HPLC analysis of ammonium glycyrrhizate listed in the European, United States, and Japanese Pharmacopoeias under reported and modified conditions: revision of the peak assignment for 18α-glycyrrhizin in the European and United States Pharmacopoeias.

We examined ammonium glycyrrhizate listed in the monographs of the European Pharmacopoeia (EP) and United States Pharmacopoeia (USP) as well as in the reagents and solutions used in the general test of the Japanese Pharmacopoeia by performing HPLC on their sample standards or reference reagents under reported and modified conditions. Comparative experiments involving five authentic samples, namely, 18ß-glycyrrhizin (1), 18α-glycyrrhizin (2), licorice-saponin G2 (3), licorice-saponin H2 (4), and galacturonic acid-replaced glycyrrhizin (the 4″-epimer of 18ß-glycyrrhizin) (5), led us to propose the revision of the peak assignment of 18α-glycyrrhizin (2) and postscript a possible co-existence of galacturonic acid-replaced glycyrrhizin (5) as a hidden component in the EP and USP. We also proposed that the α-configuration used in the nomenclature of the glycosidic bond between aglycone and the sugar units of ammonium glycyrrhizate and impurities in the EP and USP should be revised to the ß-configuration.

Is 18α-Glycyrrhizin a real natural product? Improved preparation of 18α-Glycyrrhizin from 18ß-Glycyrrhizin as a positive standard for HPLC analysis of licorice extracts.

18α-Glycyrrhizin is an epimer of 18ß-glycyrrhizin, a major component of licorice (Glycyrrhiza sp.), which is widely used as a traditional medicine. Whether 18α-glycyrrhizin is a real natural product has been debated in the long history of glycyrrhizin chemistry because 18ß-glycyrrhizin is epimerizable to a more thermodynamically stable 18α-glycyrrhizin under aqueous alkali conditions. We improved the preparation of 18α-glycyrrhizin from 18ß-glycyrrhizin by successive epimerization reactions of 18ß-glycyrrhizin, trimethyl esterification of the resulting epimerized mixture, and alkaline hydrolysis of a purified 18α-glycyrrhizin trimethyl ester. Approaches to the possible presence of 18α-glycyrrhizin in licorice extracts by HPLC using synthetic 18α-glycyrrhizin as a positive standard strongly suggested that 18α-glycyrrhizin could naturally exist as a minor congener of glycyrrhizin derivatives in Glycyrrhiza species.

Quantification of terpene trilactones in Ginkgo biloba with a 1H NMR method.

Ginkgo biloba L. has been used as a herbal medicine in the traditional treatment of insufficient blood flow, memory deficits, and cerebral insufficiency. The terpene trilactone components, the bioactive agents of Ginkgo biloba L., have also been reported to exhibit useful functionality such as anti-inflammatory and neuroprotective effects. Therefore, in the present research, we attempted to analyze quantitatively the terpene trilactone components in Ginkgo biloba leaf extract, with quantitative 1H NMR (qNMR) and obtained almost identical results to data reported using HPLC. Application of the qNMR method for the analysis of the terpene trilactone contents in commercial Ginkgo extract products, such as soft gel capsules and tablets, produced the same levels noted in package labels. Thus, qNMR is an alternative method for quantification of the terpene trilactone components in commercial Ginkgo extract products.

Antidepressant-like effect of Butea superba in mice exposed to chronic mild stress and its possible mechanism of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Butea superba (BS) is a Thai medicinal plant that has been used as a folk medicine to improve physical and mental conditions and to prevent impaired sexual performance in middle-aged or elderly males. We have previously reported that this plant extract could improve cognitive deficits and depression-like behavior in olfactory bulbectomized mice, an animal model of dementia and depression. AIM OF THE STUDY: In this study we examined the effect of BS on depression-like behavior in mice subjected to unpredictable chronic mild stress (UCMS) to clarify the antidepressant-like activity of BS and the molecular mechanism underlying this effect. MATERIALS AND METHODS: UCMS mice were administered BS daily (300 mg of dried herb weight/kg, p.o.) or a reference drug, imipramine (IMP, 10 mg/kg, i.p.), 1 week after starting the UCMS procedure. We employed the sucrose preference test and the tail suspension test to analyze anhedonia and depression-like behavior of mice, respectively. Serum and brain tissues of mice were used for neurochemical and immunohistochemical studies. The UCMS procedure induced anhedonia and depression-like behavior, and BS treatment, as well as IMP treatment, attenuated these symptoms. UCMS caused an elevation of serum corticosterone level, an index of hyper-activation of the hypothalamic-pituitary-adrenal (HPA) axis, in a manner attenuated by BS and IMP treatment. BS treatment also attenuated UCMS-induced decrease in the expression levels of brain-derived neurotrophic factor (BDNF) mRNA, cyclic AMP-responsive element binding protein (CREB) and a phosphorylated form of N-methyl-d-aspartate receptor subunit NR1, synaptic plasticity-related signaling proteins. Moreover, the UCMS procedure reduced doublecortin-positive cells in the dentate gyrus region of the hippocampus. BS administration reversed these UCMS-induced neurochemical and histological abnormalities. CONCLUSION: These results suggest that BS can ameliorate chronic stress-induced depression-like symptoms and that the effects of BS are mediated by restoring dysfunctions of the HPA axis and synaptic plasticity-related signaling systems and neurogenesis in the hippocampus.

Butea superba-induced amelioration of cognitive and emotional deficits in olfactory bulbectomized mice and putative mechanisms underlying its actions.

This study investigated the effects of alcoholic extract of Butea superba (BS) on cognitive deficits and depression-related behavior using olfactory bulbectomized (OBX) mice and the underlying molecular mechanisms of its actions. OBX mice were treated daily with BS (100 and 300 mg/kg, p.o.) or reference drugs, tacrine (2.5 mg/kg, i.p.) and imipramine (10 mg/kg, i.p.) from day 3 after OBX. OBX impaired non-spatial and spatial cognitive performances, which were elucidated by the novel object recognition test and modified Y maze test, respectively. These deficits were attenuated by tacrine and BS but not imipramine. OBX animals exhibited depression-like behavior in the tail suspension test in a manner reversible by imipramine and BS but not tacrine. OBX down-regulated phosphorylation of synaptic plasticity-related signaling proteins: NMDA receptor, AMPA receptor, calmodulin-dependent kinase II, and cyclic AMP-responsive element-binding protein. OBX also reduced choline acetyltransferase in the hippocampus. BS and tacrine reversed these neurochemical alterations. Moreover, BS inhibited ex vivo activity of acetylcholinesterase in the brain. These results indicate that BS ameliorates not only cognition dysfunction via normalizing synaptic plasticity-related signaling and facilitating central cholinergic systems but also depression-like behavior via a mechanism differing from that implicated in BS amelioration of cognitive function in OBX animals.

Secondary metabolites from the root wood of Zanthoxylum wutaiense and their antitubercular activity.

Bioassay-guided fractionation of the root wood of Zanthoxylum wutaiense led to the isolation of five new compounds, wutaipyranol A (1), 8-methoxywutaipyranol A (2), demethoxywutaiensal (3), demethoxywutaiensol (4), and dihydrodemethoxywutaiensol (5), together with six known compounds. Their structures were elucidated by 1D- and 2D-NMR, as well as MS analyses. Among the isolates, wutaipyranol A (1), 8-methoxywutaipyranol A (2), and demethoxywutaiensal (3) exhibited antitubercular activities against Mycobacterium tuberculosis H(37) Rv in vitro, with MIC values of 52.4, 55.6, and 45.8 µg/ml, respectively.

In vitro leishmanicidal activity of benzophenanthridine alkaloids from Bocconia pearcei and related compounds.

Leishmanicidal activities of benzophenanthridine alkaloids isolated from fruits of Bocconia pearcei and their derivatives were examined. Seven benzophenanthridine compounds were isolated from the methanolic extracts of B. pearcei. Among them, dihydrosanguinarine showed the most potent leishmanicidal activities (IC(50) value: 0.014 microg/ml, respectively). To examine the structure-activity relationship of the benzophenanthridine skeleton, the leishmanicidal activities for 32 synthetic samples were examined. The existence of bulky groups at the C(7)-C(8) position was found to enhance the activity. On the other hand, the bulkiness at the C(2)-C(3) position on the D-ring, a carbonyl group at C-6, substitution at C-6 and cleavage or saturation of the C(5)-C(6) bond reduced activity. A methyl group on nitrogen of the C-ring was thought to be necessary for significant activity.

Insight into estrogenicity of phytoestrogens using in silico simulation.

Phytoestrogens, including miroestrol and deoxymiroestrol, have the ability to act through competition with estrogen for binding to the estrogen receptor (ER). Here, we utilize manual ligand docking followed by molecular dynamics simulations and binding free energy calculations with the linear interaction energy method to predict the binding modes and the binding affinities of phytoestrogens on the ligand binding domain of ER (ERalpha-LBD). The calculations brought about the good correlation between the calculated binding free energy and the bioassays. Furthermore, consideration of Lennard-Jones and Coulomb interaction energies of miroestrol and deoxymiroestrol on ERalpha-LBD provided the information to develop the phytoestrogen derivatives as the preferred drug for ER positive breast cancer treatment.

Inhibitory effect of the extracts from Thai medicinal plants on iNOS expression in mouse macrophage RAW 264.7.

In the present study, we screened the inhibitory effect of the extract from 50 Thai medicinal plants on an inducible-nitric oxide synthase (iNOS) expression induced by lipopolysaccharide (LPS) in mouse macrophages RAW 264.7. From this screening, the extracts of root bark of Clausena guillauminii, C. lunulata, and C. excavata (Rutaceae) were found as the extracts which showed potent inhibitory effect on the iNOS protein expression in concentration-dependent manner. Furthermore, hydrophobic active components may exist in C. guillauminii.

The structure-activity relationship between oxycoumarin derivatives showing inhibitory effects on iNOS in mouse macrophage RAW264.7 cells.

We have investigated the structure-activity relationship between 63 natural oxycoumarin derivatives and their effects on the expression of inducible-nitric oxide synthase (iNOS) induced by lipopolysaccharide. The protein expression of iNOS was screened by Western blot analysis, and four 5,7-dimethoxycoumarins were selected as potent inhibitors of iNOS expression. In terms of structural specificity, the methoxyl group on C-5 and C-7 and the short alkyl chain (1-5 carbons) on C-6 may be essential for the potent activities. These compounds also showed inhibitory effects on nitric oxide generation and mRNA expression of inflammatory mediators, namely, iNOS and COX-2. Interestingly, the inhibitory effect on mRNA expression was specific for iNOS and was not detected for neuronal NOS. It is expected that these compounds will show anti-inflammatory activities via inhibition of the expressions of iNOS and COX-2.

Inhibitory effect of oxycoumarins isolated from the Thai medicinal plant Clausena guillauminii on the inflammation mediators, iNOS, TNF-alpha, and COX-2 expression in mouse macrophage RAW 264.7.

In the present study, we investigated the inhibitory effect of the known oxycoumarins poncitrin (3), osthol (4), and xanthoxyletin (5), newly isolated from Clausena guillauminii (Rutaceae), together with the known carbazoles heptaphylline (1) and 7-methoxyheptaphylline (2) on inducible-nitric oxide synthase (iNOS) expression induced by lipopolysaccharide (LPS) and the NO generation in RAW 264.7 mouse macrophages. Isolation of active oxycoumarins was guided by Western blot analysis of iNOS protein expression. These oxycoumarins showed an inhibitory effect on iNOS protein expression at 10 microM. Further examination of the inhibitory effects of these compounds on inflammation mediators revealed that the synthesis of nitric oxide (NO) and the protein expression of tumor necrosis factor-alpha (TNF-alpha) and cyclooxygenase-2 (COX-2) were inhibited by 5. It was expected that these compounds show anti-inflammatory activities.

Pueraria mirifica phytoestrogens improve dyslipidemia in postmenopausal women probably by activating estrogen receptor subtypes.

Impaired lipid metabolism is an important health problem in postmenopausal women with insufficient estrogens, because dyslipidemia is a risk factor for development of atherosclerosis and the incidence of cardiovascular disease markedly increases after menopause. Pueraria mirifica (PM), a Thai herb, has been noticed as a source of phytoestrogens, estrogen-mimicking plant compounds. However, the clinical effects of PM on lipid metabolism and the underlying molecular mechanisms remain undetermined. Therefore, we examined the effects of PM on serum lipid parameters in a randomized, double-blind, placebo-controlled clinical trial. Nineteen postmenopausal women were randomly assigned to receive oral administration of PM powder or placebo. After 2 months of treatment, the PM group showed a significant increase in serum concentrations of high-density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-1 (34% and 40%, respectively), and a significant decrease in low-density lipoprotein (LDL) cholesterol and apo B (17% and 9%, respectively), compared with baseline measurements. Moreover, significant decreases were observed in the ratios of LDL cholesterol to HDL cholesterol (37%) and apo B to apo A-1 (35%). Next, we determined the effects of PM phytoestrogens on the activation of estrogen receptor (ER)-mediated transactivation by transient expression assays of a reporter gene in cultured cells. Among PM phytoestrogens, miroestrol and coumestrol enhanced both ERalpha- and ERbeta-mediated transactivation, whereas other phytoestrogens, including daidzein and genistein, preferentially enhanced ERbeta-mediated transactivation. In conclusion, PM has a beneficial effect on lipid metabolism in postmenopausal women, which may result from the activation of gene transcription through selective binding of phytoestrogens to ERalpha and ERbeta.

Chemical constituents of Aristolochia constricta: antispasmodic effects of its constituents in guinea-pig ileum and isolation of a diterpeno-lignan hybrid.

Twenty constituents were isolated from the n-hexane and chloroform extracts of Aristolochia constricta, a plant whose aerial parts have been used empirically in folk medicine for various purposes. The inhibitory effects of these constituents on smooth muscle contraction in isolated guinea-pig ileum were studied in order to observe their antispasmodic effects. 3,4-Dibenzyldihydrofuran-type lignans [(-)-cubebin, (-)-hinokinin, and (-)-pluviatolide] and a kaurene-type diterpene [(-)-kaur-16-en-19-oic acid] were isolated as active principals. They inhibited electrically induced and acetylcholine-induced contraction in the isolated guinea-pig ileum. In addition, 9- O-[(-)-kaur-15-en-17-oxyl]cubebin was isolated as a new diterpeno-lignan hybrid, although this constituent did not exhibit antispasmodic activity.

New cytotoxic cyclobutanoid amides, a new furanoid lignan and anti-platelet aggregation constituents from Piper arborescens.

Three new cyclobutanoid amides with trans-trans-trans configurations, piperarborenine C, piperarborenine D and piperarborenine E, and a new furanoid lignan, (+)-arborone, together with twelve known compounds, were isolated from the stems of Piper arborescens. The structures of these new compounds were determined by means of spectral analyses. Piperarborenine C, (+)-diayangambin, piplartine, piperolactam B, piperolactam C, aristolactam BIII, goniothalactam, and methyl trans-3,4,5-trimethoxycinnamate possessed anti-platelet aggregation activity in vitro. Among them, piplartine showed the most potent anti-platelet aggregation activity induced by collagen and showed an IC50 value of 21.5 microM. Piperarborenines A - E, piperarborenine, aristololactam BIII and goniothalactam showed significant cytotoxic activity (IC50 values < 4 microg/mL) against P-388, HT-29 and A549 cell lines in vitro.

The first total synthesis of kwakhurin, a characteristic component of a rejuvenating plant, "kwao keur": toward an efficient synthetic route to phytoestrogenic isoflavones.

A convergent synthesis of kwakhurin (5), a characteristic estrogen-like isoflavone of Pueraria mirifica(Leguminosae), is described. Isoflavone skeleton 31 was constructed by Suzuki-Miyaura coupling of 3-bromochromone 26 (AC-ring) and arylboronic acid 30 (B-ring) in the presence of TBAB as an additive. Microwave-assisted coupling was also examined, but did not improve the yield. Baeyer-Villiger oxidation, followed by propargylation and reduction afforded 1,1-dimethylallyl ether 37. 6'-Prenylisoflavone 34 was obtained in high yield by Claisen rearrangement of 37 in N,N-diethylaniline. On the other hand, 1,3-rearrangement of prenyl ether 33 with clay gave 34 in poor yield. Successive methylation of 34 and deprotection yielded the target kwakhurin (5) in 12% overall yield from 2,4-dihydroxybenzaldehyde (23).

ent-halimane diterpenes and a guaiane sesquiterpene from Cladogynos orientalis.

Four new ent-halimane diterpenes (1-4) and one new guaiane sesquiterpene (5) were isolated from the CHCl(3) extract of the roots of Cladogynos orientalis, together with six known compounds. The structures of compounds 1-5 were established using spectroscopic methods, and the stereochemistry of chettaphanin I (6) was confirmed by X-ray crystallography.

Isolation of salicin derivatives from Homalium cochinchinensis and their antiviral activities.

The chemical constituents of Homalium cochinchinensis were examined. From the root bark, in addition to the previously reported cochinolide and its beta-glucopyranoside, cochinchiside A (1) and tremulacinol (4) were isolated together with three known compounds [benzoic acid, tremulacin (2), and tremuloidin (3)]. From the leaves, cochinchiside B (5) was isolated as new compound. The structures of the new compounds (1, 4, 5) were determined by spectroscopic and/or chemical methods. Antiviral testing of compounds 2-5 against HSV-1 and HSV-2 showed that tremulacin (2) and cochinchiside B (5) were weakly active. Tremulacin (2) was also weakly active against HIV-1.

Anti-platelet aggregation and chemical constituents from the rhizome of Gynura japonica.

A novel quinonoid terpenoid, (-)- alpha-tocospirone, a new chromanone, (-)-gynuraone, as well as three new steroids, (22 E,24 S)-7 alpha-hydroperoxystigmasta-5,22-dien-3 beta-ol, (22 E,24 S)-stigmasta-1,4,22-trien-3-one, and (24 R)-stigmasta-1,4-dien-3-one, together with 15 known components, were isolated from the rhizome of Formosan Gynura japonica. Structures of the new compounds were determined through spectral analyses and chemical evidence. Several of the isolated compounds, caryophyllene oxide, 6-acetyl-2,2-dimethylchroman-4-one, vanillin, 2,6-dimethoxy-1,4-benzoquinone, and benzoic acid exhibited significant anti-platelet aggregation activity in vitro.

Cytotoxic activity of natural labdanes and their semi-synthetic modified derivatives from Croton oblongifolius.

Labda-7,12( E),14-triene-17-oic acid, previously isolated from Croton oblongifolius, and its derivatives were investigated for cytotoxicity against five human tumor cell lines. Six of these compounds, labda-7,12( E),14-triene-17-al, 17-hydroxylabda-7,12( E),14-triene, 17-acetoxylabda-7,12( E),14-triene, 15-hydroxylabda-7,13( E)-diene-17,12-olide, labda-7,13( E)-diene-17,12-olide, and 12,17-dihydroxylabda-7,13( E)-diene showed non-specific, moderate, cytotoxicity against all cell lines, whereas the other compounds were inactive.

Studies on the chemical constituents of stem bark of Millettia leucantha: isolation of new chalcones with cytotoxic, anti-herpes simplex virus and anti-inflammatory activities.

Four new chalcone derivatives (1, 4, 7, 10) were isolated from the stem bark of Millettia leucantha KURZ (Leguminosae) along with two known ones (2, 6) and five known flavones (3, 5, 8, 9, 11). Structure elucidation and unambiguous assignment of the isolates were achieved with the aid of 1D and 2D NMR extensive studies. Correlation of 10 to 4 was successfully done by reduction with Et(3)SiH/CF(3)CO(2)H. Moderate cytotoxic activity was observed in chalcones (1, 10), whereas dihydrochalcones (4, 6) showed moderate anti-Herpes Simplex Virus (HSV) activity. Interestingly, flavone 8 showed significant anti-inflammatory effects inhibiting both cyclooxygenase (COX)-1 and -2.