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Dab1 is an intracellular adaptor protein essential for brain formation during development. Tyrosine phosphorylation in Dab1 plays important roles in neuronal migration, dendrite development, and synapse formation by affecting several downstream pathways. Reelin is the best-known extracellular protein that induces Dab1 phosphorylation. However, whether other upstream molecule(s) contribute to Dab1 phosphorylation remains largely unknown. Here, we found that EphA4, a member of the Eph family of receptor-type tyrosine kinases, induced Dab1 phosphorylation when co-expressed in cultured cells. Tyrosine residues phosphorylated by EphA4 were the same as those phosphorylated by Reelin in neurons. The autophosphorylation of EphA4 was necessary for Dab1 phosphorylation. We also found that EphA4-induced Dab1 phosphorylation was mediated by the activation of the Src family tyrosine kinases. Interestingly, Dab1 phosphorylation was not observed when EphA4 was activated by ephrin-A5 in cultured cortical neurons, suggesting that Dab1 is localized in a different compartment in them. EphA4-induced Dab1 phosphorylation may occur under limited and/or pathological conditions in the brain.

The polarity of the biological membrane, or lipid order, regulates many cellular events. It is generally believed that the plasma membrane polarity is regulated according to cell type and function, sometimes even within a cell. Neurons have a variety of functionally specialized subregions, each of which bears distinct proteins and lipids, and the membrane polarity of the subregions may differ accordingly. However, no direct experimental evidence of it has been presented to date. In the present study, we used a cell-impermeable solvatochromic membrane probe NR12A to investigate the local polarity of the plasma membrane of neurons. Both in hippocampal and cerebellar granule neurons, growth cones have higher membrane polarity than the cell body. In addition, the overall variation in the polarity value of each pixel was greater in the growth cone than in cell bodies, suggesting that the lateral diffusion and/or dynamics of the growth cone membrane are greater than other parts of the neuron. These tendencies were much less notably observed in the lamellipodia of a non-neuronal cell. Our results suggest that the membrane polarity of neuronal growth cones is unique and this characteristic may be important for its structure and function.

Puerarin (daidzein 8-C-glucoside) is an isoflavone C-glucoside contained in the roots of Pueraria lobata OHWI. We have previously isolated the human intestinal bacterium, strain PUE, which metabolizes puerarin to daidzein, though the enzyme which cleaves C-glycosidic bond has not been clarified. Here, we identified one of the intermediates of enzymatic puerarin C-deglycosylation reaction as 3″-oxo-puerarin (1): C-3 in the glucose moiety connecting to hydroxyl is oxidized to ketone group. 1 was easily isomerized to the mixture of 1, 2″-oxo-puerarin (2a) and cyclic acetal (2b) of 2a in non-enzymatic condition. We identified the putative puerarin-metabolizing operon of strain PUE composed of 8 genes (dgpA-H). Among them, DgpB-C complex was expressed in Escherichia coli, which cleaved the C-glycosidic bond in 1 but not puerarin. These results suggested that the puerarin C-deglycosylation reaction is a two-step enzymatic reaction, including the oxidation reaction at C-3″ in puerarin to give 1, and the subsequent C-deglycosylation of 1 to provide daidzein.

Reelin is a secreted protein that antagonizes the deposition and toxicity of amyloid β peptide (Aβ). Therefore, augmentation of Reelin activity may ameliorate Alzheimer's disease (AD). We have recently reported that a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS-3) cleaves and inactivates Reelin in the mouse brain. In the present study, we investigated the effect of reducing ADAMTS-3 on deposition of Aβ by crossbreeding drug-inducible ADAMTS-3 conditional knock-out (cKO) mice with "next-generation" AD model mice. We found that reducing ADAMTS-3 inhibited deposition of Aβ significantly in App NL-F mice, which produce human wild-type Aβ. On the other hand, reducing ADAMTS-3 had no effect in App NL-G-F mice, which produce the Arctic mutant Aβ (E22G) that forms protofibrils more efficiently than does wild-type Aβ. Thus, the findings suggest that the administration of an inhibitor against ADAMTS-3 will prevent the progression of AD pathology caused by deposition of wild-type Aβ.

We previously isolated the human intestinal bacterium, strain PUE, which can cleave the C-glucosidic bond of puerarin to yield its aglycone daidzein and glucose. In this study, we partially purified puerarin C-glucosidic bond cleaving enzyme from the cell-free extract of strain PUE and demonstrated that the reaction was catalyzed by at least three proteins, Mn(2+), and oxidized form of nicotinamide adenine dinucleotide (NAD(+)). We completely purified one of the proteins, called protein C, by chromatographic separation in three steps. The molecular mass of protein C was approximately 40 kDa and the amino acid sequence of its N-terminal region shows high homology to those of two putative proteins which belong to Gfo/Idh/MocA family oxidoreductase. Protein C catalyzed hydrogen-deuterium exchange reaction of puerarin to 2"-deuterated puerarin in D(2)O condition, which closely resembles those of glycoside hydrolase family 4 and 109.

Baicalin (BG) and its aglycone, baicalein (B) are strong antioxidants that exert various pharmacological actions and show unique metabolic fates in the rat. The aim of the present study was to identify major metabolite(s) besides BG in rat plasma after oral administration of BG or B. The main metabolite was detected by HPLC equipped with an electrochemical detector at a potential of +500 mV and identified as baicalein 6-O-β-D-glucopyranuronoside (B6G) by HPLC/MS/MS. When BG at a dose of 20 mg/kg was administered orally to Wistar rats, the level of B6G in plasma was higher than that of BG. Cmax and the area under the concentration-curve from 0 to 24 h (AUC0-24 h) values of the plasma B6G were 1.66 ± 0.34 µM and 19.8 3.9 ± µM · h, respectively, whereas those of BG were 0.853 ± 0.065 µM and 10.0 ± 3.1 µM · h, respectively. When B was administered, similar results were also obtained. B6G-producing activities from B were found in microsomes of both rat jejunum and liver, in spite of the low activity. Rat everted jejunal sacs formed B6G after application of B, but only in a small amount that was excreted into the mucosal side, and not the serosal side, indicating little contribution to the appearance of B6G in plasma. On the other hand, when B was injected into the rat portal vein, B6G was detected at a higher level than BG in the systemic circulation, demonstrating the hepatic contribution to the appearance of plasma B6G.

The present study was to study the hepatoprotective effects of phloridzin (PHL) on hepatic fibrosis induced by carbon tetrachloride (CCl₄) in rats, on the basis of this investigation, the possible mechanism of PHL was elucidated. Male Sprague Dawley (SD) rats were randomly divided into six groups: control, model, PHL-L, PHL-M, PHL-H and colchine. All rats except control group were intraperitoneally injected with CCl₄, and control rats were injected with olive oil, twice a week for eight weeks. At the same time, the rats were orally given homologue drugs once a day, respectively. Hepatoprotective effects of PHL were evaluated by liver weight indexes, biochemical values, total antioxidant capacity and total-superoxide dismutase, histopathological observations, hepatic fibrosis, and the hepatic fibrosis relative gene and protein expressions. PHL significantly improved hepatic function; remarkably decreased serum hyaluronic acid (HA), transforming growth factor-β1 (TGF-β1), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and liver tissues hydroxyproline, malondialdehyde (MDA) levels, increased glutathione peroxidase (GSH-Px), total-antioxygen capacity (T-AOC) and total-superoxide dismutase (T-SOD) contents of liver tissues; Real-time polymerase chain reaction (PCR) and immunohisto-chemical results showed PHL might markedly reverse the up-regulated mRNA and protein expressions of the α-smooth muscle actin (SMA), TGF-β1 and tissue inhibitor of metalloproteinase-1 (TIMP1), up-regulate the matrix metalloproteinase-1 (MMP1) mRNA and protein expressions. Histopathological observations provided supportive evidence for biochemical analyses and the hepatic fibrosis relative gene and protein expressions, and with the dose of PHL increasing, the aforesaid improvement became more and more strong. The studies demonstrated that PHL exerted beneficially hepatoprotective effects on hepatic fibrosis induced by CCl₄, mainly enhancing antioxidant capacity of liver organizations, reduce the level of lipid peroxidation induced by CCl₄, and protect hepatocyte membranes from damage, and alleviate hepatic fibrosis.

We isolated a human intestinal bacterium, capable of cleaving the C-ring and dehydroxylating the B-ring of both (+)-catechin (2R,3S) and (-)-epicatechin (2R,3R). Although the strain was classified as Eggerthella (Eg.) lenta [named Eg. sp. CAT-1 (JF798636)] by 16S ribosomal RNA (rRNA) gene similarity, it was quite different in substrate specificity from a previously isolated strain, Eg. sp. SDG-2, which takes part in cleavage of the C-ring and dehydroxylation of the 3,4-dihydroxyphenyl moiety (B-ring) of (3R)-flavan-3-ol derivatives. On the other hand, both Eg. sp. CAT-1 and Eg. sp. SDG-2 showed the same substrate specificity against dehydroxylation of enantiomeric lignans, (+)- and (-)-dihydroxyenterodiol, and (+)- and (-)-dihydroxyenterolactone.

C-Glycosides are usually resistant against acidic hydrolysis and enzymatic treatments because C-1 of the sugar moiety is directly attached to the aglycone by C-C bonding. Nevertheless, a human intestinal bacterium, strain PUE, can cleave the C-glucosyl bond of puerarin to yield its aglycone daidzein. To clarify the mechanism of the cleaving reaction, we tried to identify the structure of the metabolite derived from the sugar moiety of puerarin. To detect it easily, deuterium labeled puerarin, [6″,6″-D(2)]puerarin, was prepared in 7 steps. Sugars contained in a metabolite mixture from [6″,6″-D(2)]puerarin was analyzed by an HPLC-electrospray ionization (ESI)-MS method after treatment of sugars with 1-phenyl-3-methyl-5-pyrazolone (PMP). Since deuterium labeled glucose was detected in the metabolite mixture of [6″,6″-D(2)]puerarin, we concluded that puerarin was metabolized to daidzein and an intact glucose by strain PUE. As C-1 of the sugar was hydroxylated instead of hydrogenating, C-glucosyl bond-cleaving reaction is not reduction but hydrolysis. This is the first report of revealing the reaction manner and the exact products of C-glucosyl bond-cleaving reaction.

In recent years, the number of patients suffering from diseases, such as cancer, apoplexy, osteoporosis, hypertension, and type 2 diabetes mellitus is increasing worldwide. Type 2 diabetes, a lifestyle-related disease, is recognized as a serious disease. Various types of pharmaceutics for diabetes have been used. Since the relationship between diabetes and biometals such as vanadium, copper, and zinc ions has been recognized for many years, we have been developing the anti-diabetic metal complexes as new candidates. We found that several zinc(II) (Zn) complexes exhibit glucose-lowering activity for treating type 2 diabetes. High doses of salicylates have been known to reverse hyperglycemia and hyperinsulinemia in type 2 diabetic patients. These findings strongly suggest that the combined use of Zn and salicylates achieves the synergism in treating type 2 diabetes. Because aspirin, acetyl salicylic acid, has a chelating ability, we used it as a ligand to Zn. Several Zn-salicylate complexes were prepared and their biological activities were examined in this study. The complexes with an electron-withdrawing group in the ligand exhibited higher in vitro insulinomimetic activity than those of Zn complexes with an electron-donating group in the ligand. When bis(aspirinato)Zn (Zn(asp)₂) complex was orally administered on KK-A(y) mice with hereditary type 2 diabetes, the diabetic state was improved. In addition, this complex exhibited normalizing effects on serum adiponectin level and high blood pressure in metabolic syndrome. In conclusion, Zn(asp)₂ complex is newly proposed as a potent anti-diabetic and anti-metabolic syndrome agent.

Reelin is a very large secreted glycoprotein that is essential for brain formation and function, but the mechanism by which it affects the dynamics and morphology of neuronal cells remains unsolved. One previous study claimed that Reelin has a proteolytic activity against extracellular matrix proteins, which might explain many of the actions of Reelin. Therefore, in this study wild-type Reelin protein and its mutant in which a supposedly critical serine residue was replaced were expressed and tested for their self-degrading and laminin-degrading activities. We found that both of these proteins generated totally the same cleaved fragments and that neither of them is capable of degrading laminin. It is thus likely that Reelin is not a serine protease and is unable to degrade extracellular matrix.

The alkaloid, rhynchophylline (RHY), from the stems and hooks of Uncaria rhynchophylla was revealed in recent years to have protective effect on neuronal damage. The present research was carried out to investigate the in vivo metabolism of this bioactive alkaloid. After administering RHY to rats, LC-MS detected RHY in plasma, bile, brain, urine and feces, the glucuronides, 11-hydroxyrhynchophylline 11-O-beta-D-glucuronide (M1) and 10-hydroxyrhynchophylline 10-O-beta-D-glucuronide (M2) in bile, and 11-hydroxyrhynchophylline (M3) and 10-hydroxyrhynchophylline (M4) in urine and feces. Within 24 h, 78.0% of RHY was excreted into the feces and 12.6% into the urine of rats after oral administration of 37.5 mg/kg. Monitoring by LC-MS showed that 9.4% of RHY was metabolized to M3 and M4 in a ratio of about 1 : 1. RHY was also detected in the brain (0.650 ng/g) at 3 h after oral administration of the same dose. Cytochrome P450 (CYP) in rat liver microsomes played a key role in RHY hydroxylation. Specific inhibition of CYP isozymes indicated that CYP2D, CYP1A1/2 and CYP2C participated in RHY hydroxylation, but not CYP3A.

Despite the health risks for postmenopausal women, the indications and ideal candidates for hormone replacement therapy remain unclear. The present study used ovariectomized rats to examine the safety and effects of the Chinese herbal formula Menoprogen (MPG), which is prescribed for menopausal syndrome. Daily oral MPG (1000 mg/kg body weight) for 2 weeks significantly recovered uterine and adrenal gland atrophy and restored serum estradiol, estrone and progesterone levels that were decreased in rats by bilateral ovariectomy. However, yeast two-hybrid and nuclear receptor cofactor assays showed that MPG did not bind estrogen receptors alpha (ERalpha) and beta, and immunohistochemical staining revealed that unlike 17beta-estradiol, MPG did not stimulate the protein expression of ERalpha, progesterone receptor, c-jun and c-fos in the uterus. No side effects of MPG were confirmed in vivo. These findings suggest that MPG would be useful for treating women with premenopausal and postmenopausal syndromes.

A human intestinal bacterium, strain END-2, which enantioselectively oxidizes (+)-enterodiol (END) to (+)-enterolactone (ENL) through enterolactol is also responsible for demethylation during plant lignan metabolism. A free hydroxyl group adjacent to the methoxy group is required for demethylation. The bacterium transformed (+)- and (-)-secoisolariciresinol to (+)-ENL and (-)-END, respectively, by co-incubation with strain ARC-1, which is responsible for dehydroxylation.

In this study, the antipruritic effect of the methanol extract of Ganoderma lucidum (MEGL) was studied in mice. Oral administration of MEGL (10-1000 mg/kg) produced a dose-dependent inhibition of scratching, an itch-related response, induced by intradermal 5-hydroxytryptamine (5-HT) (100 nmol/site), alpha-methyl-5-HT (100 nmol/site), and proteinase-activated receptor-2 (PAR(2))-activating peptide SLIGRL-NH(2) (50 nmol/site). However, MEGL (100-1000 mg/kg) did not inhibit the scratching induced by histamine (100 nmol/site), substance P (100 nmol/site), and compound 48/80 (10 microg/site). These results raise the possibility that MEGL is effective against pruritus mediated by proteinases and 5-HT and that primary afferents expressing PAR(2) and 5-HT(2A) receptors are the sites of its action.

Phloridzin, a phloretin 2'-beta-D-glucoside, belongs to dihydrochalcones and mainly exists in the fruits of Malus pumila Mill., Lithocarpus polystachyus REHD and the root skins, stems, tender leaves and fruits of Malus hupehensis. It has many pharmacological activities, such as regulating blood sugar level and blood pressure, protecting heart, scavenging of oxygen free radicals and antioxidant injuries. Thus, market demand of products containing phloridzin is increasing year by year. Our research results demonstrated that phloridzin is provided with a double directional adjusting function of estrogenic and antiestrogenic activities. It showed significant effects on the proliferation of estrogen sensitive estrogen receptor (ER) (+)MCF-7 cells in the absence of estrogen. When added with 17beta-estradiol, phloridzin showed antagonism on estradiol-induced MCF-7 cell proliferation, but it did not significantly affect proliferation of estrogen insensitive ER (-)MDA-MB-231 cells. Phloridzin induced beta-galactosidase activity in a yeast two-hybrid assay. Light increase of the uterine weight and serum estradiol content of mouse was observed when the glucoside was administered orally for 7 d. After oral administration, phloridzin was found mainly in the blood and a small part was metabolized to phloretin. Our investigation proved that phloridzin was distributed at the target organ and played the role of phytoestrogen.

Five new highly oxygenated lanostane-type triterpenoids [ganoderic acid GS-1 (1), ganoderic acid GS-2 (2), ganoderic acid GS-3 (3), 20(21)-dehydrolucidenic acid N (4) and 20-hydroxylucidenic acid A (5)] were isolated from the fruiting body of Ganoderma sinense, together with known compounds including 6 triterpenoids and 3 sterols. The structures of the new triterpenoids determined by spectroscopic means including 2D NMR were 7beta-hydroxy-3,11,15-trioxo-lanosta-8,24(E)-dien-26-oic acid (1), 7beta,15alpha-dihydroxy-3,11-dioxo-lanosta-8,24(E)-dien-26-oic acid (2), 12beta-acetoxy-3beta,7beta-dihydroxy-11,15-dioxo-lanosta-8,24(E)-dien-26-oic acid (3), 3beta,7beta-dihydroxy-11,15-dioxo-25,26,27-trinorlanosta-8,20-dien-24-oic acid (4), and 7beta,20xi-dihydroxy-3,11,15-trioxo-25,26,27-trinorlanost-8-en-24-oic acid (5), respectively. Among these, ganoderic acid GS-2, 20-hydroxylucidenic acid N, 20(21)-dehydrolucidenic acid N and ganoderiol F inhibited human immunodeficiency virus-1 protease with IC(50) values of 20-40 microM.

Puerarin and daidzein are the major naturally occurring isoflavones in leguminous plants. These two compounds are metabolized to equol by human intestinal flora. Here we isolated two intestinal bacteria capable of metabolizing puerarin and daidzein, respectively, from human feces. One of them, strain PUE, converted puerarin to daidzein by cleaving a C-glucosyl bond, whereas the other, strain DZE, converted daidzein to equol by reducing a double bond in ring C followed by elimination of an oxo group. Based on the 16S ribosomal RNA gene sequence, strain DZE showed 85% similarity with Eggerthella lenta. Equol produced by strain DZE was identified as (3S)-equol through several analytical methods. Moreover, we obtained (3S)-equol from puerarin by co-incubation with strain PUE and DZE. In addition, 5-hydroxyequol was obtained from genistein by incubation with strain DZE.

Female hormone-dependent cancers and other diseases pose a serious health threat for women, and low-risk medicines against such cancers have not yet been discovered. The present study examines the effects of the traditional Chinese herbal mixture, Tokishakuyakusan (TS) and 17beta-estradiol on the uterus of parous ovariectomized rats. Uterine atrophy that causes a reduction in uterine tissue and the uterine cavity area, was induced by ovariectomy, and slightly recovered by the daily oral administration of TS for two weeks (1000 mg/kg body weight). TS restored the decreased plasma estradiol concentration due to ovariectomy. However the yeast two-hybrid assay showed that TS did not bind estrogen receptors alpha and beta and immunohistochemical staining revealed that 17beta-estradiol stimulated the protein expression of estrogen receptor alpha, progesterone receptor, c-fos and c-jun in the uterus, whereas TS did not. These results suggest that TS might be useful for treating menopausal syndromes among women, as well as for patients when hormone replacement therapy (HRT) with estrogen is contraindicated.

Four new lanostane triterpene lactones (colossolactone I, colossolactone II, colossolactone III and colossolactone IV) were isolated from the Vietnamese mushroom Ganoderma colossum (FR.) C. F. BAKER along with five known compounds. The structures of the new compounds were determined on the basis of MS, NMR and circular dichroism.

Two new hopane type triterpenes, named dryopteric acids A (1) and B (2), were isolated from the Rhizome of Dryopteris crassirhizoma (Aspiadaceae) together with sixteen known compounds (3-18). Of isolated compounds, ursolic acid (15), and dryopteric acid A (1) and B (2) showed potent inhibitory activities against HIV-1 protease with IC50 values of 8.9-44.5 microM. In addition, acetylated compounds 1 and 2 appreciably increased inhibitory activities with their IC50 values of 1.7 and 10.8 microM, respectively.

Ergosterol peroxide (EPO, 1) is a major antitumor sterol produced by edible or medicinal mushrooms. Following oral administration of 1 to rats or anaerobic in vitro incubation of 1 with rat fecal bacteria, three metabolites were detected and their structures were identified to be 5alpha,6alpha-epoxyergosta-8(14),22-diene-3beta,7alpha-diol (M1, 2), 5alpha,6alpha-epoxyergosta-8,22-diene-3beta,7alpha-diol (M2, 3), and 5alpha,6alpha-epoxy-3beta-hydroxyergosta-22-ene-7-one (M3, 4) by spectroscopic analysis. Of these, M2 and M3 showed more potent inhibitory activity than the original compound 1 against proliferation of CACO-2, WiDr, DLD-1 and Colo320 human colorectal adenocarcinoma cells. These findings suggest that bacterial metabolites of EPO play a significant role in its cytotoxic activity against human colorectal cancer cells.

We evaluated the composition of Swertia herbs using high performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MS). Eleven peaks of 6 species were unequivocally identified by comparing their retention times, UV spectra, on-line electrospray ionization mass (ESI-MS) spectra, and collision-induced dissociation mass spectrometry/mass spectrometry (CID-MS/MS) data with those of authentic compounds. We adopted wavelengths of 254 nm, 340 nm and 230 nm to simultaneously determine these 11 compounds. By comparing the overall DAD and total ion current (TIC) profiles of various samples, the 6 species were differentiated in terms of the occurrence and/or relative concentrations of the eleven compounds. Our novel validated HPLC-DAD-MS method not only facilitates quality control and identification of Swertia herbs, but is also applicable to systematic investigations of the distribution of secoiridoids, flavonoids, and xanthones in the genus Swertia.

The interaction between growth inhibition and chirality, especially of diastereomers, has an important modifying effect on cancer cell proliferation. Previously, we have reported on the design, synthesis, and chemical properties of a series of novel, double-stranded peptides, (y-AA-x-AA)(2)-(CH(2))(12), with -y-AA-x-AA- and -z-AA-y-AA-x-AA- sequences conjugated to the spacer. Here, we extend those results by showing that (D-, L-) and (L-, D-) diastereomers are more potent inhibitors of tyrosine phosphorylation than (L-, L-). Although the replacement of the L-Phe-L-Phe sequence with L-Tyr-L-Phe produces a less active inhibitor, the double-stranded peptide conjugated with L-Tyr-D-Phe is more active than that conjugated with L-Tyr-L-Phe. In addition, we show that SDS-PAGE gel profiles of tyrosine phosphorylation following treatment with bis(y-Tyr-x-Phe)-N,N-dodecane-1,12-diamine appear very similar to profiles of tyrosine phosphorylation following treatment with an analog of the tyrosine kinase inhibitor, erbstatin. Moreover, the level of autophosphorylation of the epidermal growth factor receptor kinase domain (EGFRKD) treated with bis(L-Tyr-D-Phe)-N,N-dodecane-1,12-diamine was lower than that seen following treatment with bis(L-Phe-D-Phe)-N,N-dodecane-1,12-diamine. These data provide new insights for the control of cancer cell proliferation through drug designs which replace the less active -L-Phe-L-Phe- (and -D-Phe-L-Phe-) with the more active -L-Tyr-L-Phe- (and -L-Tyr-D-Phe-) sequence.

During the course of experiments on the transformation of lignans to phytoestrogenic substances, such as enterodiol (END) and enterolactone (ENL), a previously isolated bacterium, Eubacterium (E.) sp. strain SDG-2, capable of phenolic p-dehydroxylation in the biotransformation of secoisolariciresinol diglucoside to END and ENL, was concluded to be Eggerthella (Eg.) lenta (Eg. sp. SDG-2) on the basis of 16S rRNA gene sequence analysis. The bacterium could transform (+)-dihydroxyenterodiol (DHEND, 3a) to (+)-END (1a), but not for (-)-DHEND (3b) to (-)-END (1b) under anaerobic conditions. By incubation of a mixture of (+)- and (-)-dihydroxyenterolactone (DHENL, 4a and 4b) with Eg. sp. SDG-2, only (-)-DHENL (4b) was converted to (-)-ENL (2b), selectively. On the other hand, we isolated a different bacterium, strain ARC-1, capable of dehydroxylating (-)-DHEND (3b) to (-)-END (1b) from human feces. Strain ARC-1 could transform not only (-)-DHEND (3b) to (-)-END (1b), but also (+)-DHENL (4a) to (+)-ENL (2b). However, the bacterium could not transform (+)-DHEND (3a) and (-)-DHENL (4b). Both bacterial strains demonstrated different enantioselective dehydroxylation.

In the course of our experiments on the metabolic conversion of lignans to the estrogenic substances enterodiol (END) and enterolactone (ENL) by human intestinal flora, we isolated two anaerobes, Ruminococcus sp. END-1 and strain END-2, capable of oxidizing END. The former selectively converted (-)-END to (-)-ENL, while the latter selectively converted (+)-END to (+)-ENL, indicating enantioselective oxidation by intestinal bacteria.

Through an estrogenic activity bioassay-guided fractionation of the 70% ethanolic extract of Cassia tora seeds two new phenolic triglucosides, torachrysone 8-O-[beta-D-glucopyranosyl(1-->3)-O-beta-D-glucopyranosyl(1-->6)-O-beta-D-glucopyranoside] (1) and toralactone 9-O-[beta-D-glucopyranosyl-(1-->3)-O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranoside] (2), along with seven known compounds were isolated. The structures of the new compounds were elucidated on the basis of spectroscopic and chemical evidence. The estrogenic activity of the fractions and the isolated compounds were investigated using the estrogen-dependent proliferation of MCF-7 cells. In addition, the yeast two hybrid assay expressing estrogen receptor alpha (ERalpha) and beta (ERbeta) and the ERalpha competitor screening assay (ligand binding screen) were used to verify the binding affinities of the isolated compounds to ER. Furthermore, a naringinase pre-treatment of the 70% alcoholic extract of Cassia tora seeds resulted in a significant increase in its estrogenic activity. From the naringinase pre-treated extract six compounds were isolated, among which 6-hydroxymusizin and aurantio-obtusin showed the most potent estrogenic activity, while torachrysone, rubrofusarin and toralactone showed a significant anti-estrogenic activity. Finally, the structure requirements responsible for the estrogenic activity of the isolated compounds were studied by investigating the activity of several synthetic compounds and chemically modifying the isolated compounds. The basic nucleus 1,3,8-trihyroxynaphthalene (T(3)HN) was found to play a principal role in the binding affinity of these compounds to ER.

Through an anti-estrogenic bioassay-guided fractionation of the methanol extract of Mansonia gagei, three new coumarins, called mansorins I (1), II (2) and III (3) and a new naphthoquinone, mansonone I (4), were isolated. Their structures were determined based on their NMR data and CD spectroscopy. The anti-estrogenic activity of the fractions and the isolated compounds were investigated using a yeast two-hybrid assay method expressing estrogen receptors alpha (ERalpha) and beta (ERbeta). In addition, an ERalpha competitor screening system (ligand binding screen) was used to verify the binding affinities of the isolated compounds to the estrogen receptor. 1,2-Naphthoquinones (mansonones) showed more binding affinities to ER in both assay systems. All the tested compounds showed higher binding affinities to ERbeta than to ERalpha in the yeast two-hybrid assay. Mansonones F and S showed the most potent estrogen binding and estrogen antagonistic effects.

Plant lignans, such as pinoresinol diglucoside, secoisolariciresinol diglucoside and arctiin, are metabolized to mammalian lignans, enterolactone or enterodiol, by human intestinal bacteria. Their metabolic processes include deglucosylation, ring cleavage, demethylation, dehydroxylation and oxidation. Here we isolated an intestinal bacterium capable of demethylating arctigenin, an aglycone of arctiin, to 2,3-bis(3,4-dihydroxybenzyl)butyrolactone (1) from human feces, and identified as an Eubacterium species (E. sp. ARC-2), which is similar to Eubacterium limosum on the basis of morphological and biochemical properties and 16S rRNA gene sequencing. By incubating with E. sp. ARC-2, arctigenin was converted to 1 through stepwise demethylation. Demethylation of arctigenin by E. sp. ARC-2 was tetrahydrofolate- and ATP-dependent, indicating that the reaction was catalyzed by methyltransferase. Moreover, E. sp. ARC-2 transformed secoisolariciresinol to 2,3-bis(3,4-dihydroxybenzyl)-1,4-butanediol by demethylation.

Our aim in this study is to elucidate the correlations between inhibition and chirality, especially, diastereomer, against cell proliferation of double-stranded peptides. In previous studies, we reported on the design, synthesis, and chemical properties on a series of novel double-stranded peptides, (y-AA-x-AA)(2)-spacer(S) (AA=amino acid, S=spacer, symbols x and y represent L- or D-forms, and (y-, x-) as represent of the symbol) conjugated with -y-AA-x-AA- and -z-AA-y-AA-x-AA- sequences to a spacer of carbon number n. The inhibition of A431 and src(ts)NRK cells growth by four diastereomers of the N(1),N(12)-bis(y-Phe-x-Phe)dodecanediamines (n=12) increased in the following order: (L-, L-)<(D-, D-)<(L-, D-)<(D-, L-). A similar trend was seen in the order for the activity of (y-AA-x-AA)(2)-spacer(S) with a spacer of carbon number n=2, 3, 4, 5, 6, and 12 against the cell growth inhibition. To understand the mechanism of diasteromer selective cell growth inhibition, the correlations between chirality and cell growth inhibition were investigated from the measurement of the changes in cytosolic Ca(2+) concentration (=[Ca(2+)](c)) of A431 cells. Although less active N(1),N(12)-bis(L-Phe-L-Phe)dodecanediamine increases cytosolic [Ca(2+)](c), more active diasteromers, N(1),N(12)-bis(L-Phe-D-Phe)dodecanediamine and N(1),N(12)-bis(D-Phe-L-Phe)dodecanediamine, decrease cytosolic [Ca(2+)](c) in A431 cell. This study provides diastereomeric effected new insights - this controls the polarity of double-stranded peptides - into the control of tumor cell proliferation and design of the uptake by penetration through the cell membrane of drugs.

Mycelia of Antrodia cinnamomea were extracted with chloroform and hot water. A neutral polysaccharide named ACN2a separated from the water extract was purified using 10% CCl3COOH, and repeated column chromatography on HW-65 and DE-52 cellulose. Its structure was determined by chemical and spectroscopic analyses. ACN2a was composed of Gal, Glc, Fuc, Man and GalN (in the ratio 1:0.24:0.07:0.026:faint), in which an alpha-D-(1-->6)-Gal linkage accounted for 73% of all linkages. The ratio of branch points was about 16% of the total residual numbers, and branches were attached to C-2 of galactosyl residues of the main chain. ACN2a had an average molecular weight of 12.9x10(5) Daltons, [alpha]D25=+115 degrees (c=0.44, H2O); [eta]=0.0417dl.g-1, Cp=0.2663 cal/(g. degrees C). The hepatoprotective effect of ACN2a was evaluated using a mouse model of hepatic injury that was induced by Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS). The administration of ACN2a (0.4, 0.8 g/kg/d, p.o.), significantly prevented increases in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzyme activities in mice treated with P. acnes-LPS, indicating hepatoprotective activity in vivo.

Three new lignans, longipedunins A (1), B (2) and C (3), together with three known compounds, benzoyl-binankadsurin A (4), acetyl-binankadsurin A (5) and schisanlactone A (6), were isolated from Kadsura longipedunculata. Their structures and stereochemistry were determined by spectral and single-crystal X-ray analyses. Compounds 1 and 6 showed appreciable inhibitory activity against HIV-1 protease with IC50 values of 50 and 20 microM, respectively.

The antiviral activities of extracts from 5 species of marine algae collected at Haeundae (Pusan, Korea), were examined using plaque reduction assays. Although the activity of a methanol (MeOH) extract of Sargassum ringoldianum (Sargassaceae) was the most potent against several types of viruses, it was also cytotoxic. A MeOH extract of Symphyocladia latiuscula (Rhodomelaceae) and its fractions exhibited antiviral activities against acyclovir (ACV) and phosphonoacetic acid (PAA)-resistant (AP(r)) herpes simplex type 1 (HSV-1), thymidine kinase (TK(-)) deficient HSV-1 and wild type HSV-1 in vitro without cytotoxicity. The major component, 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (TDB) of a CH(2)Cl(2)-soluble fraction was active against wild type HSV-1, as well as AP(r) HSV-1 and TK(-) HSV-1 (IC(50) values of 5.48, 4.81 and 23.3 microg/ml, respectively). The therapeutic effectiveness of the MeOH extract and TDB from S. latiuscula was further examined in BALB/c mice that were cutaneously infected with HSV-1 strain 7401H. Three daily oral administrations of the MeOH extract and TDB significantly delayed the appearance of score 2 skin lesions (local vesicles) and limited the development of further score 6 (mild zosteriform) lesions in infected mice without toxicity compared with controls. In addition, TDB suppressed virus yields in the brain and skin. Therefore TDB should be a promising anti HSV agent.

C-Glucosides, in which sugars are attached to the aglycone by carbon-carbon bonds, are generally resistant to acid and enzyme hydrolysis. The C-glucosyl bond of mangiferin, a xanthone C-glucoside, was cleaved by anaerobic incubation with a human intestinal bacterium, Bacteroides sp. MANG, to give norathyriol. A cell-free extract obtained by sonication of B. sp. MANG demonstrated cleaving activity for mangiferin to norathyriol by adding NADH, diaphorase, and dithiothreitol. Both high molecular weight (>10 k) and low molecular weight (<10 k) fractions obtained from the cell-free extract were required for the activity. MnCl2 was necessary for the activity, but other metal ions were not. By purification of the high molecular weight fraction using DEAE-cellulose and Phenyl Sepharose column chromatography, two fractions, designated as proteins A and B, were separated and required for the activity. Neither protein A nor protein B alone showed any activity. This is the first report describing a C-glucosyl-cleaving enzyme from human intestinal bacterium that seems to involve a novel enzyme mechanism.

The C-glucosyl bond of C-glucosides generally tolerates acid and enzymatic hydrolysis. Many C-glucosides are cleaved by human intestinal bacteria. We isolated the specific bacterium involved in the metabolism of mangiferin (2-beta-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone), C-glucosyl xanthone, from a mixture of human fecal bacteria. The anaerobic Bacteroides species named MANG, transformed mangiferin to the aglycone, norathyriol, suggesting cleavage of a C-glucosyl bond. However, B. sp. MANG cleaved C-glucosyl in a dose- and time-dependent manner only when cultivated in the presence of mangiferin. Cleavage was abolished by inhibitors of RNA and protein syntheses, such as rifampicin and chloramphenicol, respectively, indicating that the enzyme that cleaves C-glucosyl is induced by mangiferin. In contrast, mangiferin did not affect bacterial alpha- and beta-glucosidase activities under any conditions. The C-glucosyl-cleavage in cell-free extracts was not altered by potent glucosidase inhibitors such as 1-deoxynojirimycin and gluconolactone. Therefore, the C-glucosyl-cleaving enzyme substantially differs from known glucosidases that cleave O-glucosides. This is the first description of a specific intestinal bacterium that is involved in the metabolism of mangiferin and which produces a novel and inducible C-glucosyl-cleaving enzyme.

For quantitative determination of 19 triterpene constituents, including six ganoderma alcohols (1-6) and 13 ganoderma acids (7-19), in the products of Ganoderma lucidum, an analytical system was developed using high-performance liquid chromatography with an ODS column. The mobile phase was a linear gradient of 1% AcOH/H(2)O-CH(3)CN and 2% AcOH/H(2)O-CH(3)CN, and the elution profile was monitored at 243 and 250 nm for ganoderma alcohols and acids, respectively. The relative standard deviations of this method were less than 2.35% and 2.18% (n=5) for intraday and interday assays, and the recoveries were 90.9-100.8% and 93.4-103.9% for constituents of alcohol and acid groups, respectively. This system was applied to a quantitative determination of the constituents in 10 different products of G. lucidum: six usual umbrella forms of the fruiting bodies, three antlered forms of the fruiting bodies and spores, and eight specimens from the same G. lucidum strain, which was parasitized on logs from different plants or different fungus beds. The analytical results indicated that the quantity and composition of these triterpenes differed appreciably among various specimens, but the relative ratio of the alcohols and acids was not significantly different when the same strain of G. lucidum was used.

In the course of our search for natural estrogenic compounds from medicinal plants, we found that the methanolic extract from the roots of Moghania philippinensis (Fabaceae) showed significant effects on the proliferation of MCF-7 cells (human breast cancer) and induction of beta-galactosidase activity in a yeast two-hybrid assay. Through estrogenic activity-guided fractionation, we isolated several active flavonoids including prenylated ones. The CHCl(3) fraction and its new constituent, 8-(1,1-dimethylallyl)genistein (9), appreciably increased the uterine weight in ovariectomized rats when administered orally for 14 consecutive days, in which compound 9 showed stronger estrogenic activity than genistein. Antiestrogenic activities were also examined based on the inhibition of MCF-7 cell proliferation and beta-galactosidase activity in the yeast two-hybrid assay, mediated by 17beta-estradiol. 5,7,3',4'-Tetrahydroxy-6,8-diprenylisoflavone (6) showed the strongest antiestrogenic activity.

Five new triterpenoid monodesmosides (sinocrassulosides I-V, 1-5) and six bisdesmosides (sinocrassulosides VI-XI, 6-11), in which 2-11 possess different acyl groups in the glycosidic moieties, were isolated from the roots of Sinocrassula asclepiadea FRANCH. Sinocrassulosides VI (4) and V (5) also contained a novel A-seco aglycone in their structures. All of the structures were determined on the basis of spectroscopic and physicochemical evidence.

A reliable enzyme immunoassay (EIA) method was developed for quantitative determination of aconitine with high sensitivity and specificity. The bovine serum albumin (BSA)- and beta-galactosidase (beta-Gal) conjugates as immunogens and enzyme-labeled antigens were prepared by coupling of their proteins with succinic acid (short chain length; n=2, where n represents the number of methylene units) and hexadecanedioic acid (long chain length; n=14) hemiesters of benzoylaconine through the respective N-hydroxysuccinimide esters as intermediates. Two types of the BSA-conjugates with short and long chains were repeatedly injected into rabbits to obtain anti-aconitine antisera (As1 and As2, respectively). All combinations of beta-Gal-labeled antigens LAg1 (n=2) and LAg2 (n=14) with antisera As1 (n=2) and As2 (n=14) showed high sensitivity to aconitine in a range of 0.1-1.0 ng. Although the combination of LAg2 (n=14) with antiserum As1 (n=2) showed high specificity to aconitine, the combination of LAg2 (n=14) and As2 (n=14) was highly specific to both aconitine and mesaconitine. When aconitine was intravenously administered to rats, the aconitine concentration in their plasma remarkably decreased within the first 60 min, and then gradually declined, suggesting a two-compartment pharmacokinetic model in (V(c) 0.41+/-0.09 l/kg, V(dss) 1.7+/-0.4 l/kg, CL(tot) 10+/-2 ml/min x kg, AUC(0-4800) 2055+/-294.3 ng x min/ml). Following oral administration of aconitine to rats at two doses of 0.1 and 1.0 mg/kg b.w., the maximum plasma concentrations (C(max)) were 0.73+/-0.08 and 3.3+/-0.6 ng/ml at times of 45+/-9 and 150+/-52 min, respectively, and the AUC(0-1440) values were 130+/-4 and 1600+/-270 ng x min/ml. The bioavailability (F) of aconitine was determined to be 0.013, where only 1.3% of the aconitine administered orally was absorbed into the body fluid.

Four known flavonoids and two galloyl glucoses isolated from the stem-bark of Juglans mandshurica (Juglandaceae), namely taxifolin (1), afzelin (2), quercitrin (3), myricitrin (4), 1,2,6-trigalloylglucose (5), and 1,2,3,6-tetragalloylglucose (6), were evaluated for their anti-complement activity against complement system. Afzelin (2) and quercitrin (3) showed inhibitory activity against complement system with 50% inhibitory concentrations (IC(50)) values of 258 and 440 microM. 1,2,6-Trigalloylglucose (5) and 1,2,3,6-tetragalloylglucose (6) exhibited anti-complement activity with IC(50) values of 136 and 34 microM. In terms of the evaluation of the structure-activity relationship of 3,5,7-trihydroxyflavone, compounds 2, 3, and 4 were hydrolyzed with naringinase to give kaempferol (2a), quercetin (3a), and myricetin (4a) as their aglycones, and these were also tested for their anti-complement activity. Of the three aglycones, kaempferol (2a) exhibited weak anti-complement activity with an IC(50) value of 730 microM, while quercetin (3a) and myricetin (4a) were inactive in this assay system. Among the compounds tested, 1,2,3,6-tetragalloylglucose (6) showed the most potent anticomplement activity (IC(50), 34 microM).

By anaerobic incubation of pinoresinol diglucoside (1) from the bark of Eucommia ulmoides with a fecal suspension of humans, eleven metabolites were formed, and their structures were identified as (+)-pinoresinol (2), (+)-lariciresinol (3), 3'-demethyl-(+)-lariciresinol (4), (-)-secoisolariciresinol (5), (-)-3-(3", 4"-dihydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butane-1, 4-diol (6), 2-(3', 4'-dihydroxybenzyl)-3-(3", 4"-dihydroxybenzyl)butane-1, 4-diol (7), 3-(3"-hydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butane-1, 4-diol (8), 2-(3', 4'-dihydroxybenzyl)-3-(3"-hydroxybenzyl)butane-1, 4-diol (9), (-)-enterodiol (10), (-)-(2R, 3R)-3-(3", 4"-dihydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butyrolactone (11), (-)-(2R, 3R)-2-(3', 4'-dihydroxybenzyl)-3-(3", 4"-dihydroxybenzyl)butyrolactone (12), (-)-(2R, 3R)-3-(3"-hydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butyrolactone (13), 2-(3', 4'-dihydroxybenzyl)-3-(3"-hydroxybenzyl)butyrolactone (14), 2-(3'-hydroxybenzyl)-3-(3", 4"-dihydroxybenzyl)butyrolactone (15) and (-)-(2R, 3R)-enterolactone (16) by various spectroscopic means, including two dimensional (2D)-NMR, mass spectrometry and circular dichroism. A possible metabolic pathway was proposed on the basis of their structures and time course experiments monitored by thin-layer chromatography. Furthermore, a bacterial strain responsible for the transformation of (+)-pinoresinol to (+)-lariciresinol was isolated from a human fecal suspension and identified as Enterococcus faecalis strain PDG-1.

After anaerobic incubation of arctiin (1) from the seeds of Arctium lappa with a human fecal suspension, six metabolites were formed, and their structures were identified as (-)-arctigenin (2), (2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3",4"-dimethoxybenzyl)butyrolactone (3), (2R,3R)-2-(3'-hydroxybenzyl)-3-(3",4"-dimethoxybenzyl)butyrolactone (4), (2R,3R)-2-(3'-hydroxybenzyl)-3-(3"-hydroxy-4"-methoxybenzyl)butyrolactone (5), (2R,3R)-2-(3'-hydroxybenzyl)-3-(3",4"-dihydroxybenzyl)butyrolactone (6), and (-)-enterolactone (7) by various spectroscopic means including two dimensional (2D)-NMR, mass spectrometry, and circular dichroism. A possible metabolic pathway was proposed on the basis of their structures and the time course of the transformation. Enterolactones obtained from the biotransformation of arctiin and secoisolariciresinol diglucoside (SDG, from the seeds of Linum usitatissium) by human intestinal bacteria were proved to be enantiomers, with the (-)-(2R,3R) and (+)-(2S,3S) configurations, respectively. Compound 6 showed the most potent proliferative effect on the growth of MCF-7 human breast cancer cells in culture among 1 and six metabolites, while it showed inhibitory activity on estradiol-mediated proliferation of MCF-7 cells at a concentration of 10 microM. These results indicate that the transformation of 1 by intestinal flora might be essential for the manifestation of the estrogenic and antiestrogenic activity of 1.

Three new eudesmane type sesquiterpenoid lindenanolides E (1), F (2) and G (3), and two new aporphine alkaloid lindechunines A (18) and B (20) were isolated from roots of Lindera chunii MERR., together with seven known sesquiterpenes including a new naturally-occurring lindenanolide H (4) and eight known aporphine alkaloids. The structures of these compounds were determined by spectroscopic means. Of the isolated compounds, hernandonine (14), laurolistine (16), 7-oxohernangerine (17) and lindechunine A (18) showed significant anti-human immunodeficiency virus type 1 (HIV-1) integrase activity with IC(50) values of 16.3, 7.7, 18.2 and 21.1 microM, respectively. The major alkaloids presented in the roots of L. chunii were quantitatively analyzed by an HPLC method.

Three new lanostante-type triterpene aldehydes, named lucialdehydes A-C (1-3), were isolated from the fruiting bodies of Ganoderma lucidum, together with ganodermanonol (4), ganodermadiol (5), ganodermanondiol (6), ganodermanontriol (7), ganoderic acid A (8), ganoderic acid B8 (9), and ganoderic acid C1 (10). The structures of the new triterpenes were determined as (24E)-3 beta-hydroxy-5 alpha-lanosta-7,9(11),24-trien-26-al (1), (24E)-3,7-dioxo-5 alpha-lanosta-8,24-dien-26-al (2), and (24E)-3 beta-hydroxy-7-oxo-5 alpha-lanosta-8,24-dien-26-al (3), respectively, by spectroscopic means. The cytotoxicity of the compounds isolated from the ganoderma mushroom was tested in vitro against Lewis lung carcinoma (LLC), T-47D, Sarcoma 180, and Meth-A tumor cell lines. Lucialdehydes B, C (2, 3), ganodermanonol (4) and ganodermanondiol (6) showed cytotoxic effects on tested tumor cells. Of the compounds, lucialdehyde C (3) exhibited the most potent cytotoxicity against LLC, T-47D, Sarcoma 180, and Meth-A tumor cells with ED(50) values of 10.7, 4.7, 7.1, and 3.8 microg/ml, respectively.

Five new withanolide derivatives (1, 9-12) were isolated from the roots of Withania somnifera together with fourteen known compounds (2-8, 13-19). On the basis of spectroscopic and physiochemical evidence, compounds 1 and 9-12 were determined to be (20S,22R)-3 alpha,6 alpha-epoxy-4 beta,5 beta,27-trihydroxy-1-oxowitha-24-enolide (1), 27-O-beta-D-glucopyranosylpubesenolide 3-O-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranoside (withanoside VIII, 9), 27-O-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranosylpubesenolide 3-O-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranoside (withanoside IX, 10), 27-O-beta-D-glucopyranosylpubesenolide 3-O-beta-D-glucopyranoside (withanoside X, 11), and (20R,22R)-1 alpha,3 beta,20,27-tetrahydroxywitha-5,24-dienolide 3-O-beta-D-glucopyranoside (withanoside XI, 12). Of the isolated compounds, 1, withanolide A (2), (20S,22R)-4 beta,5 beta,6 alpha,27-tetrahydroxy-1-oxowitha-2,24-dienolide (6), withanoside IV (14), withanoside VI (15) and coagulin Q (16) showed significant neurite outgrowth activity at a concentration of 1 microM on a human neuroblastoma SH-SY5Y cell line.

The origin of the antioxidant activity of Luobuma aqueous extract was examined by measuring the peroxynitrite (ONOO-)-eliminating activities of fractions of this extract obtained by Sephadex LH-20 column chromatography. Three of the four fractions obtained, i.e., those excluding the H2O-eluted fraction, were found to possess (ONOO-)-eliminating activity. These three fractions were combined and fractionated again by Sephadex LH-20 column chromatography, which yielded five fractions. Seven different compounds were isolated from two of these five fractions with high activity. Epigallocatechin-(4beta-8)-epicatechin showed the highest (ONOO-)-eliminating activity.

The conjugates of some dicarboxylic acid hemiesters of triterpenes which show potent inhibition against human immunodeficiency virus type 1 protease (HIV-1 PR) with a reverse transcriptase inhibitor azidothymidine (AZT) or anti-HIV alkaloid FK 3000 were prepared, and their inhibitory activities were investigated against HIV-induced cytopathic effects (CPE) and HIV-1 PR. Most of the triterpene-AZT conjugates showed potent anti-HIV activity as well as moderate to potent PR inhibitory activity, though AZT itself showed no PR inhibitory activity at all. However, the triterpene-FK 3000 conjugates showed neither PR inhibitory activity nor anti-HIV activity.

Two new flavanone glucosides, (2R)- and (2S)-5-O-beta-D-glucopyranosyl-7,4'-dihydroxy-3',5'-dimethoxyflavanone[pervianoside I (3), peruvianoside II(4)] and a new flavonol glycoside, quercetin 3-O-[beta-D-glucopyranosyl-(1-->2)-[alpha-L-rhamnopyranosyl-(1-->6)]-beta-D-galactopyranoside] (peruvianoside III, 13) were isolated from the leaves of Thevetia peruviana Schum., together with nine known flavonol glycosides and two known iridoid glucosides. The structures of all compounds were determined on the basis of chemical and spectroscopic methods. Their inhibitory effects against HIV-1 reverse transcriptase and HIV-1 integrase were also investigated.

Forty-eight derivatives of phorbol (9) and isophorbol (14) were evaluated for their inhibition of human immunodeficiency virus (HIV)-1 induced cytopathic effects (CPE) on MT-4 cells, as well as their activation of protein kinase C (PKC), as indices of anti-HIV-1 and tumor promoting activities, respectively. Of these compounds, the most potent inhibition of CPE was observed in 12-O-tetradecanoylphorbol 13-acetate (8) and 12-O-acetylphorbol 13-decanoate (6). The former also showed the strongest PKC activation activity, while the latter showed no activity at 10 ng/ml. Both activities were generally observed in those phorbol derivatives with an A/B trans configuration, but not in the isophorbol derivatives with an A/B cis configuration. Acetylation of 20-OH in the phorbol derivatives significantly reduced the inhibition of CPE, as shown in 12-O-, 20-O-diacetylphorbol 13-decanoate (6a) (IC100=15.6 microg/ml) vs. compound 6 (IC100=0.0076 microg/ml), and 12-O-tetradecanoylphorbol 13,20-diacetate (8a) (IC100=15.6 microg/ml) vs. 12-O-tetradecanoylphorbol 13-acetate (8) (IC100=0.00048 microg/ml), except in the case of 12-O-decanoylphorbol 13-(2-methylbutyrate) (4) and phorbol 12,13-diacetate (9c). The reduction of a carbonyl group at C-3 abruptly reduced the inhibition of CPE, as observed in 3beta-hydroxyphorbol 12,13,20-triacetate (9f) (IC100=500 microg/ml) vs. phorbol 12,13,20-triacetate (9d) (IC100=62.5 microg/ml). Although 8 was equipotent in the inhibition of CPE, and activation of PKC, both activities were abruptly decreased by the acetylation of 20-OH and methylation of 4-OH [as in 8a and 4-O-methyl-12-O-tetradecanoylphorbol 13,20-diacetate (8b), respectively]. On the other hand, its positional isomer (12-O-acetylphorbol 13-tetradecanoate (8c) showed neither activities. The removal of a long acyl group in 8 led to a substantial loss of both activities, as shown in phorbol 13-acetate (9b). Of the 12-O-acetyl-13-O-acylphorbol derivatives, the highest inhibition of CPE was observed in 6, which has a dodecanoyl residue at C-13. Both an increase and decrease in the number of fatty acid carbon chains resulted in significant reduction of the inhibition of CPE.

Anaerobic incubation of phorbol (1) from Croton tiglium with human intestinal bacteria afforded five metabolites: isophorbol (2), deoxyphorbol (3), 4beta,9alpha,20-trihydroxy-13,15-seco-1,6,15-tigliatriene-3,13-dione (4), 4beta,9alpha,20-trihydroxy-15,16,17-trinor-1,6-tigliadiene-3,13-dione (5) and 4beta,9a,20-trihydroxy-14(13-->12)-abeo-12alphaH-1,6-tigliadiene-3,13-dione (6). All these metabolites (2-6) were identified and characterized by spectroscopic means, including two-dimensional (2D)-NMR. Nine defined strains from the human intestine showed an ability to transform 1 to these metabolites.