ALKALOIDS FROM OXYTROPIS MYRIOPHYLLA (PALL) DC.

Five alkaloids were isolated from the epigeal part of Oxytropis myriophylla. Three alkaloids were identified as N-benzoyl-ß-phenylethylamine, N-trans-cinnamoyl-ß-phenylethylamine, N-cis-cinnamoyl-ß-phenylethylamine and the structures of two new alkaloids were elucidated to be N-benzoyl-ß-hydroxyphenylethylarnine(2), N-trans-cinnamoyl-ß-hydroxy-phenylethylamine(5). The absolu.te structures were established by modified Mosher method.

HPLC profiling of Phellinus linteus.

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.

Effects of two Prunella species on lymphocyte proliferation and nitric oxide production.

Water extracts of Prunella vulgaris and P. laciniata have been investigated for their immunomodulatory and antiinflammatory activities concerning their effect on the mitogenic response of murine splenocytes and nitric oxide production by murine peritoneal macrophages in vitro. It was found that both extracts stimulated the proliferation of T-lymphocytes and suppressed NO production in lipopolysaccharide-stimulated macrophages dose dependently without any cytotoxicity.

Stimulation of lymphocyte proliferation and inhibition of nitric oxide production by aqueous Urtica dioica extract.

The immunomodulatory and antiinflammatory activities of aqueous Urtica dioica extract were investigated for their effect on the mitogenic response of murine splenocytes and nitric oxide production by murine peritoneal macrophages in vitro. It was found that this extract stimulated the proliferation of T-lymphocytes and suppressed NO production in lipopolysaccharide-stimulated macrophages without affecting cell viability.

Dermal mast cells play a central role in the incidence of scratching behavior in mice induced by multiple application of the hapten, 2,4,6-trinitrochlorobenzene.

Repeated application of 1% 2,4,6-trinitrochlorobenzene (TNCB) in acetone solution causes chronic skin inflammation in BALB/c mice. Associated scratching behavior gradually appeared, and chronic scratching behavior was established over 40 days after the initial application of TNCB. In order to explore the possible involvement of T cells and mast cells in the appearance of pruritus, we examined the response of athymic nude mice and genetically mast-cell-deficient mice. We could not detect either severe skin inflammation or immunoglobulin (Ig)E production in T-cell-deficient BALB/c nu/nu mice even after 80 days of TNCB treatment, whereas typical severe skin inflammation and IgE production were observed in mast-cell-deficient WBB6F1-W/Wv and WBB6F1-Sl/Sld mice. Furthermore, we observed persistent scratching behavior in WBB6F1-W/Wv mice, but not in BALB/c nu/nu and WBB6F1-Sl/Sld mice. Histological examination of TNCB-treated animals revealed the development of dermal mast cells in W/Wv mice but not in Sl/Sld mice. Degranulation of dermal mast cells was observed in the WBB6F1-W/Wv genotype, but most mast cells remained intact in TNCB-treated BALB/c nu/nu mice. These results suggest that mast cells play a pivotal role in the incidence of scratching behavior in this chronic pruritus model.

Suppressive effect of Kanzo-bushi-to, a Kampo medicine, on collagen-induced arthritis.

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.

Sho-saiko-to and Saiko-keisi-to, the traditional Chinese and Japanese herbal medicines, altered hepatic drug-metabolizing enzymes in mice and rats when administered orally for a long time.

As the consumption of herbal remedies has increased, the opportunity that such herbal medicines are co-administered with other drugs has also risen gradually and we are, therefore, very much concerned about herb-drug interactions. We examined the effects of pre-administration of Kampo medicines (Sho-saiko-to, Saiko-keishi-to, Shigyaku-san and Dai-saiko-to) on the pentobarbital-induced sleeping time in mice and rats, to clarify the possibility that they could affect the drug-metabolizing enzymes. The administration of Sho-saiko-to and Saiko-keishi-to for 4 weeks significantly shortened the pentobarbital-induced sleeping time in mice and the administration of Sho-saiko-to for 2 weeks significantly reduced the sleeping time in rats. Furthermore, we tried to identify the molecular species of rat cytochrome P450s (CYPs) affected by Sho-saiko-to and Saiko-keishi-to by competitive RT-PCR. The oral administration of Sho-saiko-to for 2 weeks upregulated the mRNA expression of CYP2B, CYP3A1, CYP2E1 and CYP4A1 in rats. The treatment with Saiko-keishi-to for 2 weeks also up-regulated the mRNA expression of CYP2B, CYP3A1 and CYP4A1. Sho-saiko-to and Saiko-keishi-to may potentially influence the drug-metabolizing enzymes in man, and would thus require much attention when used in the clinical situation.

Iridoid glucosides from Veronica pectinata var. glandulosa.

A new highly oxygenated iridoid glucoside, urphoside B (1) was isolated from the Veronica pectinata var. glandulosa together with seven known iridoid glucosides, aucubin, catalpol, veronicoside, catalposide, verproside, amphicoside and 6-O-veratroyl catalpol. The planar as well as the stereo structures of the isolated compounds were determined by means of extensive 1D- and 2D-NMR spectroscopy and confirmed by HR-Mass.

New lignan glycosides from Chinese medicinal plant, Sinopodophillum emodi.

Two new podophillotoxin glucosides, L-picropodophillotpxin 7'-O-(beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside) (2) and L-picropodophillotpxin 7'-O-beta-D-glucopyranoside (3), were isolated from Chinese medicinal plant, Sinopodophillum emodi, together with 4 known compounds, podophillotoxin (1), podorhizol 4'-O-beta-D-glucopyranoside (4), deoxypodophillotoxin (5), and dehydropodophillotoxin (6). The structures of 2 and 3 were finally determined by the extensive decouping and nuclear Overhauser effect (NOE) experiments in NMR spectra and circular dichroism (CD) spectra. Compound 2 is the second example of podophillotoxin diglucoside, and both the first one and 2 were isolated from S. emodi. X-ray crystal structure analysis of 1, 5, and 6 was carried out. Compounds 1 and 5 showed the different conformations from those reported.

Nuatigenin-type steroidal saponins from Veronica fuhsii and V. muldtfida.

A new nuatigenin-type steroidal saponin, multifidoside (2), was isolated from the aerial parts of Veronica fuhsii and V multifida and its structure was identified as 3-O-([alpha-L-rhamnopyranosyl-(1-->2glu)]-[beta-D-glucopyranosyl-(1-->4rha)-alpha-L-rhamnopyranosyl (1-->4glu)]-beta-D-glucopyranosyl]nuatigenin 26-O-beta-D-glucopyranoside. Additionally, a known steroidal saponoside, aculeatiside A (1), from V. fuhsii, a phenylethanoid glycoside, verpectoside A (3), and a flavon glycoside, isoscutellarein 7-O-(2"-O-6"-O-acetyl-beta-D-allopyranosyl-beta-D-glucopyranoside) (4) from V. multifida were isolated.

Iridoid glucosides from Veronica hederifolia.

A new iridoid glucoside, urphoside A, and six known iridoid glucosides, pikuroside, aucubin, veronicoside, catalposide, amphicoside, and verminoside, were isolated from Veronica hederifolia together with a known megastigmane glucoside, 3-hydroxy-5,6-epoxy-beta-ionol-9-O-beta-D-glucopyranoside, and a hexitol, dulcitol. The structures of the isolated compounds were established by the extensive 1D- and 2D-NMR spectroscopy.

Suppressive effect of Shichimotsu-koka-to (Kampo medicine) on pulmonary metastasis of 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.

Suppression of macrophage function by substances with a molecular weight lower than 3000 Da in B16 melanoma-conditioned medium.

In the present study, B16 melanoma cells were found to produce inhibitory and cytotoxic substances with a molecular weight lower than 3000 Da against macrophages in a conditioned medium. The B16 melanoma-conditioned medium suppressed nitric oxide (NO) production only by mouse peritoneal macrophages and the mouse macrophage-like cell line, RAW264.7 cells, but not by rat peritoneal macrophages. In addition, it showed cytotoxicity against mouse peritoneal macrophages and mouse macrophage-like cell lines, RAW264.7 and J774A.1 cells, but not against rat cells (peritoneal macrophages, 3Y1, hepatocytes), human cells (HeLa, KB, MCF-7), or mouse 3T3-L1 cells. The inhibitory activity of NO production was not affected by trypsin treatment or arginine supplementation, but it was abolished by heat treatment at 95 degrees C for 3 min. On the other hand, the cytotoxicity was not influenced by these treatments. Inducible NO synthase induction following lipopolysaccharide stimulation was reduced by treatment of mouse peritoneal macrophages with B16 melanoma-conditioned medium. These results suggest that metastatic B16 melanoma cells produce two distinct substances: to suppress NO production by macrophages and to kill macrophages and macrophage-like cell lines. We propose that these activities may help metastatic B16 melanoma cells to escape a host immunosurveillance system and to metastasize to target organs.

Phenylethanoid and iridoid glycosides from Veronica persica.

A new phenylethanoid glycoside, persicoside (1) and three known phenylethanoid glycosides, acteoside (2), isoacteoside (3) and lavandulifolioside (4) were isolated from the aerial parts of Veronica persica. On the basis of spectral analyses, the structure of the new compound was elucidated to be 3,4-dihydroxy-beta-phenylethoxy-O-[beta-D-glucopyranosyl-(1-->2)]-[beta-D-glucopyranosyl-(1-->3)]-4-O-caffeoyl-beta-D-glucopyranoside. Persicoside (1) and acteoside (2) exhibited radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Beside phenylethanoid glycosides, a hexitol, dulcitol (5) and seven known iridoid glucosides, aucubin (6), veronicoside (7), amphicoside (8), 6-O-veratroyl-catalpol (9), catalposide (10), verproside (11) and verminoside (12) were isolated.

New phenylethanoid glycosides from Veronica pectinata var. glandulosa and their free radical scavenging activities.

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.

New sesquiterpenes from Ferula ferulaeoides (Steud.) Korovin. VI. Isolation and identification of three new dihydrofuro[2,3-b]chromones.

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.

Antimetastatic activity of acteoside, a phenylethanoid glycoside.

We examined the antimetastatic effect of acteoside, a phenylethanoid glycoside widely distributed in the plant kingdom, on lung metastasis using a mouse model injected with B16 melanoma cells intravenously. Male C57BL/6 mice were injected intravenously with 2 x 10(5) of B16 melanoma cells, while acteoside at a dose of 50 mg/kg was administered intraperitoneally every other day from 13 d before B16 melanoma cell injection until all mice had succumbed to the metastatic tumor burden in the lung. Administration of acteoside prolonged survival time significantly and the average survival time was 63.3 +/- 3.4d compared with 52.1 +/- 2.5d in control mice. This result suggests that acteoside showed suppressive effect on lung metastasis of B16 melanoma cells.

Anti-inflammatory and cytotoxic activities of five Veronica species.

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.

CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4'-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis.

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-4'-hydroxyphenyllactic 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 biosynthesis.

Comparative study of the high molecular mass fraction and low molecular mass fraction of Sho-saiko-to in a murine immunologically induced liver injury model.

We compared the pharmacological actions of the high and low molecular mass fractions of Sho-saiko-to using a murine immunologically induced liver injury model to estimate the roles of these fractions in the expression of the pharmacological action. In a Bacillus Calmette-Guerin (BCG)/lipopolysaccharide (LPS)-induced liver injury model, Sho-saiko-to and both of its fractions significantly reduced the increases in the aminotranseferase levels in serum. They also reduced the increase in the nitric oxide (NOx) level in serum. On the other hand, Sho-saiko-to and its high molecular mass fraction suppressed the increase in plasma NOx level in an LPS-induced endotoxin shock model but its low molecular mass fraction did not. These results suggest the possibility that both fractions act hepatoprotectively in a different manner. We believe that these results can help to elucidate the mechanism of action of ingredients in Sho-saiko-to.