Elucidation of Chemical Interactions between Crude Drugs Using Quantitative Thin-Layer Chromatography Analysis.

To elucidate the interactions between crude drugs in Kampo medicines (traditional Japanese medicines), it is important to determine the content of the constituents in a cost-effective and simple manner. In this study, we quantified the constituents in crude drug extracts using thin-layer chromatography (TLC), an inexpensive and simple analytical method, to elucidate the chemical interactions between crude drugs. We focused on five crude drugs, for which quantitative high-performance liquid chromatography (HPLC) methods are stipulated in the Japanese Pharmacopoeia XVIII (JP XVIII) and compared the analytical data of HPLC and TLC, confirming that the TLC results corresponded with the HPLC data and satisfied the criteria of JP XVIII. (Z)-ligustilide, a major constituent in Japanese Angelica Root, for which a method of quantification has not been stipulated in JP XVIII, was also quantitatively analyzed using HPLC and TLC. Furthermore, Japanese Angelica Root was combined with 26 crude drugs to observe the variation in the (Z)-ligustilide content from each combination by TLC. The results revealed that combinations with Phellodendron Bark, Citrus Unshiu Peel, Scutellaria Root, Coptis Rhizome, Gardenia Fruit, and Peony Root increased the (Z)-ligustilide content. Quantifying the constituents in crude drug extracts using the inexpensive and simple TLC method can contribute to elucidating interactions between crude drugs in Kampo medicines, as proposed by the herbal-pair theory.

Changes in the extracted amounts and seasonally variable constituents of Diospyros kaki at different growth stages.

Persimmon Calyx is a crude drug derived from the persistent calyx of mature fruit of Diospyros kaki Thunberg (Ebenaceae) and is used for the treatment of intractable hiccups. Although there are several reports on the isolation of constituents from Persimmon Calyx, its active constituents have not been elucidated. In this study, by focusing on the medicinal part of Persimmon Calyx, calyx on mature fruit of D. kaki, we examined the changes in the extraction amounts of 3 cultivars of D. kaki ('Hiratanenashi', 'Jiro', and 'Tonewase') to identify and quantify seasonally variable constituents during the maturation process by analysing their chemical compositions. We found that the extraction weight of the calyx, fruit of persimmons, and total tannin content in calyxes were significantly increased during maturation. Lupeol (1), betulinic acid (2), pomolic acid (3), ursolic acid (4), ß-sitosterol (5), rotungenic acid (6), barbinervic acid (7), catechin (8), gallocatechin (9), and sucrose (10) were identified in the calyx of D. kaki. Compounds 1, 6, and 7 were isolated from Persimmon Calyx for the first time. Moreover, the isolated compounds (1-7) and their analogue (oleanolic acid) were quantitatively analysed, and the results showed that the amounts of 4 and oleanolic acid were reduced during maturation, whereas that of 2, 3, 6, and 7 were increased.

Quality Evaluation and Characterization of Fractions with Biological Activity from Ephedra Herb Extract and Ephedrine Alkaloids-Free Ephedra Herb Extract.

Previously, we reported that the c-Met inhibitory effect of Ephedra Herb extract (EHE) is derived from ingredients besides ephedrine alkaloids. Moreover, analgesic and anti-influenza activities of EHE and ephedrine alkaloids-free Ephedra Herb extract (EFE) have been reported recently. In this study, we examined the fractions containing c-Met kinase inhibitory activity from EHE and the fractions with analgesic and anti-influenza activities from EFE, and elucidated the structural characteristics of the active fractions. Significant c-Met kinase activity was observed in 30, 40, and 50% methanol (MeOH) eluate fractions obtained from water extract of EHE using Diaion HP-20 column chromatography. Similarly, 20 and 40% MeOH, and MeOH eluate fractions obtained from water extract of EFE were found to display analgesic and anti-influenza activities. Reversed phase-HPLC analysis of the active fractions commonly showed broad peaks characteristic of high-molecular mass condensed tannin. The active fractions were analyzed using 13C-NMR and decomposition reactions; the deduced structures of active components were high-molecular mass condensed tannins, which were mainly procyanidin B-type and partly procyanidin A-type, including pyrogallol- and catechol-type flavan 3-ols as extension and terminal units. HPLC and gel permeation chromatography (GPC) analyses estimated that the ratio of pyrogallol- and catechol-type was approximately 9 : 2, and the weight-average molecular weight based on the polystyrene standard was >45000. Furthermore, GPC-based analysis was proposed as the quality evaluation method for high-molecular mass condensed tannin in EHE and EFE.

Structures and Inhibitory Activities for Interleukin-2 Production of Seasonally Variable Constituents in Flower Parts of Magnolia kobus at Different Growth Stages.

Magnolia Flower is a crude drug used for the treatment of headaches, toothaches, and nasal congestion. Here, we focused on Magnolia kobus, one of the botanical origins of Magnolia Flower, and collected the flower parts at different growth stages to compare chemical compositions and investigate potential inhibitory activities against interleukin-2 (IL-2) production in murine splenic T cells. After determining the structures, we examined the inhibitory effects of the constituents of the bud, the medicinal part of the crude drug, against IL-2 production. We first extracted the flower parts of M. kobus from the bud to fallen bloom stages and analysed the chemical compositions to identify the constituents characteristic to the buds. We found that the inhibitory activity of the buds against IL-2 production was more potent than that of the blooms. We isolated two known compounds, tiliroside (1) and syringin (2), characteristic to the buds from the methanol (MeOH) extract of Magnolia Flower. Moreover, we examined the inhibitory activities of both compounds against IL-2 production and found that tiliroside (1) but not syringin (2), showed strong inhibitory activity against IL-2 production and inhibited its mRNA expression. Thus, our strategy to examine the relationship between chemical compositions and biological activities during plant maturation could not only contribute to the scientific evaluation of medicinal parts of crude drugs but also assist in identifying biologically active constituents that have not yet been reported.

[The Adverse Effects of Ephedra Herb and the Safety of Ephedrine Alkaloids-free Ephedra Herb Extract (EFE)].

Ephedra Herb is defined in the 17th edition of the Japanese Pharmacopoeia (JP) as the terrestrial stem of Ephedra sinica Stapf., Ephedra intermedia Schrenk et C.A. Meyer, or Ephedra equisetina Bunge (Ephedraceae). The stems of Ephedra Herb contain greater than 0.7% ephedrine alkaloids (ephedrine and pseudoephedrine). Despite its high effectiveness, Ephedra Herb exert several adverse effects, including palpitation, excitation, insomnia, and dysuria. Both the primary and adverse effects of Ephedra Herb have been traditionally believed to be mediated by these ephedrine alkaloids. However, our study found that several pharmacological actions of Ephedra Herb were not associated with ephedrine alkaloids. We prepared an ephedrine alkaloid-free Ephedra Herb extract (EFE) by eliminating ephedrine alkaloids from Ephedra Herb extract (EHE) using ion-exchange column chromatography. EFE exerted analgesic, anti-influenza, and anticancer activities in the same manner as EHE. Moreover, EFE did not induce adverse effects due to ephedrine alkaloids, such as excitation, insomnia, and arrhythmias, and showed no toxicity. Furthermore, we evaluated the safety of EFE in healthy volunteers. The number of adverse event cases was higher in the EHE-treated group than in the EFE-treated group, although the difference was not significant. Our evidence suggested that EFE was safer than EHE.

Analgesic Effects of Ephedra Herb Extract, Ephedrine Alkaloids-Free Ephedra Herb Extract, Ephedrine, and Pseudoephedrine on Formalin-Induced Pain.

The analgesic effect of Ephedra Herb (EH) is believed to be derived from the anti-inflammatory action of pseudoephedrine (Pse). We recently reported that ephedrine alkaloids-free EH extract (EFE) attenuates formalin-induced pain to the same level as that achieved by EH extract (EHE), which suggests that the analgesic effect of EH may not be due to ephedrine alkaloids (EAs). To examine the contribution of EAs to the analgesic effect of EH, mice were injected with formalin to induce a biphasic pain reaction (first phase, 0-5 min; second phase, 10-45 min) at various time points after oral administration of the following test drugs: ephedrine (Eph), Pse, "authentic" EHE from Tsumura & Co. (EHE-Ts), EFE, and EHE that was used as the source of EFE (EHE-To). Biphasic pain was suppressed at 30 min after administration of Eph, EHE-Ts, and EHE-To. At 6 h after administration of EFE, EHE-To, and Pse-and at 4 to 6 h after administration of EHE-Ts-only second-phase pain was suppressed; however, the effect of Pse at 6 h was not significant. These results suggested that EHE has a biphasic analgesic effect against biphasic formalin-induced pain: in the first phase of analgesia (30 min after administration), biphasic pain is suppressed by Eph; in the second phase of analgesia (4-6 h after administration), second-phase pain is alleviated by constituents other than EAs, although Pse may partially contribute to the relief of second-phase pain.

Non-alkaloidal composition of Ephedra Herb is influenced by differences in habitats.

Ephedra Herb is a crude drug defined as the terrestrial stem of Ephedra sinica, E. intermedia, or E. equisetina. It is often used to treat headaches, bronchial asthma, nasal inflammation, and the common cold. In this study, we isolated characteristic non-alkaloidal constituents from the extracts and identified them in relation to the habitat of Ephedra Herb. Extracts were prepared from Ephedra Herb collected from Inner Mongolia and Gansu. High-performance liquid chromatography was performed to quantitatively analyse the amount of ephedrine alkaloids in each extract. We compared the chemical compositions of the extracts by thin layer chromatography (TLC) to find spot characteristics depending on the habitat. 1H-NMR, 13C-NMR, and 2D-NMR spectra of the samples were also examined. The ephedrine content of all extracts satisfied the quality standard stated in the Japanese Pharmacopoeia. Nonetheless, we found each notable constituent characteristic to the Ephedra Herbs from both habitats. In order to identify them, Ephedra Herb extracts were separated by column chromatography, resulting in the isolation of (±)-α-terpineol-ß-D-O-glucopyranoside (1) and (E)-7-hydroxy-3,7-dimethyloct-2-en-1-yl-ß-D-O-glucopyranoside (2) as the characteristic constituents in Ephedra Herb from Inner Mongolia. Epheganoside (3), a new eudesmane-type sesquiterpene glycoside, and scopoletin (4) were found to be the characteristic constituents in Ephedra Herb from Gansu. The results obtained from this study can be used to distinguish between the habitats of Ephedra Herb.

Quantitative analysis of the anti-inflammatory activity of orengedokuto II: berberine is responsible for the inhibition of NO production.

Orengedokuto is a Kampo formula that has been used for removing "heat" and "poison" to treat inflammation, hypertension, gastrointestinal disorders, and liver and cerebrovascular diseases. We report here our analysis of the anti-inflammatory effect of the component crude drugs of orengedokuto and their constituents using the inhibition of nitric oxide (NO) production in the murine macrophage-like cell line J774.1. An initial comparison of NO production inhibitory activities of the extracts of the component crude drugs and their combinations revealed that the activity could be attributed to Phellodendron Bark and Coptis Rhizome. Berberine (1), the major constituent of these crude drugs, showed potent activity (IC50 4.73 ± 1.46 µM). Quantitative analysis of 1 in the extracts of all combinations of component crude drugs revealed that the amount of 1 in each extract of the combination of Scutellaria Root with either Phellodendron Bark and/or Coptis Rhizome was lower than that in the corresponding mixtures of the extracts of the individual crude drugs and that 1 was present in the precipitates formed during the decoction process. To the contrary, the differences in the amounts of 1 were smaller in the extracts containing Gardenia Fruit. These results indicated that the constituents of Scutellaria Root precipitated with 1 and that the constituents of Gardenia Fruit dissolved the precipitates. To identify the constituents affecting the solubility of 1, we fractionated the hot-water extracts of Scutellaria Root based on solubility tests of 1 to give baicalin (2), wogonin (3) and oroxyloside (4), which formed precipitates with 1.

Ephedrine Alkaloids-Free Ephedra Herb Extract, EFE, Has No Adverse Effects Such as Excitation, Insomnia, and Arrhythmias.

Ephedrine alkaloids-free Ephedra Herb extract (EFE) has been developed to eliminate the adverse effects caused by ephedrine alkaloid-induced sympathetic hyperactivation. Previously, we reported that EFE possesses analgesic, anti-influenza, and cancer metastatic inhibitory effects at comparable levels to that of Ephedra Herb extract (EHE). However, it has not yet been demonstrated that EFE is free from the known side effects of EHE, such as excitation, insomnia, and arrhythmias. In this study, the incidence of these adverse effects was compared between mice administered EHE and those administered EFE. Increased locomotor activity in an open-field test, reduced immobility times in a forced swim test, and reduced sleep times in a pentobarbital-induced sleep test were observed in EHE-treated mice, when compared to the corresponding values in vehicle-treated mice. In contrast, EFE had no obvious effects in these tests. In electrocardiograms, atrial fibrillation (i.e., irregular heart rhythm, absence of P waves, and appearance of f waves) was observed in the EHE-treated mice. It was suggested that this atrial fibrillation was induced by stimulation of adrenaline ß1 receptors, but not by hypokalemia. However, EFE did not affect cardiac electrophysiology. These results suggest that the abovementioned side effects are caused by ephedrine alkaloids in EHE, and that EFE is free from these adverse effects, such as excitation, insomnia, and arrhythmias. Thus, EFE is a promising new botanical drug with few adverse effects.

Synergistic effect of baicalein, wogonin and oroxylin A mixture: multistep inhibition of the NF-κB signalling pathway contributes to an anti-inflammatory effect of Scutellaria root flavonoids.

Scutellaria root, the root of Scutellaria baicalensis Georgi, is a crude drug used for inflammatory diseases. In our previous report, the combination of flavonoids contained in Scutellaria root have been found to inhibit PGE2 production more strongly than individual flavonoids. Here, to investigate the mechanism of the synergistic effect, we examined the effects of an equimolar mixture (F-mix) of baicalein (1), wogonin (2) and oroxylin A (3) on the production of PGE2 in LPS-treated J774.1 cells. Although 1 and 3 inhibited COX-2 activity, the F-mix showed no synergistic effect on COX-2 inhibition. Therefore, we investigated the steps leading to the activation of COX-2 protein. Compounds 1-3 and F-mix inhibited the expression of COX-2 protein. However, only 2 inhibited the expression of COX-2 mRNA among the flavonoids, and the F-mix showed no synergistic effect. Only 1 inhibited NF-κB translocation into the nucleus, and the F-mix showed no synergistic effect. Although 2 did not affect NF-κB translocation, it strongly inhibited NF-κB-dependent transcriptional activity, and the F-mix inhibited the activity slightly more than 2. Compounds 1-3 also inhibited NO production, and the F-mix showed a synergistic effect. However, the effects of each flavonoid on the expression of iNOS mRNA were not consistent with those on COX-2 mRNA. Because the flavonoids inhibit different steps in the production of PGE2 and NO, and their mixture did not show apparent synergistic effects in each step, we conclude that the synergistic effect of the flavonoid mixture reflects the total effect of the flavonoids inhibiting different steps in the NF-κB signalling pathway.

Two flavone C-glycosides as quality control markers for the manufacturing process of ephedrine alkaloids-free Ephedra Herb extract (EFE) as a crude drug preparation.

As part of our continuing study of ephedrine alkaloids-free Ephedra Herb extract (EFE) in pursuit of its approval as a crude drug preparation, we identified two quantitative markers for the quality control of the manufacturing process of EFE and sought to establish cost-effective and simple methods for quantitative analyses. We analysed Ephedra Herb extracts grown in different habitats and collection years by liquid chromatography/high-resolution mass spectrometry (LC/HRMS) and detected two notable peaks common to each extract. These peaks were identified as vicenin-2 (1) and isovitexin 2″-O-rhamnoside (2). Quantitative analyses using the isocratic condition of LC/MS showed that the content percentages of 1 and 2 in EFE were 0.140-0.146% and 0.350-0.411%, respectively. We concluded that 1 and 2 were adequate quality control markers for quantitative analysis of EFE. Furthermore, we quantitatively analysed apigenin (3), an aglycon common to 1 and 2, and found that the conversion factors of 1 to 3 and 2 to 3 were 1.3 and 1.5, respectively. Therefore, we concluded that 3 was a secondary standard for quantifying the contents of 1 and 2 in EFE. A series of results obtained from this study will be valuable for the quality control of EFE.

Construction of Prediction Models for the Transient Receptor Potential Vanilloid Subtype 1 (TRPV1)-Stimulating Activity of Ginger and Processed Ginger Based on LC-HRMS Data and PLS Regression Analyses.

To construct a model formula to evaluate the thermogenetic effect of ginger (Zingiber officinale Roscoe) from the ingredient information, we established transient receptor potential vanilloid subtype 1 (TRPV1)-stimulating activity prediction models by using a partial least-squares projections to latent structures (PLS) regression analysis in which the ingredient data from liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and the stimulating activity values for TRPV1 receptor were used as explanatory and objective variables, respectively. By optimizing the peak extraction condition of the LC-HRMS data and the data preprocessing parameters of the PLS regression analysis, we succeeded in the construction of a TRPV1-stimulating activity prediction model with high precision ability. We then searched for the components responsible for the TRPV1-stimulating activity by analyzing the loading plot and s-plot of the model, and we identified [6]-gingerol (1) and hexahydrocurcumin (3) as TRPV1-stimulating activity components.

Efficient Preparation of Ephedrine Alkaloids-free Ephedra Herb Extract and Its Antitumor Effect and Putative Marker Compound.

Ephedra Herb is a crude drug for the treatment of headache, bronchial asthma, nasal inflammation, and the common cold. Although it has been considered that ephedrine alkaloids (EAs) are the principal active ingredients of Ephedra Herb, EAs are known to induce palpitations, hypertension, insomnia, and dysuria as major side effects. Therefore, the administration of EAs-containing drugs to patients with cardiovascular-related diseases is strongly contraindicated. Previously, we isolated herbacetin 7-O-neohesperidoside from Ephedra Herb. In addition, we found that herbacetin, a flavonoid aglycone in Ephedra Herb, had antiproliferative and analgesic effects. Therefore, the prospect of preparing safer natural medicines without the adverse effects associated with EAs was appealing. In this symposium review, to achieve the aim of producting a clinically useful Ephedra Herb extract with none of the adverse effects associated with EAs, I present an efficient preparation method of EAs-free Ephedra Herb extract, together with its chemical composition, antiproliferative effects, and a putative marker for quality control.

Efficiently prepared ephedrine alkaloids-free Ephedra Herb extract: a putative marker and antiproliferative effects.

Ephedrine alkaloids (EAs) have been considered the main pharmacologically active substances in Ephedra Herb (, Mao; EH) since they were first identified by Prof. N. Nagai, and are known to induce palpitation, hypertension, insomnia, and dysuria as side effects. Therefore, the administration of drugs containing EH to patients with cardiovascular-related diseases is severely contraindicated. While our previous studies suggest that some of the effects of EH may not be due to EAs, considering their side effects would be expedient to develop a new EAs-free EH extract (EFE). Here, we established a preparation method for EFE and revealed its chemical composition, including the content of herbacetin, a flavonoid aglycon present in EH and a potential putative marker for EFE quality control. In addition, we showed the antiproliferative effects of EFE against the H1975 non-small cell lung cancer (NSCLC) cell line. EFE was prepared from EH extract using the ion exchange resin SK-1B. LC/Orbitrap MS analysis revealed the removal of EAs, 6-methoxykynurenic acid, and 6-hydroxykynurenic acid from the original extract. Quantitative analysis of herbacetin using LC/MS in acid-hydrolyzed EFE showed that its content was 0.104 %. Although several alkaloidal constituents were removed from EH extract, the antiproliferative effect of EFE against H1975 cells was comparable to that of EH extract. These results indicate that EFE retained the anticancer effect of EH and demonstrated its potential for future development as a new herbal medicine with reduced side effects.

Ephedrine alkaloids-free Ephedra Herb extract: a safer alternative to ephedra with comparable analgesic, anticancer, and anti-influenza activities.

It is generally accepted that the primary pharmacological activities and adverse effects of Ephedra Herb are caused by ephedrine alkaloids. Interestingly, our research shows that Ephedra Herb also has ephedrine alkaloid-independent pharmacological actions, such as c-MET inhibitory activity. This study describes the preparation of an ephedrine alkaloids-free Ephedra Herb extract (EFE) by ion-exchange column chromatography, as well as in vitro and in vivo evaluation of its pharmacological actions and toxicity. We confirmed that EFE suppressed hepatocyte growth factor (HGF)-induced cancer cell motility by preventing both HGF-induced phosphorylation of c-Met and its tyrosine kinase activity. We also investigated the analgesic effect of EFE. Although the analgesic effect of Ephedra Herb has traditionally been attributed to pseudoephedrine, oral administration of EFE reduced formalin-induced pain in a dose-dependent manner in mice. Furthermore, we confirmed the anti-influenza virus activity of EFE by showing inhibition of MDCK cell infection in a concentration-dependent manner. All assessments of toxicity, even after repeated oral administration, suggest that EFE would be a safer alternative to Ephedra Herb. The findings described here suggest that EFE has c-Met inhibitory action, analgesic effect, and anti-influenza activity, and that it is safer than Ephedra Herb extract itself. Therefore, EFE could be a useful pharmacological agent.

Identification of New Diterpenes as Putative Marker Compounds Distinguishing Agnus Castus Fruit (Chaste Tree) from Shrub Chaste Tree Fruit (Viticis Fructus).

Agnus Castus Fruit is defined in the European Pharmacopoeia as the dried ripe fruit of Vitex agnus-castus. In Europe it is used as a medicine targeting premenstrual syndrome and climacteric disorder. In Japan, Agnus Castus Fruit is becoming popular as a raw material for over-the-counter drugs and health food products, though its congenic species, Vitex rotundifolia and Vitex trifolia, have been used as Shrub Chaste Tree Fruit in traditional medicines. Therefore, it is important to discriminate these Vitex plants from the viewpoint of regulatory science. Here we tried to identify putative marker compounds that distinguish between Agnus Castus Fruit and Shrub Chaste Tree Fruit. We analyzed extracts of each crude drug by liquid chromatography-mass spectrometry, and performed differential analysis by comparison of each chromatogram to find one or more peaks characteristic of Agnus Castus Fruit. A peak was isolated and identified as an equilibrium mixture of new compounds named chastol (1) and epichastol (1a). The planar structures of 1 and 1a were determined spectroscopically. Their relative configurations were revealed by nuclear Overhauser effect spectroscopy and differential nuclear Overhauser effect-NMR data. Since avoiding contamination from closely related species is needed for the quality control of natural pharmaceuticals, this information will be valuable to establish a method for the quality control of both, Agnus Castus Fruit and Shrub Chaste Tree Fruit products.

Identification of marker compounds for Japanese Pharmacopoeia non-conforming jujube seeds from Myanmar.

Jujube seed is a crude drug defined as the seed of Ziziphus jujuba Miller var. spinosa Hu ex H.F. Chou (Rhamnaceae) in the Japanese Pharmacopoeia (JP). Most of the jujube seed in the Japanese markets is imported from China, with the rest obtained from other Asian countries. Here we confirmed the botanical origins of jujube seeds from both China and Myanmar by a DNA sequencing analysis. We found that the botanical origins of the crude drugs from China and Myanmar were Z. jujuba and Z. mauritiana, respectively. Although the jujube seed from China conforms to the JP, that from Myanmar does not. A method for discriminating jujube seeds from China and Myanmar using a chemical approach is thus desirable, and here we sought to identify a compound specific to Z. jujuba. Jujuboside A (1) was identified as a compound specific to Z. jujuba. To establish a purity test of Jujube Seed in the JP against Z. mauritiana, we fractionated the extract of Z. mauritiana seeds and identified frangufoline (2) and oleanolic acid (4) as the marker compounds specific to Z. mauritiana. Thin-layer chromatography (TLC) and gas chromatography-mass spectrometry analyses revealed that the latter compound was useful for testing by TLC analysis. The established TLC conditions were as follows: chromatographic support, silica gel; developing solvent, n-hexane:EtOAc:HCOOH = 10:5:1; developing length, 7 cm; visualization, diluted sulfuric acid; R f value, 0.43 (oleanolic acid).

Quantitative analysis of anti-inflammatory activity of orengedokuto: importance of combination of flavonoids in inhibition of PGE2 production in mouse macrophage-like cell line J774.1.

Orengedokuto is a Kampo formula which has been used for removing "heat" and "poison" to treat inflammation, hypertension, gastrointestinal disorders, and liver and cerebrovascular diseases. In this report, we quantitatively analyzed the anti-inflammatory effect of the component crude drugs of orengedokuto and their constituents, using inhibition of prostaglandin E2 (PGE2) production in the murine macrophage-like cell line J774.1. First, we compared PGE2 production inhibitory activities of extracts of combinations of the component crude drugs, which showed that the activity could be ascribed to Scutellaria Root. Next, as baicalin (1), one of the major constituents of Scutellaria Root, did not show any activity, and baicalein (2), the aglycon of 1, showed only weak activity (IC50 92 µM), a hot-water extract of Scutellaria Root was fractionated under the guidance of the activity to give wogonin (3) (IC50 28 µM), 6-methoxywogonin (4) (IC50 7.2 µM) and oroxylin A (5) (IC50 45 µM) from the most active fraction. However, the activities of these compounds at concentrations equivalent to those in the extract were weaker than that of the extract, and none of these compounds alone could explain the activity of the extract. Therefore, we examined the activity of combinations of compounds 2-5. Comparison of all combinations of the four compounds in a ratio which is the same as in the extract revealed that wogonin (3) had an essential role in the activity, and a combination of baicalein (2) and wogonin (3), together with 6-methoxywogonin (4) or oroxylin A (5), was necessary to show activity equivalent to that of the extract.

Collagenase inhibitors from Viola yedoensis.

Fractionation of acetone and methanol extracts of Viola yedoensis, under the guidance of inhibition against Clostridium histolyticum collagenase (ChC), resulted in the isolation of esculetin (1) (IC(50) 12 µM) and scopoletin (2) (IC(50) 1.8 µM) as the active constituents, together with trans-p-coumaric acid (3), cis-p-coumaric acid (4), 3-O-ß-D-glucosyl-7-O-α-L-rhamnosylkaempferol (5), rutin (6), isovitexin (7), isoorientin (8), vicenin-2 (9), isoscoparin (10), vanillic acid (11) and adenosine (12). Modification of phenolic hydroxy groups of 1 showed that small O-alkyl groups largely increased the activity, whereas larger O-alkyl groups decreased the activity, and 6,7-dimethoxycoumarin (scoparone 13) potently inhibited ChC (IC(50) 24 nM).