbia hirta, known locally as “tawa–tawa”, is used in folk medicine to cure dengue fever by people in rural areas [53]. Practitioners of traditional medicines believe that decoction of tawa–tawa leaves can reverse viral infection and prevent the fever from moving into critical stages, although there are no scientific studies proving its effectiveness [54]. Sometimes, tawa–tawa is prepared together with papaya leaves since papaya leaf extract has a function as an antibiotic to cure fever. While papaya leaf extract kills the bacterial infection that caused the fever, tawa–tawa extract prevents bleeding. In addition, unpublished research has found that Psidium guava leaves are a good way to increase platelets, thus helping to avoid bleeding [55]. A water decoction of guava leaves contains quercetin, which acts to inhibit the formation of enzyme mRNA in the virus [56].Table 1 Plants with reported anti-dengue activity, according to family

lished research has found that Psidium guava leaves are a good way to increase platelets, thus helping to avoid bleeding [55]. A water decoction of guava leaves contains quercetin, which acts to inhibit the formation of enzyme mRNA in the virus [56].Table 1 Plants with reported anti-dengue activity, according to family Family Species Local/common name Part(s) used Compound isolated References Acanthaceae Andrographis paniculata Hempedu Bumi (Malaysia) Leaves [7] Amaranthaceae Alternanthera philoxeroides Alligator weed Whole plants [57] Caricaceae Carica papaya Papaya Leaves [9] Chordariaceae Cladosiphon okamuranus Brown seaweed Whole plants Fucoidan (3) [59] Cucurbitacea Momordica charanthia Bitter Melon, Peria (Malaysia) Fruit [7] Elaeagnaceae Hippophae rhamnoides Sea Buckthorn Leaves [11] Euphorbiaceae Cladogynos orientalis Chettaphangkhee (Thailand) Whole plants [2] Euphorbia hirta a Gatas–gatas Leaves [53, 54] Fabaceae Leucaena leucocephala White Leadtree, Petai Belalang (Malaysia) Seeds Galactomanan (7) [12, 62] Mimosa scabrella – Seeds Galactomanan (7) [12] Tephrosia madrensis – Leaves and flowers Glabranine (8), 7-O-methylglabranine (9) [10] Tephrosia crassifolia – Leaves and flowers [10]

Euphorbia hirta a Gatas–gatas Leaves [53, 54] Fabaceae Leucaena leucocephala White Leadtree, Petai Belalang (Malaysia) Seeds Galactomanan (7) [12, 62] Mimosa scabrella – Seeds Galactomanan (7) [12] Tephrosia madrensis – Leaves and flowers Glabranine (8), 7-O-methylglabranine (9) [10] Tephrosia crassifolia – Leaves and flowers [10] Tephrosia viridiflora – Leaves and flowers [10] Fagaceae Quercus lusitanica Gall Oak Seeds [39] Flagellariaceae Flagellaria indica Whip Vine Whole plants [2] Halymeniaceae Cryptonemia crenulata Red seaweed Whole plants Galactan (4) [60] Labiatae Ocimum sanctum Holy Basil, Tulsi (India) Leaves [7, 64] Meliaceae Azidarachta indica Neem Leaves [8] Myrtaceae Psidium guajava a Guava, Jambu Batu (Malaysia) Leaves [55, 56, 66] Piperaceae Piper retrofractum Dīplī (Thailand), Long Pepper Whole plants [2, 65] Phyllophoraceae Gymnogongrus torulosus Red seaweed Whole plants Galactan (4) [61] Gymnogongrus griffithsiae Red seaweed Whole plants Kappa carrageenan (5) [60] Poaceae Cymbopogon citratus Lemon Grass Whole plants [7] Rhizophoraceae Rhizophora apiculata Bakau (Malaysia) Whole plants [2] Rubiaceae Uncaria tomentosa Cat’s Claw Stem barks [67, 68] Saururaceae Houttuynia cordata Pak Kan Thong (Thailand), Chameleon Plant Whole plants, aerial stem and leaves Hyperoside (6) [2, 5] Solieriaceae Meristiella gelidium – Whole plants Kappa carrageenan (5) [63] Verbenaceae Lippia alba Pronto Alivio (Colombia), Bushy Matgrass Whole plants [23, 50]

Claw Stem barks [67, 68] Saururaceae Houttuynia cordata Pak Kan Thong (Thailand), Chameleon Plant Whole plants, aerial stem and leaves Hyperoside (6) [2, 5] Solieriaceae Meristiella gelidium – Whole plants Kappa carrageenan (5) [63] Verbenaceae Lippia alba Pronto Alivio (Colombia), Bushy Matgrass Whole plants [23, 50] Lippia citriodora Verbena Olorosa (Colombia), Lemon Verbena Whole plants [23] Zingiberaceae Boesenbergia rotunda Finger Root, Chinese Ginger Rhizoms 4-hydroxypanduratin A (1), panduratin A (2) [58] Zosteraceae Zostera marina Marine eelgrass Whole plants Zosteric acid (10) [47] aPlants species as yet uninvestigated for anti-dengue activity Overview of studies on plant species used as anti-dengue The use of herbal-based medicine and medicinal plants to treat many diseases is growing worldwide as they has few or no adverse effects. The following sections describe some species of medicinal plants from various families that have been investigated for anti-dengue activity (Table 1). In addition, we describe species used as traditional treatment for dengue together with their isolated compound. Alternanthera philoxeroides Alternanthera philoxeroides belongs to family Amaranthaceae. A. philoxeroides is also called “Alligator Weed”, and is an immersed aquatic plant. It originated from South America but is currently invading Australia.

Overview of studies on plant species used as anti-dengue The use of herbal-based medicine and medicinal plants to treat many diseases is growing worldwide as they has few or no adverse effects. The following sections describe some species of medicinal plants from various families that have been investigated for anti-dengue activity (Table 1). In addition, we describe species used as traditional treatment for dengue together with their isolated compound. Alternanthera philoxeroides Alternanthera philoxeroides belongs to family Amaranthaceae. A. philoxeroides is also called “Alligator Weed”, and is an immersed aquatic plant. It originated from South America but is currently invading Australia. The effect of A. philoxeroides extracts against dengue virus was investigated in vitro [57]. An MTT assay was carried out to determine the cytotoxicity of A. philoxeroides on C6/36 cell lines. Coumarin extract of A. philoxeroides showed lowest toxicity on cells (TD50 = 535.91), whereas a petroleum ether extract of A. philoxeroides had the strongest inhibitory effect on dengue virus (ED50 = 47.43). Andrographis paniculata Andrographis paniculata belongs to family Acanthaceae. It is an erect annual herb native to India and Sri Lanka and cultivated widely in Southern and Southeastern Asia. In Malaysia, it is called “Hempedu Bumi”, which has a bitter taste.

The effect of A. philoxeroides extracts against dengue virus was investigated in vitro [57]. An MTT assay was carried out to determine the cytotoxicity of A. philoxeroides on C6/36 cell lines. Coumarin extract of A. philoxeroides showed lowest toxicity on cells (TD50 = 535.91), whereas a petroleum ether extract of A. philoxeroides had the strongest inhibitory effect on dengue virus (ED50 = 47.43). Andrographis paniculata Andrographis paniculata belongs to family Acanthaceae. It is an erect annual herb native to India and Sri Lanka and cultivated widely in Southern and Southeastern Asia. In Malaysia, it is called “Hempedu Bumi”, which has a bitter taste. The maximum nontoxic dose (MNTD) of methanolic extract of A. paniculata against Vero E6 cells in vitro was investigated [7]. A. paniculata recorded the maximal dose, which was not toxic to cells at 0.050−1. The methanolic extract of A. paniculata showed the highest antiviral inhibitory effect on DENV-1 by antiviral assay based on cytopathic effects. Azidarachta indica Azidarachta indica belongs to the family Meliaceae. It is fast-growing tree with a final height in the range of 15–20 m. It is native to India and Pakistan and grows throughout tropical and semi-tropical regions.

The maximum nontoxic dose (MNTD) of methanolic extract of A. paniculata against Vero E6 cells in vitro was investigated [7]. A. paniculata recorded the maximal dose, which was not toxic to cells at 0.050−1. The methanolic extract of A. paniculata showed the highest antiviral inhibitory effect on DENV-1 by antiviral assay based on cytopathic effects. Azidarachta indica Azidarachta indica belongs to the family Meliaceae. It is fast-growing tree with a final height in the range of 15–20 m. It is native to India and Pakistan and grows throughout tropical and semi-tropical regions. The in vitro and in vivo inhibitory potential of aqueous extract of Azidarachta indica (neem) leaves on the replication of DENV-2 was evaluated [8]. Cytotoxicity studies were carried out to determine the MNTD in a virus inhibition assay. The aqueous extract of neem leaves (NL) completely inhibited 100–10,000 tissue culture infective dose (TCID)50 of virus as indicated by the absence of cytopathic effects at its maximum non-toxic concentration of 1.897 mg mL−1. An in vivo study on the inhibitory effects on virus of NL aqueous extract in day-old suckling mice was carried out by intracerebral inoculation. It was shown that the aqueous extract inhibited the virus at non-toxic doses in the range of 120–30 mg mL−1 as indicated by the absence of 511-bp dengue group specific amplicons upon RT-PCR. Boesenbergia rotunda Boesenbergia rotunda belongs to family Zingiberaceae. It is a medicinal and culinary herb known as Chinese ginger. It is found throughout China and Southeast Asia.

reatment, 40 and 80 μmol/L of 18 inhibited the proliferation of NEC-2 cells by 41.21 ± 0.25 and 94.11 ± 0.37 %, respectively. Both doses could block more than 70 % of cells at G0/G1. After 24 h of treatment with 18 (40 μmol/L), cell atrophy appeared, cell adhesion decreased, and some cells died from fragmentation [64]. Protective activity in neuronal cells I. latifolia has been used in Chinese folk medicine to treat headaches and various inflammatory diseases. The protective activity of I. latifolia against glutamate-induced neurotoxicity was tested using cultured rat cortical neurons in order to understand the mechanism of its inhibitory effect on ischemic brain damage, and several potentially active compounds were identified. The exposure of cultured cortical neurons to 500 μM glutamate for 12 h triggered neuronal cell death. I. latifolia (10–100 μg/mL) inhibited glutamate-induced neuronal death, elevation of intracellular calcium ([Ca2+]i), generation of reactive oxygen species (ROS), increase of a pro-apoptotic protein, BAX, and decrease of an anti-apoptotic protein, BcL-2. Hypoxia-induced neuronal cell death was also inhibited by I. latifolia. Compounds 98, 99, and 101 isolated from I. latifolia also inhibited the glutamate-induced increase in [Ca2+]i, generation of ROS, the change of apoptosis-related proteins, and neuronal and hypoxia-induced neuronal cell deaths. The results suggested that I. latifolia and its active compounds prevented glutamate-induced neuronal cell damage by inhibiting the increase of [Ca2+]i, generation of ROS, and, resultantly, the apoptotic pathway. In addition, the neuroprotective effects on ischemia-induced brain damage might be associated with the anti-excitatory and anti-oxidative actions, and could be attributable to these active compounds, CQAs [54].

The in vitro and in vivo inhibitory potential of aqueous extract of Azidarachta indica (neem) leaves on the replication of DENV-2 was evaluated [8]. Cytotoxicity studies were carried out to determine the MNTD in a virus inhibition assay. The aqueous extract of neem leaves (NL) completely inhibited 100–10,000 tissue culture infective dose (TCID)50 of virus as indicated by the absence of cytopathic effects at its maximum non-toxic concentration of 1.897 mg mL−1. An in vivo study on the inhibitory effects on virus of NL aqueous extract in day-old suckling mice was carried out by intracerebral inoculation. It was shown that the aqueous extract inhibited the virus at non-toxic doses in the range of 120–30 mg mL−1 as indicated by the absence of 511-bp dengue group specific amplicons upon RT-PCR. Boesenbergia rotunda Boesenbergia rotunda belongs to family Zingiberaceae. It is a medicinal and culinary herb known as Chinese ginger. It is found throughout China and Southeast Asia. The activity of some compounds extracted from B. rotunda for the inhibition of dengue virus protease has been tested on DENV-2 [58]. The cyclohexenyl chalcone derivatives of B. rotunda, 4-hydroxypanduratin A (1) and panduratin A (2) showed good competitive inhibitory activities towards DENV-2 NS3 protease with Ki values of 21 μM and 25 μM, respectively. The small value of Ki shows the potential of 4-hydroxypanduratin A to inhibit DENV-2 NS3 protease in vitro.

exenyl chalcone derivatives of B. rotunda, 4-hydroxypanduratin A (1) and panduratin A (2) showed good competitive inhibitory activities towards DENV-2 NS3 protease with Ki values of 21 μM and 25 μM, respectively. The small value of Ki shows the potential of 4-hydroxypanduratin A to inhibit DENV-2 NS3 protease in vitro. Carica papaya Carica papaya belongs to family Caricaceae. It is an erect, fast-growing and unbranched tree or shrub indigenous to Central America and cultivated in Mexico and most tropical countries for its edible fruits. C. papaya leaf has been used traditionally in the treatment of DF [55]. The leaf has been investigated for its potential against DF. The aqueous extract of leaves of this plant exhibited potential activity against DF by increasing the platelet (PLT) count, white blood cells (WBC) and neutrophils (NEUT) in blood samples of a 45-year-old patient bitten by carrier mosquitoes [9]. After 5 days of oral administration of 25 mL aqueous extract of C. papaya leaves to the patient twice daily, the PLT count increased from 55 × 103/μL to 168 × 103/μL, WBC from 3.7 × 103/μL to 7.7103/μL and NEUT from 46.0 to 78.3 %. Increased platelets could lead to reduced bleeding, thus avoiding progression to the severe illness of DHF. Cladogynos orientalis Cladogynos orientalis belongs to family Euphorbiaceae. It is a white-stellate-hairy shrub about 2 m high found in Southeast Asia, Malaysia and Thailand.

C. papaya leaf has been used traditionally in the treatment of DF [55]. The leaf has been investigated for its potential against DF. The aqueous extract of leaves of this plant exhibited potential activity against DF by increasing the platelet (PLT) count, white blood cells (WBC) and neutrophils (NEUT) in blood samples of a 45-year-old patient bitten by carrier mosquitoes [9]. After 5 days of oral administration of 25 mL aqueous extract of C. papaya leaves to the patient twice daily, the PLT count increased from 55 × 103/μL to 168 × 103/μL, WBC from 3.7 × 103/μL to 7.7103/μL and NEUT from 46.0 to 78.3 %. Increased platelets could lead to reduced bleeding, thus avoiding progression to the severe illness of DHF. Cladogynos orientalis Cladogynos orientalis belongs to family Euphorbiaceae. It is a white-stellate-hairy shrub about 2 m high found in Southeast Asia, Malaysia and Thailand. The in vitro activity of Cladogynos orientalis—a Thai medicinal plant—against dengue virus was evaluated [2]. The dichloromethane ethanol extract of C. orientalis was tested for anti-dengue activities against DENV-2 in Vero cells by the MTT method. The results showed that the ethanol extract of C. orientalis at a concentration of 12.5 μg mL−1 exhibited inhibitory activity on DENV-2 with 34.85 %. In addition, C. orientalis at a concentration of 100 μg mL−1 exhibited an inactivated viral particle activity of 2.9 %. Cladosiphon okamuranus Cladosiphon okamuranus belongs to family Chordariaceae. It is a brown seaweed found naturally in Okinawa, Japan.

The in vitro activity of Cladogynos orientalis—a Thai medicinal plant—against dengue virus was evaluated [2]. The dichloromethane ethanol extract of C. orientalis was tested for anti-dengue activities against DENV-2 in Vero cells by the MTT method. The results showed that the ethanol extract of C. orientalis at a concentration of 12.5 μg mL−1 exhibited inhibitory activity on DENV-2 with 34.85 %. In addition, C. orientalis at a concentration of 100 μg mL−1 exhibited an inactivated viral particle activity of 2.9 %. Cladosiphon okamuranus Cladosiphon okamuranus belongs to family Chordariaceae. It is a brown seaweed found naturally in Okinawa, Japan. A sulfated polysaccharide named fucoidan (3) from Cladosiphon okamuranus was found to potentially inhibit DENV-2 infection [59]. The virus infection was tested in BHK-21 cells in a focus-forming assay. Fucoidan reduced infectivity by 20 % at 10 μg mL−1 as compared with untreated cells. However, a carboxy-reduced fucoidan in which glucuronic acid was converted to glucose attenuated the inhibitory activity on DENV2 infection. Cryptonemia crenulata Cryptonemia crenulata belongs to family Halymeniaceae. It is a marine species found throughout the Atlantic Islands, North America, Caribbean Islands, Western Atlantic, South America, Africa, Indian Ocean Islands, Southeast Asia and Pacific Islands.

A sulfated polysaccharide named fucoidan (3) from Cladosiphon okamuranus was found to potentially inhibit DENV-2 infection [59]. The virus infection was tested in BHK-21 cells in a focus-forming assay. Fucoidan reduced infectivity by 20 % at 10 μg mL−1 as compared with untreated cells. However, a carboxy-reduced fucoidan in which glucuronic acid was converted to glucose attenuated the inhibitory activity on DENV2 infection. Cryptonemia crenulata Cryptonemia crenulata belongs to family Halymeniaceae. It is a marine species found throughout the Atlantic Islands, North America, Caribbean Islands, Western Atlantic, South America, Africa, Indian Ocean Islands, Southeast Asia and Pacific Islands. The sulfated polysaccharides from Cryptonemia crenulata, i.e., galactan (4), were selective inhibitors of DENV-2 multiplication in Vero cells with IC50 values of 1.0 μg mL−1, where the IC50 values for the reference polysaccharides heparin and DS8000 were 1.9 and 0.9 μg mL−1, respectively [60]. However, the compound has lower antiviral effect against DENV-3 and DENV-4, and was totally inactive against DENV-1. The inhibitory effect of C2S-3 against DENV-2 was slightly higher when treatment was by adsorption (EC50 = 2.5 ± 0.1 μg mL−1) with respect to treatment only during internalization (EC50 = 5.5 ± 0.7 μg mL−1) [1]. Thus, the inhibitory effect was increased when C2S-3 was included at both stages of adsorption and internalization. Cymbopogon citratus Cymbopogon citratus belongs to family Poaceae. It is a grass species known as lemon grass and is a tropical plant from Southeast Asia.

The sulfated polysaccharides from Cryptonemia crenulata, i.e., galactan (4), were selective inhibitors of DENV-2 multiplication in Vero cells with IC50 values of 1.0 μg mL−1, where the IC50 values for the reference polysaccharides heparin and DS8000 were 1.9 and 0.9 μg mL−1, respectively [60]. However, the compound has lower antiviral effect against DENV-3 and DENV-4, and was totally inactive against DENV-1. The inhibitory effect of C2S-3 against DENV-2 was slightly higher when treatment was by adsorption (EC50 = 2.5 ± 0.1 μg mL−1) with respect to treatment only during internalization (EC50 = 5.5 ± 0.7 μg mL−1) [1]. Thus, the inhibitory effect was increased when C2S-3 was included at both stages of adsorption and internalization. Cymbopogon citratus Cymbopogon citratus belongs to family Poaceae. It is a grass species known as lemon grass and is a tropical plant from Southeast Asia. The antiviral activity of Cymbopogon citratus was determined based on cytopathic effects shown by the degree of inhibition of DENV-1 infected Vero E6 cells [7]. The methanolic extract of C. citratus showed a slight inhibition effect on DENV-1. This result was further confirmed with an inhibition assay by the MTT method. However, C. citrates showed no significant inhibition. Moreover, C. citratus showed the lowest of MNTD at concentration of 0.001 mg mL−1. C. citratus was found to be quite a cytotoxic plant as it showed maximum cytotoxicity at 0.075 mg mL−1.

This result was further confirmed with an inhibition assay by the MTT method. However, C. citrates showed no significant inhibition. Moreover, C. citratus showed the lowest of MNTD at concentration of 0.001 mg mL−1. C. citratus was found to be quite a cytotoxic plant as it showed maximum cytotoxicity at 0.075 mg mL−1. Euphorbia hirta Euphorbia hirta belongs to family Euphorbiaceae. It is a common weed in garden beds, garden paths and wastelands and is found throughout Java, Sunda, Sumatra, Peninsular Malaysia, the Philippines and Vietnam. The water decoction of leaves from Euphorbia hirta, locally known as gatas–gatas, is used in the Philippines as a folk medicine to treat DF [54]. Internal haemorrhaging will stop and dengue fever will be cured after 24 h. However, the mechanism of action is still unknown and the antiviral properties and its ability to increase blood platelets are currently investigated. The tea obtained from boiled leaves of E. hirta is used to cure DF [53]. Flagellaria indica Flagellaria indica belongs to family Flagellariaceae. It is robust perennial climber that grows in many of the tropical and subtropical regions of the Old World, India, Southeast Asia, Polynesia and Australia.

The water decoction of leaves from Euphorbia hirta, locally known as gatas–gatas, is used in the Philippines as a folk medicine to treat DF [54]. Internal haemorrhaging will stop and dengue fever will be cured after 24 h. However, the mechanism of action is still unknown and the antiviral properties and its ability to increase blood platelets are currently investigated. The tea obtained from boiled leaves of E. hirta is used to cure DF [53]. Flagellaria indica Flagellaria indica belongs to family Flagellariaceae. It is robust perennial climber that grows in many of the tropical and subtropical regions of the Old World, India, Southeast Asia, Polynesia and Australia. Flagellaria indica was investigated for its anti-dengue properties in Vero cells [2]. The antiviral assay results show that 45.52 % inhibition of DENV-2 was observed in vitro in the presence of 12.5 μg mL−1 of ethanol extract of the plant. By conducting MTT assays, the cytotoxicity of F. indica was determined. The CC50 of ethanol extract of F. indica were 312 μg mL−1. Thus, this study indicates that F. indica has a significant potential effect on DENV. Gymnogongrus griffithsiae Gymnogongrus griffithsiae belongs to family Phyllophoraceae. It is a red seaweed found in Ireland, Europe, Atlantic Islands, North America, South America, Caribbean Islands, Africa, Southwest and Southeast Asia and Australia and New Zealand.

ell damage by inhibiting the increase of [Ca2+]i, generation of ROS, and, resultantly, the apoptotic pathway. In addition, the neuroprotective effects on ischemia-induced brain damage might be associated with the anti-excitatory and anti-oxidative actions, and could be attributable to these active compounds, CQAs [54]. Effect on pulmonary symptoms, including cough, asthma, and expectoration The effects of I. latifolia on isolated guinea pig tracheal smooth muscle cells were evaluated by obtaining CaCl2 and histamine accumulative dose–response curves in vitro. After incubation with the aqueous extract of I. latifolia, the dose–response curves of CaCl2 and histamine were significantly shifted to the right and the maximal contractile force was reduced. The extract could inhibit the isolated tracheal strip contraction induced by acetylcholine and histamine (IC50 pf 0.16 and 0.21 mg/mL, respectively). It also had a significant dilating effect on tracheal smooth muscle, which indicated that I. latifolia had an important effect on pulmonary symptoms [55].

Flagellaria indica was investigated for its anti-dengue properties in Vero cells [2]. The antiviral assay results show that 45.52 % inhibition of DENV-2 was observed in vitro in the presence of 12.5 μg mL−1 of ethanol extract of the plant. By conducting MTT assays, the cytotoxicity of F. indica was determined. The CC50 of ethanol extract of F. indica were 312 μg mL−1. Thus, this study indicates that F. indica has a significant potential effect on DENV. Gymnogongrus griffithsiae Gymnogongrus griffithsiae belongs to family Phyllophoraceae. It is a red seaweed found in Ireland, Europe, Atlantic Islands, North America, South America, Caribbean Islands, Africa, Southwest and Southeast Asia and Australia and New Zealand. The inhibitory properties against DENV-2 of the sulfated polysaccharide from Gymnogongrus griffithsiae, kappa carrageenan (5) was evaluated in Vero cells [60]. The compound effectively inhibits DENV-2 multiplication at the IC50 value of 0.9 μg mL−1, which is the same as the IC50 value for the commercial polysaccharides DS8000. However, the compound has lower antiviral effect against DENV-3 and DENV-4, and was totally inactive against DENV-1. Gymnogongrus torulosus Gymnogongrus torulosus belongs to family Phyllophoraceae. It is a red seaweed found in Australia and New Zealand. Gymnogongrus torulosus was investigated for its in vitro antiviral properties against DENV-2 in Vero cells [61]. Galactan (4) extracted from this plant was active against DENV-2, with IC50 values in the range of 0.19–1.7 μg mL−1.

Gymnogongrus torulosus Gymnogongrus torulosus belongs to family Phyllophoraceae. It is a red seaweed found in Australia and New Zealand. Gymnogongrus torulosus was investigated for its in vitro antiviral properties against DENV-2 in Vero cells [61]. Galactan (4) extracted from this plant was active against DENV-2, with IC50 values in the range of 0.19–1.7 μg mL−1. Hippophae rhamnoides Hippophae rhamnoides belongs to family Elaeagnaceae. It is a deciduous shrub occurring throughout Europe including Britain, from Norway south and east to Spain, and in Asia to Japan and the Himalayas. The anti-dengue activity of extracts of Hippophae rhamnoides leaves was investigated against dengue virus type-2 (DENV-2) in infected blood-derived human macrophages [11]. The findings showed that cells treated with H. rhamnoides leaf extracts was able to maintain cell viability of dengue-infected cells on par with Ribavirin, a commercial anti-viral drug along with a decrease and increase in TNF-α and IFN-γ, respectively. Moreover, H. rhamnoides leaf extract proved its anti-dengue activity as indicated by a decrease in plaque numbers after the treatment of infected cells. Houttuynia cordata Houttuynia cordata belongs to family Saururaceae. It is herbaceous perennial flowering plants growing between 20 and 80 cm, and is native to Japan, Korea, Southern China and Southeast Asia.

The anti-dengue activity of extracts of Hippophae rhamnoides leaves was investigated against dengue virus type-2 (DENV-2) in infected blood-derived human macrophages [11]. The findings showed that cells treated with H. rhamnoides leaf extracts was able to maintain cell viability of dengue-infected cells on par with Ribavirin, a commercial anti-viral drug along with a decrease and increase in TNF-α and IFN-γ, respectively. Moreover, H. rhamnoides leaf extract proved its anti-dengue activity as indicated by a decrease in plaque numbers after the treatment of infected cells. Houttuynia cordata Houttuynia cordata belongs to family Saururaceae. It is herbaceous perennial flowering plants growing between 20 and 80 cm, and is native to Japan, Korea, Southern China and Southeast Asia. Ethanol extract from Houttuynia cordata revealed an anti-dengue activity with 35.99 % inhibition against DENV-2 in Vero cells at a concentration of 1.56 μg mL−1 [2]. Aqueous extract of H. cordata showed effective inhibitory action against DENV-2 through direct inactivation of viral particles before infection of the cells [5]. A concentration of 100 μg mL−1 also effectively protects the cells from viral entry and inhibits virus activities after adsorption. HPLC analysis of H. cordata extract indicated that hyperoside (6) was the predominant bioactive compound, and was likely to play a role in this inhibition.

fore infection of the cells [5]. A concentration of 100 μg mL−1 also effectively protects the cells from viral entry and inhibits virus activities after adsorption. HPLC analysis of H. cordata extract indicated that hyperoside (6) was the predominant bioactive compound, and was likely to play a role in this inhibition. Leucaena leucocephala Leucaena leucocephala belongs to family Fabaceae. It is a species of Mimosoid tree indigenous throughout Southern Mexico and Northern Central America and the West Indies from the Bahamas and Cuba to Trinidad and Tobago. Galactomannans (7) extracted from seeds of Leucaena leucocephala have demonstrated activity against yellow fever virus (YFV) and DENV-1 in vitro and in vivo [12]. Galactomannans are polysaccharides consisting of a mannose backbone with galactose side groups, more specifically their structure consists of a main chain of (1 → 4)-linked β-d-mannopyranosyl units substituted by α-d-galactopyranosyl units [62]. L. leucocephala show protection against death in 96.5 % of YFV-infected mice. In vitro experiments with DENV-1 in C6/36 cell culture assays showed that the concentration producing a 100-fold decrease in virus titer of DENV-1 was 37 mg L−1. Lippia alba and Lippia citriodora Lippia alba and Lippia citriodora belong to family Verbenaceae. They are flowering plants native to Southern Texas, Mexico, the Caribbean, Central and South America.

Galactomannans (7) extracted from seeds of Leucaena leucocephala have demonstrated activity against yellow fever virus (YFV) and DENV-1 in vitro and in vivo [12]. Galactomannans are polysaccharides consisting of a mannose backbone with galactose side groups, more specifically their structure consists of a main chain of (1 → 4)-linked β-d-mannopyranosyl units substituted by α-d-galactopyranosyl units [62]. L. leucocephala show protection against death in 96.5 % of YFV-infected mice. In vitro experiments with DENV-1 in C6/36 cell culture assays showed that the concentration producing a 100-fold decrease in virus titer of DENV-1 was 37 mg L−1. Lippia alba and Lippia citriodora Lippia alba and Lippia citriodora belong to family Verbenaceae. They are flowering plants native to Southern Texas, Mexico, the Caribbean, Central and South America. Essential oils from Lippia alba and Lippia citriodora showed a considerable inhibitory effect on dengue virus serotype replication in Vero cells [23]. A 50 % reduction in virus plaque number values was found with L. alba oil at between 0.4–32.6 μg mL−1 whereas for L. citriodora oil, the IC50 values were between 1.9 and 33.7 μg mL−1. L. alba essential oil was more effective against DENV-2 than other serotypes, while for L. citriodora essential oil, the virucidal action against DENV-1, 2 and 3 were similar but lower than against DENV-4. Essential oil of L. alba was observed to produce a 100 % reduction of YFV yield at 100 μg mL−1 [50].

33.7 μg mL−1. L. alba essential oil was more effective against DENV-2 than other serotypes, while for L. citriodora essential oil, the virucidal action against DENV-1, 2 and 3 were similar but lower than against DENV-4. Essential oil of L. alba was observed to produce a 100 % reduction of YFV yield at 100 μg mL−1 [50]. Meristiella gelidium Meristiella gelidium belongs to family Solieriaceae. It is a marine species found in Atlantic Islands, North America, Caribbean Islands and South America. The antiviral activity of kappa carragenan (5) in Meristiella gelidium was evaluated against DENV-2 [63]. The IC50 of carragenans isolated from M. gelidium was in the range of 0.14–1.6 μg mL−1. The results show that the extract and the fraction derived from M. gelidium were more effective inhibitors of DENV-2 when compared with reference polysaccharides (heparin and DS 8000). Mimosa scabrella Mimosa scabrella belongs to family Fabaceae. It is a fast-growing, 15–20 m high and up to 50 cm diameter tree native to the cool, subtropical plateaus of Southeastern Brazil. Galactomannans (7) extracted from seeds of Mimosa scabrella have demonstrated activity against YFV and DENV-1 in vitro and in vivo [12]. M. scabrella showed protection against death in 87.7 % of YFV-infected mice. In vitro experiments with DENV-1 in C6/36 cell culture assays showed that a concentration of 347 mg L−1 produced a 100-fold decrease in virus titer of DENV-1.

abrella have demonstrated activity against YFV and DENV-1 in vitro and in vivo [12]. M. scabrella showed protection against death in 87.7 % of YFV-infected mice. In vitro experiments with DENV-1 in C6/36 cell culture assays showed that a concentration of 347 mg L−1 produced a 100-fold decrease in virus titer of DENV-1. Momordica charantia Momordica charantia belongs to family Cucurbitaceae. It is also known as bitter melon or peria (Malaysia), a tropical and subtropical vine found throughout Asia, Africa and the Caribbean. The MNTD of the methanolic extract of Momordica charantia against Vero E6 cells was investigated in vitro [7]. M. charantia recorded a maximal dose that was not toxic to cells of 0.20 mg mL−1. The methanolic extract of M. charantia showed inhibitory effect on DENV-1 by antiviral assay based on cytopathic effects. Ocimum sanctum Ocimum sanctum belongs to family Labiatae. It is an aromatic herb and shrub native to the tropical regions of Asia and the Americas. Tea, which is traditionally prepared by using Ocimum sanctum boiled leaves, acts as a preventive medicament against DF [64]. The MNTD of methanolic extract of O. sanctum against Vero E6 cells in vitro was investigated [7]. However, no significant difference in MNTD for O. sanctum was recorded. The methanolic extract of O. sanctum showed a slight inhibitory effect on DENV-1 based on cytopathic effects. Piper retrofractum Piper retrofractum belongs to family Piperaceae. It is a flowering vine native to Southeast Asia and cultivated in Indonesia and Thailand mostly for its fruit.

Tea, which is traditionally prepared by using Ocimum sanctum boiled leaves, acts as a preventive medicament against DF [64]. The MNTD of methanolic extract of O. sanctum against Vero E6 cells in vitro was investigated [7]. However, no significant difference in MNTD for O. sanctum was recorded. The methanolic extract of O. sanctum showed a slight inhibitory effect on DENV-1 based on cytopathic effects. Piper retrofractum Piper retrofractum belongs to family Piperaceae. It is a flowering vine native to Southeast Asia and cultivated in Indonesia and Thailand mostly for its fruit. In vitro anti-dengue activity of Piper retrofractum in Vero cells was investigated [2]. The inhibitory activity against DENV-2 infected cells was determined on dichloromethane ethanol extract by the MTT method. The ethanol extract of P. retrofractum exhibited an inactivated viral particle activity or 84.93 % at a concentration of 100 μg mL−1. Previous study has shown that an aqueous extract of long pepper, P. retrofractum, gives the highest level of activity against mosquito larvae [65]. Psidium guajava Psidium guajava belongs to family Myrtaceae. It is an evergreen shrub or small tree indigenous to Mexico, the Caribbean and Central and South America. It is cultivated widely in tropical and subtropical regions around the world.

In vitro anti-dengue activity of Piper retrofractum in Vero cells was investigated [2]. The inhibitory activity against DENV-2 infected cells was determined on dichloromethane ethanol extract by the MTT method. The ethanol extract of P. retrofractum exhibited an inactivated viral particle activity or 84.93 % at a concentration of 100 μg mL−1. Previous study has shown that an aqueous extract of long pepper, P. retrofractum, gives the highest level of activity against mosquito larvae [65]. Psidium guajava Psidium guajava belongs to family Myrtaceae. It is an evergreen shrub or small tree indigenous to Mexico, the Caribbean and Central and South America. It is cultivated widely in tropical and subtropical regions around the world. Psidium guajava leaf extract has been tested in vitro and showed to inhibit the growth of dengue virus [66]. Water boiled with guava leaves was used to avoid bleeding in DHF, and increased platelet counts to 100.000/mm3 within a period of approximately 16 h [56]. P. guajava ripe fruit or juice has healing properties in cases of DF by improving the declining levels of platelets [55]. Quercus lusitanica Quercus lusitanica belongs to family Fagaceae. It is a species of oak native to Morocco, Portugal and Spain.

Psidium guajava leaf extract has been tested in vitro and showed to inhibit the growth of dengue virus [66]. Water boiled with guava leaves was used to avoid bleeding in DHF, and increased platelet counts to 100.000/mm3 within a period of approximately 16 h [56]. P. guajava ripe fruit or juice has healing properties in cases of DF by improving the declining levels of platelets [55]. Quercus lusitanica Quercus lusitanica belongs to family Fagaceae. It is a species of oak native to Morocco, Portugal and Spain. Quercus lusitanica extract was found to have a good inhibitory effect on the replication of DENV-2 in C6/36 cells [39]. The methanol extract of the seeds completely inhibited (10–1,000 fold) the TCID50 of virus at its maximum non-toxic concentration of 0.25 mg mL−1 as indicated by the absence of cytopathic effects. A low dose of Q. lusitanica (0.032 mg mL−1) showed 100 % inhibition with 10 TCID50 of virus. Proteomics techniqueswere used to demonstrate that the effect of Q. lusitanica was to downregulate NS1 protein expression in infected c6/36 cells after treatment with the extract. Rhizophora apiculata Rhizophora apiculata belongs to family Rhizophoraceae. It is a mangrove tree up to 20 m tall that grows in Australia (Queensland and Northern Territory), Guam, India, Indonesia, Malaysia, Micronesia, New Caledonia, Papua New Guinea, the Philippines, Singapore, the Solomon Islands, Sri Lanka, Taiwan, Maldives, Thailand and Vietnam.

piculata belongs to family Rhizophoraceae. It is a mangrove tree up to 20 m tall that grows in Australia (Queensland and Northern Territory), Guam, India, Indonesia, Malaysia, Micronesia, New Caledonia, Papua New Guinea, the Philippines, Singapore, the Solomon Islands, Sri Lanka, Taiwan, Maldives, Thailand and Vietnam. Anti-dengue properties of the ethanolic extract of Rhizophora apiculata in DENV-2 in Vero cells have been reported [2]. R. apiculata exhibited inhibitory activity and an inactivated viral particle activity of 56.14 % and 41.5 % at concentrations of 12.5 and 100 μg mL−1, respectively. Tephrosia crassifolia, Tephrosia madrensis and Tephrosia viridiflora Tephrosia crassifolia, Tephrosia madrensis and Tephrosia viridiflora belong to family Fabaceae. Genus Tephrosia is an herb, undershrub or shrub, distributed mainly in tropical and subtropical regions of the world. Three species from this family (Tephrosia crassifolia, Tephrosia madrensis and Tephrosia viridiflora) were investigated [10]. The flavonoids isolated from T. madrensis, glabranine (8) and 7-O-methyl-glabranine (9) exert strong inhibitory effects on dengue virus replication in LLC-MK2 cells. Methyl-hildgardtol A isolated from T. crassifolia exhibited a moderate to low inhibitory effect, while hildgargtol A from T. crassifolia and elongatine from T. viridiflora had no effect on viral growth. Uncaria tomentosa Uncaria tomentosa belongs to family Rubiaceae. It is a woody vine growing in the tropical jungles of Central and South America.

Three species from this family (Tephrosia crassifolia, Tephrosia madrensis and Tephrosia viridiflora) were investigated [10]. The flavonoids isolated from T. madrensis, glabranine (8) and 7-O-methyl-glabranine (9) exert strong inhibitory effects on dengue virus replication in LLC-MK2 cells. Methyl-hildgardtol A isolated from T. crassifolia exhibited a moderate to low inhibitory effect, while hildgargtol A from T. crassifolia and elongatine from T. viridiflora had no effect on viral growth. Uncaria tomentosa Uncaria tomentosa belongs to family Rubiaceae. It is a woody vine growing in the tropical jungles of Central and South America. Uncaria tomentosa is a large wood vine native to the Amazon and Central American rainforests [67]. It is used widely as traditional medicine by native people of the Peruvian rainforest [68]. The antiviral activity of U. tomentosa was revealed by viral antigen (DENV-Ag) detection in monocytes by flow cytometry in C6/36 cells [67]. The most effective activity emerged from the alkaloidal fraction of U. tomentosa. The pentacyclic oxindole alkaloid-enriched fraction of U. tomentosa was observed as most effective at decreasing DENV-Ag detection in monocytes at concentrations of 1 μg mL−1, whereas the crude hydro-ethanolic extract demonstrates inhibitory activity at concentrations of 10 μg mL−1. Zostera marina Zostera marina belongs to family Zosteraceae. It is an aquatic plant known as eelgrass and is native to North America and Eurasia.

Uncaria tomentosa is a large wood vine native to the Amazon and Central American rainforests [67]. It is used widely as traditional medicine by native people of the Peruvian rainforest [68]. The antiviral activity of U. tomentosa was revealed by viral antigen (DENV-Ag) detection in monocytes by flow cytometry in C6/36 cells [67]. The most effective activity emerged from the alkaloidal fraction of U. tomentosa. The pentacyclic oxindole alkaloid-enriched fraction of U. tomentosa was observed as most effective at decreasing DENV-Ag detection in monocytes at concentrations of 1 μg mL−1, whereas the crude hydro-ethanolic extract demonstrates inhibitory activity at concentrations of 10 μg mL−1. Zostera marina Zostera marina belongs to family Zosteraceae. It is an aquatic plant known as eelgrass and is native to North America and Eurasia. A compound from the temperate marine eelgrasss Zostera marina has been identified as possessing anti-dengue virus activity in a focus-forming unit assay in LLC-MK2 cells [47]. The anti-adhesive compound p-sulfoxy-cinnamic acid, zosteric acid, ZA (10), derived from Z. marina showed a modest IC50 of approximately 2.3 mM against DENV-2. The other compound with related chemistries, CF 238, showed the most activity, with IC50 values of 24, 46, 14 and 47 μM against DENV-1, DENV-2, DENV-3 and DENV-4, respectively.

e compound p-sulfoxy-cinnamic acid, zosteric acid, ZA (10), derived from Z. marina showed a modest IC50 of approximately 2.3 mM against DENV-2. The other compound with related chemistries, CF 238, showed the most activity, with IC50 values of 24, 46, 14 and 47 μM against DENV-1, DENV-2, DENV-3 and DENV-4, respectively. Summary of medicinal plants tested for their anti-dengue activity Plants from which extracts have been prepared and tested to detect inhibition activity against DENV are listed in Table 2. This list consists of 16 plant species (from 12 families) that show high anti-dengue activity with high IC50 less than 5 μg mL−1 on four serotypes of DENV. The plants shown in Table 2 need to be studied further to identify and isolate potential bioactive compounds.Table 2 Some medicinal plants tested for their anti-dengue activity Family Species Part(s) used Extracts tested Stage of validation References Amaranthaceae Alternanthera philoxeroides Whole plants Petroleum ether, ethyl ether, ethyl acetate and coumarin extract In vitro [57] Chordariaceae Cladosiphon okamuranus Whole plants Ethanol extract In vitro [59] Euphorbiaceae Cladogynos orientalis Whole plants Ethanol extract In vitro [2] Fabaceae Leucaena leucocephala Seeds Aqueous extract In vivo and in vitro [12, 62] Tephrosia crassifolia Leave and flowers Flavonoid extract In vitro [10] Tephrosia madrensis Leaves and flowers Flavonoid extract In vitro [10]

Family Species Part(s) used Extracts tested Stage of validation References Amaranthaceae Alternanthera philoxeroides Whole plants Petroleum ether, ethyl ether, ethyl acetate and coumarin extract In vitro [57] Chordariaceae Cladosiphon okamuranus Whole plants Ethanol extract In vitro [59] Euphorbiaceae Cladogynos orientalis Whole plants Ethanol extract In vitro [2] Fabaceae Leucaena leucocephala Seeds Aqueous extract In vivo and in vitro [12, 62] Tephrosia crassifolia Leave and flowers Flavonoid extract In vitro [10] Tephrosia madrensis Leaves and flowers Flavonoid extract In vitro [10] Tephrosia viridiflora Leave and flowers Flavonoid extract In vitro [10] Fagaceae Quercus lusitanica Seeds Methanol extract In vitro and proteomics technique [39] Halymeniaceae Cryptonemia crenulata Whole plants Polysaccharide extract In vitro [60] Phyllophoraceae Gymnogongrus griffithsiae Whole plants Polysaccharide extract In vitro [60] Piperaceae Piper retrofractum Whole plants Dichloromethane and ethanol extract In vitro [2, 65] Rhizophoraceae Rhizophora apiculata Whole plants Ethanol extract In vitro [2] Solieraceae Meristiella gelidium Whole plants Polysaccharide extract In vitro [63] Verbenaceae Lippia alba Whole plants Essential oils In vitro [23, 50] Lippia citriodora Whole plants Essential oils In vitro [23] Zosteraceae Zostera marina – – In vitro [47]

Tephrosia viridiflora Leave and flowers Flavonoid extract In vitro [10] Fagaceae Quercus lusitanica Seeds Methanol extract In vitro and proteomics technique [39] Halymeniaceae Cryptonemia crenulata Whole plants Polysaccharide extract In vitro [60] Phyllophoraceae Gymnogongrus griffithsiae Whole plants Polysaccharide extract In vitro [60] Piperaceae Piper retrofractum Whole plants Dichloromethane and ethanol extract In vitro [2, 65] Rhizophoraceae Rhizophora apiculata Whole plants Ethanol extract In vitro [2] Solieraceae Meristiella gelidium Whole plants Polysaccharide extract In vitro [63] Verbenaceae Lippia alba Whole plants Essential oils In vitro [23, 50] Lippia citriodora Whole plants Essential oils In vitro [23] Zosteraceae Zostera marina – – In vitro [47] Potential of plant bioactive compounds to combat dengue The active compounds showed a wide range of activity against DENV. The isolated products belong to various chemical classes such as sulfated polysaccharides, flavonoids, quercetin and natural chalcone compounds. The chemical structures of ten of these different phytochemicals, isolated from 11 plants, are shown in Fig. 5. The secondary metabolites of medicinal plants comprise a variety of compounds with a wide range of biological activities [68]. There are reports on medicinal plants extracts and essential oils possessing potential to new antiviral properties [41, 42]. Many plant extracts in different solvents have been reported to exhibit activity against a vector of dengue fever, Ae. Aegypti [20, 69].Fig. 5 Structure of some potential compounds for treatment of dengue fever (DF) isolated from medicinal plants (1–10)

al oils possessing potential to new antiviral properties [41, 42]. Many plant extracts in different solvents have been reported to exhibit activity against a vector of dengue fever, Ae. Aegypti [20, 69].Fig. 5 Structure of some potential compounds for treatment of dengue fever (DF) isolated from medicinal plants (1–10) Conclusion and future directions This review has covered only 31 potential plants that could be used in the treatment of dengue and about ten isolated active phytochemicals. The available research highlights the information available for various parts and extract types of medicinal plants for the treatment of dengue. However, some of the plants that have not yet been fully explored may have a broad range of potential therapeutic applications. The development of new anti-dengue products from bioactive compounds is necessary in order to find more effective and less toxic anti-dengue drugs. Therefore, any extensive study on the potential of plants with isolated active compounds that have shown anti-dengue activity should go through additional in vitro and in vivo animal testing followed by toxicity and clinical tests. This route may reveal a promising compound to be optimized and thus be suitable for application in the production of new anti-dengue compounds. If pursued from drugs derived from medicinal plants around the continents, this work may prove valuable to the health of individuals and to nations. Moreover, such discoveries may lead to the development of highly efficient and safe anti-dengue treatments. However, to identify potential anti-dengue plants or compounds, knowledge of the mechanisms of virus infection need to be understood inorder to facilitate the search for and development of the most appropriate drugs. Further research is needed to determine how to target the most appropriate stages to prevent the spread of virus infection. Focusing on each phase in the life cycle of the virus, new compounds could prevent (1) infection of host cells, (2) the viral maturation process, (3) synthesis of viral RNA, or (4) the spread of viral particles.

h is needed to determine how to target the most appropriate stages to prevent the spread of virus infection. Focusing on each phase in the life cycle of the virus, new compounds could prevent (1) infection of host cells, (2) the viral maturation process, (3) synthesis of viral RNA, or (4) the spread of viral particles. Abbreviations CC50Cytotoxicity concentration DENVDengue virus DFDengue fever DHFDengue hemorrhagic fever DSSDengue shock syndrome HPLCHigh performance liquid chromatography IC50Inhibitory concentration MNTDMaximum non-toxic dose MTT3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide TCID50Median tissue culture infective dose The authors would like to thank the World Health Organization for permission to use copyrighted material, and the Institute of Bioproduct Development, Universiti Teknologi Malaysia for financial assistance for this research work.

Introduction Type-2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by β-cell dysfunction and insulin resistance, and has emerged as a major health care burden around the world [1–3]. Protein tyrosine phosphatase 1B (PTP1B) is an enzyme found in the important insulin-targeted tissues such as liver, muscle, and fat cells. PTP1B plays a key role as a negative regulator in insulin signal transduction [4] by dephosphorylating activated insulin receptors (IR) or insulin receptor substrates (IRS) [5, 6]. An excess of PTP1B will impair insulin down-regulation [7–9], leading to type II diabetes mellitus. Based on the above research results, the inhibition of PTP1B has been sought as a novel therapeutic strategy, and much attention has been paid to PTP1B inhibitors using small molecules for the treatment of type II diabetes [10–12]. In our screening program to search for PTP1B inhibitors, we have tested the ethanol extracts of Indonesian marine organisms such as marine sponges and ascidians, and the extract of a marine sponge Lamellodysideaherbacea exhibited significant inhibitory activity against PTP1B. Bioassay-guided separation of the extract led to the isolation of a bioactive component, and the structure was assigned as 2-(3′,5′-dibromo-2′-methoxyphenoxy)-3,5-dibromophenol (1) [13]. We described herein the PTP1B inhibitory activity and cytotoxicity against two human cancer cell lines, HCT-15 (colon) and Jurkat (T-cell lymphoma), of compound 1 and its methyl ether (2) and ester derivatives (3–6).

nent, and the structure was assigned as 2-(3′,5′-dibromo-2′-methoxyphenoxy)-3,5-dibromophenol (1) [13]. We described herein the PTP1B inhibitory activity and cytotoxicity against two human cancer cell lines, HCT-15 (colon) and Jurkat (T-cell lymphoma), of compound 1 and its methyl ether (2) and ester derivatives (3–6). Materials and methods General experimental procedure EI–MS was performed by a JMS-MS 700 mass spectrometer (JEOL, Tokyo, Japan). 1H- and 13C-NMR spectra were recorded on a JNM-AL-400 NMR spectrometer (JEOL) at 400 MHz for 1H and 100 MHz for 13C in CDCl3 (δH 7.26, δC 77.0). Preparative HPLC was carried out using the L-6200 system (Hitachi Ltd., Tokyo, Japan). Materials Fetal bovine serum (FBS) and other culture materials were purchased from Invitrogen (Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals including organic solvent were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Marine sponge The marine sponge was collected by scuba diving in the coral reef at Manado, Indonesia, in 2010 and identified as Lamellodysideaherbacea. The voucher specimen is deposited at the Faculty of Fisheries and Marine Science, Sam Ratulangi University and the Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University as 10-09-16=2-6.

was collected by scuba diving in the coral reef at Manado, Indonesia, in 2010 and identified as Lamellodysideaherbacea. The voucher specimen is deposited at the Faculty of Fisheries and Marine Science, Sam Ratulangi University and the Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University as 10-09-16=2-6. Extraction and isolation The marine sponge (94 g wet weight) was thawed, cut into small pieces, and extracted three times with ethanol. The ethanol extract was evaporated to dryness (284.3 mg) and 20 mg of the crude extract was subjected to HPLC separation (90 % MeOH; detection, UV 210 nm; flow rate, 2.0 mL/min) using an ODS column (PEGASIL ODS, 10 mm × 250 mm, Senshu Scientific Co., Tokyo, Japan) to give 5.4 mg of 2-(3′,5′-dibromo-2′-methoxyphenoxy)-3,5-dibromophenol (1). 2-(3′,5′-Dibromo-2′-methoxyphenoxy)-3,5-dibromophenol (1) Obtained as a viscous oil; 1H-NMR (CDCl3) δ 4.03 (s, 3H), 6.80 (d, 1H, J = 4.0), 7.18 (d, 1H, J = 4.0), 7.35 (d, 1H, J = 4.0), 7.45 (d, 1H, J = 4.0); 13C-NMR (CDCl3) δ 61.5, 117.3, 117.3, 118.7, 119.0, 119.9, 120.1, 127.4, 130.5, 139.0, 145.9, 150.5, 150.7; EI–MS m/z 528, 530 532, 534, and 526 [M+]; HREI–MS m/z 527.7180 (Δ −2.7 mmu, calcd for C13H879Br4O3: 527.7207), 529.7203 (Δ +1.7 mmu, calcd for C13H879Br381Br1O3: 529.7186), 531.7159 (Δ −0.7 mmu, calcd for C13H879Br281Br2O3: 531.7166), 533.7137 (Δ −0.9 mmu, calcd for C13H879Br181Br3O3: 533.7146), 535.7103 (Δ −2.2 mmu, calcd for C13H881Br4O3: 535.7125).

26 [M+]; HREI–MS m/z 527.7180 (Δ −2.7 mmu, calcd for C13H879Br4O3: 527.7207), 529.7203 (Δ +1.7 mmu, calcd for C13H879Br381Br1O3: 529.7186), 531.7159 (Δ −0.7 mmu, calcd for C13H879Br281Br2O3: 531.7166), 533.7137 (Δ −0.9 mmu, calcd for C13H879Br181Br3O3: 533.7146), 535.7103 (Δ −2.2 mmu, calcd for C13H881Br4O3: 535.7125). Preparation of methyl derivative (2) TMS-diazomethane (73 μL, 0.064 mmol) was added to a MeOH solution of 1 (3.8 mg, 0.0071 mmol in 300 μL) and stirred at room temperature for 14 h. The reaction mixture was concentrated in vacuo to give a brown material, and a product was purified by preparative HPLC (90 % MeOH) using ODS column (PEGASIL ODS) to give 3,5-dibromo-2-(3′,5′-dibromo-2′-methoxyphenoxy)-1-methoxybenzene (2, 2.0 mg, 0.0037 mmol, 52 %). 3,5-Dibromo-2-(3′,5′-dibromo-2′-methoxyphenoxy)-1-methoxybenzene (2) Obtained as a viscous oil; 1H-NMR (CDCl3) δ 3.76 (s, 3H), 4.00 (s, 3H), 6.46 (d, 1H, J = 4.0), 7.09 (d, 1H, J = 4.0), 7.36 (d, 1H, J = 4.0), 7.42 (d, 1H, J = 4.0); 13C-NMR (CDCl3) δ 57.2, 61.7, 116.4, 117.2, 119.3, 119.4, 119.6, 128.2, 129.5, 137.2, 140.1, 146.2, 152.1, 154.2; EI–MS m/z 542, 544 546, 548, and 550 [M+]; HREI–MS m/z 541.7386 (Δ +2.2 mmu, calcd for C14H1079Br4O3: 541.7364), 543.7319 (Δ −2.4 mmu, calcd for C14H1079Br381Br1O3: 543.7343), 545.7318 (Δ −0.5 mmu, calcd for C14H1079Br281Br2O3: 545.7323), 547.7288 (Δ −1.4 mmu, calcd for C14H1079Br181Br3O3: 547.7302), 549.7262 (Δ −2.0 mmu, calcd for C14H1081Br4O3: 549.7282).

+]; HREI–MS m/z 541.7386 (Δ +2.2 mmu, calcd for C14H1079Br4O3: 541.7364), 543.7319 (Δ −2.4 mmu, calcd for C14H1079Br381Br1O3: 543.7343), 545.7318 (Δ −0.5 mmu, calcd for C14H1079Br281Br2O3: 545.7323), 547.7288 (Δ −1.4 mmu, calcd for C14H1079Br181Br3O3: 547.7302), 549.7262 (Δ −2.0 mmu, calcd for C14H1081Br4O3: 549.7282). Preparation of derivatives 3–6 Acetic anhydride (100 μL, 1.1 mmol) and 4-(dimethylamino)pyridine (1.0 mg, 0.0080 mmol) were added to a solution of 1 (3.0 mg, 0.056 mmol) in pyridine (100 μL), and the resulting solution was stirred at room temperature for 12 h. The reaction mixture was concentrated in vacuo to dryness, and a product was purified by preparative HPLC (column; PEGASIL ODS, 10 mm × 250 mm; solvent, 90 % MeOH; detection, UV at 220 nm; flow rate, 2.0 mL/min) to give 3,5-dibromo-2-(3′,5′-dibromo-2′-methoxyphenoxy)phenyl ethanoate (3, 1.2 mg, 0.0022 mmol, 30 %). The other derivatives (4–6) were prepared using the following regents instead of acetic anhydride: n-butyric anhydride (4, 1.4 mg, 0.0023 mmol, 32 %), n-hexanoic anhydride (5, 1.1 mg, 0.0018 mmol, 25 %), and benzoyl chloride (6, 1.5 mg, 0.0023 mmol, 33 %).

methoxyphenoxy)phenyl ethanoate (3, 1.2 mg, 0.0022 mmol, 30 %). The other derivatives (4–6) were prepared using the following regents instead of acetic anhydride: n-butyric anhydride (4, 1.4 mg, 0.0023 mmol, 32 %), n-hexanoic anhydride (5, 1.1 mg, 0.0018 mmol, 25 %), and benzoyl chloride (6, 1.5 mg, 0.0023 mmol, 33 %). 3,5-Dibromo-2-(3′,5′-dibromo-2′-methoxyphenoxy)phenyl ethanoate (3) Obtained as a viscous oil; 1H-NMR (CDCl3) δ 2.08 (s, 3H), 3.95 (s, 3H), 6.78 (d, 1H, J = 2.4), 7.18 (d, 1H, J = 2.4), 7.34 (d, 1H, J = 2.4), 7.44 (d, 1H, J = 2.4); EI–MS m/z 570, 572 574, 576, and 578 [M+]; HREI–MS m/z 569.7316 (Δ +0.3 mmu, calcd for C15H1079Br4O4: 569.7313), 571.7296 (Δ +0.4 mmu, calcd for C15H1079Br381Br1O4: 571.7292), 573.7283 (Δ +1.1 mmu, calcd for C15H1079Br281Br2O4: 573.7272), 575.7247 (Δ −0.4 mmu, calcd for C15H1079Br181Br3O4: 575.7251), 577.7216 (Δ −1.5 mmu, calcd for C15H1081Br4O4: 577.7231). 3,5-Dibromo-2-(3′,5′-dibromo-2′-methoxyphenoxy)phenyl butanoate (4) Obtained as a viscous oil; 1H-NMR (CDCl3) δ 0.89 (t, 3H, J = 7.2), 1.57 (m, 2H), 2.29 (t, 2H, J = 7.2), 3.95 (s, 3H), 6.58 (d, 1H, J = 1.9), 7.36 (d, 1H, J = 2.4), 7.40 (d, 1H, J = 1.9), 7.71 (d, 1H, J = 2.4); EI–MS m/z 598, 600 602, 604, and 606 [M+]; HREI–MS m/z 597.7625 (Δ −0.1 mmu, calcd for C17H1479Br4O4: 597.7626), 599.7580 (Δ −2.5 mmu, calcd for C17H1479Br381Br1O4: 599.7605), 601.7569 (Δ −1.6 mmu, calcd for C17H1479Br281Br2O4: 601.7585), 603.7591 (Δ +2.7 mmu, calcd for C17H1479Br181Br3O4: 603.7564), 605.7518 (Δ −2.6 mmu, calcd for C17H1481Br4O4: 605.7544).

+]; HREI–MS m/z 597.7625 (Δ −0.1 mmu, calcd for C17H1479Br4O4: 597.7626), 599.7580 (Δ −2.5 mmu, calcd for C17H1479Br381Br1O4: 599.7605), 601.7569 (Δ −1.6 mmu, calcd for C17H1479Br281Br2O4: 601.7585), 603.7591 (Δ +2.7 mmu, calcd for C17H1479Br181Br3O4: 603.7564), 605.7518 (Δ −2.6 mmu, calcd for C17H1481Br4O4: 605.7544). 3,5-Dibromo-2-(3′,5′-dibromo-2′-methoxyphenoxy)phenyl hexanoate (5) Obtained as a viscous oil; 1H-NMR (CDCl3) δ 0.87 (t, 3H, J = 6.8), 1.25 (m, 4H), 1.51 (m, 2H), 2.30 (t, 2H, J = 7.8), 3.95 (s, 3H), 6.57 (d, 1H, J = 2.0), 7.36 (d, 1H, J = 2.0), 7.40 (d, 1H, J = 2.0), 7.71 (d, 1H, J = 2.4); EI–MS m/z 626, 628 630, 632, and 634 [M+]; HREI–MS m/z 625.7952 (Δ +1.3 mmu, calcd for C19H1879Br4O4: 625.7939), 627.7924 (Δ +0.6 mmu, calcd for C19H1879Br381Br1O4: 627.7918), 629.7881 (Δ −1.7 mmu, calcd for C19H1879Br281Br2O4: 629.7898), 631.7874 (Δ −0.4 mmu, calcd for C19H1879Br181Br3O4: 631.7878), 633.7856 (calcd for C19H1881Br4O4: 633.7856). 3,5-Dibromo-2-(3′,5′-dibromo-2′-methoxyphenoxy)phenyl benzoate (6) Obtained as a viscous oil; 1H-NMR (CDCl3) δ 3.80 (s, 3H), 6.68 (d, 1H, J = 2.4), 7.27 (d, 1H, J = 1.9), 7.41 (t, 2H, J = 7.7), 7.52 (d, 1H, J = 1.9), 7.59 (t, 1H, J = 7.3), 7.76 (d, 1H, J = 2.4), 7.83 (d, 2H, J = 7.2); EI–MS m/z 632, 634, 636, 638, and 640 [M+]; HREI–MS m/z 631.7455 (Δ −1.4 mmu, calcd for C20H1279Br4O4: 631.7469), 633.7468 (Δ +1.9 mmu, calcd for C20H1279Br381Br1O4: 633.7449), 635.7433 (Δ +0.5 mmu, calcd for C20H1279Br281Br2O4: 635.7428), 637.7430 (Δ +2.3 mmu, calcd for C20H1279Br181Br3O4: 637.7407), 639.7379 (Δ −0.9 mmu, calcd for C20H1281Br4O4: 639.7388).

+]; HREI–MS m/z 631.7455 (Δ −1.4 mmu, calcd for C20H1279Br4O4: 631.7469), 633.7468 (Δ +1.9 mmu, calcd for C20H1279Br381Br1O4: 633.7449), 635.7433 (Δ +0.5 mmu, calcd for C20H1279Br281Br2O4: 635.7428), 637.7430 (Δ +2.3 mmu, calcd for C20H1279Br181Br3O4: 637.7407), 639.7379 (Δ −0.9 mmu, calcd for C20H1281Br4O4: 639.7388). PTP1B inhibitory assay Protein tyrosine phosphatase 1B (PTP1B) inhibitory activity was determined by measuring the rate of hydrolysis of a substrate, p-nitrophenyl phosphate (pNPP, Sigma, St. Louis, MO, USA) according to the published method with a slight modification [14]. Briefly, PTP1B (100 μL of 0.5 μg/mL stock solution, Enzo Life Sciences, Farmingdale, NY, USA) in 50 mM citrate buffer (pH 6.0) containing 0.1 M NaCl, 1 mM dithiothreitol (DTT), and 1 mM N,N,N′,N′-ethylenediamine tetraacetate (EDTA) were added to each well of a 96-well plastic plate (Corning Inc., Corning, NY, USA). A sample (2.0 μL in MeOH) was added to each well to make the final concentrations from 0 to 4.7–5.6 μM and incubated for 10 min at 37 °C. The reaction was initiated by the addition of pNPP (100 μL of 4.0 mM stock solution) in the citrate buffer, incubated at 37 °C for 30 min, and terminated with the addition of 10 μL of a stop solution (10 M NaOH). The optical density of each well was measured at 405 nm using an MTP-500 microplate reader (Corona Electric Co., Ltd., Ibaraki, Japan). PTP1B inhibitory activity (%) is defined as [1 − (ABSsample − ABSblank)/(ABScontrol − ABSblank)] × 100, where ABSblank is the absorbance of wells containing only the buffer and pNPP, ABScontrol is the absorbance of p-nitrophenol liberated by the enzyme in the assay system without a test sample, and ABSsample is that with a test sample. The assays were performed in two duplicate experiments for all test samples. Oleanolic acid (Tokyo Chemical Industry, Tokyo, Japan), a known phosphatase inhibitor [15], was used as a positive control.

itrophenol liberated by the enzyme in the assay system without a test sample, and ABSsample is that with a test sample. The assays were performed in two duplicate experiments for all test samples. Oleanolic acid (Tokyo Chemical Industry, Tokyo, Japan), a known phosphatase inhibitor [15], was used as a positive control. Cytotoxicity assay against HCT-15 and Jurkat cells HCT-15 and Jurkat cells were obtained from the Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University (Miyagi, Japan). The cell lines were cultured in RPMI-1640 medium. The medium was supplemented with 10 % fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Exponentially growing cells, cultured in a humidified chamber at 37 °C containing 5.0 % CO2, were used for the experiments. Cytotoxic activity was evaluated using the colorimetric MTT assay [16]. HCT-15 (1.0 × 104 cells in 100 μL) or Jurkat cells (2.0 × 104 cells in 100 μL) were added to each well of a 96-well plastic plate. A sample (1.0 μL in MeOH) was added to each well to make the final concentrations from 0 to 39–47 μM, and the cells were incubated for 48 h at 37 °C. MTT (10 μL of 5.5 mg/mL stock solution) and a cell lysate solution (90 μL, 40 % N,N-dimethylformamide, 20 % sodium dodecyl sulfate, 2.0 % CH3COOH and 0.030 % HCl) were added to each well, and the plate was shaken thoroughly by agitation at room temperature for overnight. The optical density of each well was measured at 570 nm using an MTP-500 microplate reader.

a cell lysate solution (90 μL, 40 % N,N-dimethylformamide, 20 % sodium dodecyl sulfate, 2.0 % CH3COOH and 0.030 % HCl) were added to each well, and the plate was shaken thoroughly by agitation at room temperature for overnight. The optical density of each well was measured at 570 nm using an MTP-500 microplate reader. Cytotoxicity assay against Huh-7 cells Cytotoxic activity against Huh-7 cells was assessed by the MTT assay, a modification of our previously described method [17]. Following the treatment of cells with test samples, 10 μL of MTT (5.0 mg/mL saline) was added to each well, the samples were incubated for 90 min at 37 °C and centrifuged (300g for 5 min), and the supernatant was aspirated off. The cells were lysed and solubilized by the addition of 100 μL of 0.040 N HCl in 2-propanol. The absorbance of each well was determined at 590 nm using an Inter-med model NJ-2300 Microplate Reader (Cosmo Bio Co., Ltd., Tokyo, Japan). Survival (%) was calculated relative to the control. Results and discussion Among the ethanol extracts of about 90 marine sponges and ascidians collected in the coral reefs at North Sulawesi, Indonesia, the extract of a marine sponge Lamellodysidea herbacea showed potent inhibitory activity (IC50 = 0.58 μg/mL) against PTP1B in the screening bioassay. Bioassay-guided isolation by HPLC yielded compound 1 as an inhibitor of PTP1B. The other fractions obtained after separation of 1 did not show an inhibitory activity against PTP1B.

t of a marine sponge Lamellodysidea herbacea showed potent inhibitory activity (IC50 = 0.58 μg/mL) against PTP1B in the screening bioassay. Bioassay-guided isolation by HPLC yielded compound 1 as an inhibitor of PTP1B. The other fractions obtained after separation of 1 did not show an inhibitory activity against PTP1B. The EI–MS spectrum of 1 showed the presence of four Br atoms, and the molecular formula C13H8Br4O3 was deduced from HREI–MS data. The 13C NMR spectrum of 1 revealed 13 carbon signals, and the signals due to two sets of meta-coupled aromatic protons (δ 6.80, 7.18, 7.35, and 7.45) and OMe protons (δ 4.03) were detected in the 1H NMR spectrum. The positions of an OMe, OH, and four Br atoms were assigned by the analysis of 2D NMR (1H–1H COSY, HMQC, and HMBC) data for 1 and confirmed by the NOE experiments on the methyl derivative (2). The NMR data for 1 were identical with those of the reported values for 2-(3′,5′-dibromo-2′-methoxyphenoxy)-3,5-dibromophenol (Fig. 1) [13].Fig. 1 Structures of compounds 1 and 2

ce was reduced. The extract could inhibit the isolated tracheal strip contraction induced by acetylcholine and histamine (IC50 pf 0.16 and 0.21 mg/mL, respectively). It also had a significant dilating effect on tracheal smooth muscle, which indicated that I. latifolia had an important effect on pulmonary symptoms [55]. Toxicity Kudingcha has been historically consumed as a functional tea, reflecting the philosophy of medicine–food homology in traditional Chinese medicine, with little or no acute toxicity. Acute and long-term toxicity of I. latifolia was studied in rats using aqueous extracts. The maximum tolerable dose was 168 g/kg, indicating that there is no acute toxicity. There were also no effects on body weight, hematopoiesis, and biochemical index in the blood of rats intragastrically treated with aqueous extracts at 4.5, 9, or 18 g/kg for 90 days [56]. Clinical usage Studies have shown that drinking tea from both I. kudingcha and I. latifolia for 2 months is sufficient to reduce hypertension nearly as well as or better than common hypertension medications such as nifedipine [57, 58]. More importantly, no adverse reaction was observed. These studies suggested that I. latifolia and I. kudingcha have potential use in the treatment of hypertension.

assigned by the analysis of 2D NMR (1H–1H COSY, HMQC, and HMBC) data for 1 and confirmed by the NOE experiments on the methyl derivative (2). The NMR data for 1 were identical with those of the reported values for 2-(3′,5′-dibromo-2′-methoxyphenoxy)-3,5-dibromophenol (Fig. 1) [13].Fig. 1 Structures of compounds 1 and 2 Compounds 1 and 2 inhibited the PTP1B activity (Fig. 2) with IC50 values of 0.85 and 1.7 μM, respectively, which were almost the same efficacy as that of oleanolic acid (1.1 μM), a positive control (Table 1). Oleanolic acid is a ubiquitous triterpene detected in various plants, most of which are used as crude Asian drugs for the treatments of inflammation, cancers, hepatitis, and diabetes [15, 18, 19], and has recently been reported to have a significant inhibitory activity against PTP1B [20]. Oleanolic acid derivatives were demonstrated to promote cellular insulin signaling by increasing the level of insulin receptor phosphorylation [20]. The highest concentration of compound 2 did not show a dose-dependent effect (Fig. 2). This will be due to a solubility problem of 2 at higher concentration in this bioassay system.Fig. 2 Inhibitory activity of 1 (a) and 2 (b) against PTP1B and two human cancer (HCT-15 and Jurkat) cells. Data are shown as the mean ± SD (n = 4) of two duplicate experiments Table 1 Inhibitory activity of compounds 1–6 against PTP1B and three human cancer cell lines Compound IC50 (μM) PTP1B Cytotoxicity Huh-7 HCT-15 Jurkat 1 0.85 32 12 9.5 2 1.7 48 >46 >46 3 0.62 NT 10.3 6.0 4 0.68 NT 14.3 9.6 5 0.69 NT 7.1 8.1 6 0.97 NT 4.3 20 Oleanolic acid 1.1 NT NT NT

Compounds 1 and 2 inhibited the PTP1B activity (Fig. 2) with IC50 values of 0.85 and 1.7 μM, respectively, which were almost the same efficacy as that of oleanolic acid (1.1 μM), a positive control (Table 1). Oleanolic acid is a ubiquitous triterpene detected in various plants, most of which are used as crude Asian drugs for the treatments of inflammation, cancers, hepatitis, and diabetes [15, 18, 19], and has recently been reported to have a significant inhibitory activity against PTP1B [20]. Oleanolic acid derivatives were demonstrated to promote cellular insulin signaling by increasing the level of insulin receptor phosphorylation [20]. The highest concentration of compound 2 did not show a dose-dependent effect (Fig. 2). This will be due to a solubility problem of 2 at higher concentration in this bioassay system.Fig. 2 Inhibitory activity of 1 (a) and 2 (b) against PTP1B and two human cancer (HCT-15 and Jurkat) cells. Data are shown as the mean ± SD (n = 4) of two duplicate experiments Table 1 Inhibitory activity of compounds 1–6 against PTP1B and three human cancer cell lines Compound IC50 (μM) PTP1B Cytotoxicity Huh-7 HCT-15 Jurkat 1 0.85 32 12 9.5 2 1.7 48 >46 >46 3 0.62 NT 10.3 6.0 4 0.68 NT 14.3 9.6 5 0.69 NT 7.1 8.1 6 0.97 NT 4.3 20 Oleanolic acid 1.1 NT NT NT NT not tested Interestingly, the methylation of a phenol in 1 reduced the cytotoxicity against HCT-15 and Jurkat cells (Fig. 2; Table 1). Compound 1 had a moderate cytotoxicity against HCT-15 and Jurkat cells with IC50 values of 12 and 9.5 μM, respectively. On the other hand, 2 did not show an apparent cytotoxicity at 18 μM.

NT not tested Interestingly, the methylation of a phenol in 1 reduced the cytotoxicity against HCT-15 and Jurkat cells (Fig. 2; Table 1). Compound 1 had a moderate cytotoxicity against HCT-15 and Jurkat cells with IC50 values of 12 and 9.5 μM, respectively. On the other hand, 2 did not show an apparent cytotoxicity at 18 μM. Therefore, the ester derivatives (3–6) were prepared from 1 (Scheme 1) and tested for their activity against PTP1B and two cancer cell lines (Table 1). Compound 3–6 revealed comparable to stronger inhibitory activity against PTP1B than that of 1, but cytotoxicity against HCT-15 and Jurkat cells were observed. From these results, 2 is found to be the most interesting compound among these compounds as it possessed potent inhibitory activity against PTP1B and showed much reduced cytotoxicity.Scheme 1 Semisynthetic preparation of 3–6 The inhibitory activity of 1 and 2 on cell proliferation of human hepatoma Huh-7 cells was therefore examined. Since PTP1B is located in the insulin-targeted tissues such as liver, muscle, and fat cells, Huh-7 cells are used for cell-based experiments to investigate the mechanism of action of PTP1B inhibitors. Compound 2 showed weaker cytotoxicity (IC50 = 48 μM) than 1 (32 μM) (Table 1). Cell-based experiments are now in progress using Huh-7 cells and compound 2.

n-targeted tissues such as liver, muscle, and fat cells, Huh-7 cells are used for cell-based experiments to investigate the mechanism of action of PTP1B inhibitors. Compound 2 showed weaker cytotoxicity (IC50 = 48 μM) than 1 (32 μM) (Table 1). Cell-based experiments are now in progress using Huh-7 cells and compound 2. Polybrominated diphenyl ethers have been isolated from marine organisms, such as sponges, ascidians, and algae, and are reported to exhibit a variety of biological activities: antibacterial and antifungal activities [21–24], brine shrimp toxicity [23], antimicroalgal activity [25], anti-inflammatory activity [26], maturation of starfish oocytes [27], and inhibitory activities against several enzymes [27–29]. In this study, we demonstrated that a known bromodiphenyl ether (1) was a potent inhibitor of PTP1B, an important target enzyme for the treatment of type II diabetes, and that the methoxy derivative (2) is more useful than the original phenol and the ester derivatives. Compound 2 will be a new lead compound for PTP1B inhibitors.

udy, we demonstrated that a known bromodiphenyl ether (1) was a potent inhibitor of PTP1B, an important target enzyme for the treatment of type II diabetes, and that the methoxy derivative (2) is more useful than the original phenol and the ester derivatives. Compound 2 will be a new lead compound for PTP1B inhibitors. This work was supported in part by a Grant-in-Aid for Scientific Research (21603012) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan to M.N. and by a Colab. Res. & Int. Pub. Project No. 492/SP 2H/PL/2011 from DGHE, Ministry of National Education of Indonesia to R.E.P.M. We are grateful to the Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University for kindly providing human cancer cell lines. We express our thanks to Dr. K. Ogawa of Z. Nakai Laboratory for identification of the marine sponge and Mr. T. Matsuki and S. Sato for mass spectra.

Introduction Kudingcha is a particularly bitter-tasting tea that has been widely used in China throughout history. It is known to disperse wind-heat, clear toxins from the blood, and refresh the mentalities. It has also been used to cure the common cold, rhinitis, itching eyes, conjunctival congestion, and headache; it is helpful for digestion and alleviating the adverse effects of alcohol [1, 2]. Today, nearly 20 plants from different families with similarities in appearance, flavor, and traditional usage in different areas of China are all named “Kudingcha” [3]. However, the plants most commonly found in the markets of China can be divided into 2 groups: one group, including species Ilex latifolia Thunb and I. kudingcha C.J. Tseng, was named the “large-leaved Kudingcha”; the other group, containing the species Ligustrum robustum (syn. Ligustrum purpurascens), was named the “small-leaved Kudingcha.” The large-leaved Kudingcha was certified to be the original Kudingcha species and has obvious antioxidant, anti-inflammatory, lipid metabolism, hepatoprotective, and anti-tumor activities [4–7], similar to the popular green tea (Camellia sinensis) and the Yerba maté tea (Ilex paraguariensis) from South America. Over the last several years, Kudingcha has been considered as a dietetic beverage and is gaining popularity with names like “beauty-slimming tea,” “longevity tea,” “green-golden tea,” and “clearing-heat tea.”

r to the popular green tea (Camellia sinensis) and the Yerba maté tea (Ilex paraguariensis) from South America. Over the last several years, Kudingcha has been considered as a dietetic beverage and is gaining popularity with names like “beauty-slimming tea,” “longevity tea,” “green-golden tea,” and “clearing-heat tea.” In the current review, we present and analyze the plant characteristics, ethnobotanical usages, phytochemistry, and pharmacological activities of the large-leaved Kudingcha, as well as its relationship with green tea and Yerba maté tea. These up-to-date research observations will be helpful in understanding the characteristics and superiorities of this special tea and would be applicable in developing new products and herbal medicines in the future. Historical and ethnopharmacological investigation of Kudingcha in China In the book “Tong Jun Lu” (A.D. 25–225; E. Han Dynasty), Kudingcha was described as having a bitter flavor and reportedly kept people awake all night [8]. The original species in “Tong Jun Lu” were investigated and certified to be I. latifolia, I. kudingcha, and I. cortia [9]. At present, I. cortia is called “Gougucha” and is classified separately from Kudingcha due to its different botanical characters, chemical ingredients, and medicinal usages [11]. On the basis of our investigation of the literature, plant resources, and chemical and molecular analyses conducted since 2006, it appears that the large-leaved Kudingcha from the genus Ilex (I. latifolia and I. kudingcha) is the original Kudingcha species [2, 8, 11].

s, chemical ingredients, and medicinal usages [11]. On the basis of our investigation of the literature, plant resources, and chemical and molecular analyses conducted since 2006, it appears that the large-leaved Kudingcha from the genus Ilex (I. latifolia and I. kudingcha) is the original Kudingcha species [2, 8, 11]. The medicinal properties of Kudingcha were reported in “Ben Cao Shi Yi” of the Tang Dynasty, including clearing the thirst and phlegm, resolving hydropsia, refreshing the mind, and improving the eyesight. In the encyclopedia “Ben Cao Gang Mu” of the Ming Dynasty, Kudingcha was described as an effective herb that contained no toxins and had medicinal properties that could scatter wind-heat, refresh the mind and spirit, and promote fluid production to quench thirst [3]. The records in ancient books were all about the large-leaved Kudingcha from the genus Ilex. The current application of I. latifolia is limited to a few provinces of Eastern China. The supply and market share are limited compared to that of I. kudingcha.

ind and spirit, and promote fluid production to quench thirst [3]. The records in ancient books were all about the large-leaved Kudingcha from the genus Ilex. The current application of I. latifolia is limited to a few provinces of Eastern China. The supply and market share are limited compared to that of I. kudingcha. The other important group (the small-leaved Kudingcha) from the genus Ligustrum was first reported in the book of “Guizhou Min Jian Fang Yao Ji” in 1958. Some investigators put forward that the small-leaved Kudingcha originated from the mountains of Dalou and the river valley of Wujiang in Guizhou province. It has been used in the civilian Tujia ethnic group and Miao ethnic group for a long time, so was named as one of the four famous old tea species locally [2, 10]. No literal records have been found in ancient books to date. The small-leaved Kudingcha (L. robustum) was initially used because of its similarities in plant morphology, bitter flavor, and ethnobotanical use in folk medicine.

group for a long time, so was named as one of the four famous old tea species locally [2, 10]. No literal records have been found in ancient books to date. The small-leaved Kudingcha (L. robustum) was initially used because of its similarities in plant morphology, bitter flavor, and ethnobotanical use in folk medicine. Plant characteristics and distribution of Kudingcha species Two species of the large-leaved Kudingcha from the genus Ilex have very similar botanical appearances. Both come from trees that are evergreen, and the primary difference between the species is found at the base of the leaves [4]. The leaf characteristics of Kudingcha species are listed in Table 1. The small-leaved Kudingcha from the genus Ligustrum are shrubs or small trees, deciduous or evergreen. The leaves are much smaller and thinner than the large-leaved Kudingcha, and the flavor is not as bitter as, which makes it more suitable to drink routinely [8]. According to Table 1, the essential leaf characteristics of each species are useful to distinguish Kudingcha original plants. While these Kudingcha species are distributed across different locations over China, they have similar ethnopharmacological properties (Table 2).Table 1 Leaf characteristics of the most commonly used Kudingcha [65, 66]

e essential leaf characteristics of each species are useful to distinguish Kudingcha original plants. While these Kudingcha species are distributed across different locations over China, they have similar ethnopharmacological properties (Table 2).Table 1 Leaf characteristics of the most commonly used Kudingcha [65, 66] Leaf characteristics I. kudingcha I. latifolia L. robustum Blade Oblong to oblong-elliptic Oblong or ovate oblong Lanceolate to subovate or elliptic Petiole 2–2.2 cm, abaxially subrounded, rugose, adaxially sulcate, puberulent Subterete, abaxially rugose, adaxially slightly impressed 2–8 mm, pubescent, groovy Margin Doubly serrate or densely serrate Sparsely serrate – Base Obtuse or cuneate Rounded or broadly cuneate Broadly cuneate or subrounded Apex Acute or short acuminate Short or long acuminate Long acuminate Texture Leathery Thickly leathery Papery Mid vein Raised and keeled abaxially, impressed and sparsely puberulent adaxially Impressed adaxially – Lateral vein Evident on both surfaces Obscure abaxially, obvious adaxially – Vein-pair 5–7 – – Vein-angle About 50° – – Table 2 Distribution and medical uses of the most commonly used Kudingcha

hickly leathery Papery Mid vein Raised and keeled abaxially, impressed and sparsely puberulent adaxially Impressed adaxially – Lateral vein Evident on both surfaces Obscure abaxially, obvious adaxially – Vein-pair 5–7 – – Vein-angle About 50° – – Table 2 Distribution and medical uses of the most commonly used Kudingcha Common name Species Distribution Recorded uses [8] Large-leaved Kudingcha I. kudingcha Guangxi, Guangdong, Hannan, Hunan Clearing pathogenic summer-heat and detoxifying, treatment of the dehydration and abdominal pain I. latifolia Zhejiang, Jiangxi, Jiangsu Removing the excessive fire and noxious heat from the lung and spleen, removing the damp-heat pathogens in the large intestine to eliminate the dysentery, mental refreshing Small-leaved Kudingcha L. robustum (syn. L. purpurascens) Guizhou, Yunnan, Sichuan Mental refreshing, treatment of the pathogenic summer-heat, polydipsia and thirst, headache, conjunctival congestion Secondary metabolites of large-leaved Kudingcha Triterpenoids, phenolic acids, flavonoids, and essential oils were isolated and identified from the large-leaved Kudingcha. Among different types of constituents, triterpenoids and polyphenols were considered to be the most important metabolites, with various bioactivities (summarized in Fig. 1).Fig. 1 The chemical structures of compounds isolated from the large-leaved Kudingcha

were isolated and identified from the large-leaved Kudingcha. Among different types of constituents, triterpenoids and polyphenols were considered to be the most important metabolites, with various bioactivities (summarized in Fig. 1).Fig. 1 The chemical structures of compounds isolated from the large-leaved Kudingcha Triterpenoids The ursane-type triterpenoids with lactone at the position of C20 and C28 are called α-kudinlactone (48–57, 113), β-kudinlactone (58–69), and γ-kudinlactone (74–78) (Fig. 1), and are considered to be the most characteristic chemicals in the Kudingcha species. The chemical analysis showed that I. kudingcha had the highest total triterpenoid content. The β-kudinlactone was the main type of triterpenoid present in I. latifolia, while γ-kudinlactone and α-kudinlactone were not present in I. latifolia. At the same time, none of these characteristic kudinosides were detected in the supposititious species I. pentagona and I. cornuta of China, and the similar Yerba maté of South America [11]. In addition, oleanane-type (1–13) and lupane-type triterpenes (81–87) and their glycosides were also found in I. latifolia and I. kudingcha.

time, none of these characteristic kudinosides were detected in the supposititious species I. pentagona and I. cornuta of China, and the similar Yerba maté of South America [11]. In addition, oleanane-type (1–13) and lupane-type triterpenes (81–87) and their glycosides were also found in I. latifolia and I. kudingcha. Phenolic acids Phenolic acids with antioxidant activities have been found in recent years. The main types of phenolic acids found in large-leaved Kudingcha were polyphenols and related compounds [30, 32]. High-performance liquid chromatography (HPLC) analysis showed the presence of 6 of these derivatives, but no caffeine, in female and male leaves of I. latifolia. However, the levels of these derivatives varied from season to season. The current-year spring leaves showed the highest levels of caffeic acid derivatives (147–235 mg/g), while the current-year autumn leaves, old spring leaves, and old autumn leaves had lower amounts of the derivatives of 36, 24–44, and 31 mg/g, respectively. The levels of 4-O-caffeoyl quinic acid increased 3–6 times in current-year leaves from spring to autumn. The contents of the caffeic acid derivatives in the female plant were similar to those in the old leaves of the male plant. These results suggest that the new spring leaves of I. latifolia would be a good source for antioxidants [33]. Flavonoids Compounds 90–92 and 119–121 were the main flavones isolated from the large-leaved Kudingcha. They are also the primary constituents in the popular green tea.

Phenolic acids Phenolic acids with antioxidant activities have been found in recent years. The main types of phenolic acids found in large-leaved Kudingcha were polyphenols and related compounds [30, 32]. High-performance liquid chromatography (HPLC) analysis showed the presence of 6 of these derivatives, but no caffeine, in female and male leaves of I. latifolia. However, the levels of these derivatives varied from season to season. The current-year spring leaves showed the highest levels of caffeic acid derivatives (147–235 mg/g), while the current-year autumn leaves, old spring leaves, and old autumn leaves had lower amounts of the derivatives of 36, 24–44, and 31 mg/g, respectively. The levels of 4-O-caffeoyl quinic acid increased 3–6 times in current-year leaves from spring to autumn. The contents of the caffeic acid derivatives in the female plant were similar to those in the old leaves of the male plant. These results suggest that the new spring leaves of I. latifolia would be a good source for antioxidants [33]. Flavonoids Compounds 90–92 and 119–121 were the main flavones isolated from the large-leaved Kudingcha. They are also the primary constituents in the popular green tea. Essential oils and other secondary metabolites The essential oils have been studied by using gas chromatography coupled to mass spectrometry (GC/MS), which indicated the presence of alcohol, aldehyde, ketone, ether, fatty acids, and fatty acid ester [34, 35]. Other secondary metabolites such as phytosterols and polysaccharides were also isolated and identified [26, 41].

ssential oils have been studied by using gas chromatography coupled to mass spectrometry (GC/MS), which indicated the presence of alcohol, aldehyde, ketone, ether, fatty acids, and fatty acid ester [34, 35]. Other secondary metabolites such as phytosterols and polysaccharides were also isolated and identified [26, 41]. Biological activities Regularly drinking Kudingcha as a herbal tea has a positive role in the prevention and treatment of cardiovascular and cerebrovascular diseases, diabetes, pharyngitis, and cancer, particularly for the relevant troublesome conditions of arteriosclerosis, hypertension, dizziness, insomnia, palpitations, and chest tightness caused by cardiovascular diseases [8]. The most important biological activity of the tea is lipid metabolism activity. Moreover, it has antioxidant and anti-tumor activities, and has effects on the hypoglycemic and vascular system, similar to green tea and Yerba maté [2].

ss, insomnia, palpitations, and chest tightness caused by cardiovascular diseases [8]. The most important biological activity of the tea is lipid metabolism activity. Moreover, it has antioxidant and anti-tumor activities, and has effects on the hypoglycemic and vascular system, similar to green tea and Yerba maté [2]. Lipid metabolism activity An acyl CoA cholesteryl acyl transferase (ACAT) catalyzes the intracellular esterification of cholesterol in various tissues, and inhibitors of ACAT may serve as new types of medicines to treat arteriosclerosis and obesity. Compounds 85, 89, 104, 114, and 20–22 isolated from I. kudingcha showed potent inhibitory activity in the ACAT assay. Compounds 104 (IC50 0.044 mM), 20 (IC50 0.064 mM), and 21 (IC50 0.073 mM) showed higher inhibitory activity than the other compounds (IC50 0.154–0.468 mM) and those of lignans (IC50 25–207 mM) isolated from Schisandra chinensis, Machilus thunbergii, Magnolia denudate, Magnolia ovate, and the polyacetylenes (IC50 42–86 mM) from Panax ginseng [5]. Seventeen triterpenoids isolated from the aqueous extract of I. kudingcha were examined for inhibitory activity in the ACAT assay. Compounds 4 (the inhibitory activities were 64.3, 36.5, 15.7 % at 1.0, 0.2, 0.05 mg/mL, respectively) and 36 (the inhibitory activities were 63.9, 29.0, 31.7 % at 1.0, 0.2, 0.05 mg/mL, respectively) showed clear inhibitory activity [6].

us extract of I. kudingcha were examined for inhibitory activity in the ACAT assay. Compounds 4 (the inhibitory activities were 64.3, 36.5, 15.7 % at 1.0, 0.2, 0.05 mg/mL, respectively) and 36 (the inhibitory activities were 63.9, 29.0, 31.7 % at 1.0, 0.2, 0.05 mg/mL, respectively) showed clear inhibitory activity [6]. A cell-based screening model using THP-1 macrophages was initially applied to aggregate LDL (aggLDL)-induced lipid deposition on macrophages to test the inhibitory effects of the 12 triterpenoids from I. kudingcha. Compounds 63, 64, 49–53, 55, and 62 inhibited the formation of foam cells and reduced intracellular total cholesterol and triglyceride contents. The δ-lactone ring in the aglycone was indispensable to the biological activity. The OH group at the C-12 position, the number of monosaccharides in the sugar chain, and the presence of a terminal rhamnose residue in the sugar chain might improve this inhibitory effect. Moreover, compound 64 was considered as a potential candidate for the treatment of arteriosclerosis [53]. Medium and high doses of total saponins (150 and 300 mg kg−1 day−1, respectively) from I. kudingcha reduced the level of serum total cholesterol (TC) in ApoE-knockout mice (30–35 %), a reduction that was similar to the group treated with atorvastatin. It was likely that the saponins reduced the level of TC through the inhibition of ACAT activity. In addition, Kudingcha treatment led to greater inhibition of malondialdehyde (MDA) activity than atorvastatin [6, 36].

) in ApoE-knockout mice (30–35 %), a reduction that was similar to the group treated with atorvastatin. It was likely that the saponins reduced the level of TC through the inhibition of ACAT activity. In addition, Kudingcha treatment led to greater inhibition of malondialdehyde (MDA) activity than atorvastatin [6, 36]. Protection of the vascular system Various Ilex species were used in Chinese folk medicine to treat coronary heart diseases. The mode of action was considered to be mediated by their coronary vasodilative effects. The water extract of I. latifolia was shown to increase the contractility and decrease the frequency of contraction in an isolated rat heart perfusion system. The extract inhibited Na+/K+-ATPase activities in rat heart sarcolemma, rat brain microsome, and a purified enzyme from porcine cerebral cortex. It also inhibited Ca2+-dependent ATPase at a similar dose. Following exposure of the isolated rat heart to the extract at a dose that produces pronounced cardiac effects, the inhibition of Na+/K+-ATPase activity can be readily detected in the heart [7]. The water extract of I. kudingcha was able to increase coronary blood flow in isolated guinea pig heart and increase cerebral blood flow in anesthetized rabbits, prolong the survival time of mice under hypoxia, and protect rats from myocardial ischemia induced by pituitrin. The effects were beneficial in the prevention and treatment of coronary artery disease and angina pectoris [37].

od flow in isolated guinea pig heart and increase cerebral blood flow in anesthetized rabbits, prolong the survival time of mice under hypoxia, and protect rats from myocardial ischemia induced by pituitrin. The effects were beneficial in the prevention and treatment of coronary artery disease and angina pectoris [37]. Intravenous injection (0.05 g/kg) of the I. kudingcha extract remarkably lowered the blood pressure of normal anesthetized dogs. This hypotensive effect was also seen when the extracts (0.75 and 0.94 g/kg, respectively) were administered to 2-kidney, 1-clip hypertensive rats and spontaneously hypertensive rats by gastric gavage. The subcutaneous abdominal fatty tissue of normal and obese rats decreased significantly when the animals were given the extract (1.5 g/kg) by gastric gavage. The results suggested that I. kudingcha had potential as a resource for the treatment of hypertension and obesity [38].

usly hypertensive rats by gastric gavage. The subcutaneous abdominal fatty tissue of normal and obese rats decreased significantly when the animals were given the extract (1.5 g/kg) by gastric gavage. The results suggested that I. kudingcha had potential as a resource for the treatment of hypertension and obesity [38]. Hypoglycemic effect Caco-2 cells are derived from a human colon adenocarcinoma and are used widely as a model of intestinal absorption by epithelial cells and to study the absorption of glucose in vitro. Glucose absorption by Caco-2 cells was significantly inhibited by the aqueous extract of I. kudingcha under both Na+-dependent conditions and Na+-free conditions, indicating an effect on SGLT1 and GLUT transporters. Analysis of the polyphenols in the extracts suggested that dicaffeoylquinic acids and flavanols may be particularly important in producing these effects. I. kudingcha was the most effective compared to the other plants tested, suggesting that it may merit evaluation in a clinical setting [39]. The water extract of I. latifolia showed marked inhibition of hyperglycemia in the epinephrine hyperglycemia rat model. The blood sugar levels of rats given either a low dose or a high dose of the extract (5 or 10 g/kg, respectively) were significantly reduced compared to the control group, indicating that I. latifolia has potential as a hypoglycemic drug [40].

rked inhibition of hyperglycemia in the epinephrine hyperglycemia rat model. The blood sugar levels of rats given either a low dose or a high dose of the extract (5 or 10 g/kg, respectively) were significantly reduced compared to the control group, indicating that I. latifolia has potential as a hypoglycemic drug [40]. Antioxidant activity The antioxidant activities of I. kudingcha have been evaluated in vitro. The crude extract (CE), as well as the 4 fractions of chloroform (CfF), ethyl acetate (EaF), n-butanol (nBF), and water (WtF), were evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay, Trolox equivalent antioxidant capacity (TEAC), and ferric ion reducing antioxidant power (FRAP). The activities decreased in the order of EaF > nBF > CE > WtF > CfF, according to the DPPH and FRAP assays, with the exception of the rank order of CfF and WtF in the TEAC assay. The extracts were certified to contain a large number of caffeoylquinic acids (CQAs) (93–98), which contributed to the antioxidant activity. From a health point of view, Kudingcha is a beneficial herbal drink due to its antioxidant activity [32].

s, with the exception of the rank order of CfF and WtF in the TEAC assay. The extracts were certified to contain a large number of caffeoylquinic acids (CQAs) (93–98), which contributed to the antioxidant activity. From a health point of view, Kudingcha is a beneficial herbal drink due to its antioxidant activity [32]. The ethyl acetate-soluble fraction of I. kudingcha, which contained an abundance of phenolic compounds, displayed remarkable free radical-scavenging activities against DPPH (IC50 16.3 μg/mL), ·OH (IC50 87.5 μg/mL for non-site-specific and 27.3 μg/mL for site-specific assays) and O2− (IC50 1.3 μg/mL). The fraction also showed a strong suppressive effect on rat liver mitochondrial peroxidation (IC50 7.1 μg/mL) and significantly protected against the oxidation of LDL mediated by either Cu2+ or AAPH free radicals (IC50 were 14 and 4.8 μg/mL, respectively) [30]. The crude polysaccharide KPS II of I. kudingcha and 2 refined polysaccharides, KPS IIIa and KPS IIIb from KPS II, have concentration-dependent hydroxyl radical-scavenging activity. KPS IIIa and KPS IIIb were both composed of arabinose, xylose, mannose, galactose, and glucose, with molar ratios of 10.2:1.0:1.2:8.0:5.4 and 17.7:1.0:1.1:1.8:4.1, respectively. The scavenging capacity of the crude polysaccharide was higher than that of the refined polysaccharides [41].

xyl radical-scavenging activity. KPS IIIa and KPS IIIb were both composed of arabinose, xylose, mannose, galactose, and glucose, with molar ratios of 10.2:1.0:1.2:8.0:5.4 and 17.7:1.0:1.1:1.8:4.1, respectively. The scavenging capacity of the crude polysaccharide was higher than that of the refined polysaccharides [41]. The contents of total polyphenols and flavonoids of I. latifolia and I. kudingcha were detected by DPPH assay, ABTS+ assay, OH-free radical-scavenging assay, reducing power assay, and FRAP assay, and were found to be correlated with their antioxidant activities. It was found that the water extract of I. kudingcha had both higher polyphenol content and better antioxidant performance than that of I. latifolia [42]. Antimicrobial activity The studies of the anti-bacterial effect of I. latifolia showed an inhibitory effect on the growth of Streptococcus A [minimum inhibitory concentration (MIC) 1,215 mg/mL), Pneumococcus (MIC 25 mg/mL), Streptococcus B (MIC 25 mg/mL), Bacillus diphtheria (MIC 50 mg/mL), Bacillus dysenteriae (MIC 50 mg/mL), Staphylococcus aureus (MIC 100 mg/mL), Pseudomonas aeruginosa (MIC 100 mg/mL), Escherichia coli (MIC 100 mg/mL), and Micrococcus catarrhalis (MIC 200 mg/mL) by the agar dilution method in vitro. Intragastric administration can significantly improve the survival rate of mice infected by E. coli, B. dysenteriae, Pneumobacillus, and Streptococcus B [43].

/mL), Pseudomonas aeruginosa (MIC 100 mg/mL), Escherichia coli (MIC 100 mg/mL), and Micrococcus catarrhalis (MIC 200 mg/mL) by the agar dilution method in vitro. Intragastric administration can significantly improve the survival rate of mice infected by E. coli, B. dysenteriae, Pneumobacillus, and Streptococcus B [43]. The crude polysaccharides of I. kudingcha were tested against 7 bacterial and fungal strains commonly known to cause food spoilage, and the results showed that the polysaccharides had more significant activities against bacteria than fungi. The MIC against S. aureus was 5 mg/mL [44]. I. kudingcha was reported to have strong anti-bacterial activity against S. aureus, Salmonella typhosa, and β-hemolytic streptococci, as seen by the agar diffusion method. It could also enhance the action of hypoxia tolerance, hypothermia tolerance, and sports tolerance in the mouse, where it was shown to have anti-stress activities [45]. Antiviral activity The anti-HSV-1 activities of different extracts of I. kudingcha were tested by cytopathogenic effect (CPE) observation and plaque reduction assay. The results showed that the aqueous extract had a more pronounced anti-HSV-1 effect (IC50 108.24 μg/mL) and lower cytotoxicity (non-toxic concentration 500 μg/mL) than the ethyl acetate and methanol extract. It is suggested that the extract acts by binding to the HSV-1 receptor and prevents the virus from entering the cells [46].

showed that the aqueous extract had a more pronounced anti-HSV-1 effect (IC50 108.24 μg/mL) and lower cytotoxicity (non-toxic concentration 500 μg/mL) than the ethyl acetate and methanol extract. It is suggested that the extract acts by binding to the HSV-1 receptor and prevents the virus from entering the cells [46]. Studies were also conducted to measure the antiviral effects of I. kudingcha water extract against Coxsackie virus B3 (CVB3) cytotoxicity in HeLa cells by CPE and MTT methods. The results showed that this extract had a significant anti-CVB3 effect in vitro, yielding TC50 values of 45700.3 μg/mL, IC50 values of 119.18 μg/mL, and TI of 383.46 in HeLa cells [47]. Enhanced immunity Aqueous extracts of I. latifolia were found to have beneficial effects on humoral and cellular immunity. The samples enhanced the phagocyte function of macrophages in the mouse abdominal cavity and increased the number of plaque-forming cells (PFCs), indicating that I. latifolia had potency as a new medicine to adjust and enhance the immune function of an organism [48]. To further study this effect, mice were immunocompromised by intragastric treatment with dexamethasone (DEX) for 7 days. The results showed that I. latifolia could improve the phagocytic index of macrophages, and high doses could distinctly enhance the number of PFCs and the percentage of phagocytic macrophages [49].

Enhanced immunity Aqueous extracts of I. latifolia were found to have beneficial effects on humoral and cellular immunity. The samples enhanced the phagocyte function of macrophages in the mouse abdominal cavity and increased the number of plaque-forming cells (PFCs), indicating that I. latifolia had potency as a new medicine to adjust and enhance the immune function of an organism [48]. To further study this effect, mice were immunocompromised by intragastric treatment with dexamethasone (DEX) for 7 days. The results showed that I. latifolia could improve the phagocytic index of macrophages, and high doses could distinctly enhance the number of PFCs and the percentage of phagocytic macrophages [49]. Anti-tumor activity The anti-tumor activity of the ethanol extract and the volatile oils of the old leaves and fresh leaves of I. latifolia were tested by MTT assays. The results showed that the ethanol extract from the old leaves and the volatile oil from the fresh leaves had the most potent anti-tumor activity. For the ethanol extract of old leaves, the inhibition rate against human gastric cancer cells SGC-7901 was 62.40 %. For the volatile oil from the fresh leaves, the inhibition rates against human lung cancer cells NCI-H460 (IC50 42.86 mg/mL) and SGC-7901 were 93.33 and 32.44 %, respectively. The results provided a scientific basis for the use of the tea in the development of new anti-tumor medicines [50].

cells SGC-7901 was 62.40 %. For the volatile oil from the fresh leaves, the inhibition rates against human lung cancer cells NCI-H460 (IC50 42.86 mg/mL) and SGC-7901 were 93.33 and 32.44 %, respectively. The results provided a scientific basis for the use of the tea in the development of new anti-tumor medicines [50]. The growth inhibitory activities of compounds 105, 106, 109, 110, 21, and 22 were evaluated in MCF-7 (estrogen receptor-positive) and MDA-MB-231 (estrogen receptor-negative) human breast cancer cells. Compounds 105 and 106 showed a growth inhibitory effect against MCF-7 cells, with IC50 values of 29.51 ± 3.44 and 38.49 ± 3.16 μM, respectively. Compound 21 (IC50 4.58 ± 0.56 μM) was the most potent among all the tested compounds, indicating that the p-(E)-coumaroyl moiety at the C-27 position may contribute to improving the growth inhibitory potential when compared with the triterpene with the p-(Z)-coumaroyl moiety (22, IC50 12.65 ± 0.94 μM). All the tested triterpenoids showed more potent growth inhibitory activity in MCF-7 cells than in MDA-MB-231 cells, implying that their growth inhibitory activities may be partly dependent on the status of the estrogen receptor [51].

mpared with the triterpene with the p-(Z)-coumaroyl moiety (22, IC50 12.65 ± 0.94 μM). All the tested triterpenoids showed more potent growth inhibitory activity in MCF-7 cells than in MDA-MB-231 cells, implying that their growth inhibitory activities may be partly dependent on the status of the estrogen receptor [51]. The human nasopharyngeal carcinoma cell line NCE was divided into groups A (treated with 40 μmol/L compound 18), B (treated with DMSO 10 μL), and C (without treatment), and the inhibition of cellular proliferation were detected by Western blotting at 12, 24, 48, 72, 96, and 120 h after treatment. The cellular proliferation in group A was remarkably inhibited at all time points, and the inhibitory rate increased from 16.3 to 96.4 %. Under an optical microscope, the growth of NCE cells was inhibited, and the morphology changed after treatment. The Western blotting results showed that 18 could downregulate the expression of ERK and Cyclin D1 in a time-dependent manner. The data suggest that 18 could be used for treating nasopharyngeal carcinoma by downregulating the expression of ERK and Cyclin D1 in nasopharyngeal carcinoma cells [52]. In another study, an MTT assay was used to detect the effect of 18 on cellular proliferation, microscopy was used to observe cytotoxicity, and flow cytometry was used to detect apoptosis and cell cycle inhibition. The results showed that, within 24 h of treatment, 40 and 80 μmol/L of 18 inhibited the proliferation of NEC-2 cells by 41.21 ± 0.25 and 94.11 ± 0.37 %, respectively. Both doses could block more than 70 % of cells at G0/G1. After 24 h of treatment with 18 (40 μmol/L), cell atrophy appeared, cell adhesion decreased, and some cells died from fragmentation [64].

I. latifolia for 2 months is sufficient to reduce hypertension nearly as well as or better than common hypertension medications such as nifedipine [57, 58]. More importantly, no adverse reaction was observed. These studies suggested that I. latifolia and I. kudingcha have potential use in the treatment of hypertension. The relationship between the large-leaved Kudingcha and related teas Interestingly, the large-leaved Kudingcha from the genus Ilex has many similarities to Yerba maté tea (I. paraguariensis) from South America and the worldwide popular green tea (C. sinensis), a member of the Theaceae family [59]. These 3 teas have all been historically used and have shown antioxidant, lipid-lowering, anti-inflammatory, weight-reducing, anti-tumor, and anti-glycation properties [2, 5, 6]. These similarities could be ascribed to their similar phylogeny and chemical compositions.

C. sinensis), a member of the Theaceae family [59]. These 3 teas have all been historically used and have shown antioxidant, lipid-lowering, anti-inflammatory, weight-reducing, anti-tumor, and anti-glycation properties [2, 5, 6]. These similarities could be ascribed to their similar phylogeny and chemical compositions. In order to better understand the relationship among these teas, further comparison of the main chemical constituents of the large-leaved Kudingcha and the other 2 teas have been discussed. Most notably, the common chemical characters in regular tea are caffeine, polyphenols, and catechins. Caffeine, which is related to weight reduction, is the highest in Yerba maté tea, medium in the green tea, and could not be found in the large-leaved Kudingcha [60, 61]. Polyphenols with conformed antioxidant activities are also the highest in Yerba maté tea, followed by green tea and large-leaved Kudingcha. The primary polyphenols in the large-leaved Kudingcha are 3,5-di-O-caffeoylquinic acid and related dicaffeoylquinic acids, whereas chlorogenic acid is the main polyphenol in Yerba maté tea. Catechins, which have anti-tumor properties, are the highest in green tea, followed by the large-leaved Kudingcha and Yerba maté tea [32, 62, 63]. Moreover, the saponins were thought to be the source of the bitter flavor and one of the important characteristics in the large-leaved Kudingcha and Yerba maté teas. The aroma components in Ilex and green tea also had several similarities.

een tea, followed by the large-leaved Kudingcha and Yerba maté tea [32, 62, 63]. Moreover, the saponins were thought to be the source of the bitter flavor and one of the important characteristics in the large-leaved Kudingcha and Yerba maté teas. The aroma components in Ilex and green tea also had several similarities. The long history, similar chemical structure, and common pharmaceutical activities suggest a close genetic relationship among the large-leaved Kudingcha, Yerba maté tea, and green tea. Although the large-leaved Kudingcha is not as popular worldwide, we should pay more attention to its possible benefits and advantages, such as the lack of caffeine that is found in more ordinary teas. Conclusions A large number of phytochemical studies have led to the identification of multiple constituents of the large-leaved Kudingcha that confer properties such as lipid metabolism, vascular system protection, antioxidant, anti-asthma, anti-tussive, and anti-tumor activities. A critical assessment of the results presented in this review may provide scientific evidence for reasonable utilization of the original plants of the large-leaved Kudingcha and promote further investigation for the development of new herbal medicine and tea products. More pharmacological activities need to be undertaken and relevant mechanisms understood in order to enhance the utilization and quality of Kudingcha species in the future. This research was supported by the National Natural Science Foundation of China (no. 81274188).

Introduction Gypsum is a crude mineral drug used in traditional Japanese kampo medicine and traditional Chinese medicine. In the Japanese Pharmacopoeia XVI Edition (JPXVI) [1], gypsum is defined as natural hydrous calcium sulfate and is prescribed in 13 of the over-the-counter kampo formulas permitted by the Ministry of Health, Labour and Welfare of Japan. According to the theory of kampo medicine, gypsum clears “heat”, drains “fire”, and promotes “fluid” production, which leads to treatment of symptoms including profuse sweating, oral dryness, severe thirst, feeling of heat or burning skin, and itching [2]. Ikarashi et al. [3] reported that in mice given a diet containing 0.3 % gypsum powder for 4 weeks, the expression levels of aquaporin 3 mRNA and protein in the skin were increased. Since aquaporin 3 plays an important role in maintaining skin hydration, the traditional usage of gypsum may be related to this pharmacological effect.

al. [3] reported that in mice given a diet containing 0.3 % gypsum powder for 4 weeks, the expression levels of aquaporin 3 mRNA and protein in the skin were increased. Since aquaporin 3 plays an important role in maintaining skin hydration, the traditional usage of gypsum may be related to this pharmacological effect. In kampo medicine, prescribed crude drugs in formulas are mostly boiled in water, and the resulting decoction is administered to the patient after removing the residues by filtration. Since the solubility of CaSO4 in water is low (~1.6 g/l at 100 °C) and since gypsum contains few organic compounds, the pharmacological role of gypsum prescribed in kampo formulas remains unknown, and some researchers have attempted to solve it. There have been some reports describing the interaction of gypsum with other crude drugs prescribed together in kampo formulas. For example, gypsum counteracted the thermogenous effect of Ephedra Herb (terrestrial stem of Ephedra sinica) when administered simultaneously to rats [4]. The suppressive effect of byakkokaninjinto, a kampo formula consisting of Gypsum and four other crude drugs, on immunoglobulin E (IgE)-mediated triphasic skin reaction in mice disappeared when gypsum was removed from the formula [5]. It was also reported that gypsum affected the extraction efficiency of organic compounds from the crude drugs prescribed in the formula. When chotosan, a kampo formula containing gypsum, Uncaria Hook (hook of Uncaria rhynchophylla), and nine other crude drugs, was decocted without gypsum, the content of alkaloids derived from Uncaria Hook in the filtered decoction was significantly decreased [6]. In makyokansekito, a kampo formula containing gypsum, Ephedra Herb, Apricot Kernel (seed of Prunus armeniaca), and Glycyrrhiza (root and stolon of Glycyrrhiza uralensis), the elution of alkaloids from Ephedra Herb significantly decreased when the formula was boiled without gypsum [7].

n was significantly decreased [6]. In makyokansekito, a kampo formula containing gypsum, Ephedra Herb, Apricot Kernel (seed of Prunus armeniaca), and Glycyrrhiza (root and stolon of Glycyrrhiza uralensis), the elution of alkaloids from Ephedra Herb significantly decreased when the formula was boiled without gypsum [7]. The dosage of gypsum administered as kampo formulas is usually 5–15 g/day. However, physicians using kampo medicines have found clinically that a much higher gypsum dosage markedly enhances the “heat”-clearing effect of the kampo formula. Imai et al. [8] reported that inflammatory skin lesions in patients with atopic dermatitis recovered when the gypsum dosage was increased from 15 to 100 g/day in byakkokakeishito. However, there is no experimental evidence for the anti-allergic effects of byakkokakeishito or for the effectiveness of increasing the gypsum dosage in byakkokakeishito. In the present study, we compared the anti-allergic activities of byakkokakeishito extracts (BKTs) prepared with varied amounts of gypsum to clarify the pharmacological role of gypsum in kampo formulas using three different murine models of allergy: contact dermatitis induced by painting hapten onto skin; allergic dermatitis induced by cutaneous injection of mite antigen; and skin passive cutaneous anaphylaxis (PCA) reaction using ovalbumin (OVA) as antigen.

larify the pharmacological role of gypsum in kampo formulas using three different murine models of allergy: contact dermatitis induced by painting hapten onto skin; allergic dermatitis induced by cutaneous injection of mite antigen; and skin passive cutaneous anaphylaxis (PCA) reaction using ovalbumin (OVA) as antigen. Materials and methods Preparation of herbal formulas Gypsum (lot No. 7H16 M, natural hydrous calcium sulfate), Anemarrhena Rhizome (0E22, the rhizome of Anemarrhena asphodeloides), Cinnamon Bark (6K20 M, the bark of the trunk of Cinnamomum cassia, Chinese cinnamon), and Oriza Seed (5G29, the seed of Oryza sativa, Asian rice) were purchased from Daiko Shoyaku (Nagoya, Japan), and Glycyrrhiza (23040711, the root and stolon of Glycyrrhiza uralensis, Chinese liquorice) was from Tsumura (Tokyo, Japan). These crude drugs were of JPXVI grade and used as small pieces prepared by cutting or crushing of the whole crude drugs. The voucher specimens are deposited in our laboratory.

ku (Nagoya, Japan), and Glycyrrhiza (23040711, the root and stolon of Glycyrrhiza uralensis, Chinese liquorice) was from Tsumura (Tokyo, Japan). These crude drugs were of JPXVI grade and used as small pieces prepared by cutting or crushing of the whole crude drugs. The voucher specimens are deposited in our laboratory. Tables 1 and 2 show the components of BKT samples used in the present study. The standard gypsum dosage administered as BKT is 15 g/day according to an authentic textbook on kampo medicine [9]. The crude drugs corresponding to their daily dosage were packed together in paper bags and boiled in 600 ml of distilled water for 60 min. The crude drugs were then removed from the paper bags, and the decoctions were lyophilized. The dried extracts were stored at room temperature in a desiccator until use. Tables 1 and 2 also show the yields of the extracts.Table 1 Components of crude drugs in various byakkokakeishito extract (BKT) samples BKT0a BKT7.5 BKT15b BKT30 BKT60 Gypsum – 7.5 gc 15 g 30 g 60 g Oriza seed 8.0 g 8.0 g 8.0 g 8.0 g 8.0 g Anemarrhena rhizome 5.0 g 5.0 g 5.0 g 5.0 g 5.0 g Cinnamon bark 4.0 g 4.0 g 4.0 g 4.0 g 4.0 g Glycyrrhiza 2.0 g 2.0 g 2.0 g 2.0 g 2.0 g Ratio of yieldd 18.3 % 14.8 % 13.2 % 9.4 % 6.0 % Daily dosage of extract administered to micee 0.70 g/kg – 0.90 g/kg – 0.95 g/kg aName of the extract prepared by decocting a mixture of four or five crude drugs as shown in the Table bThis formula is recognized as the standard byakkokakeishito formula in the textbook of kampo medicine [9] cWeight of each crude drug when administered at the human daily dosage

BKT0a BKT7.5 BKT15b BKT30 BKT60 Gypsum – 7.5 gc 15 g 30 g 60 g Oriza seed 8.0 g 8.0 g 8.0 g 8.0 g 8.0 g Anemarrhena rhizome 5.0 g 5.0 g 5.0 g 5.0 g 5.0 g Cinnamon bark 4.0 g 4.0 g 4.0 g 4.0 g 4.0 g Glycyrrhiza 2.0 g 2.0 g 2.0 g 2.0 g 2.0 g Ratio of yieldd 18.3 % 14.8 % 13.2 % 9.4 % 6.0 % Daily dosage of extract administered to micee 0.70 g/kg – 0.90 g/kg – 0.95 g/kg aName of the extract prepared by decocting a mixture of four or five crude drugs as shown in the Table bThis formula is recognized as the standard byakkokakeishito formula in the textbook of kampo medicine [9] cWeight of each crude drug when administered at the human daily dosage dRatio of yield of the extract calculated as % of dried weight of the extract to the weight of a mixture of crude drugs eCalculated as ten times the human daily dosage. For example, for BKT15, sum of crude drugs (34 g) times ratio of yield (13.2 %) divided by human body weight (50 kg) × 10 = 0.898 g/kg = 0.90 g/kg Table 2 Components of crude drugs in the various byakkokakeishito extracts (BKT) BKT60a BKT-O BKT-A BKT-C BKT-G A A+G60 G60 Gypsum (G) 60 gb 60 g 60 g 60 g 60 g – 60 g 60 g Oriza seed (O) 8.0 g – 8.0 g 8.0 g 8.0 g – – – Anemarrhena rhizome (A) 5.0 g 5.0 g – 5.0 g 5.0 g 5.0 g 5.0 g – Cinnamon bark (C) 4.0 g 4.0 g 4.0 g – 4.0 g – – – Glycyrrhiza (G) 2.0 g 2.0 g 2.0 g 2.0 g – – – – Ratio of yieldc 6.0 % 6.1 % 3.6 % 5.2 % 6.0 % 45.2 % 5.1 % 0.90 % Dosage of extract administered to miced 0.47 g/kg 0.43 g/kg 0.27 g/kg 0.39 g/kg 0.46 g/kg 0.23 g/kg 0.33 g/kg 0.054 g/kg

g 5.0 g – 5.0 g 5.0 g 5.0 g 5.0 g – Cinnamon bark (C) 4.0 g 4.0 g 4.0 g – 4.0 g – – – Glycyrrhiza (G) 2.0 g 2.0 g 2.0 g 2.0 g – – – – Ratio of yieldc 6.0 % 6.1 % 3.6 % 5.2 % 6.0 % 45.2 % 5.1 % 0.90 % Dosage of extract administered to miced 0.47 g/kg 0.43 g/kg 0.27 g/kg 0.39 g/kg 0.46 g/kg 0.23 g/kg 0.33 g/kg 0.054 g/kg aName of the extract prepared by decocting a mixture of four or five crude drugs as shown in the Table bWeight of each crude drug when administered at the human daily dosage cRatio of yield of the extract calculated as % of dried weight of the extract to the weight of a mixture of crude drugs dCalculated as five times the human daily dosage. For example, for BKT60, sum of crude drugs (79 g) times ratio of yield (6.0 %) divided by human body weight (50 kg) × 5 = 0.47 g/kg Determination of calcium in BKT samples The lyophilized BKT samples (10 mg) were dissolved in 100 ml distilled water, and the calcium concentrations were measured using an atomic absorption photometer (AA-660, Shimadzu, Kyoto, Japan). The measurement was repeated three times, and data are represented as mean ± S.E.

dCalculated as five times the human daily dosage. For example, for BKT60, sum of crude drugs (79 g) times ratio of yield (6.0 %) divided by human body weight (50 kg) × 5 = 0.47 g/kg Determination of calcium in BKT samples The lyophilized BKT samples (10 mg) were dissolved in 100 ml distilled water, and the calcium concentrations were measured using an atomic absorption photometer (AA-660, Shimadzu, Kyoto, Japan). The measurement was repeated three times, and data are represented as mean ± S.E. HPLC analysis An amount of each BKT sample corresponding to 1 % of the human daily dosage was sonicated in MeOH (5 ml) for 30 min and centrifuged at 12,000g for 7 min. The supernatant fraction (25 μl) was subjected to HPLC analysis using the following conditions: system, Shimadzu LC-10AVP; column, TSK-GEL ODS-80TS (4.6 × 250 mm; Tosoh, Tokyo, Japan); mobile phase, 0.05 M AcONH4 (pH 3.6)/CH3CN 90:10 → 45:55 (0 → 30 min), linear gradient; flow rate, 1.0 ml/min; column temperature, 40 °C; detection, 200–400 nm using a photodiode array detector. Peaks were identified on the basis of the comparison with the retention times and UV spectra of authentic compounds.

Japan); mobile phase, 0.05 M AcONH4 (pH 3.6)/CH3CN 90:10 → 45:55 (0 → 30 min), linear gradient; flow rate, 1.0 ml/min; column temperature, 40 °C; detection, 200–400 nm using a photodiode array detector. Peaks were identified on the basis of the comparison with the retention times and UV spectra of authentic compounds. Quantitative analysis of timosaponin A-III An amount of each BKT sample corresponding to 4 % of the human daily dosage was sonicated in MeOH (40 ml) for 30 min. A 100-μl aliquot of the sample or the standard solution of timosaponin A-III (Wako Pure Chemicals, Osaka, Japan) was mixed with 100 μl MeOH containing 1 ng/ml astragaloside IV (Wako Pure Chemicals) as an internal standard. Timosaponin A-III concentration was determined using an LC/MS/MS system (Waters Quattro Premier XE; Milford, MA, USA) with an electrospray ionization source in the positive ion mode and multiple reaction monitoring. HPLC separation was performed under the following conditions: column, Inertosil ODS-3 (4.6 × 250 mm, GL Science, Tokyo, Japan); mobile phase, a linear gradient elution system, 0.1 % AcOH in H2O (solvent A):0.1 % AcOH in acetonitrile (solvent B) (B/A) = 40/60 → 40/60 for 0 → 2 min; 40/60 → 90/10 for 2 → 4 min; 90/10 → 90/10 for 4 → 6 min at a flow rate of 200 μl/min. The injection volume of the sample was 10 μl. Both quadrupoles were maintained at the unit resolution and the transitions (precursor to daughter) monitored were m/z 741.5–84.8 for timosaponin A-III (retention time, 5.5 min) and m/z 785.4–143.0 for astragaloside IV (2.8 min). Linear regression in a concentration range of 0.1–5 ng/ml for timosaponin A-III was calibrated by the peak area ratio of these compounds to astragaloside IV by the least-squares method (r2 > 0.999).

d were m/z 741.5–84.8 for timosaponin A-III (retention time, 5.5 min) and m/z 785.4–143.0 for astragaloside IV (2.8 min). Linear regression in a concentration range of 0.1–5 ng/ml for timosaponin A-III was calibrated by the peak area ratio of these compounds to astragaloside IV by the least-squares method (r2 > 0.999). Animal experiments Balb/c and ddY mice were purchased from Japan SLC (Hamamatsu, Japan). NC/Jic mice were obtained from Clea Japan Inc. (Tokyo, Japan). They were housed in a temperature-controlled room (at 23 ± 1 °C) with lighting from 7 a.m. to 7 p.m. and allowed free access to food (CE-2; Oriental Yeast, Tokyo, Japan) and water under conventional conditions. Lyophilized BKT samples were suspended in distilled water at prescribed concentrations and orally administered to mice. The experimental procedures were approved by the Animal Care Committee in Graduate School of Pharmaceutical Sciences, Nagoya City University.

st, Tokyo, Japan) and water under conventional conditions. Lyophilized BKT samples were suspended in distilled water at prescribed concentrations and orally administered to mice. The experimental procedures were approved by the Animal Care Committee in Graduate School of Pharmaceutical Sciences, Nagoya City University. Contact dermatitis model The contact dermatitis model was prepared according to the method of Yamashita et al. [10] with slight modifications. Male Balb/c mice (6–7 weeks old) were initially sensitized by painting 100 μl of 2,4,6-trinitrochlorobenzene (TNCB; Nacalai Tesque, Kyoto, Japan) solution in acetone (50 mg/ml) onto their abdomens. 7 days after sensitization, the mice were challenged by painting 10 μl of TNCB solution in olive oil (10 mg/ml) on both sides of their right ears. In the normal group, each vehicle was painted on murine abdomens and ears. The thicknesses of both ears were measured using a dial thickness gauge (Peacok model G-1A, Ozaki MFJ; Tokyo, Japan) 24 h after challenge, and data are expressed as the difference in the thickness of the two ears. The BKT samples were suspended in water and administered once a day from 3 days before until 6 days after sensitization.

s of both ears were measured using a dial thickness gauge (Peacok model G-1A, Ozaki MFJ; Tokyo, Japan) 24 h after challenge, and data are expressed as the difference in the thickness of the two ears. The BKT samples were suspended in water and administered once a day from 3 days before until 6 days after sensitization. Mite antigen-induced atopic dermatitis-like skin lesion model The model was prepared as described previously [11]. In brief, Dermatophagoides farinae allergenic extract (Greer, Lenoir, NC, USA) (mite antigen) was diluted to 1 mg/ml in saline, and intracutaneously injected into the right ears of the mice (10 μl/ear) on days 0 (first injection), 2, 4, 7, 9, 11, 14, and 16. The BKT samples were suspended in water and orally administered to the mice once a day from 7 days before until 16 days after the first injection. Ear thickness was measured using a dial thickness gauge 1 and 24 h after injection, and data are expressed as the difference from the values before the first injection. The mice were killed by CO2 inhalation on day 17, and blood was collected. The area under the curve (AUC) from day 0 to day 17 after the first injection was calculated using the trapezoidal rule. IgE in the serum was measured by sandwich enzyme-linked immunosorbent assay.

nce from the values before the first injection. The mice were killed by CO2 inhalation on day 17, and blood was collected. The area under the curve (AUC) from day 0 to day 17 after the first injection was calculated using the trapezoidal rule. IgE in the serum was measured by sandwich enzyme-linked immunosorbent assay. PCA reaction model The PCA reaction model was prepared as described previously [12]. In brief, OVA (Seikagaku Corporation, Tokyo, Japan) was suspended in 20 mg/ml Al(OH)3 solution (final concentration of OVA, 10 μg/ml), and 0.2 ml of this suspension (OVA, 2 μg) was injected intraperitoneally into 4-week-old female Balb/c mice five times at 2-week intervals. 10 days after the fifth injection of OVA, whole blood was collected to obtain anti-OVA serum. A 10-μl aliquot of 30-fold diluted anti-OVA serum was injected intracutaneously into each ear of 6-week-old male ddY mice After 48 h, OVA (10 mg/kg body weight) in saline containing 0.5 % Evans blue (Wako Pure Chemicals) was injected intravenously to induce PCA reaction (antigen challenge). The BKT samples were suspended in water and administered orally to overnight-fasted mice 2 h before antigen challenge. The mice were killed by cervical dislocation 30 min after the antigen challenge. Their ears were removed, immersed in 0.3 ml of 1 M KOH overnight at 37 °C, and Evans blue in the ears was extracted with 0.7 ml of acetone/3.3 M phosphoric acid (67:3). Evans blue concentration was measured colorimetrically at 620 nm. Data are expressed as quantity of Evans blue per gram fresh weight of ears.

hallenge. Their ears were removed, immersed in 0.3 ml of 1 M KOH overnight at 37 °C, and Evans blue in the ears was extracted with 0.7 ml of acetone/3.3 M phosphoric acid (67:3). Evans blue concentration was measured colorimetrically at 620 nm. Data are expressed as quantity of Evans blue per gram fresh weight of ears. Statistical analyses Statistical analyses were performed by repeated one-way analysis of variance and Bonferroni/Dunnett’s multiple t test using PASW Statistics (version 18, SPSS; IBM, Armonk, NY, USA). A probability value <0.05 was considered statistically significant.

hallenge. Their ears were removed, immersed in 0.3 ml of 1 M KOH overnight at 37 °C, and Evans blue in the ears was extracted with 0.7 ml of acetone/3.3 M phosphoric acid (67:3). Evans blue concentration was measured colorimetrically at 620 nm. Data are expressed as quantity of Evans blue per gram fresh weight of ears. Statistical analyses Statistical analyses were performed by repeated one-way analysis of variance and Bonferroni/Dunnett’s multiple t test using PASW Statistics (version 18, SPSS; IBM, Armonk, NY, USA). A probability value <0.05 was considered statistically significant. Results Constituents of BKT samples Various BKT samples were prepared by changing the amount of gypsum (Table 1). The BKT sample prepared in order to provide the equivalent of the human daily dosage of gypsum (15 g/day) was recognized as a standard in a textbook of kampo medicine. The calcium content in the BKT samples increased as gypsum content increased (Fig. 1a). No apparent differences in HPLC profiles were observed between the BKT samples prepared with various amounts of gypsum (Fig. 1b). The amount of gypsum present in BKT did not affect the timosaponin A-III content, and BKT prepared without gypsum (BKT0), BKT15, and BKT prepared with 60 g/kg of gypsum (BKT60) contained 3.56, 3.43, and 3.39 mg of timosaponin A-III, respectively, in the amount of human daily dosage.Fig. 1 Calcium content (a) and HPLC chromatograms (b) of byakkokakeishito extracts (BKTs) containing different contents of gypsum. a Modified BKT samples were prepared by decocting crude drugs as shown in Table 1, and calcium contents were measured. Each column represents mean ± S.E. of three samples. b Each BKT sample was dissolved in MeOH and analyzed by HPLC as described in “Materials and Methods”. Chromatograms are shown as the wavelength at 254 nm. Peaks were identified as mangiferin-7-O-glucoside (1), mangiferin (2), liquiritin (3), glycyroside (4), isoliquiritin (5), liquiritigenin (6), glycyrrhizin (7), and cinnamaldehyde (8)

was dissolved in MeOH and analyzed by HPLC as described in “Materials and Methods”. Chromatograms are shown as the wavelength at 254 nm. Peaks were identified as mangiferin-7-O-glucoside (1), mangiferin (2), liquiritin (3), glycyroside (4), isoliquiritin (5), liquiritigenin (6), glycyrrhizin (7), and cinnamaldehyde (8) Contact dermatitis model Daily oral administration of BKT15 significantly suppressed ear swelling induced by TNCB in a dose-dependent manner (Fig. 2). We then compared the effect of modified BKT samples containing varying amounts of gypsum. The BKT samples were administered orally to mice at dosages corresponding to the BKT15 dosage (0.9 g/kg/day). The suppressive effects of BKT on the contact dermatitis model were not significantly different between the BKT samples containing different amounts of gypsum, and gypsum itself did not show any effects on murine contact dermatitis (data not shown).Fig. 2 Effect of byakkokakeishito extracts (BKTs) on 2,4,6-trinitrochlorobenzene (TNCB)-induced contact hypersensitivity in male Balb/c mice. Ear swelling was measured 24 h after the sensitization. The daily dosage of BKT15 (0.90 g/kg) corresponds to ten times that of the human daily dose. Each column represents mean ± S.E. of 8 mice. *p < 0.05 vs. control by Bonferroni/Dunnett’s multiple t test

ne (TNCB)-induced contact hypersensitivity in male Balb/c mice. Ear swelling was measured 24 h after the sensitization. The daily dosage of BKT15 (0.90 g/kg) corresponds to ten times that of the human daily dose. Each column represents mean ± S.E. of 8 mice. *p < 0.05 vs. control by Bonferroni/Dunnett’s multiple t test Mite antigen-induced atopic dermatitis-like skin lesion model Daily oral administration of BKT15 resulted in a significant decrease in ear swelling on days 15 and 17 after the first injection of the mite antigen (Fig. 3a), and BKT15 (0.90 g/kg/day) significantly suppressed the level of ear swelling evaluated as AUC from day 0 to day 17 (Fig. 3b). BKT15 suppressed serum IgE levels on day 17 in a dose-dependent manner, and mice treated with BKT15 (0.90 g/kg/day) exhibited a significant difference (Fig. 3c). We then compared the effect of modified BKT samples containing varying amounts of gypsum at a dosage corresponding to the BKT15 dosage (0.90 g/kg/day). The suppressive effects on ear swelling and serum IgE levels were not significantly different between the BKT samples containing varying amounts of gypsum (data not shown).Fig. 3 Effect of byakkokakeishito extracts (BKTs) on the ear swelling of NC/Jic mice topically treated with mite antigen. Time course of ear swelling in the NC/Jic mice (a), its area under the curve (AUC) for 17 days (b), and serum IgE levels on day 17 (c) are shown. The daily dose of BKT15 (0.90 g/kg) corresponds to ten times that of the human daily dose. Each column represents mean ± S.E. of 8 mice. *p < 0.05 vs. control by Bonferroni/Dunnett’s multiple t test

ing in the NC/Jic mice (a), its area under the curve (AUC) for 17 days (b), and serum IgE levels on day 17 (c) are shown. The daily dose of BKT15 (0.90 g/kg) corresponds to ten times that of the human daily dose. Each column represents mean ± S.E. of 8 mice. *p < 0.05 vs. control by Bonferroni/Dunnett’s multiple t test PCA reaction model A single oral administration of BKT15 to mice significantly suppressed the ear PCA reaction in a dose-dependent manner (Fig. 4a). We then compared the effect of BKT containing varying amounts of gypsum at a dosage corresponding to the BKT15 dosage (0.45 g/kg). BKT0 did not suppress the PCA reaction, while BKT60 significantly reduced dye elution in the ears of mice (Fig. 4b).Fig. 4 Effect of byakkokakeishito extracts (BKTs) on the PCA reaction in ddY mice. Dose-dependent suppressive effects of BKT15 on the PCA reaction were evaluated (a). The daily dose of BKT15 (0.45 g/kg) corresponds to five times that of the human daily dose. The suppressive effects of modified BKT samples shown in Table 1 on the PCA reaction at the dose corresponding to the dose of BKT15 (0.45 g/kg) were evaluated (b). The suppressive effects of modified BKT samples shown in Table 2 on the PCA reaction at the dose corresponding to the dose of BKT60 (0.47 g/kg) were evaluated (c and d). Each column represents mean ± S.E. of 7 or 8 mice. *p < 0.05 vs. control by Bonferroni/Dunnett’s multiple t test

BKT15 (0.45 g/kg) were evaluated (b). The suppressive effects of modified BKT samples shown in Table 2 on the PCA reaction at the dose corresponding to the dose of BKT60 (0.47 g/kg) were evaluated (c and d). Each column represents mean ± S.E. of 7 or 8 mice. *p < 0.05 vs. control by Bonferroni/Dunnett’s multiple t test We prepared modified BKT60 samples by removing one crude drug from the byakkokakeishito formula at a dosage corresponding to the BKT60 dosage (0.47 g/kg/day) (Table 2). BKT60 prepared without Oriza Seed exhibited a suppressive effect similar to that shown by the original BKT60. BKT60 prepared without Cinnamon Bark or Glycyrrhiza exhibited slightly less suppressive effect compared with the original BKT60. BKT60 prepared without Anemarrhena Rhizome lost its suppressive effect on the PCA reaction, and the dye elution was same as that in the control group (Fig. 4c). Although neither Anemarrhena Rhizome nor gypsum significantly suppressed the PCA reaction, the extract prepared from a mixture of Anemarrhena Rhizome and gypsum exhibited an effect comparable to that of BKT60 (Fig. 4d).

ive effect on the PCA reaction, and the dye elution was same as that in the control group (Fig. 4c). Although neither Anemarrhena Rhizome nor gypsum significantly suppressed the PCA reaction, the extract prepared from a mixture of Anemarrhena Rhizome and gypsum exhibited an effect comparable to that of BKT60 (Fig. 4d). Discussion In the present study, by using three different murine models of allergy, we have unambiguously shown for the first time that byakkokakeishito exhibited significant anti-allergic activities. It has been reported that byakkokaninjinto, another kampo formula containing gypsum, ameliorated symptoms of atopic dermatitis and inflammation in mice [5, 13]. The suppressive effect of byakkokaninjinto on IgE-mediated triphasic skin reaction in mice disappeared when byakkokaninjinto prepared without gypsum was administered [5]. These results may indicate that gypsum participates in the anti-allergic effects of the kampo formulas. Therefore, we evaluated how the anti-allergic activity of BKT in the animal model is affected by changing the amount of gypsum in BKT.

e disappeared when byakkokaninjinto prepared without gypsum was administered [5]. These results may indicate that gypsum participates in the anti-allergic effects of the kampo formulas. Therefore, we evaluated how the anti-allergic activity of BKT in the animal model is affected by changing the amount of gypsum in BKT. In the PCA reaction model, the anti-allergic effect of BKT was significantly augmented by increasing the amount of gypsum. This effect of gypsum may be due to the increased extraction of low-molecular-weight organic compounds from other crude drugs prescribed in BKT together with gypsum. In fact, the concentration of Ephedra alkaloids of makyokansekito, a kampo formula comprising gypsum, Ephedra Herb, Armenia Seed, and Glycyrrhiza, was significantly decreased when prepared without gypsum [7]. However, in the present investigation neither the HPLC profiles nor the timosaponin A-III content in BKT was affected by the amount of gypsum in the formula.

s of makyokansekito, a kampo formula comprising gypsum, Ephedra Herb, Armenia Seed, and Glycyrrhiza, was significantly decreased when prepared without gypsum [7]. However, in the present investigation neither the HPLC profiles nor the timosaponin A-III content in BKT was affected by the amount of gypsum in the formula. Some physicians believe that there is no benefit in increasing the gypsum dosage to more than 20 g/day in the prescription because calcium concentration in water reaches saturation at this dosage [14]. However, the present study revealed that the calcium concentration in BKT was increased dose-dependently up to at least 60 g/day of gypsum when the crude drugs were boiled in 600 ml water, and the concentration did not reach the saturation level. The increased calcium concentration in BKT may have enhanced the anti-allergic effect of BKT in the PCA reaction when the amount of gypsum was increased. It has been reported that calcium promotes passive transport of drugs through the intestinal epithelial membrane in rats [15]. Mace et al. [16] revealed that a high calcium concentration in the intestinal lumen induces glucose transporter 2 (GLUT2) insertion into the apical membrane of intestinal endothelial cells and stimulates glucose absorption in a rat jejunal loop model. It is suggested that some types of glycosides in plant materials might pass through the intestinal membrane passively via GLUT2 [17]. In BKT, calcium may promote intestinal absorption of some anti-allergic compounds derived from the four other prescribed crude drugs forming BKT, apart from gypsum.

t jejunal loop model. It is suggested that some types of glycosides in plant materials might pass through the intestinal membrane passively via GLUT2 [17]. In BKT, calcium may promote intestinal absorption of some anti-allergic compounds derived from the four other prescribed crude drugs forming BKT, apart from gypsum. Increasing the amount of gypsum did not affect the anti-allergic activity of BKT in the contact dermatitis model or the mite antigen-induced atopic dermatitis model. In the PCA reaction model, the drug was administered to mice as a single dose 2 h before antigen challenge, and dye efflux was estimated. In contrast, in the other two models, BKT was administered to mice once a day for 9 days (contact dermatitis model) or 23 days (mite antigen-induced atopic dermatitis model), and anti-allergic activity was evaluated about 24 h after the previous administration of BKT. Therefore, the blood concentration of active ingredients in BKT at the evaluation of the activity in the latter two models would be stable and at a lower level than that in PCA reaction model. Hence, the promotion of intestinal absorption of active ingredients by the increased calcium concentration might not affect the blood concentration at the time of evaluation in the latter two models, whereas in the PCA reaction model, increased intestinal absorption of the active compounds by calcium might lead to elevated blood concentrations of the active ingredients and thereby to enhanced anti-allergic activity of BKT.

might not affect the blood concentration at the time of evaluation in the latter two models, whereas in the PCA reaction model, increased intestinal absorption of the active compounds by calcium might lead to elevated blood concentrations of the active ingredients and thereby to enhanced anti-allergic activity of BKT. The modified BKT samples prepared without Anemarrhena Rhizome or gypsum did not show significant anti-allergic effects in the PCA model. Furthermore, the extract prepared by decocting a mixture of these two crude drugs exhibited an anti-allergic effect comparable to that of BKT, but neither Anemarrhena Rhizome nor gypsum alone exhibited this activity. These results suggest that some of the constituents derived from Anemarrhena Rhizome participate in the anti-allergic effects of BKT, and that calcium derived from gypsum would enhance the suppressive effect of BKT probably by enhancing intestinal absorption of the constituents. Lee et al. [18] estimated anti-allergic effects of several steroidal saponins isolated from Anemarrhena Rhizome and demonstrated that timosaponin A-III exhibits inhibitory activity in the PCA reaction and pruritus. We have shown in the present investigation that the timosaponin A-III content did not differ between BKT samples containing different gypsum contents. It would be interesting to examine whether gypsum promotes intestinal absorption of timosaponin A-III, and this awaits further investigation.

PCA reaction and pruritus. We have shown in the present investigation that the timosaponin A-III content did not differ between BKT samples containing different gypsum contents. It would be interesting to examine whether gypsum promotes intestinal absorption of timosaponin A-III, and this awaits further investigation. In conclusion, BKT exhibits an anti-allergic effect in several murine models. This may provide experimental evidence for the clinical use of BKT in allergic diseases. Gypsum may augment the anti-allergic activity of BKT, presumably by increasing intestinal absorption of Anemarrhena Rhizome-derived active constituents. The authors are grateful to Dr. Sumio Imai, Mr. Nobuyoshi Goto, and Mr. Hideaki Tani for providing us with the interesting clinical case successfully treated with BKT.

Introduction Herbs play an important role in our health and our food and have a variety of culinary and medicinal uses. Although herbs have been in use in the human diet and traditional medicine since antiquity, they have recently become the center of attention of the nutrition-science world because of their potential health benefits and detoxification properties. There are many herbal benefits: they have hypotensive or antihypertensive effects [1, 2] and contain unique anti-oxidants [3], essential oils, vitamins, phytosterols and many other plant-derived nutrients, which help the immune system defend the body against viruses, toxins, bacteria and other germs [4]. In general, medicinal plants and herbal materials may be found with various kinds of microbial contaminants, of which bacterial and fungal infections are regarded as the most common [5]. Beside biological contaminants, herbs may be contaminated by toxic chemical substances such as mycotoxins, heavy metals, pesticides and deposited pesticide residues.

herbal materials may be found with various kinds of microbial contaminants, of which bacterial and fungal infections are regarded as the most common [5]. Beside biological contaminants, herbs may be contaminated by toxic chemical substances such as mycotoxins, heavy metals, pesticides and deposited pesticide residues. Similar to other crops, herbal plants are attacked by insects and diseases both in the field and during storage, and therefore pesticides are widely used for their protection. Attention is focused on pesticide contamination due to its high toxicity and persistence in the environment. Although the use of organochlorine pesticides (OCPs) has been restricted or forbidden by legislation for many years, these compounds are still being detected [6]. Pesticide contaminants may be related to the origin of these herbal plants, such as when they are growing in a contaminated environment, e.g. in soil where banned pesticides, such as DDT [7], have been deposited for many years. During the growing and post-harvest periods herbs can be protected against agrophages through the controlled use of plant protection products (insecticides and herbicides) [8]. This is the first source of pesticide residues. The second source is the uncontrolled application of biopesticides against mosquitoes on large areas of forests.

g and post-harvest periods herbs can be protected against agrophages through the controlled use of plant protection products (insecticides and herbicides) [8]. This is the first source of pesticide residues. The second source is the uncontrolled application of biopesticides against mosquitoes on large areas of forests. It is well known that there are many contaminants and residues that may cause harm to the consumers of herbal medicines [9]. Herbal materials and medicinal plants are also often used as food, functional food, and nutritional and dietary supplements. Thus, medicinal plants and herbal products must be safe for patients and consumers. It is, therefore, essential to establish a convenient quality control method to assure the safety of herbal products. To prevent and screen for pesticide residues and to ensure safety and conformity of quality standards, medicinal herbs and herbal products should be included in the appropriate regulatory framework. Herbs are classified as foodstuffs of plant origin by Regulation (EC) 396/2005 [10] (a herb can be a leaf, flower, stem, seed, root, fruit, bark or any other part of a plant) and as herbal drugs according to the European Pharmacopoeia [11].

and herbal products should be included in the appropriate regulatory framework. Herbs are classified as foodstuffs of plant origin by Regulation (EC) 396/2005 [10] (a herb can be a leaf, flower, stem, seed, root, fruit, bark or any other part of a plant) and as herbal drugs according to the European Pharmacopoeia [11]. In order to ensure consumer safety, authorities in Europe have set maximum residue limits (MRLs) for some pesticides in herbs [12]. Because of the widespread use of plant protection products to protect herbs during cultivation, control of their residues has become a necessity. In cases when herbs are used as medicinal drugs there is a need of guarantee in a form of certificate for pesticide residues. The analytical determination of pesticides in herbs with an unidentified pesticide treatment history is a formidable task, because it involves the identification and quantification of several hundred possible single or combinations of compounds in the presence of complex matrices.

In order to ensure consumer safety, authorities in Europe have set maximum residue limits (MRLs) for some pesticides in herbs [12]. Because of the widespread use of plant protection products to protect herbs during cultivation, control of their residues has become a necessity. In cases when herbs are used as medicinal drugs there is a need of guarantee in a form of certificate for pesticide residues. The analytical determination of pesticides in herbs with an unidentified pesticide treatment history is a formidable task, because it involves the identification and quantification of several hundred possible single or combinations of compounds in the presence of complex matrices. Only a few analytical methods for the determination of pesticide residues in herbs have been described in the recent literature and they are limited to selected compounds or groups [13–15]. In the case of herbs, no more than 30 pesticides were included in a single method. Therefore, it was considered desirable to devise a novel procedure that would allow for screening a much broader range of pesticides (approximately 163) to assure the production of good quality herbal products. The published studies are based on Soxhlet extraction [16], microwave-assisted extraction (MAE) [13] or on the QuChERS method [15], and are very often followed by gas chromatography–mass spectrometry (GC–MS) [17, 18]. Some research works have studied pesticide residues in herbal material and were mainly based on surveying and monitoring market samples [16].

let extraction [16], microwave-assisted extraction (MAE) [13] or on the QuChERS method [15], and are very often followed by gas chromatography–mass spectrometry (GC–MS) [17, 18]. Some research works have studied pesticide residues in herbal material and were mainly based on surveying and monitoring market samples [16]. Herb sample preparation is a crucial step in pesticide residue analysis. In recent times, research has been focusing on those methods which allow for reduction of the organic solvent, and the elimination of the additional sample clean-up and pre-concentration steps before chromatographic analysis [19]. The complexity of the herb matrix is due to the presence of phenolic compounds, carotenoids, chlorophyll and essential oils [20]. In order to eliminate the effects of interference and to avoid the matrix effect it is necessary to develop a sensitive method. To the best of our knowledge, no analytical method has been developed able to simultaneously determine multi-pesticide residues in herbs like linden, lungwort, melissa and peppermint using matrix solid phase dispersion (MSPD).

Herb sample preparation is a crucial step in pesticide residue analysis. In recent times, research has been focusing on those methods which allow for reduction of the organic solvent, and the elimination of the additional sample clean-up and pre-concentration steps before chromatographic analysis [19]. The complexity of the herb matrix is due to the presence of phenolic compounds, carotenoids, chlorophyll and essential oils [20]. In order to eliminate the effects of interference and to avoid the matrix effect it is necessary to develop a sensitive method. To the best of our knowledge, no analytical method has been developed able to simultaneously determine multi-pesticide residues in herbs like linden, lungwort, melissa and peppermint using matrix solid phase dispersion (MSPD). Matrix solid phase dispersion has been used for performing the extraction of a variety of matrices from a number of compounds, e.g. caffeine in green tea leaves [21], rutin in Sambucus nigra L. (elderberry) [22], polybrominated diphenyl ethers and polychlorinated biphenyls in biota samples [23], phenolic compounds in fruit-green tea [24], free fatty acids in chocolate [25] and pesticides in fruits and vegetables [26, 27], soil [28] or bees [29]. However, little is known about the application of MSPD as a sample preparation method for the analysis of various groups of pesticides in herbs. Previous papers adopting this extraction approach refer to only a few pesticides in herbs by GC [30–32].

pesticides in fruits and vegetables [26, 27], soil [28] or bees [29]. However, little is known about the application of MSPD as a sample preparation method for the analysis of various groups of pesticides in herbs. Previous papers adopting this extraction approach refer to only a few pesticides in herbs by GC [30–32]. The main objective of this work was to optimize the process of preparation, extraction and purification of herbal samples using MSPD and liquid–solid extraction (LSE) for qualitative and quantitative analysis of a wide spectrum of pesticide residues. The analytical novelty of this work is the validation of an efficient, sensitive, interference-free, fast and simple MSPD method that would allow determination of over 160 pesticides representing a wide range of physicochemical properties in complex herb matrices. In addition, this paper shows the potential of MSPD as a convenient method for the analysis of a wide range of pesticides in various herbs.

terference-free, fast and simple MSPD method that would allow determination of over 160 pesticides representing a wide range of physicochemical properties in complex herb matrices. In addition, this paper shows the potential of MSPD as a convenient method for the analysis of a wide range of pesticides in various herbs. Experimental Chemicals and materials Acetone, acetonitrile, dichloromethane, diethyl ether, n-hexane and methanol for pesticides residue analysis were provided by J.T. Baker (Deventer, Holland), and Florisil (60–100 mesh), anhydrous sodium sulfate, Celite and octadecyl silica gel C18 (200–400 mesh) were purchased from Fluka (Seelze-Hannover, Germany). Silica gel (230–400 mesh) and neutral aluminum oxide (0.063–0.200 mm) were obtained from Merck (Darmstadt, Germany). Sorbents were activated at 600 °C, with the exception of the neutral aluminum oxide which was activated at 130 °C. Deactivated sorbents were prepared by adding the appropriate amount of distilled water to activated sorbents (for preparation of 5 % neutral aluminum oxide and 4 % Florisil, 5 ml and 4 ml of water was added to obtain 100 g, respectively).

, with the exception of the neutral aluminum oxide which was activated at 130 °C. Deactivated sorbents were prepared by adding the appropriate amount of distilled water to activated sorbents (for preparation of 5 % neutral aluminum oxide and 4 % Florisil, 5 ml and 4 ml of water was added to obtain 100 g, respectively). The 163 pesticide standards were purchased from Dr. Ehrenstorfer Laboratory (Germany). The purities of the standard pesticides ranged from 95 to 99.8 %. Each stock solution at various concentrations was prepared in acetone and stored at 4 °C for further dilution. Multicompound standard working solutions (M1–M4, each containing about forty active substances) were prepared by dissolving 0.2–4.0 ml of each stock solution in an n-hexane/acetone (9:1, v/v) mixture to give a final concentration range of 0.05–1.0 μg/ml. The stock and working solutions were stored in completely filled vials closed with parafilm at −20 °C until analysis.

h containing about forty active substances) were prepared by dissolving 0.2–4.0 ml of each stock solution in an n-hexane/acetone (9:1, v/v) mixture to give a final concentration range of 0.05–1.0 μg/ml. The stock and working solutions were stored in completely filled vials closed with parafilm at −20 °C until analysis. Samples The following herbs were used in the experiment: chamomile (Matricaria chamomilla L.), linden (Tilia), lungwort (Pulmonaria L.), melissa (Melissa L.), peppermint (Mentha piperita L.) and thyme (Thymus vulgaris L.). All of them were cultivated in north-eastern Poland (cultivation year 2010). These samples were used for blanks, fortified samples for recovery assays and matrix-matched standards for calibration in the comparison of methods. About 1 kg portions of the herbs were air-dried (at a temperature of approximately 40 °C), cut and ground. Samples were stored until the moment of extraction at 4 °C, then the plant material was ground and its portion was used in the applied sample preparation procedure. Samples (n = 15) of herbs for the monitoring study were purchased from local producers: chamomile (n = 1), linden (n = 3), lungwort (n = 3), melissa (n = 3), peppermint (n = 3) and thyme (n = 2).

extraction at 4 °C, then the plant material was ground and its portion was used in the applied sample preparation procedure. Samples (n = 15) of herbs for the monitoring study were purchased from local producers: chamomile (n = 1), linden (n = 3), lungwort (n = 3), melissa (n = 3), peppermint (n = 3) and thyme (n = 2). Sample preparation Matrix solid phase dispersion—MSPD (Procedure 1) Two grams of the ground herb sample were put in a mortar with 4 g of solid support (Florisil), and manually blended using a pestle to obtain a homogeneous mixture. After homogenization, the blend was quantitatively transferred with a spatula to a glass macro column packed with anhydrous sodium sulfate (5 g), octadecyl C18 (1 g) and silica gel (2.5 g). The absorbed analytes were then eluted using 25 ml acetone/methanol (9:1, v/v).

ed using a pestle to obtain a homogeneous mixture. After homogenization, the blend was quantitatively transferred with a spatula to a glass macro column packed with anhydrous sodium sulfate (5 g), octadecyl C18 (1 g) and silica gel (2.5 g). The absorbed analytes were then eluted using 25 ml acetone/methanol (9:1, v/v). Liquid–solid extraction—LSE (Procedure 2) Two grams of the ground herb sample were weighed in an Erlenmeyer flask. Extraction was carried out by placing the sample with 50 ml of hexane/diethyl ether/acetone (1:2:2, v/v/v) as an extracting solvent on a rotary shaker (Ika Shaker KS 501 digital) at high speed (2500 rpm) for 30 min. The extract was filtered through a filter with 5 g of Celite and 5 g of anhydrous sodium sulfate, then a 20 ml portion of hexane/diethyl ether/acetone (1:2:2, v/v/v) was added and shaken for another 30 min. Extracts were then combined in the same flask and evaporated until dry using a rotary evaporator at a temperature of about 40 °C. The dry residue was then dissolved in 2 ml of hexane/acetone (9:1, v/v). The extract was cleaned on a chromatography column containing sodium sulfate (2 g), 5 % neutral aluminum oxide (2.5 g) and 4 % Florisil (2 g) using 30 ml hexane/dichloromethane (7:3, v/v).

using a rotary evaporator at a temperature of about 40 °C. The dry residue was then dissolved in 2 ml of hexane/acetone (9:1, v/v). The extract was cleaned on a chromatography column containing sodium sulfate (2 g), 5 % neutral aluminum oxide (2.5 g) and 4 % Florisil (2 g) using 30 ml hexane/dichloromethane (7:3, v/v). The extracts obtained from Procedures 1 and 2 were evaporated until dry using a rotary evaporator at a temperature of approximately 40 °C and the dry residue was re-dissolved in 2 ml of hexane/acetone (9:1, v/v) and then transferred to 2 ml vials for further GC-NP/EC analysis. The stages of both preparation procedures are shown in Fig. 1.Fig. 1 Sample preparation procedures and dual system of detection Preparation of spiked herb samples For both procedures, matrix-matched standards were prepared at concentration levels ranging from 0.05 to 0.5 mg/kg. Blank herb samples (previously tested for the absence of pesticide residues) were used for fortification experiments. Spiked samples were prepared by adding an appropriate volume of spiking solution to exactly weighed portions of milled plant material (2 g) and left for 1 h (to allow pesticide absorption by the sample). Sample preparation was carried out using the two techniques, MSPD and LSE. The main purpose of this step was to calculate the average of the recovery percent of the investigated pesticides through both extraction techniques.

of milled plant material (2 g) and left for 1 h (to allow pesticide absorption by the sample). Sample preparation was carried out using the two techniques, MSPD and LSE. The main purpose of this step was to calculate the average of the recovery percent of the investigated pesticides through both extraction techniques. Chromatographic analysis Pesticide analysis was performed using an Agilent 7890 A gas chromatograph (Santa Clara, CA, USA) equipped with an automatic split–splitless injector Model HP 7683, a 63Ni micro-electron capture detector (μEC) and a nitrogen phosphorous detector (NP). The flux at the end of the GC column was divided into two branches by means of a “Y” press-tight connector connected at one end to the GC column and at the other to the two detectors (Fig. 1). Chemstation chromatography manager data acquisition and processing system (Hewlett-Packard, version A.10.2) was used. Chromatographic separation was performed on an Agilent HP-5 column (30 m, 0.32 mm I.D., 0.25 μm film thickness; Little Falls, DE, USA). When positive peaks of pesticides were detected above LODs, the results were confirmed by analysis on the different polarity column. The DB-35, a midpolarity column (35 %-phenyl-methylpolysiloxane) with low bleed (30 m–0.32 mm I.D., 0.5 μm film thickness) supplied by Agilent (Little Falls, DE, USA), was found ideal for conformational analysis. The operating conditions for GC analysis are given in Table 1. Quantification was performed by comparing the heights of peaks obtained in samples with those found in standards (±0.005 min for positive match).Table 1 Conditions for the injection and GC analysis

lls, DE, USA), was found ideal for conformational analysis. The operating conditions for GC analysis are given in Table 1. Quantification was performed by comparing the heights of peaks obtained in samples with those found in standards (±0.005 min for positive match).Table 1 Conditions for the injection and GC analysis Injection mode EC detector NP detector Column HP-5 DB-35 HP-5 DB-35 Injector temperature program 210 °C 210 °C 210 °C 210 °C Carrier gas (flow-rate) Helium 3.0 ml/min Nitrogen 1.9 ml/min Helium 3.0 ml/min Nitrogen 1.9 ml/min Detector temperature 300 °C 300 °C 300 °C 300 °C Make up gas (flow-rate) Nitrogen 57 ml/min Nitrogen 60 ml/min Nitrogen 8 ml/min, hydrogen 3.0 ml/min, air 60 ml/min Nitrogen 8 ml/min, hydrogen 3.0 ml/min, air 60 ml/min Splitless period (min) 2 2 2 2 Oven temperature program 120–190 °C at 16 °C/min, 230 °C at 8 °C/min to 285 °C at 18 °C/min (13 min) 120–190 °C at 13 °C/min, 240 °C at 8 °C/min to 295 °C at 16 °C/min (15 min) 120–190 °C at 16 °C/min, to 230 °C at 8 °C/min to 285 °C at 18 °C/min (13 min) 120–190 °C at 13 °C/min, 240 °C at 8 °C/min to 295 °C at 16 °C/min (15 min) Injection volume of final extract (μl) 2  2 2 2 Total time for analysis (min) 25.431 30.070 25.431 30.070 Equilibration time (min) 2 2 2 2 Validation of method Blank samples of six different herbs were used to validate the applied methods in accordance with Document SANCO [33].

Injection mode EC detector NP detector Column HP-5 DB-35 HP-5 DB-35 Injector temperature program 210 °C 210 °C 210 °C 210 °C Carrier gas (flow-rate) Helium 3.0 ml/min Nitrogen 1.9 ml/min Helium 3.0 ml/min Nitrogen 1.9 ml/min Detector temperature 300 °C 300 °C 300 °C 300 °C Make up gas (flow-rate) Nitrogen 57 ml/min Nitrogen 60 ml/min Nitrogen 8 ml/min, hydrogen 3.0 ml/min, air 60 ml/min Nitrogen 8 ml/min, hydrogen 3.0 ml/min, air 60 ml/min Splitless period (min) 2 2 2 2 Oven temperature program 120–190 °C at 16 °C/min, 230 °C at 8 °C/min to 285 °C at 18 °C/min (13 min) 120–190 °C at 13 °C/min, 240 °C at 8 °C/min to 295 °C at 16 °C/min (15 min) 120–190 °C at 16 °C/min, to 230 °C at 8 °C/min to 285 °C at 18 °C/min (13 min) 120–190 °C at 13 °C/min, 240 °C at 8 °C/min to 295 °C at 16 °C/min (15 min) Injection volume of final extract (μl) 2  2 2 2 Total time for analysis (min) 25.431 30.070 25.431 30.070 Equilibration time (min) 2 2 2 2 Validation of method Blank samples of six different herbs were used to validate the applied methods in accordance with Document SANCO [33]. Calibration curve and linearity Calibration standards for the analysis of pesticides were prepared in a matrix solution (by adding respective spiking solutions to a blank herb matrix) to produce final concentrations between 0.005 and 2.5 mg/kg. Linearity was determined from the coefficients of determination (R2).

Validation of method Blank samples of six different herbs were used to validate the applied methods in accordance with Document SANCO [33]. Calibration curve and linearity Calibration standards for the analysis of pesticides were prepared in a matrix solution (by adding respective spiking solutions to a blank herb matrix) to produce final concentrations between 0.005 and 2.5 mg/kg. Linearity was determined from the coefficients of determination (R2). Precision and accuracy; LOD and LOQ Repeatability (precision) was calculated for five consecutive days using three replicates of three different concentration levels. Precision and accuracy were evaluated by performing recovery studies of each extraction technique and are expressed as relative standard deviation (RSD, %) and mean recovery, respectively. The limits of detection (LOD) and quantification (LOQ) were calculated using signal-to-noise ratio (S/N) criteria in all cases; LOD = 3 S/N and LOQ = 10 S/N. Recovery study Samples without pesticides were used for fortification experiments. Recovery data was obtained at three different concentrations within the range in the matrix. Blank samples were spiked through the addition of an appropriate volume of a mixture of standard pesticide solution, then the sample was left for 1 h to allow pesticide absorption. The samples were then prepared according to Procedures 1 and 2 described above.

ee different concentrations within the range in the matrix. Blank samples were spiked through the addition of an appropriate volume of a mixture of standard pesticide solution, then the sample was left for 1 h to allow pesticide absorption. The samples were then prepared according to Procedures 1 and 2 described above. Estimation of uncertainty The actions performed during the uncertainty estimation of the analytical result were in accordance with the Guide to the Expression of Uncertainty in Measurement [34]: defining the measuring procedure and determining the measured value; developing a mathematical model to be used for calculating analytical results based on the measured parameters; finding values for all possible parameters that can influence the final results, and estimating the associated standard uncertainties; applying the law of propagation of uncertainty in order to calculate the combined standard uncertainty of the final results. The combined standard uncertainty was determined using ProNP3 (PROLAB) software.

ll possible parameters that can influence the final results, and estimating the associated standard uncertainties; applying the law of propagation of uncertainty in order to calculate the combined standard uncertainty of the final results. The combined standard uncertainty was determined using ProNP3 (PROLAB) software. Results and discussion In this study 163 pesticides (6 acaricides, 62 fungicides, 18 herbicides and 77 insecticides) which may be found in herb samples were investigated using the MSPD and LSE procedures. Because these target analytes represent various substance groups (Table 2) with different physico-chemical properties, development of a simple and reliable multiresidue analytical method to determine pesticide residues in a complex herb matrix was a considerable challenge.Table 2 MSPD validation parameters for 163 active substances of four different herbs (sorted by substance group)

(Table 2) with different physico-chemical properties, development of a simple and reliable multiresidue analytical method to determine pesticide residues in a complex herb matrix was a considerable challenge.Table 2 MSPD validation parameters for 163 active substances of four different herbs (sorted by substance group) No. Substance group Active substance Pesticide type Detector R 2 LOD (mg/kg) LOQ (mg/kg) Linden (Tilia) Lungwort (Pulmonaria L.) Melissa (Melissa L.) Peppermint (Mentha piperita L.) Rec. (n = 3) (%) RSD (%) Rec. (n = 3) (%) RSD (%) Rec. (n = 3) (%) RSD (%) Rec. (n = 3) (%) RSD (%) 1. Acylalanine Benalaxyl F NP 0.99986 0.010 0.020 105 8 104 8 102 2 102 4 2. Alkanamide Napropamide H NP 0.99983 0.010 0.020 95 7 99 9 105 14 100 8 3. Amine Diphenylamine F NP 0.99998 0.010 0.020 86 5 105 3 83 3 100 4 4. Anilinopyrimidine (3) Cyprodinil F NP 0.99600 0.005 0.007 105 7 103 6 106 5 107 5 5. Mepanipyrim F NP 0.99994 0.010 0.020 97 6 98 10 101 2 103 3 6. Pyrimethanil F NP 0.99980 0.005 0.007 84 10 96 12 99 8 97 10 7. Aryloxyphenoxypropionate Fluazifop-p-butyl H NP 0.99738 0.010 0.020 88 13 98 5 92 9 87 6 8. Benzamide Propyzamide H EC/NP 0.99998 0.010 0.020 85 5 100 6 111 8 106 7 9. Benzilate Bromopropylate A EC 0.99988 0.005 0.010 91 8 115 4 107 2 105 6 10. Benzonitrile Dichlobenil H EC/NP 0.99994 0.005 0.010 72 9 105 2 90 11 103 4 11. Bridged diphenyl Tetradifon A EC 0.99686 0.007 0.010 74 2 95 8 103 6 93 8 12. Carbamate (7) Carbaryl I NP 0.99545 0.020 0.030 70 9 103 16 96 1 91 11 13. Carbofuran I, A NP 0.99876 0.010 0.020 77 6 95 10 86 3 85 9 14. Chlorpropham H NP 0.99997 0.008 0.010 103 7 108 3 104 7 101 6 15. Iprovalicarb F NP 0.99998 0.010 0.020 82 5 104 5 106 8 100 3 16. Pirimicarb I NP 1.00000 0.005 0.008 90 6 94 9 98 10 91 7 17. Propham H NP 0.99999 0.008 0.010 78 8 105 12 109 6 103 10 18. Propoxur I, A NP 0.99908 0.008 0.010 80 7 92 14 101 7 92 11 19. Carboxamide (2) Boscalid F EC/NP 0.99953 0.005 0.007 83 10 96 5 97 9 89 3 20. Hexythiazox A EC/NP 0.99976 0.030 0.040 82 5 96 2 91 8 86 5 21. Chlorinated aromatic hydrocarbon HCB F EC 0.99998 0.005 0.007 101 9 96 4 107 13 93 11 22. Chlorinated hydrocarbon Dieldrin I EC 0.99984 0.004 0.008 80 4 87 13 86 14 77 11 23. Chloroacetamide (3) Acetochlor H EC/NP 0.99996 0.010 0.015 108 9 103 5 102 7 106 6 24. Metazachlor H EC/NP 0.99970 0.009 0.010 71 8 77 7 74 3 80 4 25. Propachlor H EC/NP 0.99989 0.010 0.015 105 7 110 9 105 10 104 7 26. Chloronitrile Chlorothalonil F EC/NP 0.99653 0.008 0.010 89 9 85 11 95 15 100 5 27.

3 86 14 77 11 23. Chloroacetamide (3) Acetochlor H EC/NP 0.99996 0.010 0.015 108 9 103 5 102 7 106 6 24. Metazachlor H EC/NP 0.99970 0.009 0.010 71 8 77 7 74 3 80 4 25. Propachlor H EC/NP 0.99989 0.010 0.015 105 7 110 9 105 10 104 7 26. Chloronitrile Chlorothalonil F EC/NP 0.99653 0.008 0.010 89 9 85 11 95 15 100 5 27. Chlorophenyl (5) Dicloran F EC 0.99568 0.008 0.010 74 14 91 16 86 14 94 3 28. Quintozene F EC 0.99942 0.004 0.006 81 4 109 15 72 11 99 2 29. Tecnazene F EC 0.99743 0.005 0.009 98 2 115 2 84 4 104 5 30. Tetrachlorvinphos I, A EC/NP 0.99969 0.008 0.010 92 16 92 3 91 9 96 4 31. Tolclofos-methyl F EC/NP 0.99983 0.005 0.009 83 9 103 5 116 5 93 6 32. Cyanoacetamide ozime Cymoxanil F EC/NP 0.99866 0.040 0.050 98 5 108 7 96 6 102 7 33. Cyclodiene Aldrin I EC 0.99947 0.003 0.005 83 8 86 5 82 13 85 8 34. Dicarboximide (2) Iprodione F EC/NP 0.99967 0.008 0.010 108 8 101 9 115 15 99 6 35. Procymidone F EC/NP 0.99939 0.006 0.010 104 4 113 3 108 5 106 4 36. Dinitroaniline (2) Pendimethalin H EC/NP 0.99999 0.008 0.010 99 9 103 6 79 8 102 4 37. Trifluralin H EC/NP 0.99996 0.007 0.010 108 2 101 2 99 3 100 7 38. Diphenyl ether Nitrofen H EC 0.99913 0.005 0.007 109 3 99 1 89 12 95 3 39. Hydroxyanilide Fenhexamid F EC/NP 0.99742 0.009 0.010 96 1 108 1 106 8 89 5 40. Imidazole (3) Fenamidone F EC/NP 0.99999 0.010 0.020 74 7 98 1 93 7 98 4 41. Imazalil F EC 0.99254 0.009 0.010 53 3 55 12 66 4 67 8

Chlorophenyl (5) Dicloran F EC 0.99568 0.008 0.010 74 14 91 16 86 14 94 3 28. Quintozene F EC 0.99942 0.004 0.006 81 4 109 15 72 11 99 2 29. Tecnazene F EC 0.99743 0.005 0.009 98 2 115 2 84 4 104 5 30. Tetrachlorvinphos I, A EC/NP 0.99969 0.008 0.010 92 16 92 3 91 9 96 4 31. Tolclofos-methyl F EC/NP 0.99983 0.005 0.009 83 9 103 5 116 5 93 6 32. Cyanoacetamide ozime Cymoxanil F EC/NP 0.99866 0.040 0.050 98 5 108 7 96 6 102 7 33. Cyclodiene Aldrin I EC 0.99947 0.003 0.005 83 8 86 5 82 13 85 8 34. Dicarboximide (2) Iprodione F EC/NP 0.99967 0.008 0.010 108 8 101 9 115 15 99 6 35. Procymidone F EC/NP 0.99939 0.006 0.010 104 4 113 3 108 5 106 4 36. Dinitroaniline (2) Pendimethalin H EC/NP 0.99999 0.008 0.010 99 9 103 6 79 8 102 4 37. Trifluralin H EC/NP 0.99996 0.007 0.010 108 2 101 2 99 3 100 7 38. Diphenyl ether Nitrofen H EC 0.99913 0.005 0.007 109 3 99 1 89 12 95 3 39. Hydroxyanilide Fenhexamid F EC/NP 0.99742 0.009 0.010 96 1 108 1 106 8 89 5 40. Imidazole (3) Fenamidone F EC/NP 0.99999 0.010 0.020 74 7 98 1 93 7 98 4 41. Imazalil F EC 0.99254 0.009 0.010 53 3 55 12 66 4 67 8 42. Prochloraz F EC/NP 0.99706 0.008 0.010 84 2 79 10 73 8 72 9 43. Isoxazolidinone Clomazone H NP 1.00000 0.020 0.030 81 8 98 5 95 4 82 6 44. Morpholine (2) Dimethomorph F NP 0.99507 0.010 0.020 80 7 100 1 93 12 97 4 45. Fenpropimorph F NP 0.99991 0.010 0.020 84 4 104 14 96 4 100 10 46. Neonicotinoid Acetamiprid I EC/NP 0.99562 0.009 0.010 48 5 51 15 45 5 55 10

42. Prochloraz F EC/NP 0.99706 0.008 0.010 84 2 79 10 73 8 72 9 43. Isoxazolidinone Clomazone H NP 1.00000 0.020 0.030 81 8 98 5 95 4 82 6 44. Morpholine (2) Dimethomorph F NP 0.99507 0.010 0.020 80 7 100 1 93 12 97 4 45. Fenpropimorph F NP 0.99991 0.010 0.020 84 4 104 14 96 4 100 10 46. Neonicotinoid Acetamiprid I EC/NP 0.99562 0.009 0.010 48 5 51 15 45 5 55 10 47. Organochlorine (15) Alpha-HCH I EC 0.99461 0.004 0.005 96 4 93 3 92 6 87 10 48. Beta-HCH I EC 0.99963 0.007 0.009 94 9 93 2 97 6 88 9 49. Dicofol A EC 0.99937 0.005 0.008 107 7 105 15 114 4 118 1 50. Endrin I EC 0.99934 0.004 0.005 82 1 89 3 87 2 85 8 51. Gamma-HCH (lindane) I, A EC 0.99898 0.005 0.007 102 10 103 11 101 3 109 8 52. Endosulfan-alpha I, A EC 0.99956 0.005 0.008 84 14 99 8 95 5 92 12 53. Endosulfan-beta I, A EC 0.99794 0.005 0.008 82 6 99 3 93 9 83 5 54. Endosulfan-sulfate I, A EC 0.99779 0.005 0.008 88 6 94 14 96 2 91 14 55. Heptachlor I EC 0.99998 0.004 0.005 92 5 97 4 99 4 96 3 56. Heptachlor-epoxide I EC 0.99997 0.004 0.005 89 2 101 7 107 2 91 16 57. Methoxychlor (DMDT) I EC 0.99967 0.006 0.010 116 2 114 9 113 3 117 8 58. op’ -DDT I EC 0.99989 0.004 0.005 76 2 77 5 70 9 72 3 59. pp’ -DDD I EC 0.99995 0.004 0.005 86 2 94 2 95 2 90 7 60. pp’ -DDE I EC 0.99890 0.003 0.004 91 14 95 8 91 9 96 6 61. pp’ -DDT I EC 0.99920 0.006 0.007 71 9 75 12 80 6 73 7 62. Organophosphate (31) Azinphos-ethyl I, A EC/NP 0.99990 0.008 0.010 94 1 112 5 92 7 101 4 63. Azinphos-methyl I, A EC/NP 0.99771 0.008 0.010 81 7 91 6 89 10 96 7 64. Chlorfenvinphos I, A EC/NP 0.99989 0.007 0.010 85 8 109 13 94 7 99 11 65. Chlorpyrifos I EC/NP 0.99964 0.005 0.007 97 8 100 10 101 8 98 6 66. Chlorpyrifos-methyl I, A EC/NP 0.99999 0.005 0.007 105 7 107 8 103 5 100 5 67. Coumaphos I EC/NP 0.99997 0.008 0.010 77 10 102 6 105 6 99 5 68. Diazinon I, A EC/NP 0.99998 0.005 0.008 105 8 108 1 115 10 100 3 69. Dimethoate I, A EC/NP 0.99999 0.008 0.010 62

pyrifos I EC/NP 0.99964 0.005 0.007 97 8 100 10 101 8 98 6 66. Chlorpyrifos-methyl I, A EC/NP 0.99999 0.005 0.007 105 7 107 8 103 5 100 5 67. Coumaphos I EC/NP 0.99997 0.008 0.010 77 10 102 6 105 6 99 5 68. Diazinon I, A EC/NP 0.99998 0.005 0.008 105 8 108 1 115 10 100 3 69. Dimethoate I, A EC/NP 0.99999 0.008 0.010 62 8 66 2 69 4 68 3 70. Ethion I, A EC/NP 0.99999 0.009 0.010 76 8 78 15 86 12 88 5 71. Ethoprophos I EC/NP 0.99994 0.007 0.010 72 10 114 6 113 13 111 8 72. Fenitrothion I EC/NP 0.99985 0.005 0.009 89 7 98 5 106 6 99 7 73. Fenthion I NP 0.99997 0.009 0.010 58 10 63 3 54 1 66 7 74. Heptenophos I NP 0.99998 0.008 0.010 78 4 87 15 96 7 83 11 75. Isofenphos I EC/NP 1.00000 0.006 0.010 74 13 73 10 89 6 86 6 76. Isofenphos-methyl I EC/NP 0.99992 0.006 0.010 106 9 108 2 109 16 100 3 77. Malaoxon I, A NP 1.00000 0.005 0.009 92 15 103 1 108 7 98 2 78. Malathion I, A EC/NP 0.99992 0.008 0.010 81 12 103 5 95 7 81 7 79. Mecarbam I, A EC/NP 0.99995 0.008 0.010 88 5 85 8 97 11 87 9 80. Methidathion I, A EC/NP 0.99994 0.007 0.010 81 2 102 9 98 16 92 8 81. Paraoxon-ethyl I EC/NP 0.99997 0.005 0.010 74 8 74 6 90 2 84 5 82. Paraoxon-methyl I EC/NP 0.99945 0.005 0.010 100 4 103 5 98 7 93 4 83. Parathion I, A EC/NP 0.99932 0.008 0.010 96 6 101 10 109 10 91 11 84. Parathion-methyl I EC/NP 0.99967 0.008 0.010 105 3 105 12 117 9 102 10 85. Phorate I, A EC/NP 0.99995 0.009 0.010 55 4 65 13 49 8 61 11

74. Heptenophos I NP 0.99998 0.008 0.010 78 4 87 15 96 7 83 11 75. Isofenphos I EC/NP 1.00000 0.006 0.010 74 13 73 10 89 6 86 6 76. Isofenphos-methyl I EC/NP 0.99992 0.006 0.010 106 9 108 2 109 16 100 3 77. Malaoxon I, A NP 1.00000 0.005 0.009 92 15 103 1 108 7 98 2 78. Malathion I, A EC/NP 0.99992 0.008 0.010 81 12 103 5 95 7 81 7 79. Mecarbam I, A EC/NP 0.99995 0.008 0.010 88 5 85 8 97 11 87 9 80. Methidathion I, A EC/NP 0.99994 0.007 0.010 81 2 102 9 98 16 92 8 81. Paraoxon-ethyl I EC/NP 0.99997 0.005 0.010 74 8 74 6 90 2 84 5 82. Paraoxon-methyl I EC/NP 0.99945 0.005 0.010 100 4 103 5 98 7 93 4 83. Parathion I, A EC/NP 0.99932 0.008 0.010 96 6 101 10 109 10 91 11 84. Parathion-methyl I EC/NP 0.99967 0.008 0.010 105 3 105 12 117 9 102 10 85. Phorate I, A EC/NP 0.99995 0.009 0.010 55 4 65 13 49 8 61 11 86. Phosalone I, A EC/NP 0.99981 0.007 0.010 94 7 103 5 99 13 97 10 87. Phosmet I, A EC/NP 0.99987 0.009 0.010 96 3 104 8 102 7 100 6 88. Pirimiphos-ethyl I, A NP 0.99999 0.005 0.008 74 4 70 1 82 3 76 2 89. Pirimiphos-methyl I, A NP 0.99996 0.005 0.008 95 7 104 2 111 1 103 3 90. Profenofos I, A EC/NP 0.99995 0.008 0.010 73 3 72 6 88 5 75 8 91. Quinalphos I, A EC/NP 0.99608 0.005 0.006 75 1 107 3 113 16 107 5 92. Triazophos I, A NP 0.99999 0.007 0.010 112 7 106 4 116 10 101 3 93. Organothiophosphate (5) Bromophos-ethyl I EC/NP 0.99956 0.005 0.007 86 5 104 8 100 14 84 9 94. Bromophos-methyl I EC/NP 0.99996 0.005 0.007 93 5 104 5 97 7 94 3 95. Fenchlorphos I EC/NP 0.99987 0.009 0.010 100 10 104 6 94 4 99 5 96. Formothion I, A EC/NP 0.99984 0.008 0.010 68 15 58 8 65

86. Phosalone I, A EC/NP 0.99981 0.007 0.010 94 7 103 5 99 13 97 10 87. Phosmet I, A EC/NP 0.99987 0.009 0.010 96 3 104 8 102 7 100 6 88. Pirimiphos-ethyl I, A NP 0.99999 0.005 0.008 74 4 70 1 82 3 76 2 89. Pirimiphos-methyl I, A NP 0.99996 0.005 0.008 95 7 104 2 111 1 103 3 90. Profenofos I, A EC/NP 0.99995 0.008 0.010 73 3 72 6 88 5 75 8 91. Quinalphos I, A EC/NP 0.99608 0.005 0.006 75 1 107 3 113 16 107 5 92. Triazophos I, A NP 0.99999 0.007 0.010 112 7 106 4 116 10 101 3 93. Organothiophosphate (5) Bromophos-ethyl I EC/NP 0.99956 0.005 0.007 86 5 104 8 100 14 84 9 94. Bromophos-methyl I EC/NP 0.99996 0.005 0.007 93 5 104 5 97 7 94 3 95. Fenchlorphos I EC/NP 0.99987 0.009 0.010 100 10 104 6 94 4 99 5 96. Formothion I, A EC/NP 0.99984 0.008 0.010 68 15 58 8 65 10 59 9 97. Methacrifos I, A EC/NP 0.99979 0.009 0.010 82 7 94 10 95 9 84 12 98. Oxadiazine Indoxacarb I EC/NP 0.99965 0.009 0.010 93 8 101 12 91 7 91 11 99. Oxazole Vinclozolin F EC/NP 0.99971 0.009 0.010 92 8 104 13 94 4 102 9 100. Phenylamide (2) Metalaxyl F NP 0.99986 0.008 0.010 89 9 95 5 103 5 97 4 101. Oxadixyl F EC/NP 0.99995 0.020 0.030 70 10 75 6 77 3 71 5 102. Phenylpyrazole Fipronil I EC/NP 0.99988 0.004 0.005 84 4 83 9 92 5 83 7 103. Phenylpyrrole Fludioxonil F NP 0.99908 0.008 0.010 100 5 107 5 87 4 88 6 104. Phosphorothiolate Pyrazophos F EC/NP 1.00000 0.009 0.010 73 7 76 14 76 16 79 11 105. Phthalimide (2) Captan F EC 0.99917 0.008 0.010 59 13 68 16 50 12 67 6 106. Folpet F EC 0.99889 0.008 0.010 69 15 64 12 56 10 59 10

97. Methacrifos I, A EC/NP 0.99979 0.009 0.010 82 7 94 10 95 9 84 12 98. Oxadiazine Indoxacarb I EC/NP 0.99965 0.009 0.010 93 8 101 12 91 7 91 11 99. Oxazole Vinclozolin F EC/NP 0.99971 0.009 0.010 92 8 104 13 94 4 102 9 100. Phenylamide (2) Metalaxyl F NP 0.99986 0.008 0.010 89 9 95 5 103 5 97 4 101. Oxadixyl F EC/NP 0.99995 0.020 0.030 70 10 75 6 77 3 71 5 102. Phenylpyrazole Fipronil I EC/NP 0.99988 0.004 0.005 84 4 83 9 92 5 83 7 103. Phenylpyrrole Fludioxonil F NP 0.99908 0.008 0.010 100 5 107 5 87 4 88 6 104. Phosphorothiolate Pyrazophos F EC/NP 1.00000 0.009 0.010 73 7 76 14 76 16 79 11 105. Phthalimide (2) Captan F EC 0.99917 0.008 0.010 59 13 68 16 50 12 67 6 106. Folpet F EC 0.99889 0.008 0.010 69 15 64 12 56 10 59 10 107. Pyrazole Tebufenpyrad A NP 0.99998 0.009 0.010 103 11 106 9 105 8 102 6 108. Pyrethroid (13) Acrinathrin I, A EC 0.99976 0.010 0.020 106 5 96 6 108 2 98 9 109. Alpha-cypermethrin I EC/NP 0.99892 0.006 0.010 93 13 99 6 100 10 95 7 110. Beta-cyfluthrin I EC/NP 0.99962 0.009 0.010 86 2 101 8 99 9 88 9 111. Bifenthrin I, A EC 0.99980 0.009 0.010 94 8 105 3 96 6 94 9 112. Cyfluthrin I EC/NP 0.99920 0.009 0.010 74 6 94 6 96 13 93 5 113. Cypermethrin I EC 0.99993 0.009 0.010 73 16 85 12 81 5 86 11 114. Deltamethrin I EC/NP 0.99993 0.009 0.010 87 9 93 5 94 8 89 8 115. Esfenvalerate I EC/NP 0.99044 0.008 0.010 76 5 104 3 94 11 84 4 116. Fenpropathrin I, A EC/NP 0.99997 0.007 0.010 80 10 94 5 88 7 79 8 117. Fenvalerate I, A EC/NP 0.99965 0.008 0.010 74 3 97 14 83 7 87 12 118. Lambda-cyhalothrin I EC/NP 0.99933 0.008 0.010 77 5 94 16 74 5 92 15 119. Permethrin I EC 0.99983 0.009 0.010 75 10 97 10 101 2 90 11 120. Zeta-cypermethrin I EC 0.99977 0.008 0.010 97 5 98 6 102 8 96 7 121. Pyridazinone Pyridaben I, A EC/NP 0.99992 0.010 0.020 77 2 80 9 100 4 83 8 122. Pyrimidine Fenarimol F EC/NP 0.99995 0.009 0.010 76 4 85 7 88 9 81 8 123. Pyrimidinol Bupirimate F EC/NP 0.99974 0.008 0.010 88 8 97 8 118 8 97 7 124. Quinoline Quinoxyfen F EC/NP 0.99996 0.009 0.010 103 1 99 2 107 3 100 3 125. Strobilurin (5) Azoxystrobin F EC/NP 0.99960 0.009 0.010 91 6 105 5 107 7 97 4 126. Dimoxystrobin F EC/NP 0.99999 0.008 0.010 78 5 92 6 92 11 96 5 127. Kresoxim-methyl F EC/NP 0.99994 0.007 0.010 99 2 108 4 103 5 97 8 128. Pyraclostrobin F EC/NP 1.00000 0.010 0.020 84 2 82 10 108 16 81 11 129. Trifloxystrobin F EC/NP 0.99993 0.005 0.008 78 2 83 6 104 6 85 8 130. Strobilurin type-methoxyacrylate Picoxystrobin F EC/NP 0.99998 0.008 0.010 77 11 79 9 96 3 77 9 131. Sulphamide (2) Dichlofluanid F EC/NP 0.99407 0.006 0.010 77 5 98 6 74 6 89 8 132. Tolylfluanid F EC/NP 0.99987 0.008 0.010 76 2 89 1 74 2 99 2 133. Triazine (3) Atrazine H NP 1.00000 0.007 0.010 107 7 100 5 96 1 103 6 134. Prometrine H NP 0.99998 0.009 0.010 72 3 104 6 93 2 84 9 135. Simazine H NP 0.99999 0.006 0.010 77 8 101 5 113 1 91 7 136. Triazinone Metribuzin H EC/NP 0.99979 0.008 0.010 76 8 96 2 81 2 94 3 137.

NP 0.99987 0.008 0.010 76 2 89 1 74 2 99 2 133. Triazine (3) Atrazine H NP 1.00000 0.007 0.010 107 7 100 5 96 1 103 6 134. Prometrine H NP 0.99998 0.009 0.010 72 3 104 6 93 2 84 9 135. Simazine H NP 0.99999 0.006 0.010 77 8 101 5 113 1 91 7 136. Triazinone Metribuzin H EC/NP 0.99979 0.008 0.010 76 8 96 2 81 2 94 3 137. Triazole (21) Azaconazole F EC/NP 0.99995 0.009 0.010 73 8 78 2 81 5 82 4 138. Biterthanol F NP 0.99959 0.009 0.010 103 3 104 5 75 5 77 5 139. Cyproconazole F EC/NP 0.99999 0.009 0.010 97 8 103 6 101 3 90 8 140. Difenoconazole F EC/NP 0.99996 0.008 0.010 75 7 72 3 77 8 72 4 141. Diniconazole F EC/NP 0.99972 0.007 0.010 86 1 87 6 110 11 87 5 142. Epoxiconazole F EC/NP 0.99999 0.009 0.010 96 10 90 5 111 2 95 6 143. Fenbuconazole F NP 0.99976 0.009 0.010 90 5 91 7 98 4 95 5 144. Fluquinconazole F EC/NP 0.99975 0.009 0.010 74 8 75 6 106 5 87 7 145. Flusilazole F NP 0.99985 0.007 0.010 78 1 107 3 115 9 102 6 146. Flutriafol F EC/NP 0.99994 0.009 0.010 71 3 88 4 86 10 76 5 147. Hexaconazole F EC/NP 0.99971 0.006 0.010 100 9 104 5 92 8 101 3 148. Imibenconazole F EC/NP 0.99990 0.008 0.010 87 10 86 6 98 3 84 9 149. Metconazole F NP 0.99993 0.008 0.010 95 7 105 8 93 5 90 9 150. Myclobutanyl F EC/NP 0.99992 0.009 0.010 101 9 106 10 109 4 103 11 151. Paclobutrazol F EC/NP 0.99995 0.015 0.020 94 4 110 5 114 8 101 6 152. Penconazole F EC/NP 0.99946 0.009 0.010 95 6 109 6 104 8 91 7 153. Propiconazole F EC/NP 0.99977 0.008 0.010 92 8 99 4 105 5 94 8 154. Tebuconazole F NP 0.99990 0.007 0.010 100 9 103 2 110 7 109 3 155. Tetraconazole F EC/NP 0.99987 0.008 0.010 101 4 102 6 106 8 100 7 156. Triadimefon F EC/NP 0.99998 0.008 0.010 88 6 90 4 92 5 95 5 157. Triadimenol F EC/NP 1.00000 0.009 0.010 106 11 106 6 105 11 101 7 158. Triazole (isomer mix) Bromuconazole F EC/NP 0.99996 0.008 0.010 97 13 104 2 93 8 91 3 159. Unclassified (4) Fenazaquin A, I NP 0.99990 0.010 0.015 102 5 100 2 102 3 104 3 160. Buprofezin I,A EC/NP 0.99995 0.008 0.009 81 2 85 5 88 8 85 6 161. DEET I NP 0.99999 0.015 0.020 106 4 106 6 100 9 106 7 162. Pyriproxyfen I NP 0.99995 0.020 0.025 73 11 78 5 71 10 78 6 163. Uracyl Lenacil H NP 0.99998 0.010 0.020 74 5 75 4 76 4 75 5 Pesticides in bold have recovery below 70 % (values in italics)

3 160. Buprofezin I,A EC/NP 0.99995 0.008 0.009 81 2 85 5 88 8 85 6 161. DEET I NP 0.99999 0.015 0.020 106 4 106 6 100 9 106 7 162. Pyriproxyfen I NP 0.99995 0.020 0.025 73 11 78 5 71 10 78 6 163. Uracyl Lenacil H NP 0.99998 0.010 0.020 74 5 75 4 76 4 75 5 Pesticides in bold have recovery below 70 % (values in italics) A acaricide, F fungicide, H herbicide, I insecticide, EC electron capture, NP nitrogen phosphorus, R 2 correlation coefficient, RSD relative standard deviation, Rec. mean recovery Optimization of extraction techniques The studies were carried out by varying different parameters: sorbent, sample to sorbent mass ratio, extracting solvent, extraction time and clean-up sorbent. Conditions for the best extraction efficiency were used for the rest of the study.

A acaricide, F fungicide, H herbicide, I insecticide, EC electron capture, NP nitrogen phosphorus, R 2 correlation coefficient, RSD relative standard deviation, Rec. mean recovery Optimization of extraction techniques The studies were carried out by varying different parameters: sorbent, sample to sorbent mass ratio, extracting solvent, extraction time and clean-up sorbent. Conditions for the best extraction efficiency were used for the rest of the study. Preliminary studies were performed to evaluate the efficiency of MSPD. Various sorbents such as Florisil and silica gel, activated and deactivated, were tested. The use of deactivated sorbents gave recoveries below 40 %. The optimum extraction conditions were obtained with activated Florisil (activation temperature 600 °C). The determination of the plant matrix to sorbent (Florisil) mass ratio was the second step of the optimization procedure. The following mass ratios were examined: 1:1, 1:2, 1:4; a herb to sorbent mass ratio of 1:2 was found to be the most satisfactory. The selection of a dispersing solvent and its volume was the third step of the MSPD optimization procedure. Acetone, diethyl ether, hexane, methanol and its mixtures in different ratios were tested in this step. The most appropriate extraction solvent was acetone/methanol (9:1, v/v). The experiment revealed that 25 ml of the mixture was sufficient for effective elution of pesticide residues. Although 10 ml of methanol produced similar yields, it did not evaporate as quickly as the solvent mixture mentioned above.

in this step. The most appropriate extraction solvent was acetone/methanol (9:1, v/v). The experiment revealed that 25 ml of the mixture was sufficient for effective elution of pesticide residues. Although 10 ml of methanol produced similar yields, it did not evaporate as quickly as the solvent mixture mentioned above. Our experiments showed that the final MSPD extract contained a large amount of matrix co-extracts (Fig. 2a). These can impact the analyte identification by GC-NP/EC. Interfering peaks with retention times close to those of the target residue are the main factors which reduce the achievement of low detection limits. To protect the GC system as much as possible, we focused on reducing the level of the co-extracted matrix. Several cleaning sorbents such as PSA, GCB and C18 were tested. The addition of 1 g of octadecyl C18 as a clean-up sorbent at the bottom of the chromatography column was necessary to minimize interference and produced the best recoveries. The optimum extraction conditions with high recovery were conducted with a 2 g herb sample and 4 g of Florisil as a sorbent, along with a simultaneous stage of clean-up with C18. A chromatogram of a blank linden sample where octadecyl sorbent was used for the preparation of a MSPD extract is shown in Fig. 2b. The MSPD method proposed for the analysis of pesticides in herbs provided clean blank extracts and therefore no additional clean-up step was necessary.Fig. 2 Chromatogram of blank linden sample obtained from MSPD extract: a without C18; b with C18

bent was used for the preparation of a MSPD extract is shown in Fig. 2b. The MSPD method proposed for the analysis of pesticides in herbs provided clean blank extracts and therefore no additional clean-up step was necessary.Fig. 2 Chromatogram of blank linden sample obtained from MSPD extract: a without C18; b with C18 In preliminary tests with LSE, the solvent and extraction time were tested. Acetone, acetonitrile, hexane, diethyl ether and their mixtures were tested. During experiments we found that a decrease of solvent polarity (acetonitrile → hexane) led to reduced solubility of polar co-extracts in the hexane extract. Unfortunately, poor recoveries were obtained for polar pesticides. Finally, most of the pesticides were recovered from a 2 g sample shaken with 70 ml of hexane/diethyl ether/acetone (1:2:2, v/v/v) (50 ml and an additional 20 ml portion). An increase in the extraction mixture volume up to 100 ml resulted in no significant improvement in analyte recoveries. Additionally, pesticide recoveries increased when the extraction time was extended to 1 h; however, further increase in the extraction time to 2 h provided slightly lower values. Therefore, 1 h was selected as the extraction time for this procedure.

o 100 ml resulted in no significant improvement in analyte recoveries. Additionally, pesticide recoveries increased when the extraction time was extended to 1 h; however, further increase in the extraction time to 2 h provided slightly lower values. Therefore, 1 h was selected as the extraction time for this procedure. Due to the presence of interfering peaks from the matrix, further clean-up stages were necessary (Fig. 3a) to reduce the amounts of matrix co-extracts. Purification of the extract was carried out using a chromatography column packed with sodium sulfate (1 g), 5 % neutral aluminum oxide (2 g) and 4 % Florisil (2 g). Analyte recoveries were calculated against extraction volume at different hexane/dichloromethane ratios: 9:1, 8:2 and 7:3. The best results were achieved using 30 ml of a mixture of hexane/dichloromethane (7:3, v/v) (Fig. 3b).Fig. 3 Chromatogram of blank linden sample extract obtained from liquid–solid extraction (LSE): a before clean-up; b after clean-up In summarizing the above optimization steps for both procedures, we observed that the MSPD extraction offers important savings in time (extraction up to 15 min), requires less volume and toxic solvent for efficient isolation of analyzed compounds and was faster and simpler to perform when compared with LSE.

Due to the presence of interfering peaks from the matrix, further clean-up stages were necessary (Fig. 3a) to reduce the amounts of matrix co-extracts. Purification of the extract was carried out using a chromatography column packed with sodium sulfate (1 g), 5 % neutral aluminum oxide (2 g) and 4 % Florisil (2 g). Analyte recoveries were calculated against extraction volume at different hexane/dichloromethane ratios: 9:1, 8:2 and 7:3. The best results were achieved using 30 ml of a mixture of hexane/dichloromethane (7:3, v/v) (Fig. 3b).Fig. 3 Chromatogram of blank linden sample extract obtained from liquid–solid extraction (LSE): a before clean-up; b after clean-up In summarizing the above optimization steps for both procedures, we observed that the MSPD extraction offers important savings in time (extraction up to 15 min), requires less volume and toxic solvent for efficient isolation of analyzed compounds and was faster and simpler to perform when compared with LSE. Comparison of extraction techniques Considering the amounts of co-isolated matrix compounds, but also the recovery of the target analytes as an important performance characteristic of the analytical method, MSPD extraction was investigated for the extraction of multiple pesticide residues (163) from herb samples at spiking levels ranging from 0.05 to 0.5 mg/kg in a subsequent experiment.

ated matrix compounds, but also the recovery of the target analytes as an important performance characteristic of the analytical method, MSPD extraction was investigated for the extraction of multiple pesticide residues (163) from herb samples at spiking levels ranging from 0.05 to 0.5 mg/kg in a subsequent experiment. The pesticides studied covered a wide range of polarities, from the polar propoxur (logKow = 0.14) to non-polar lambda-cyhalothrin (logKow = 6.9). Data in Fig. 4 for both methods were obtained from linden samples spiked at levels ranging from 0.05 to 0.5 mg/kg. The values of octanol–water partition coefficients were found in databases [35, 36]. Comparing the results obtained in Fig. 4, it can be observed that MSPD successfully recovered 155 pesticides, with recoveries >70 %, whereas LSE was effective with only 24 pesticides (hexane extract) and 118 pesticides (hexane/diethyl ether/acetone extract). As shown by Fig. 4a, pesticides with logKow <4 were poorly extracted through the use of LSE. Lower recoveries of these pesticides may be explained by the use of a non-polar solvent (hexane) required for the elimination of most of the matrix co-extracts. On the other hand, liphophilic pesticides (logKow >4) had acceptable recoveries (>40 %) but they represent only 33 % of all compounds analyzed. Better recoveries were obtained using more polar mixtures of solvents: hexane/diethyl ether/acetone (1:2:2, v/v/v) Fig. 4b. However, the results was not satisfactory enough because recoveries in the range 70–120 % comprised 72 % pesticides of all tested, and many active substances resulted in recoveries <70 and >120 %.Fig. 4 Recoveries (%) of pesticides tested vs. their logK ow. a LSE hexane extract b LSE hexane/diethyl ether/acetone extract c MSPD

the results was not satisfactory enough because recoveries in the range 70–120 % comprised 72 % pesticides of all tested, and many active substances resulted in recoveries <70 and >120 %.Fig. 4 Recoveries (%) of pesticides tested vs. their logK ow. a LSE hexane extract b LSE hexane/diethyl ether/acetone extract c MSPD Using the MSPD method represents the best choice for most pesticides (Fig. 4c): satisfactory recoveries (70–120 %) for most pesticides were obtained with this method. Several exceptions (acetamiprid, captan, dimethoate, fenthion, folpet, formothion, imazalil and phorate) (<70 %) were observed. Finally, the MSPD extraction technique provided better results in terms of recovery of target analytes and the amount of isolated matrix co-extracts. Matrix effect The response of the detectors to certain pesticides may be affected by the presence of co-extractives from the sample. These matrix effects may be observed as an increase or decrease in response compared with those produced by solvent solutions of the analyte. The effect of the matrix can be variable and unpredictable in the occurrence of measurable effects. The matrix effect on the detector (EC and NP) response for the pesticides and matrices studied was evaluated in the present work. To determine if there was a different response between matrix-matched standards and standards in solvent, matrix-matched standards were used.

ictable in the occurrence of measurable effects. The matrix effect on the detector (EC and NP) response for the pesticides and matrices studied was evaluated in the present work. To determine if there was a different response between matrix-matched standards and standards in solvent, matrix-matched standards were used. Validation for the analysis of pesticides The MSPD optimization procedure was investigated to determine conditions which would be general for various herbs. The procedure involving MSPD extraction was validated with six different herb samples fortified at three spiking levels: the first ranging between 0.005 and 0.05 mg/kg, the second at 0.05–0.5 mg/kg and the third at 0.25–2.5 mg/kg (HP-5 column). The GC-NP/EC analytical conditions in this study allow for the analysis of all target compounds in a single chromatographic run of 25.431 min. All pesticides were satisfactorily separated with high sensitivity and selectivity. The applicability of the MSPD for different kinds of herbs was examined in the present experiment. The validation parameters of linden, lungwort, melissa and peppermint are given in Table 2.

The GC-NP/EC analytical conditions in this study allow for the analysis of all target compounds in a single chromatographic run of 25.431 min. All pesticides were satisfactorily separated with high sensitivity and selectivity. The applicability of the MSPD for different kinds of herbs was examined in the present experiment. The validation parameters of linden, lungwort, melissa and peppermint are given in Table 2. Response linearity of the method was found in the concentration range studied, with correlation coefficients between 0.99254 and 1.00000. Calibration curves were obtained from matrix matching calibration solutions. The precision of the method was evaluated and expressed as RSD (%) at three concentration levels. Table 2 shows the results with RSD values (RSDs ≤16 %). Accuracy was also evaluated at three concentration levels. As seen in Table 2, the mean recovery values were in the range of 70–119 % for most pesticides. There were several exceptions: acetamiprid, captan, dimethoate, fenthion, folpet, formothion, imazalil and phorate, where recoveries were below 70 %. Most results for MSPD were within the acceptable range (70–120 %) and indicate that this method was both accurate and precise. LODs and LOQs of all tested pesticide residues extracted using the MSPD technique and analyzed through GC-EC/NP were determined in order to evaluate the efficiency and availability of the method. The LODs and LOQs ranged from 0.003 to 0.03 mg/kg and 0.005 to 0.04 mg/kg, respectively.

thod was both accurate and precise. LODs and LOQs of all tested pesticide residues extracted using the MSPD technique and analyzed through GC-EC/NP were determined in order to evaluate the efficiency and availability of the method. The LODs and LOQs ranged from 0.003 to 0.03 mg/kg and 0.005 to 0.04 mg/kg, respectively. The above results prove that MSPD fulfilled the requirements in all herbs tested. MSPD was found to be adequate for the analysis of herbs with differing amounts of essential oil components. Chromatograms of a selected multicompound standard mixture (containing 43 active substances) in the matrix and a linden sample spiked with this mixture (extracted using MSPD) are presented in Fig. 5a, b, respectively.Fig. 5 Chromatogram of: a selected multicompound standard mixture in matrix; b linden sample fortified with selected multicompound standard mixture: 1 propachlor, 2 trifluralin, 3 alpha-HCH, 4 HCB, 5 beta-HCH, 6 gamma-HCH, 7 chlorothalonil, 8 chlorpyrifos methyl, 9 heptachlor, 10 fenchlorphos, 11 aldrine, 12 chlorpyrifos, 13 dicofol, 14 heptachlor epoxide, 15 procymidone, 16 alpha-endosulfan, 17 pp′-DDE, 18 dieldrin, 19 myclobutanyl, 20 krezoxim-methyl, 21 endrin, 22 beta-endosulfan, 23 pp′-DDD, 24 op′-DDT, 25 pp′-DDT, 26 bifenthrin, 27 DMDT, 28 phosalone, 29 prochloraz, 30 boscalid, 31 deltamethrin (isomers), 32 azoxystrobin, 33 imibenconazole, 34 chlorpropham, 35 cyprodinil, 36 mepanipyrim, 37 fludioxonil, 38 cyproconazole, 39 benalaxyl, 40 tebuconazole, 41 fenazaquin, 42 bitertanol, 43 fenbuconazole