The cytochalasin class of fungal metabolites was analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for their rapid identification in microbial extracts. ESI-MS analyses of reference cytochalasins were performed and several product ions were produced in MS/MS experiments on parent ions that are structurally characteristic. A precursor ion search was performed to detect cytochalasins in an ethyl acetate extract of fungal strain RK97-F21. Three cytochalasins were detected and one of the components was identified as epoxycytochalasin H by comparing the tandem mass spectra of the product ions with those of reference compounds. This finding was further validated by LC/MS and LC/MS/MS experiments.

A taxine, 5 alpha O-(3'-dimethylamino-3'-phenylpropionyl) taxinine M (1) together with two known compounds 7-O-acetyltaxine A (2) and 2 alpha-acetoxy-2' beta-deacetylaustrospicatine (3) were isolated from the needles of the Himalayan yew, Taxus wallichiana Zucc. Their structures were elucidated on the basis of the NMR spectral data, ESI-MS/MS analysis and chemical methods. Compounds 1 and 3 showed moderate cytotoxic activity against the lung cancer cell line A549 in vitro.

A sensitive method for the detection and characterization of basic taxoids from the ethyl acetate extract of Taxus wallichiana has been described. A combined analysis of the fragmentation spectra of 3 purified standard basic taxoids and the substructure analysis of 139 previously reported taxoids provided information on typical primary and secondary product ions that are generated by CID mass spectrometry of basic taxoids. Precursor-scan analysis of selected product ions allowed for the detection of 57 basic taxoids from the ethyl acetate extract of T. wallichiana, 45 of which have not been reported. The method describe in this paper provides a fast method for the "dereplication" of natural products. The mass spectrometric data derived by this method was sufficient for the partial structure elucidation of novel basic taxoids. The method presented in this paper can be easily adapted into a high-throughput screening protocol for the identification and characterization of bioactive natural products.

The seeds of Alpinia blepharocalyx K. Schum. (Zingiberaceae) is used in Chinese traditional medicine for the treatment of stomach disorders. From the ether fraction of a 95% ethanolic extract, which showed hepatoprotective and antiproliferative activities, we isolated 16 novel diarylheptanoids bearing a chalcone or a flavanone moiety [calyxins A-H; epicalyxins B-D, G, and H; 6-hydroxycalyxin F; and blepharocalyxins A and B] together with seven known compounds, while the residual fraction of the ethanolic extract gave 32 novel diarylheptanoids namely, calyxins A, E-G, and I-M; epicalyxins B, F, I-K, and M; deoxycalyxin A; blepharocalyxins C-E; neocalyxins A and B; (3S,5S)- and (3S,5R)-3-hydroxy-1-(4-hydroxyphenyl)-5-methoxy-7-phenyl-6E-heptene, (3S,5S)- and (3S,5R)-3-hydroxy-1-(4-hydroxyphenyl)-5-ethoxy-7-phenyl-6E-heptene, (3S)-3-methoxy-1,7-bis(4-hydroxyphenyl)-6E-hepten-5-one, 1,7-bis(4-hydroxyphenyl)-hepta-4E,6E-dien-3-one, (3S,7R)-5,6-dehydro-1,7-bis(4-hydroxy-phenyl)-4"-de-O-methyl-centrolobine, (3S,5S,6S,7R)-5,6-dihydroxy-1,7-bis(4-hydroxyphenyl)-4"-de-O-me-thylcentrolobine, (3S,5R,6S,7R)- and (3S,5S,6R,7R)-5,6-dihydroxy-1,7-bis(4-hydroxyphenyl)-4"-de-O-methyl-centrolobine, 1,2- dihydro-bis(de-O-methyl)curcumin, and (3S,7S)-5,6-dehydro-4"-de-O-methylcentrolobine, and one known diarylheptanoid [(3S,5S)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane] together with 12 other known phenolic compounds. Moreover, in vitro NO inhibitory and antiproliferative activities of the isolated compounds were also tested and the active constituents identified.

The absorption, distribution and excretion of butylidenephthalide after dermal application to hairless mouse have been examined with [8–14C]butylidenephthalide. By the investigation of the whole body autoradiogram and liquid scintillation analysis, it was indicated that the transdermally applied butylidenephthalide quickly permeate into peripheral circulation system without accumulation in the skin and then distribute into lung, liver, bile and kidney. The total radioactivity, however, was decreased due to excretion into urine, and in the case of i.v.-administration, 80% of the administered butylidenephthalide was excreted into urine within 24 h, while only 5% was excreted into feces within 24 h. Then, the metabolite in urine was determined to be a cysteine conjugate by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method. Thus, it has been concluded that after dermal application butylidenephthalide quickly permeates through skin into peripheral circulation system; distributes to lung, liver, bile and kidney; and then excreted into urine as a cysteine adduct.

Alpinia blepharocalyx K. Schum. (Zingiberaceae) is used in traditional Chinese medicine for the treatment of stomach disorder (1). We have recently reported fifteen novel diar-ylheptanoids together with six known phenolic compounds (2-5). Further investigation on the ether soluble fraction of the seeds of A. blepharocalyx has led to the isolation of a new diarlyheptanoid, 5,6-dehydro-4"-de-O-methylcentrolobin (1)

Four unprecedented diarylheptanoids-calyxin H (1) and epicalyxin H (2), possessing a diarylheptanoid unit and a chalcone moiety, and blepharocalyxins A (3) and B (4), possessing two diarylheptanoid units and a chalcone moiety-were isolated from the seeds of Alpiniablepharocalyx. The structures of 1-4, including absolute stereochemistry, were elucidated by spectroscopic means and after a consideration of their biogenesis.

Thirteen novel diarylheptanoids bearing a chalcone or a flavanone moiety (1-13), a new curcumin derivative, 1,2-dihydrobis(de-O-methyl)curcumin (14), and two known flavonoids (15 and 16) isolated from the seeds of Alpinia blepharocalyx K. Schum. were tested for their inhibitory effects on nitric oxide (NO) production in lipopolysaccaride (LPS)-activated murine macrophages J774.1 in vitro. All the tested compounds inhibited NO production in a concentration-dependent manner (IC50=36-568 microM). Among the compounds examined, blepharocalyxin B (13) was the most potent inhibitor of NO production (IC50=36 microM). Analysis of the structure activity relationship among these novel diarylheptanoids led to the conclusion that the position of attachment of a chalcone or a flavanone to a diarylheptanoid does not affect their inhibitory potency although their presence in association causes a substantial enhancement of the inhibitory activity. Moreover, a conjugated double bond in a chalcone moiety potentiated the inhibitory activity. On the other hand, hexamethoxydeoxycalyxin A (17) and pentamethoxycalyxin B (18), a methylated product of calyxin A (1) and an epimeric mixture of calyxin B, showed greatly reduced activity suggesting that phenolic hydroxyl groups are involved in the inhibitory activity.

Hypoglycemic activity-guided fraction led to the isolation of the known compound, 4-hydroxybenzoic acid, from Pandanus odorus Ridl. (Thai name: Toei-hom, Pandanaceae). This compound showed a hypoglycemic effect in normal rats after the oral administration of 5 mg/kg. Additionally, the compound increased serum insulin levels and liver glycogen content in normal rats.

The hypoglycemic effect of the water extract of the whole plant of Piper sarmentosum Roxb. (Piperaceae, Thai name: Chaplu) was examined in normal and streptozotocin-diabetic rats. In an oral glucose tolerance test, a single oral administration of the water extract at doses of 0.125 and 0.25 g/kg significantly lowered the plasma glucose level in the normal rats. A reference drug, glibenclamide, at a dose of 5 mg/kg (per os, p.o.) also showed a significant hypoglycemic effect in the normal rats. In contrast, a single oral administration of the water extract at these doses and glibenclamide did not significantly lower the plasma glucose level in the diabetic rats. However, the repeated oral administration of the water extract at a dose of 0.125 g/kg for 7 days produced a significant hypoglycemic effect in the diabetic rats. Glibenclamide (5 mg/kg, p.o.) also caused significant hypoglycemia in the diabetic rats. These results demonstrated that the water extract of whole plant of Piper sarmentosum has a hypoglycemic effect in rats.

Calyxins A (1), E (2) and F (3), 6-hydroxycalyxin F (4), Calyxin G and epicalyxin G (5 and 6), novel diarylheptanoids having a chalcone or a flavanone moiety, were isolated from Alpinia blepharocalyx K. Schum. and their structures, including the corrected one of calyxin A (1), were elucidated by spectroscopic methods.

We previously found that a methanolic extract of the stems of Sambucus sieboldiana inhibited bone resorption in organ culture. In this study, we further fractionated the methanol extract guided by the activity towards bone resorption stimulated by parathyroid hormone (PTH) in vitro. The ethyl acetate fraction (EtOAc Fr.) of the methanolic extract inhibited PTH-stimulated bone resorption of neonatal mouse bones, and the inhibitory activity was more potent than those of other fractions. Oral administration of the EtOAc Fr. (50 and 100 mg/kg/d) to ovariectomized (OVX) rat prevented the decrease in bone mineral density (BMD) of the lumbar (L2-4) vertebra, indicating that the EtOAc Fr. is effective in vivo. Furthermore, the EtOAc Fr. (50, 100 and 150 mg/kg/d) decreased the serum calcium level elevated in low calcium dietary rats. The phenolic constituents of the EtOAc fraction were examined for their inhibitory effect on bone resorption stimulated by PTH in neonatal mouse bone. Among them, vanillic acid, vanillin and coniferyl alcohol showed significant inhibitory effects on bone resorption. Of the compounds examined, vanillic acid was found to have a significant inhibitory effect on the decrease of BMD in OVX mice. Therefore, the EtOAc Fr. of S. sieboldiana showed a suppressive effect on bone resorption in vitro and in vivo. In addition, the inhibitory effects of the EtOAc Fr. on bone resorption may be at least partly due to the inhibitory action of vanillic acid.

1 2 cis Tribulus terrestris N-trans 3 4 N-trans 5 1 5 1 2 D D

Blepharocalyxins A and B (1 and 2), novel natural products having two diarylheptanoids and a chalcone moiety were isolated from the seeds of Alpinia blepharocalyx K. Schum. The structures of these compounds were elucidated by intensive study of the 2D NMR spectroscopy. These compounds inhibited nitric oxide production in endotoxin-activated murine macrophages, J 774.1.

Six novel diarylheptanoids bearing chalcone or flavanone moiety (1–6) were isolated from the seeds of Alpinia blepharocalyx K. Schum. Their structures were determined by spectroscopic analysis. Stereo-chemical assignment of diarylheptanoid part of these compounds was done by NMR spectral analysis of their MTPA esters. These compounds inhibited nitric oxide (NO) production in endotoxin-activated murine macrophages, J774.1.

The hepatoprotective effects of the methanol and water extracts of the roots of Panax notoginseng were studied on various animal models. The methanol and water extracts of P. notoginseng showed a significant activity in carbon tetrachloride-induced liver injury in rats. However, the methanol extract was found to be more active. The hepatoprotective effects of the methanol extract was further evaluated using D-galactosamine as well as heat-killed Propionibacterium acnes/lipopolysaccharide-induced liver injury models. Two major isolates from the methanol extract, ginsenosides-Re and -Rgl, showed a significant hepatoprotective effect on D-galactos-amine/lipopolysaccharide-induced liver injury in mice.