In Vitro Metabolism And Drug Interaction Of Ginkgolides And Bilobalide

The Ginkgo biloba represents a distinct and ancient lineage of gymnosperms, native to China, but the Ginkgo biloba has a history ranging back into the Jurassic. At present Ginkgo biloba is regarded as valuable herbal medicine. Standardized extract from Ginkgo biloba L. is a complex mixture containing flavoids and terpene lactones. The terpene lactones ginkgolide A, B, C (abbreviated as GA, GB, GC, respectively) and bilobalide (abbreviated as BB) are the most characteristic. The ginkgolides are diterpenes with a C₂₀ cage skeleton consisting of six-5 members rings and bilobalide is a sesquiterpene trilactone with four-5members rings. Ginkgolides and bilobalide are unique components of Ginkgo biloba. They are reported to possess various pharmacological activities, such as protecting liver, stomach and kidney;antivirus, anticancer and antioxidation, et al. Pharmacological study indicated that ginkgolides are potent antagonists of platelet activating factor (PAF), and therefore, could pro.ect the central-nerve system (CNS) and blood-brain barrier (BBB). Ginkgolides become the reasonable choice treating cerebral and vascular insufficiency because of this pharmacological activity.Of all the compounds classes present in Ginkgo biloba, the terpene lactones have received by for the most attention. This is due to their chemical uniqueness and the analytical challenge, the Ginkgo terpene lactones are poor chromophors with veryweak absorption in the 200-220nm range. Be relative to flavoids, little review on terpene lactones has appeared recently, especially the review on their absorption, distribution, metabolism and elimination in vivo. The text was divided into three parts to evaluate the established HPLC-ELSD method for determination of ginkgolides and bilobalide in rat hepatic microsomes, the in vitro metabolism of ginkgolides and bilobalide, and the metabolism interaction of ginkgolides and bilobalide.1. A HPLC-ELSD method for determination of ginkgolides and bilobalide in rat hepatic microsomesAIM: To establish a HPLC- ELSD method for determination of ginkgolides and bilobalide in rat hepatic microsomal incubates. METHODS: ginkgolides and bilobalide in rat hepatic microsomal incubates was extracted by diethylether. The determination was performed on a Diamonsil ODS-Cis column (250mmx4.6mm 5 urn) with mobile phase of methanol-water(37:63) at a flow rate of l.OmL-min"1. ELSD temperature was 60°C, gain was 9 and pressure was 2.5bar. RESULTS: The calibration curve was good linearity over the range from 6.80 to 136.0 ug-mL"1 of ginkgolide A, 5.40 109-O^g-mL"1 of ginkgolide B, 3.60 72.0ug-mL"' of ginkgolide C, 3.30¹33.60ug-mL'' of bilobalide. The limit of detection was 32ng of ginkgolide A, 20ng of ginkgolide B, 16ng of ginkgolide C , 12ng of bilobalide (S/N=3) and the limit of quantification was 3.30ug-mL^' of bilobalide , 6.80ug-mL"' of ginkgolide A, 5.40 og-mL"1 of ginkgolide B, 3.60(igmL"' of ginkgolide C (RSD<10%, n=3) . The average recoveries and RSD were in the range of 86.0 102.9% and 1.84—10.6%. CONCLUSION: The method is simple, accurate and can be used to study the metabolism of ginkgolides and bilobalide in the rat hepatic microsomes.2. In vitro metabolism of ginkgolides and bilobalide in rat liver microsome.AIM: To obtain the information about the phase I and phase II metabolism of ginkgolides and bilobalide in rat liver microsomes. METHODS: The metabolism of ginkgolides and bolobalide was investigated in four kinds of rat liver microsomal incubates pretreated with phenobarbital (PB), dexamethasone (Dex) , P-naphthoflavone (BNF) and control. Ginkgolides and bilobalide were incubated with rat liver microsomes, extracted by diethylether, and the remaining concentration were determined by HPLC-ELSD. RESULTS: After 80 min incubation, the phaseI metabolism of ginkgolides and bilobalide almost not took place. However, the phase II metabolism took place significantly in the microsomes treated with BNF, PB, Dex and control group. The concentration of ginkgolide A was reduced for 24.3%, 25.9% 28.7% and 23.1% in above microsomal incubates, respectively, ginkgolide B for 21.1%, 22.0%, 28.5% and 19.0%, ginkgolide C for 46.1%, 51.5%, 52.1% and 41.6%, and bilobalide for 30.3%, 27.1%, 28.2% and 20.0%. The Km of ginkgolide A in the four microsomes (control group, BNF group, PB group, Dex group) was 14.66±1.85, 12.50*1.64, 13.60±1.53, 48.75±3.26ymol-L"1, and the Vmax was 7.132 ±0.735, 6.323±0.609, 6.064±0.712, 9.760±0.814 nmol-mm'-mg protein1;The Km of ginkgolide B in the four microsomes was 63.98+6.09, 66.44+5.87, 42.61±3.24, 51.67±5.77mnol-L"1, and the Vmax was 8.350 ±0.661, 8.685±0.709, 9.188±0.659, 7.517+0.534 nmol-imV-mg protein"1. The Km of ginkgolide C in the four microsomes was 9.56±1.07, 26.8±2.54, 7.93±1.25, 8.17±1.12(.imol-L"1, and the Vmax was 7.261 ±0.513, 7.100±0.488, 6.879±0.502, 7.591+0.647 nmol-muV'-mg protein"1. The Km of bilobalide in the four microsome was 38.84±2.99, 37.29±3.27, 35.76±2.85, 32.18±2.88nmol-L"1, and the Vmax was 7.010 ±0.498, 6.465±0.546, 8.516+0.693, 9.050±0.701 nmol-min^mg protein"1. CONCLUSION: The phaseI metabolism of ginkgolides and bilobalide hardly took place, however the phase II metabolism took place indeed .The metabolism rate of ginkgolide C was greater than others.3. The metabolism interaction of ginkgolides and bilobalideAIM: To observe the metabolism interaction of ginkgolides and bilobalide, and provide some useful information for clinical medcines. METHODS: ginkgolides and bilobalide were incubated with each other in the microsomes pretreated with dexamethasone, the interaction was observed by monitoring the loss of substrate. RESULTS: 1) The Ki of ginkgolide A to ginkgolide B, ginkgolide C and bilobalide was lH^umol-L"1, 43.4umol-L"',159.3umol-L"1, and IC50 was 135-Oumol-L"1, 120.7umol-L"', 345.0umol-L^', respectively. 2) The Ki of ginkgolide B to ginkgolide A, ginkgolide C and bilobalide was 35.8|4.mol-L"', 197.8^mol-L'1,212.8nmol-L'1, and IC50 was 67.8umol-L"', 302.0umol-L/', 365.0\imol-L^\ respectively. 3) The Ki of ginkgolide C to ginkgolide A, ginkgolide B and bilobalide was SO.OumolL"1, 43.8umol-I/1,137.3umoH/1, and IC50 was 73.3nmol-L"1, 71.0umoH/1, 157.7 p.mol-L"1, respectively. 4) The Ki of bilobalide to ginkgolide A, ginkgolide B and Ginkgolide C was 60.5umol-L"', 144.5^molL"1,180.8|imol-L'1 , and IC50 was 98.7nmol-L"1, 68.6^mol-L"', 69.5^mol-L'', respectively. CONCLUSION: The metabolism interaction of ginkgolides and bilobalide each other was weak.