Keiji Wada

Bilobalide, a sesquiterpene trilactone from Ginkgo biloba, is an antagonist at recombinant α1β2γ2L GABAA receptors

Abstract The sesquiterpene trilactone bilobalide is one of the active constituents of the 50:1 Ginkgo biloba leaf extract widely used to enhance memory and learning. Bilobalide was found to antagonise the direct action of γ-aminobutyric acid (GABA) on recombinant α1β2γ2L GABAA receptors. The effect of bilobalide on the direct action of GABA at α1β2γ2L GABAA receptors expressed in Xenopus laevis oocytes using two-electrode voltage-clamp method was evaluated and compared with the effects of the classical GABAA receptor competitive antagonist bicuculline and noncompetitive antagonist picrotoxinin. Bilobalide (IC50=4.6±0.5 μM) was almost as potent as bicuculline and pictrotoxinin (IC50=2.0±0.1 and 2.4±0.5 μM, respectively) at α1β2γ2L GABAA receptors against 40 μM GABA (GABA EC50). While bilobalide and picrotoxinin were clearly noncompetitive antagonists, the potency of bilobalide decreased at high GABA concentrations suggesting a component of competitive antagonism.

Effects of extract of Ginkgo biloba leaves and its constituents on carcinogen-metabolizing enzyme activities and glutathione levels in mouse liver

The effects of a standardized extract of Ginkgo biloba L. leaves (EGb) and its terpene constituents, bilobalide and ginkgolides, on the activities of detoxification enzymes, i.e., glutathione S-transferases (GSTs) and DT-diaphorase, and glutathione contents, were investigated in the mouse liver. Oral treatment with EGb (100-1,000 mg/kg) and bilobalide (10-30 mg/kg) once a day for 4 days caused a dose-dependent elevation in GST activity. Ginkgolide A (30 mg/kg, for 4 days) also significantly elevated GST activity, whereas ginkgolide B and ginkgolide C at the same dose had no effects. EGb significantly increased the protein level of GST pi, and bilobalide significantly increased those of GST alpha and GST mu Moreover, EGb-treatment and bilobalide-treatment caused significant elevations in DT-diaphorase activity and in hepatic glutathione contents.

In Vitro Absorption and Metabolism of a Citrus Chemopreventive Agent, Auraptene, and Its Modifying Effects on Xenobiotic Enzyme Activities in Mouse Livers

Abstract: We previously reported that auraptene (7-geranyloxycoumarin, AUR), widely occurring in citrus fruit, is a structurally novel type of effective cancer-preventive agent, as manifested in several rodent models. However, its bioavailability and metabolism in biological systems have yet to be investigated. In the present study, we examined the chemical stability of AUR at pH 1.57 and 37°C (as a stomach digestion model) and observed its stoichiometric conversion to umbelliferone [7-hydroxycoumarin, UMB; half-life (t1/2) = 15 h; 7-ethoxycoumarin (ETC) was stable for 24 h]. Differentiated Caco-2 cells, a human colorectal adenocarcinoma cell line, were used as a small intestine model. ETC permeated the basolateral (portal vein) side of Caco-2 cells in a time-dependent manner; AUR slightly permeated the cells, but with an intracellular accumulation. Epoxyauraptene and UMB were detected when AUR was treated with the rat liver S-9 mixture. ETC was also converted to UMB, but its t1/2 of two hours was much shorter than that of AUR (≥24 h). This suggests that AUR, bearing a geranyloxyl side chain, is a relatively metabolism-resistant substrate for cytochrome P-450 enzymes and, thus, is stable in the liver compared with ETC. Oral administration of AUR by gavage at 50-200 mg/kg body wt dose dependently induced glutathione S-transferase (GST) activity in mouse livers without affecting cytochrome P-450 activity. Using 10 coumarin-related compounds, we found that only those coumarins having a 7-alkyloxyl group induced GST, but not cytochrome P-450, activity. The present study presumes that AUR accumulates in the epithelial cells of the small intestine and then gradually permeates into the portal vein. Stable localizability of AUR in the colon and liver may be associated with the induction of GST activity, which is important as the action mechanism for suppression of rodent chemical carcinogenesis.

A bitter diterpenoid furanolactone columbin from Calumbae Radix inhibits azoxymethane-induced rat colon carcinogenesis

The modifying effect of dietary administration of a diterpenoid furanolactone columbin isolated from the crude drug Calumbae Radix (the root of Jateorhiza columba MIERS, Menispermacea) on azoxymethane (AOM)-induced was investigated in male F344 rats. Animals were initiated with AOM (three weekly subcutaneous injections of 15 mg/kg body weight) to induce colonic neoplasms. They were fed the experimental diets mixed with columbin (4, 20, and 100 ppm) for 4 weeks, starting 1 week before the first dosing of AOM and thereafter maintained on the basal diet without columbin. Additional experimental groups included the AOM alone group, the columbin alone group (100 ppm in diet for 4 weeks), and the untreated control group. Dietary feeding of columbin (4, 20, and 100 ppm) during the initiation phase of AOM-induced colon carcinogenesis reduced the incidence and multiplicity of colonic adenocarcinoma and the inhibition by feeding of 20 ppm (incidence: 20%, P=0.0242 and multiplicity: 0.20+/-0.40, P<0.02) and 100 ppm (incidence: 10%, P=0.0029 and multiplicity: 0.10+/-0.30, P<0.002) columbin was significant when compared with the AOM alone group (incidence: 55% and multiplicity: 0.55+/-0.50). Also, columbin administration in diet lowered the number of argyrophilic nucleolar organizer regions protein per nucleus in non-lesional colonic crypts and the blood polyamine content, which are reflected in cell proliferation activity. These results indicate chemopreventive ability of dietary columbin against chemically induced colon tumorigenesis when fed during the initiation phase, providing a scientific basis for chemopreventive ability of columbin against human colon cancer.

Bilobalide prevents reduction of γ-aminobutyric acid levels and glutamic acid decarboxylase activity induced by 4-O-methylpyridoxine in mouse hippocampus

We previously reported that bilobalide, a constituent of Ginkgo biloba L. leaves, protected mice against convulsions induced by 4-O-methylpyridoxine (MPN). To elucidate the mechanism of the anticonvulsant activity of bilobalide, this study examined the effect of bilobalide on MPN-induced changes in the levels of gamma-aminobutyric acid (GABA) and glutamate, and in the activity of glutamic acid decarboxylase (GAD) in the hippocampus, cerebral cortex and striatum of the mouse. GABA levels and GAD activity in the hippocampus and cerebral cortex were significantly enhanced by bilobalide treatment (30 mg/kg, p.o., for 4 days) alone. MPN significantly decreased GABA levels and GAD activity in the three brain regions tested compared with those in the control. Pretreatment with bilobalide effectively suppressed the MPN-induced reduction in GABA levels and GAD activity in the hippocampus and cerebral cortex. On the other hand, there were no significant differences in the glutamate levels in the three regions despite various treatments. These results suggested that bilobalide prevents MPN-induced reduction in GABA levels through potentiation by bilobalide of GAD activity, and this effect of bilobalide contributes to its anticonvulsant effect against MPN-induced convulsions.

Effects of bilobalide, a sesquiterpene in Ginkgo biloba leaves, on population spikes in rat hippocampal slices.

The effects of bilobalide, a sesquiterpene isolated from the leaves of Ginkgo biloba L., were investigated in a rat hippocampal slice preparation. Bilobalide (10-500 microM) significantly increased the amplitude of population spikes evoked by electrical stimulation of Schaffer collateral/commissural fibers in a concentration-dependent manner. Paired-pulse inhibition at interpulse intervals of 10-50 ms was significantly reduced in the presence of bilobalide (50 microM). The inhibitory action of muscimol (1 microM) was attenuated by bilobalide (100 microM). These results suggest that bilobalide induces an enhancement of excitability of CA1 pyramidal neurons, which involves, at least in part, a reduction in GABAergic inhibition in rat hippocampus.

The Restorative Effects of Eucommia ulmoides Oliver Leaf Extract on Vascular Function in Spontaneously Hypertensive Rats

Eucommia ulmoides Oliv. leaf is a traditional Chinese antihypertensive and antidiabetic medicine. We examined the effects of chronic Eucommia leaf extract (ELE) administration on artery function and morphology in spontaneously hypertensive rats (SHRs). ELE was orally administered via normal diet ad libitum to six-week-old male SHRs at a concentration of 5% for seven weeks. Acetylcholine (ACh)-induced endothelium-dependent relaxation, sodium nitroprusside (SNP)-induced endothelium-independent relaxation, plasma nitric oxide (NO) levels, and media thickness were assessed. ELE significantly improved ACh-induced aortic endothelium-dependent relaxation but did not affect SNP-induced endothelium-independent relaxation in the SHRs, as compared to the animals receiving normal diet. Plasma NO levels and media thickness were significantly increased and decreased, respectively, in the ELE-treated SHRs. Therefore, long-term ELE administration may effectively improve vascular function by increasing plasma NO levels and bioavailability, and by preventing vascular hypertrophy in the SHR aorta.

Mixed antagonistic effects of bilobalide at ρ1 GABAC receptor

Bilobalide was found to be a moderately potent antagonist with a weak use-dependent effect at recombinant human rho(1) GABA(C) receptors expressed in Xenopus oocytes using two-electrode voltage clamp methodology. Antagonism of bilobalide at homomeric rho(1) GABA(C) receptors appeared to be mixed. At low concentration, bilobalide (3 microM) caused a parallel right shift and surmountable GABA maximal response of the GABA dose-response curve characteristic of a competitive antagonist. At high concentrations, bilobalide (10-100 microM) caused nonparallel right shifts and reduced maximal GABA responses of GABA dose-response curves characteristic of a noncompetitive antagonist. The potency of bilobalide appears to be dependent on the concentrations of GABA and was more potent at lower GABA concentrations. The mechanism of action of bilobalide at rho(1) GABA(C) receptors appears to be similar to that of the chloride channel blocker picrotoxinin.

Evaluation of synthesized coumarin derivatives on aromatase inhibitory activity

In women across the world, the most common type of cancer is breast cancer. Among medical treatments, endocrine therapy based on aromatase inhibitors (AI) is expected to be effective against not only post-menopausal but also pre-menopausal breast cancer. In this study, we examined the structure-activity relationship between the aromatase inhibitory effects of 7-diethylaminocoumarin derivatives with a substituent at position 3 and coumarin derivatives with a substituent at position 7. Consequently, we found that 7-(pyridin-3-yl)coumarin (IC50 values 30.3nM) and 7,7-diethylamino-3,3-biscoumarin (28.7nM) are the most potent inhibitors of aromatase. These inhibitors were found to be comparable to the existing CYP19 inhibitor exemestane (42.5nM).

Food Poisoning by Ginkgo biloba Seeds

Gin-nan is the Japanese word for the seed of Ginkgo biloba L. The albumen of the seed is used as a crude drug and food in China and Japan. In particular, it is used as an antitussive and expectorant in traditional medicine. However, when this substance has been taken to excess during food shortages, “Gin-nan food poisoning” has sometimes occurred in Japan [1–35] and China [36–37]. Figure 1 shows the numbers of patients with gin-nan food poisoning in Japan in our survey [1–38]. The symptoms of this poisoning are mainly convulsions and loss of consciousness. Infants and particularly children under 6 years of age made up about 74% of all patients (Fig. 2). The consequences are not serious for survivors, but mortality is about 27% in Japan.