Yoichiro Nakai

Partial agonistic effect of yokukansan on human recombinant serotonin 1A receptors expressed in the membranes of Chinese hamster ovary cells

ETHNOPHARMACOLOGICAL RELEVANCE Yokukansan (YKS) is a traditional Japanese medicine consisted of seven medicinal herbs and has been used for treatment of neurosis, insomnia, and behavioral and psychological symptoms of dementia (BPSD) in Japan. AIM OF THE STUDY The aim of the present study is to clarify the intrinsic activity of YKS on serotonin (5-HT)1A and 5-HT2A receptors and also to determine the constituent herbs which are responsible for the effect of YKS. MATERIALS AND METHODS The dry powdered extracts of YKS, seven constituent herbs, and YKS-analogues which were produced by eliminating one of the constituent herbs from YKS in the manufacturing process, were used for the evaluation. Competitive binding assays for 5-HT receptors and [(35)S]GTPgammaS binding assays for the evaluation of agonistic/antagonistic activity were performed using Chinese hamster ovary cell membranes stably expressing human recombinant 5-HT1A or 5-HT2A receptors. RESULTS YKS (6.25-400 microg/ml) concentration-dependently inhibited the binding of [(3)H]8-OH-DPAT to 5-HT1A receptors. The IC(50) value was estimated to be 61.2 microg/ml. In contrast, YKS failed to inhibit the binding of [(3)H]ketanserin to 5-HT2A receptors. Only Uncaria hook (3.13-50 microg/ml), of the seven constituent herbal extracts, inhibited the [(3)H]8-OH-DPAT binding to 5-HT1A receptors in a concentration-dependent manner, and the IC(50) value was estimated to be 7.42 microg/ml. The extracts of YKS or Uncaria hook increased [(35)S]GTPgammaS binding to 5-HT1A receptors to approximately 50% of that of a full agonist, 5-HT. Both the competitive binding and [(35)S]GTPgammaS binding of YKS to 5-HT1A receptors were remarkably attenuated by eliminating Uncaria hook from YKS, but it was almost unchanged when one of the other constituent herbs was eliminated from YKS. CONCLUSION These results suggest that YKS has a partial agonistic effect on 5-HT1A receptors, which is mainly attributed to Uncaria hook.

Effect of the rhizomes of Atractylodes lancea and its constituents on the delay of gastric emptying.

The effect of rhizomes of Atractylodes lancea on gastric disorders, in particular, the delay in gastric emptying induced by N(G)-nitro-L-arginine in rats, was investigated. Intragastric treatment with an aqueous extract (250 mg/kg) and its lipophilic fractions (4 mg/kg) significantly improved delayed gastric emptying. The major constituents of the lipophilic fraction were two sesquiterpens, hinesol and beta-eudesmol, and four known polyacetylenic compounds, atractylodin, atractylodinol, acetylatractylodinol and 4,6,12-tetradecatriene-8,10-diyne-1,3,14-triol. The activity was found in the four polyacetylenic compounds at a similar potency, but not in the two sesquiterpens. To clarify the effect of the four polyacetylenic compounds in this extract, we attempted to evaluate the activity of atractylodin, as representative, at a dose of 0.2 mg/kg based on the total amounts (0.2 mg/250 mg aqueous extract) of the four polyacetylenic compounds. In addition, atractylodin improved the delay in gastric emptying at between 0.1 and 0.3 mg/kg in a dose-dependent manner. These results suggest that the aqueous extract improved the delayed gastric emptying, and polyacetylenic compounds contributed to its activity.

Separation and isolation methods for analysis of the active principles of Sho-saiko-to (SST) oriental medicine.

Abstract Sho-saiko-to (SST) was introduced into Japan as an oriental classical medicine from China approximately 1500 years ago, and it is currently the most representative Kampo medicine (traditional Japanese medicine). SST is manufactured in Japan as an ethical drug on a modern industrial scale in which the quality of ingredients is standardized with Good Manufacturing Practices (GMP) regulation. SST is widely used for the treatment of chronic hepatitis. Experimental and clinical studies including multi-center, placebo-controlled, double-blind studies have demonstrated the various pharmacological effects of SST. SST is prepared from the hot water extraction of seven raw materials, therefore many kinds of constituents are included. Three-dimensional (3D) HPLC analysis is useful for obtaining many kinds of constituents, especially low molecular ultraviolet (UV) quenching compounds, contained in SST as well as its fractions. Fingerprint pattern provided by 3D HPLC analysis makes possible to identify the overall-viewing of SST. Databases of UV spectra of the components of medicinal herbs obtained by reversed-phase (RP) HPLC using a photodiode array (PDA) and fingerprint patterns of crude drugs made by 3D HPLC analysis facilitate the identification, analysis and quality of herbal drugs. Studies using both PDA HPLC and an amino acid analysis with a fluorometric detector have found that SST contains fifteen major low molecular compounds (i.e. baicalin, wogonin-7-O-glucuronide, liquiritin, their three aglycons, liquiritin apioside, glycyrrhizin, saikosaponin b1, saikosaponin b2, ginsenoside Rg1, ginsenoside Rb1, (6)-gingerol, (6)-shogaol and arginine). These compounds have various pharmacological actions, and are assumed to be responsible, at least partly, for the pharmacological effects of SST. Although there have only been a few investigations on high molecular compounds with pharmacological actions contained in SST, several kinds of polysaccharides have been isolated from constituent herbs of SST. This review paper summarizes analytical methods of separation, isolation and identification of compounds with biological activities from SST, which is a mixture drug of medicinal herbs. Accordingly, this paper would not focus on methods of separation, isolation and analysis of particular compounds from each constituent herb of SST.

The herbal medicine Toki-shakuyaku-san improves the hypertension and intrauterine growth retardation in preeclampsia rats induced by Nω-nitro-L-arginine methyl ester

The chronic inhibition of nitric oxide (NO) synthesis with Nomega-nitro-L-arginine methyl ester (L-NAME) induces a preeclampsia-like syndrome including hypertension and intrauterine growth retardation (IUGR) in pregnant rats. We tested the traditional herbal medicine Toki-shakuyaku-san (TS) for beneficial effects in this model. L-NAME was infused subcutaneously into pregnant rats from day 14 of gestation. TS (1 g/kg, 2 g/kg) was administered by gavage from day 14 to 20. Systolic blood pressure was measured on day 19. On day 20, rats were sacrificed and serum NO levels, placental weight, fetal body weight, fetal cerebrum weight and the thickness of the cerebral cortex were analyzed. TS (1 g/kg, 2 g/kg) inhibited L-NAME-induced hypertension. The decrease in fetal body weight, cerebrum weight and thickness of the cerebral cortex was abrogated by TS (2 g/kg). The effect of TS on blood pressure was found only in the rats that were both pregnant and infused with L-NAME. L-arginine, at the amount equivalent to that contained in TS, showed no effect. Further, the change in serum NO levels induced by TS was only marginal. TS thus improved the hypertension and IUGR in preeclampsia rats induced by L-NAME in a NO-independent manner. These data suggested that TS may be beneficial for the treatment and prevention of preeclampsia.

Effects of Saireito, a Japanese herbal medicine, on edema via antagonistic actions against aldosterone in anti-GBM nephritic rats

BackgroundIn order to clarify the diuretic mechanisms of Saireito, a Japanese herbal medicine, the mineralcorticoid receptor antagonistic action of Saireito was evaluated in anti-glomerular basement membrane (GBM) nephritic rats.MethodsAnti-GBM nephritis was induced in rats by the intravenous, injection of anti-GBM serum, and test drugs were administered 5 days after the induction of nephritis. In addition, we also investigated aldosterone-loaded mice to clarify the effects of test drugs on aldosterone signal transduction. In an in vitro study, a mineralocorticoid receptor binding assay of the componets of Saireito was performed.ResultsSaireito and spironolactone inhibited the development of proteinuria and abdominal ascites in anti-GBM nephritic rats. Saireito and spironolactone increased the urine volume and decreased the abdominal saline content in aldosterone-loaded mice. Saikosaponin H, a component of Saireito, inhibited the receptor binding of aldosterone in the in vitro assay 50% inhibitory concentration ([IC50], 22 µmol/l). Saikosaponin H also inihibited the decrease in urine volume in aldosterone-loaded mice.ConclusionsThese results suggest that the diuretic action of Saireito may be partly due to an antagonistic action on the mineralocorticoid receptor, exerted by saikosaponin H.