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A new lignan glycoside was isolated from the n -butanol extract of the roots and rhizomes of Sinopodophyllum emodi . On the basis of spectral evidences, its structure was established and named as 4,5-didemethylpodophyllotoxin 7′- O - β - d -glucopyranoside. This compound showed cytotoxicity against four human cancer cell lines (Hela, K562, SH-SY5Y and CNE) in vitro .

HPLC chromatograms of MeOH extracts from a fruit body of the wild-grown P. linteus (natural fruit body), from cultivated fungus (cultivated fruit body), and from the cultured mycelia were compared. The extract prepared from the natural fruit bodies revealed a typical HPLC profile referred to as type 1 with a major peak corresponding to meshimakobnol A (1) together with two minor peaks of hypholomine B (3) and inoscavin A (4); the cultivated fruit bodies exhibited a profile referred to as type 2 with major peaks corresponding to 3 and 4 and a minor peak of 1, and the cultured mycelia showed a profile referred to as type 3 without any of these peaks. We also analyzed HPLC chromatograms of commercial products of P. linteus obtained in the markets. Most of the products claimed to be natural fruit bodies exhibited type 1 profiles, except for one product having an intermediate HPLC profile between type 1 and type 2. The products claimed to be cultivated fruit bodies and cultured mycelia revealed type 2 and type 3 profiles, respectively. The present results indicate that the HPLC chromatogram of the methanol extract of P. linteus can be used as a fingerprint to identify whether the product is from natural fruit bodies, cultivated fruit bodies, or cultured mycelia.

In the present study, the immunotherapeutic activity of oral intake of natural Phellinus linteus fruit body (nPF) against growth and pulmonary metastasis of injected B16/BL6 melanoma cells has been examined. The nPF powder was orally administered to B16/BL6-bearing mice, a model for the pulmonary metastasis of melanoma, at doses of 83, 415 and 2,075 mg/kg day in their diet. The dietary intake of nPF powder significantly prolonged the survival time of the B16/BL6-bearing mice. Furthermore, ingestion of nPF dose-dependently enhanced NO production by macrophages prepared from the tumor-bearing, nPF-treated animals. Simultaneously, nPF did not induce direct cytotoxicity against B16/BL6. These results suggest that the oral intake of nPF powder may be useful in immunochemotherapy for cancer because of its life prolongation through the inhibitory effect on growth and metastasis of cancer.

Acceleration of vascular smooth muscle cell (VSMC) proliferation is closely linked to the pathogenesis of vascular diseases. We, therefore, focused on traditional Japanese herbal medicines (Kampo medicines) used to ameliorate the impairment of microcirculation or blood stasis and screened them for their ability to inhibit rat VSMC proliferation. Among them, Unsei-in was found to effectively suppress VSMC proliferation, and Coptis rhizome was the responsible constituent crude drug. The extract of Coptis rhizome inhibited VSMC proliferation with the GI(50) value of 4.4 microg/ml, which was much lower than those against the proliferation of 3Y1, dRLh-84, B16, and HeLa cells. The Coptis rhizome extract inhibited the progression of VSMC arrested at G(0)/G(1) phase from G(0)/G(1) to S phase, but not that of 3Y1 cells. Biological assay-guided fractionation revealed that an alkaloid of Coptis rhizome, coptisine, was the active ingredient in selectively preventing VSMC proliferation with GI(50) of 3.3 microM (1.2 microg/ml). When the structurally-related isoquinoline alkaloids of protoberberine class were studied for their inhibitory activities, berberine decreased the VSMC proliferation with GI(50) of 95.1 microM (35.4 microg/ml), about 30 times higher concentration than coptisine, while palmatine failed to show any activity. This study provides evidence that coptisine, an ingredient of Unsei-in, prevents VSMC proliferation selectively at lower concentrations compared with various cells or other structurally related alkaloids.

Kanzo-bushi-to (KBT) is a traditional Japanese herbal medicine (Kampo medicine), which is used in Japan to treat rheumatoid arthritis. In the present study, we investigated the suppressive effect of KBT on collagen-induced arthritis (CIA) and further studied the underlying mechanism. CIA was induced in male DBA/1J mice by immunization with bovine type II collagen, followed by a booster injection 21 d later. KBT was given at a dose of 430 mg/kg/d from three days before the first immunization to the end of the experiment. KBT suppressed CIA development effectively and further protected focal bone erosion and bone destruction as evidenced by the reduced histological score. Histochemical examination revealed that KBT decreased TRAP-positive cells at the synovium-bone interface and at the sites of focal bone erosion, coincident with the findings that RANKL/OPG mRNA ratio was significantly reduced by KBT treatment. KBT also decreased mRNA levels of M-CSF and iNOS in joints and of iNOS in peritoneal macrophages. In conclusion, KBT prevented osteoclast generation by decreasing RANKL/OPG ratio and M-CSF mRNA levels, resulting in reduction in bone erosion and destruction. In addition, KBT has anti-inflammatory effect such as the suppression of iNOS expression in peritoneal macrophages and joints of CIA mice. These finding suggests that KBT is a potential new therapeutic agent for the treatment of RA.

To evaluate the usefulness of Kampo medicines (traditional herbal medicines) used clinically for the treatment of rheumatoid arthritis (RA), we selected eight of them and examined their effects on collagen-induced arthritic and pX transgenic mice. Among these, Dai-bofu-to, Kanzo-bushi-to, and Makyo-yokkan-to significantly reduced the severity of arthritis in collagen-induced arthritis (CIA) mice. The onset of arthritis was delayed by three Kampo medicines, but only the effect of Makyo-yokkan-to was statistically significant. In addition, three Kampo medicines suppressed the arthropathy of pX transgenic mice, which had developed spontaneously. The onset of arthritis was delayed by 10.7, 8.3, and 15.4 days following treatment with Dai-bofu-to, Kanzo-bushi-to, and Makyo-yokkan-to, respectively. A study of the underlying mechanism showed that Kanzo-bushi-to decreased serum antitype II collagen antibody levels, suggesting that Kanzo-bushi-to possesses immunomodulating activity. This study shows that some Kampo medicines are effective in an induced or spontaneously developed arthritis animal model of human RA.

Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4P-hydroxyphenyl- lactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid bio- synthesis. fl 2002 Federation of European Biochemical Soci- eties. Published by Elsevier Science B.V. All rights reserved.

Pancratistatin derivatives, 1-O-(3-hydroxybutyryl)pancratistatin (HBP) and 1-O-(3-O-beta-D-glucopyranosylbutyryl)pancratistatin (GBP), showed strong cytostatic activity against rat embryo fibroblast 3Y1 at concentrations less than 1 microM. When the effect on cell cycle progression was examined in 3Y1 fibroblasts arrested at G0/G1 phase by serum deprivation, HBP, GBP, and pancratistatin inhibited the progression of 3Y1 fibroblasts from G0/G1 to S phase. In addition, when the effect on cell cycle progression was studied in 3Y1 fibroblasts synchronized at late G1/early S phases by treating with hydroxyurea, HBP blocked further progression through S phase, while GBP and pancratistatin did not affect the progression, but retarded it. On the other hand, when the effect of HBP and GBP on the progression was evaluated in promyelocytic leukemia HL-60RG cells synchronized at G0/G1 phase, the cells did not progress into S phase and accumulated in sub G0/G1 phase, which indicated apoptotic cells. These findings suggest that of Amaryllidaceae alkaloids, HBP blocks the progression of cell cycle at least at G0/G1 and S phases and GBP does at least at G0/G1 phase, resulting in apoptosis induction in tumor cells.

Biological activities of five Veronica species (Scrophulariaceae), V. cymbalaria, V. hederifolia, V. pectinata var. glandulosa, V. persica and V. polita were studied for their anti-inflammatory and cytotoxic activities. Their methanol extracts showed both the inhibitory activity of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophages and cytotoxic activity against KB epidermoid carcinoma and B16 melanoma. When the methanol extracts were fractionated between water and chloroform, water fractions significantly inhibited NO production without any cytotoxicity, while chloroform fractions showed cytotoxicity dose-dependently. When the radical scavenging activity was determined using 2,2-diphenyl-1-picryl-hydrazyl (DPPH), water fractions of the five Veronica species scavenged free radicals effectively, suggesting that the inhibitory effect of this species on NO production was due to their radical scavenging activity. On the other hand, chloroform fractions of Veronica species except for V. cymbalaria showed similar cytotoxic activity against KB and B16 melanoma cells.

Shichimotsu-koka-to (SKT) is a Kampo (traditional Japanese herbal) medicine, which is used in Japan to treat hypertension and atherosclerosis. We investigated the inhibitory effect of SKT on experimental pulmonary metastasis of B16 melanoma cells. The intake of SKT at a dose of 430 mg/kg for 6 weeks from 2 weeks before tumor inoculation significantly reduced the number of metastatic surface nodules in the lung and extended the life span. When the duration of SKT intake was examined, survival time was not affected by preintake before B16 melanoma cell inoculation and was slightly extended by postintake after B16 melanoma cell inoculation, although the life span was prolonged by intake throughout the experiment. To address the mechanism underlying the antimetastatic effect of SKT, we studied whether SKT modulated macrophage function, which is involved in killing tumor cells. The intake of SKT for 6 weeks dose dependently increased nitric oxide (NO) production by macrophages following stimulation with lipopolysaccharide. The elevated NO was found to serve as a cytotoxic mediator against B16 melanoma cells in co-culture with macrophages. On the contrary, B16 melanoma-conditioned medium reduced NO production by macrophages. However, SKT treatment reversed the reduction in NO production by the conditioned medium significantly. These findings may suggest that macrophage function-modulating activity by SKT appears to underlie its antimetastatic activity, which leads to a decrease in the number of lung metastatic surface nodules and the extension of life span.

A genomic clone encoding mature karasurin-A (KRNA), a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica, was efficiently expressed in E. coli using an expression cassette vector pMAL-c2. The resultant recombinant KRNA fused with maltose-binding protein (MBP) was recovered from the soluble fraction of the bacterial cells and purified to near homogeneity after one round of the affinity chromatography. Neither the karasurin precursor retaining both N- and C-terminal peptides, nor the protein with the N-terminal peptide was successufully produced even as a MBP-fusion. The protein with its C-terminal peptide was over-produced but was recovered in an insoluble fraction. Both the recombinant MBP-KRNA fusion protein and recombinant KRNA with MBP removed were as active as the native KRNA from root tubers. The immunogenicity of the recombinant KRNA was also unaffected by fusion with MBP.

The differential display technique was used to isolate genes expressed during yeast extract-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures, and a cDNA clone whose deduced amino acid sequence is similar to plant cytosolic NADP-malic enzymes was identified and designated LeME. The heterologously-expressed LeME oxidized L-malate in presence of NADP, but it did not catalyze formation of 4-hydroxyphenyllactic acid from 4-hydroxyphenylpyruvate in presence of NADPH, one of the crucial biosynthetic steps leading to rosmarinic acid. The potential role of LeME in elicitor-inducible rosmarinic acid biosynthesis is discussed.

A known phenylethanoid glycoside, ehrenoside (1), was isolated together with three new phenylethanoid glycosides, verpectoside A (2), B (3) and C (4) from the aerial parts of Veronica pectinata var. glandulosa. On the basis of spectral analysis (UV, FAB-MS, 1H-, 13C- and 2D-NMR), compounds 2-4 were determined to be 2-(3,4-dihydroxyphenyl)ethyl-O-alpha-L-arabinopyranosyl-(1--2)-[alpha-L-rhamnopyranosyl-(1--3)]-(4-O-trans-feruloyl)-beta-D-glucopyranoside, 2-(3,4-dihydroxyphenyl)ethyl-O-beta-D-glucopyranosyl-(1--2)-[alpha-L-rhamnopyranosyl-(1--3)]-(4-O-trans-caffeoyl)-beta-D-glucopyranoside and 2-(3,4-dihydroxyphenyl)ethyl-O-beta-D-glucopyranosyl-(1--2)-[alpha-L-rhamnopyranosyl-(1--3)]-(4-O-trans-feruloyl)-beta-D-glucopyranoside, respectively. Isolated phenylethanoid glycosides exhibited potent radical scavenging activity against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical.

Monensin (1), an ionophore antibiotic agent, was converted to 25-carboxymonensin (2) and 7-O-substituted-25-carboxyl derivatives (3a–d), which have two carboxyl groups at both ends of the molecule. Ca2+ ion transport activity of the dicarboxylic monensins was evaluated by the CHCl3 liquid membrane method, and 25-carboxymonensins (2 and 3a–d) were shown to transport the Ca2+ ion through the CHCl3 liquid membrane.

Gallic acid (GA) derivatives, 3,4-methylenedioxyphenyl 3,4,5-trihydroxybenzoate (GD-1) and S-(3,4-methylenedioxyphenyl)3,4,5-trihydroxythiobenzoate (GD-3), were previously reported to induce apoptosis in tumor cells with IC50s of 14.5 microm and 3.9 microm, respectively. To elucidate the mechanism by which these gallic acid derivatives (GDs) induce apoptosis, we studied whether GD-1 and GD-3 can activate caspases. When promyelocytic leukemia HL-60RG cells were treated with GD-1 and GD-3, poly(ADP-ribose)polymerase (PARP), a substrate of caspase-3, was cleaved into 85 kDa of degradative product with increasing incubation time. GA also activated PARP cleavage, which was inhibited by catalase, N-acetyl-L-cysteine (NAC), and intracellular Ca2+ chelator 1,2-bis(2-aminophenoxyethane)-N,N,N,N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), in addition to a caspase inhibitor, Z-VAD-FMK. Its inhibitory pattern was identical with that of hypoxanthine/xanthine oxidase. On the other hand, GD-1- and GD3-induced PARP cleavage was not suppressed by catalase or NAC, but by BAPTA-AM. This suggested that the GD-elicited signaling pathway is different from GA's. Taken together, GDs activated caspase-3 following intracellular Ca2+ elevation independent of reactive oxygen species. Thus, it became evident that the signaling pathway leading to apoptosis was regulated by GDs in a different manner from GA.

The C-26 modified monensin derivatives, 26-O-benzoylmonensin (3), 26-O-benzylmonensin (4) and 26-phenylaminomonensin (5) were prepared from monensin (1). Na+ ion transport activity through biological membrane and antibacterial activity of 3-5 were evaluated and compared with the activities reported for a 26-phenylurethane derivative (2). Among these compounds, 5 showed the largest Na+ ion transport and antibacterial activities. In these compounds, the formation of head-to-tail hydrogen bonds was suggested to be an important factor for Na+ ion transport and antibacterial activities.

Ionophore antibiotic monensin ( 1 ) was converted to 25-carboxylmonensin ( 3 ), which has two carboxyl groups at both ends of the molecule. Ca 2+ ion transport activity of 25-carboxylmonensin ( 3 ) was evaluated by the CHCl 3 liquid membrane method, and the activity was 3.1 times the value of the calcium ionophore, lasalocid A.

Nine novel prenyl-dihydrofurocoumarin-type sesquiterpenoid derivatives, 2, 3-dihydro-7-hydroxy-2R*, 3R*-dimethyl-2-[4, 8-dimethyl-3(E), 7-nonadienyl]-furo[3, 2-c]coumarin, 2, 3-dihydro-7-hydroxy-2S*, 3R*-dimethyl-2-[4, 8-dimethyl-3(E), 7-nonadien-6-onyl]-furo[3, 2-c]coumarin, 2, 3-dihydro-7-hydroxy-2S*, 3R*-dimethyl-2-[4-methyl-5-(4-methyl-2-furyl)-3(E)-pentenyl]-furo[3, 2-c]coumarin, 2, 3-dihydro-7-hydroxy-2R*, 3R*-dimethyl-2-[4-methyl-5-(4-methyl-2-furyl)-3(E)-pentenyl]-furo[3, 2-c]coumarin, 2, 3-dihydro-7-methoxy-2S*, 3R*-dimethyl-2-[4, 8-dimethyl-3(E), 7-nonadienyl]-furo[3, 2-c]coumarin, 2, 3-dihydro-7-methoxy-2R*, 3R*-dimethyl-2-[4, 8-dimethyl-3(E), 7-nonadienyl]-furo[3, 2-c]coumarin, 2, 3-dihydro-7-methoxy-2S*, 3R*-dimethyl-2-[4, 8-dimethyl-3(E), 7-nonadien-6-onyl]-furo-[3, 2-c]coumarin, and 2, 3-dihydro-7-methoxy-2S*, 3R*-dimethyl-2-[4-methyl-5-(4-methyl-2-furyl)-3(E)-pentenyl]-furo[3, 2-c]coumarin, were isolated from the roots of Ferula ferulioides. The structures were established by comprehensive spectral analysis. The biosynthetic pathway leading to these prenyl-furocoumarin-type sesquiterpenoids is proposed based on their structures.

Four new triterpenoid saponins, ardisimamilloside C (1), 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->4)-[beta -D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl]-3beta,16al pha,28,30-tetrahydroxy-olean-12-en, ardisimamilloside D (2), 3-O-?alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->4)-[beta -D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl]-3beta,15al pha,28,30-tetrahydroxy-olean-12-en, ardisimamilloside E (3), 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->4)-[beta -D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosl]-13beta,2 8-epoxy-3beta,16alpha,29-oleananetriol, and ardisimamilloside F (4), 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->4)-[beta -D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl]-3beta,16al pha-dihydroxy-13beta,28-epoxy-oleanan-30-oic acid were isolated from the roots of Ardisia mamillata Hance. Structure assignments were established on the basis of highresolution (HR)-FAB-MS, 1H-, 13C-, and two-dimensional (2D)-NMR spectra, and on the chemical evidence.

We studied here the effects of 8 kinds of kampo-hozais clinically used to treat atopic dermatitis (Shofu-san, Toki-inshi, Unsei-in, Oren-gedoku-to, Ji-zuso-ippo, Jumi-haidoku-to, Juzen-taiho-to, Hochu-ekki-to) on delayed-type hypersensitivity (DTH), using three types of murine models such as picryl chloride (PC)-induced (contact hypersensitivity), sheep red blood cell (SRBC)-induced (Jones-Mote's reaction) and tuberculin-induced DTH response, in order to clarify and to compare the immunopharmacological action of kampo-hozais. Most of the kampo-hozais investigated here suppressed PC-induced contact hypersensitivity, especially at the inductive phase. Comparing the efficacies of these kampo-hozais on the three types of DTH responses in mice, they were generally divided into 4 groups. Shofu-san significantly reduced PC-induced and tuberculin-induced DTH responses but not a SRBC-induced DTH response. On the other hand, Toki-inshi reduced contact hypersensitivity, tuberculin type DTH response and Jones-Mote's reaction. Ji-zuso-ippo and Juzen-taiho-to suppressed mainly Jones-Mote's reaction, and Unsei-in, Oren-gedoku-to and Jumi-haidoku-to intensively suppressed contact hypersensitivity. We thought that these findings could help us understand how to use these kampo-hozais properly.

In this study, the cytotoxic activity of gallic acid derivatives (GDs) was studied using some cancer cell lines. Among them, 3,4-methylenedioxyphenyl 3,4,5-trihydroxybenzoate (GD-1) and S-(3,4-methylenedioxyphenyl)-3,4,5-trihydroxy-thiobenzoate (GD-3) were found to induce cell death in cancer cell lines with IC50s ranging from 2.9 to 114.4 microM, a concentration comparable with or lower than that of gallic acid. On the other hand, although gallic acid did not show any cytotoxicity against primary cultured rat hepatocytes and human keratinocytes, GD-1 and -3 showed slightly higher sensitivity against such normal cells, when compared with gallic acid. The cell death induced by gallic acid and GD-1 was accompanied by internucleosomal DNA fragmentation characteristic of apoptosis, whereas only smear DNA degradation was detected following GD-3 treatment. When the mechanism by which GD-1 and -3 caused cell death in HL-60RG cells was examined, GD-1 and -3-induced cell death was inhibited by the intracellular Ca2+ chelator, bis-(o-aminophenoxy)-N,N,N,N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), calmodulin inhibitor, W-7, and the Ca2+/Mg2+ -dependent endonuclease inhibitor zinc sulfate. In contrast, catalase, N-acetylcysteine (NAC), and ascorbic acid inhibited gallic acid-induced apoptosis in HL-60RG cells, whereas they had no effect on GD-1- and -3-induced cell death. This result suggests that GD-1 and -3 induced cell death in a different manner to gallic acid. In conclusion, esterification of gallic acid with a 3,4-methylenedioxyphenyl group yielded potent agents to treat cancer with a different signaling pathway from gallic acid, although selectivity was lost.

The metabolic behavior of orally administered gallic acid was investigated by HPLC and 4-O-methyl gallic acid was found to be the main metabolite in rat peripheral blood and urine. After oral administration of gallic acid, maximum concentration in portal vein and inferior vena cava occurred at 15 and 30 min, respectively. In portal vein, gallic acid was preferentially detected relative to 4-O-methyl gallic acid, whereas gallic acid and 4-0-methyl gallic acid were equally detected in inferior vena cava. On the other hand, 4-O-methyl gallic acid but not gallic acid was found in liver. The contents of gallic acid and 4-O-methyl gallic acid in urine were nearly 100 times higher than those in blood. The ratio of 4-O-methyl gallic acid to total gallic acid metabolites in urine was from 0.55 to 0.76, indicating that a considerable amount of gallic acid was excreted without being metabolized. In this study we found that gallic acid administered orally existed in the blood for 6 h at most, and more than half was metabolized to 4-O-methyl gallic acid, followed by excretion into urine.

Shosaikoto, which is one of the Japanese and Chinese traditional herbal medicine mixtures, has several immunomodulating activities. Especially, Shosaikoto is a potent polyclonal B cell mitogen in vivo and in vitro. In order to characterize the active substances for mitogenic activity, Shosaikoto was fractionated by means of ethanol precipitation to give two fractions, called ethanol-precipitate (EP) fraction and ethanol-soluble (ES) fraction. The EP fraction consisted mainly of polysaccharides and showed significant mitogenic activity. The ES fraction consisted of low molecular compounds and did not show the activity in vitro. The EP fraction of hot water extracts of Glycyrrhizae Radix and Bupureuri Radix showed the activities, and the ES fraction of a hot water extract of Glycyrrhizae Radix and all fractions of hot water extract of Scutellariae Radix showed the cytotoxicity. These results suggested that the crude polysaccharide fractions could play an important role in the mitogenic activity by Shosaikoto.

Two new Amaryllidaceae alkaloids, 1- O -(3-hydroxybutyryl) pancratistanin and 1-O-(3-O-β- d -glucopyranosylbutyryl) pancratistatin, were isolated from the bulbs of Zephyranthes carinata . The structures of the two alkaloids were established by comprehensive spectral analysis. The cytotoxicity of these two alkaloids and pancratistatin is appraised.

Three new hederagenin-based acetylated saponins isolated from the fruits of Gliricidia sepium, were identified by chemical and spectroscopic methods as hederagenin-3-O- (4-O-acetyl-beta-D-xylopyranosyl)-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2 ) -alpha-L-arabinopyranoside, hederagenin-3-O-(3,4-di-O-acetyl-beta-D- xylopyranosyl)-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-alpha- arabinopyranoside and hederagenin-3-O-(3,4-di-O-acetyl-alpha-L- arabinopyranosyl)-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L- arabinopyranoside.

Rubroskyrin, a modified bis-anthraquinone pigment from Penicillium islandicum Sopp, was studied on the redox interaction with NADH-linked redox system of rat liver microsomes, comparing with luteoskyrin and rugulosin. It was found that rubroskyrin was enzymatically reduced by NADH/microsomes and was immediately autoxidized by dissolved oxygen, producing hydrogen peroxide (H2O2). Luteoskyrin and rugulosin did not exhibit such a redox reaction, consuming dissolved oxygen and producing H2O2. The H2O2 production was significantly accelerated by superoxide dismutase (SOD), suggesting the production of superoxide anion during the reduction–autoxidation cycles of rubroskyrin. The thin layer chromatographic (TLC) and spectrophotometric analyses revealed that rubroskyrin was biotransformed by the NADH/microsomes system to stable product(s) which was no longer enzymatically reduced by the NADH/microsome system. From these results it was speculated that the mutagenicity of rubroskyrin might result from the generation of active oxygen by the NADH/microsome-catalyzed redox reaction, and that the redox reaction-linked biotransformation might lead to the elimination of cytotoxicity of rubroskyrin, showing significantly less toxicity than those of luteoskyrin and rugulosin in vivo. Copyright © 1998 John Wiley & Sons, Ltd.

Gallic acid (3,4,5-trihydroxybenzoic acid) is a naturally abundant plant phenolic compound and it is well known as a component of hydrolyzable tannins. We report here that gallic acid and related compounds have trypanocidal activity against Trypanosoma brucei brucei (GUTat 3.1) in both the long slender bloodstream forms and the procyclic forms, in vitro. LD50 values of gallic acid are 46.96 +/- 1.28 microM for bloodstream forms and 30.02 +/- 3.49 for procyclic forms, respectively. A study of structurally related compounds suggested that the pyrogallol moiety could be responsible for this activity.

The antihyperlipidemic action of Ogi-Keishi-Gomotsu-To-Ka-Kojin (OKGK) was examined in rats with experimentally-induced hyperlipidemia. Oral administration of OKGK at 0.69 or 1.38 g/kg/day significantly reduced the increase of serum triglycerides (TG) and phospholipids (PL). OKGK given as a nutritional supplement (1.25%) in the diet significantly suppressed the increase of serum TG and PL in rats with hypercholesterolemia induced by a cholesterol and fat-enriched diet. These results suggest the effectiveness of OKGK in the treatment of hypertriglyceridemia induced by both endogenous and exogenous TG. The mechanism by which OKGK shows antihy-pertriglyceridemic action was investigated in detail. OKGK decreased [(14)C] acetic acid incorporation into TG and PL but not fatty acids in primary cultured rat hepatocytes from rats treated with oral OKGK for 1 week. OKGK also reduced [(3)H] oleic acid into TG and PL, suggesting OKGK suppresses TG and PL syntheses in the liver. Furthermore, OKGK enhanced the activities of lipoprotein lipase (LPL) and hepatic TG lipase (HTGL) in postheparin plasma. These results suggest OKGK inhibits triglyceride synthesis in the liver and stimulates the hydrolysis of TG in lipoprotein.

Rubroskyrin, a modified bisanthraquinone pigment from an yellow rice moldPenicillium islandicum Sopp, was examined for its redox-interaction with the mitochondrial respiratory chain by using rat liver submitochondrial particles (SMP) and was compared with luteoskyrin and rugulosin.Rubroskyrin showed a redox interaction with the NAD-linked respiratory chain of SMP, promoting NADH oxidase in the presence of rotenone, a specific inhibitor to coupling site I of the respiratory chain. Rubroskyrin-mediated NADH oxidase was not inhibited by antimycin A and cyanide, inhibitors to coupling sites II and III, respectively, indicating a generation of an electron transport shunt from a rotenone-insensitive site of NADH dehydrogenase (complex I) to dissolved oxygen. An electrontransport shunt to cytochromec oxidase from complex I was also observed in the experiment with cytochromec and antimycin A. Rubroskyrin did not interact with succinate-linked respiratory chain.Such enzymatic redox response which generates electron transport shunt was not detected for luteoskyrin and rugulosin in the present study.

From the underground part of Iris tenuifolia several flavonoids have been isolated. Six new flavanones have been characterized by high resolution mass spectrometry (HR-MS), and 1H and 13C NMR as 5,2′,3′-trihydroxy-6,7-methylenedioxyflavanone, 5,2′-dihydroxy-6,7-methylenedioxyflavanone, 5,2′,3′-trihydroxy-7-methoxyflavanone, 5,3′-dihydroxy-7,2′-dimethoxyflavanone, 3,5,2′,3′-tetrahydroxy-7-methoxyflavanone and 3,5,3′-trihydroxy-7,2′-dimethoxyflavanone. The crystal and molecular structure of the first flavanone has been established by X-ray crystallography.

Hairy root cultures of Trichosanthes kirilowii var. japonica (Cucurbitaceae) were established by direct inoculation of Agrobacterium rhizogenes ATCC15834 on sterile seedlings. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis of intracellular proteins showed that karasurins, ribosome-inactivating proteins (RIPs) in root tubers of T. kirilowii var. japonica, were not accumulated in the hairy roots. A major basic protein produced by the hairy roots was tentatively identified as a class III chitinase based on N-terminal amino acid sequence homology comparisons. In spite of the apparent lack of karasurins, the basic protein fraction from the hairy roots exhibited ribosome-inactivating activity in the rabbit reticulocyte system, suggesting the presence of RIPs in the hairy roots.

Two glycopeptides, supprescins A and B, that suppress the production of pisatin, a phytoalexin of pea, were synthesized. In the synthesis of supprescin A, condensation of 3, 4, 6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosyl trichloroacetimidate or its glycosidic β isomer with N-(carbobenzoxy)-L-seryl-O-benzyl-L-seryl-glycine methyl ester was carried out in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) to give the monoglycosyl tripeptide derivatives. For the synthesis of supprescin B, glycosylation of 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide and 1, 2, 3, 6-tetra-O-benzoyl-α-D-galactopyranose was promoted by silver trifluoromethanesulfonate (AgOTf) to provide a disaccharide derivative. The coupling of diglycosyl imidate, 2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-3, 6-di-O-benzoyl-2-azido-2-deoxy-D-galactopyranosyl trichloroacetimidate, and N-(carbobenzoxy)-L-seryl-O-benzyl-L-seryl-glycyl-4-benzyl-L-aspartyl-5-benzyl-L-glutamyl-O-benzyl-L-threonine methyl ester in the presence of TMSOTf afforded the diglycosyl hexapeptide derivatives. Reduction, followed by N-acetylation, and then removal of the remaining protecting groups afforded the desired supprescin B.

A block synthesis of the model compound for the phytoalexin elicitor-active glycoprotein is described. Combination of the C -terminus free compounds, N-(9- fluorenylmethoxycarbonyl )-O-(tert- butyl )- l - seryl - l - proline (1) or N-(9- fluorenylmethoxycar bonyl )-(2,3,4,6- tetra -O- acetyl -β- d - glucopyranosyl )-(1→ 6)-(2,3,4- tri -O- acetyl -α- d - mannopyranosyl )-(1→ 6)-(2,3,4- tri -O- acetyl -α- d - mannopyranosyl )- l - seryl - l - proline (2) with the N -terminus free compounds, 2,3,4,6- tetra -O- acetyl -β- d - glucopyranosyl (1→6)-(2,3,4- tri -O- acetyl -α- d - mannopyranosyl )-(1→6)-(2,3,4- tri -O- acetyl -α- d - mannopyranosyl )- l - seryl - l - prolyl - l - seryl - l - proline methyl ester (4), O-(tert- butyl )- l - seryl - l - prolyl -(2,3,4,6- tetra -O- acetyl - β- d - glucopyranosyl )- (1→6)-(2,3,4- tri -O- acetyl -α- d - mannopyranosyl )-(1→6)-(2,3,4- tri -O- acetyl -α- d - mannopyranosyl )- l - seryl - l - proline methyl ester (6) or 2,3,4,6- tetra -O- acetyl -β- d - glucopyranosyl -(1→6)-(2,3,4- tri -O- acetyl -α- d - mannopyranosyl )-(1→6)-(2,3,4- tri -O- acetyl -α- d - mannopyranosyl )- l - seryl - l - prolyl -(2,3,4,6- tetra -O- acetyl -β- d - glucopyranosyl )-(1→6)-(2,3,4- tri -O- acetyl -α- d - mannopyranosyl )-(1→6)-(2,3,4- tri - O- acetyl -α- d - mannopyranosyl )- l - seryl - l - proline methyl ester (8), by use of N -ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) gave three hexaglycosyl hexapeptides and a nonaglycosyl hexapeptide derivatives (9, 11, 14, and 17). These N -terminus free compounds were derived from triglycosyl tetrapeptides (3, and 5) or a hexaglycosyl tetrapeptide (7) on selective deblock reaction by morpholine. The hexaglycosyl hexapeptides (10, 13, and 16) and the nonaglycosyl hexapeptide (18) have been prepared by the convergent block synthesis.

The toxic effect of rubroskyrin, a modified bis-anthraquinone pigment fromPenicillium islandicum Sopp, on mitochondrial respiration has been studied by using isolated rat liver mitochondria, comparing with those of luteoskyrin and rugulosin which are well knownislandicum toxins. It was found that rubroskyrin exerted an uncoupling effect on mitochondrial respiration, abating the respiratory control ratio (RCR) by a dose dependent manner (UD50: 10 μM) and markedly depressed state 3 respiration at high concentrations, whereas such respiration-impairing effects of luteoskyrin and rugulosin were not detected at the concentrations tested (max. 35 μM). The involvement of redox-reaction was not detected in the uncoupling effect of the quinone pigment, rubroskyrin.Spectroscopic study at various pHs revealed that rubroskyrin possessed a pK value within the range of physiological pH, suggesting that rubroskyrin is a weak acid-type uncoupler capable of conducting protons across mitochondrial inner membrane.

Four new oleanane-type saponins, papyrioside LA--LD, were isolated from the leaves of Tetrapanax papyriferum. The structures of these new compounds were elucidated as 11 alpha-hydroxy-3,21-dioxo-olean-12-en-28-oyl-alpha-L-rhamnopyranosy l- (1-->4)-(6-O-acetyl-beta-D-glucopyranosyl)-(1-->6)- beta-D-glucopyranoside, 11 alpha-methoxy-3,21-dioxo-olean-12-en-28-oyl-alpha-L-rhamnopyranosy l- (1-->4)-(6-O-acetyl-beta-D-glucopyranosyl)-(1-->6)- beta-D-glucopyranoside, 3 alpha-hydroxy-11 alpha-methoxy-21-oxo-olean-12-en-28-oyl-alpha-L-rhamnopyranosyl-(1 -->4)- (6-O-acetyl-beta-D-glucopyranosyl)-(1-->6)-beta-D-glucopyranoside and 21 alpha-hydroxy-11 alpha-methoxy-3-oxo-olean-12-en-28-oyl-alpha-L- rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)- beta-D-glucopyranoside, respectively, on the basis of spectroscopic and chemical evidence.

We investigated the anti-atherosclerotic action shown by Shosaikoto, a Kampo medicine, using hypercholesterolemic mice. Oral administration of Shosaikoto significantly suppressed the elevation of serum cholesterol in C57BL/6 mice fed a 1.25% cholesterol-enriched diet for four weeks and improved the T cell ratio in peripheral blood, which decreased with the increase of the serum cholesterol level. In addition, Shosaikoto reduced the accumulation of cholesteryl oleate, which alters macrophages into foam cells, after the treatment of macrophages with oxidized or acetylated low density lipoprotein (LDL). Enzymatic study revealed that the treatment of macrophages with oxidized LDL enhanced acyl-coenzyme A : cholesterol acyltransferase (ACAT) activity and markedly reduced neutral cholesteryl ester hydrolase (NCEase) activity. Shosaikoto treatment prevented a decrease in the NCEase activity, however due to the oxidized LDL treatment, although it slightly augmented ACAT activity. Thus, Shosaikoto, which is known to modulate the immune system, improves macrophage and lymphocyte functions diminished by hypercholesterolemia, resulting in an anti-atherosclerotic action.

Novel 1H,6H-pyranyl[4,3-c][2]benzopyrane-1,6-diones, meshimakobnol A and B, were isolated from natural Phellinus linteus fruit body. The structure elucidation of these fused aromatic compounds was achieved by a spectroscopic method including the measurement of FG-HMBC with various delay times.

Gallic acid (3,4,5-trihydroxybenzoic acid) is a naturally occurring plant phenol obtained by the hydrolysis of tannins and is know to show some pharmacological activities. In screening anti-cancer agents in traditional Chinese medicines, gallic acid was found to show cytotoxicity against all cancer cells that we examined in this study (IC50s: 4.8-13.2 micrograms/ml). Gallic acid was found to show cytotoxicity against primary cultured rat hepatocytes and macrophages, and lesser cytotoxicity against fibroblasts and endothelial cells. Cell death in dRLh-84 cells occurred within 6h after gallic acid treatment at a concentration of more than 20 micrograms/ml. A study of structurally related compounds suggested that the cytotoxicity shown by gallic acid was not a common feature in phenolic compounds, but was a fairly specific characteristic of gallic acid. That is, three adjacent phenolic hydroxyl groups of gallic acid were responsible for the cytotoxicity, and the carboxyl group was not responsible, but seemed to be implicated in distinguishing between normal cells and cancer cells.

A comparison of antihepatotoxic activities between glycyrrhizin (18 beta-GL) and its genuine aglycone, glycyrrhetinic acid (18 beta-GA), was carried out using in vivo and in vitro assay methods. The oral administration of 18 beta-GA at 1, 24, and 48 h before D-galactosamine (GalN) treatment significantly reduced the increase of serum transaminase activities 24 h after GalN treatment, whereas 18 beta-GL did not inhibit the increase of serum transaminase activities. The intraperitoneal administration of 18 beta-GA 1 h before GalN treatment restored the increase of serum transaminase activities with lower doses than 18 beta-GL. In CCl4-induced cytotoxicity of primary cultured rat hepatocytes, 18 beta-GA protected the CCl4-induced leakage of transaminase at doses of 5 to 50 micrograms/ml, whereas 18 beta-GL inhibited slightly the leakage at a dose of 1000 micrograms/ml. In the same way, 18 alpha-GA, the alpha-isomer of 18 beta-GA, reduced the CCl4-induced cytotoxicity more strongly than 18 alpha-GL. Furthermore, the adsorbability of 18 alpha, beta-GA on primary cultured rat hepatocytes was higher than that of 18 alpha, beta-GL. These results suggest that 18 alpha, beta-GA is a more potent antihepatotoxic agent than 18 alpha, beta-GL, and that the potency of the antihepatotoxic compounds parallels their adsorbability in hepatocytes.

The inhibitory effects of various synthetic oligosaccharides (1-8) on anti-lipid IV antiserum binding activity were examined by ELISA (enzyme linked immunosorbent assay). Compound 8, containing an epitope GlcA-4Me beta 1-4Fuc of lipid IV, inhibited but precursors (1-5) of lipid IV did not inhibit the binding activity. In addition to the nonreducing end disaccharide derivative (6) having a methyl group at position 4 of glucuronic acid, its analogous compound (7) having no methyl groups was synthesized and their inhibition activities were compared. Moreover, the inhibitory effects of compounds (1-5) were examined using each antiserum.

Three novel 2-prenyl-dihydrofurochromone-type sesquiterpenoid derivatives, 2,3-dihydro-7-hydroxy-2S*,3R*-dimethyl-2-[4,8-dimethyl-3(E),7-nonadienyl]-furo[2,3-b]chromone, 2,3-dihydro-7-hydroxy-2S*,3R*-dimethyl-2-[4-methyl-5-(4-methyl-2-furyl)-3(E),7-pentenyl]-furo[2,3-b]chromone, and 2,3-dihydro-7-hydroxy-2R*,3R*-dimethyl-2-[4-methyl-5-(4-methyl-2-furyl)-3(E),7-pentenyl]-furo[2,3-b]chromone, were isolated from the roots of Ferula ferulaeoides. The structures were established by comprehensive spectral analysis. The biosynthetic pathway leading to these 2-prenyl-dihydrofurochromone-type sesquiterpenoids is proposed based on their structures.

Serum concentrations of arctiin, tracheloside, and their metabolites formed in the gastrointestinal tract were investigated in the rat. Arctiin or tracheloside was not detected in the serum after oral administration (200 mg/kg). In regard to their metabolites, each metabolite 1 (AM1, TM1), their genuine genins, appeared in the serum, and the serum concentration of arctiin metabolite 1 (AM1) reached its peak at 4 h and that of tracheloside metabolite 1 (TM1) reached its peak at 8 h. On the other hand, both metabolites 2 (AM2, TM2), which each possess a catechol moiety as reported previously, were not found in the serum. Now, we have studied the detection of their metabolites in the rat large intestinal contents after oral administration. It was revealed that all metabolites reported previously were certainly formed in rat gastrointestinal tract in vivo. Thus, we presumed a possibility that metabolite 2 was converted into metabolite 1 through C-3" methylation by catechol-O-methyltransferase (COMT) in rat liver. Each metabolite 2 was incubated with rat liver cytosol in the presence of S-adenosyl-L-methionine. It was proved that metabolite 2 was rapidly converted into metabolite 1 within 3 min. We suggest that arctiin or tracheloside was transformed to at least two metabolites in the gastrointestinal tract, and after absorption from the intestine, metabolite 2 was converted into metabolite 1 through methylation by COMT in the liver, and arctiin and tracheloside existed as metabolite 1, the genuine genin, in the blood stream.

A genomic clone encoding a karasurin precursor was isolated from root tubers of Trichosanthes kirilowii var. japonica. The sequence analysis indicates that karasurin-A and karasurin-C exhibiting abortifacient and ribosome-inhibiting activities are produced from a common karasurin precursor by proteolitically cleaving at a different site. Northern hybridization analysis shows that the karasurin gene was expressed only in root tubers. An efficient system to overproduce the recombinant karasurin-A as fused protein with the maltose-binding protein was established.

Tow new aryltetralin-type lignan glycosides: methyl epipodophyllate 7'-O-beta-D-glucopyranoyl-(1-->6)-beta-D-glucopyranoside (1), 4-demethylepipodophyllotoxin 7'-O-beta-D-glucopyranoside (2), and a new phenyl ethanol glycoside: phenyl ethanol 4-O-beta-D-xylopyranosyl-(1-->6)-beta-D-glucopyranoside (3), along with three known compounds: junipetriolosides (4), 3,4-dihydroxy-phenyl ethanol (5), and 4-hydroxy-phenyl ethanol (6) were isolated and identified from the n-butanol extract of the roots and rhizomes of Sinopodophyllum emodi (Wall.) Ying. The structures of the above were established by means of spectral data and chemical methods.