【作者】 唐景玲；

【导师】 崔福德； 何仲贵；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2006， 博士

【摘要】 银杏叶提取物（Ginkgo biloba extracts，GBE）是国内外常用的中药，临床上广泛用于提高老年人的认知作用等疾病。GBE的主要活性成分是5-7％的萜内酯（银杏内酯和白果内酯）和22-27％的黄酮醇糖苷（如：苷元槲皮素、山奈素和异鼠李素）。在不同品牌的银杏叶固体制剂中，由于GBE主要活性组分的溶解度差，溶出度和生物利用度存在差异大、重现性差等问题。自乳化药物传递系统（SEDDS）是由油、表面活性剂和助表面活性剂组成的均一混合物，当它进入胃肠道遇到水相介质可在轻微搅拌和胃肠的蠕动下自发乳化形成乳剂。研究表明SEDDS的快速自发乳化和在吸收部位的高度分散能有效的提高水难溶性药物的口服吸收。GBE作为心脑血管疾病治疗与预防的常用药物，一般需长期用药，在临床上与其它药物合用的几率较大。因此，本文从这两方面入手，旨在研究制备GBE自乳化给药系统以提高主要成分的吸收，并对GBE与其他药物相互作用进行研究，为临床合理用药和预防不良反应提供理论依据。处方前研究表明，25℃时GBE在丙二醇和甘油两种助表面活性剂中的溶解度较其他辅料大；建立了GBE主要成分含量测定和溶出度测定的分析方法，采用先将黄酮苷水解为苷元，再用HPLC-UV法测定银杏黄酮苷的含量和溶出度；分别采用HPLC-ELSD法和HPLC-ESI-MS法测定银杏萜内酯的含量和溶出度。选用油酸乙酯和Miglyol 812为油相，丙二醇为助表面活性剂，Tween 80、T₅₀C₅₀（50％Tween 80-50％Cremophor EL35）为乳化剂，通过绘制假三元相图，确定自乳化区域大小。油酸乙酯为油相所形成的自乳化区域显著大于Miglyol 812，混合表面活性剂T₅₀C₅₀与Tween 80相比自乳化区域稍有增加。考察了温度、搅拌强度、介质的pH和稀释倍数等外在因素对自乳化效率的影响，最终选择37℃、低速搅拌、蒸馏水稀释500倍作为稀释介质来评价SEDDS的自乳化效率。GBE的加入对自乳化区域无影响，但使自乳化时间延长，所形成乳剂的粒径增大。最终用于溶出度和生物利用度研究的处方为GBE-油酸乙酯-T₅₀C₅₀-丙二醇（12.5：45：45：10）。所制备的GBE自乳化胶囊20min时，各主要成分的溶出度可达90％以上，而对照片剂20min时，各主要成分的溶出度不足40％。加速和室温留样试验结果表明，GBE自乳化系统稳定性良好。建立了灵敏、专属的HPLC-ESI-MS方法测定家犬血浆中银杏内酯和白果内酯的浓度，该方法的最低定量限：银杏内酯B为2.5 ng／ml，白果内酯、银杏内酯A和C为10 ng／ml，该方法的日内日间精密度和准确度均＜15％。对GBE自乳化胶囊和普通片剂进行了家犬体内药物动力学研究，结果表明与片剂相比，给予自乳化胶囊后白果内酯、银杏内酯A和B的T_max均明显缩短，C_max显著增加，分别增加了44.8％、52.4％和44.4％（p＜0.05），AUC_0-10h也显著增加。给予GBE自乳化胶囊和普通片剂后，白果内酯、银杏内酯A和银杏内酯B的相对生物利用度分别为162.1±42.5％、154.6±65.3％和155.8±26.8％。对AUC_0-t和C_max分别进行方差分析，并进一步采用双单侧检验和（1-2α）置信区间法分析，结果表明GBE SEDDS胶囊和片剂相比，白果内酯、银杏内酯A和B的吸收程度生物不等效。茶碱为咖啡因类生物碱之一、具有较强的松弛支气管平滑肌，兴奋心脏及利尿作用，也有一定的中枢兴奋作用，且治疗窗很窄（5～20μg／ml）。本文考察了大鼠连续5天口服GBE后对茶碱体内药动学的影响，大鼠静脉给予茶碱（10 mg／kg），经GBE 10 mg／kg和100 mg／kg前处理组与对照组相比，茶碱清除率分别为对照组的1.3倍和1.7倍：口服给予茶碱（10 mg/kg），茶碱清除率分别为对照组的1.5倍和1.7倍（p＜0.05），经GBE 100mg／kg前处理，茶碱注射给药和口服给药后AUC_0-24h分别下降了40％和38％。表明GBE前处理可能增加了大鼠CYP1A2的活性，从而增加了茶碱的清除率。采用外翻肠囊法，以非索非那定和噻利洛尔（这两个药物均为P-gp的典型底物）为模型药物，考察GBE及其主要成分是否对P-gp底物的转运产生影响。盐酸维拉帕米（P-gp的典型抑制剂）可显著增加非索非那定和噻利洛尔从粘膜侧至浆膜侧的转运量约2倍（p＜0.01），槲皮素可显著增加非索非那定和噻利洛尔从粘膜侧至浆膜侧的转运量（p＜0.05），这一结果与文献报道槲皮素是P-gp抑制剂的结论相一致。但GBE、芦丁和银杏内酯对非索非那定和噻利洛尔从粘膜侧至浆膜侧的转运量无显著影响。更多还原

【Abstract】 Ginkgo biloba extracts （GBE） have become a widely used herbal remedy for increasing cognitive function in elderly people in the USA, Europe, Japan and many other countries. The primary active components of GBE include 5-7% terpene lactones （ginkgolides and bilobalide） and 22-27% ginkgo flavonol glycosides （e.g., the flavones quercetin, kaempferol, and isorhamnetin）. The dissolution and bioavailability of the primary active components from the oral solid preparations of different Ginkgo biloba brands were obviously different and irreproducible, due to the lower solubility of the active components. Self-emulsifying drug delivery systems （SEDDS） are homogeneous mixtures of oils, surfactants and cosolvent, which are emulsified in aqueous media under conditions of gentle stirring and digestive motility that would be encountered in the gastrointestinal tract. It was found that SEDDS could efficiently improve oral absorption of the sparingly soluble drugs by rapid self-emulsification and subsequently dispersion in the absorption sites. GBE needs long-term administering medicine to the patients, especially to elderly patients, for treatment and prevention cerebrovascular diseases. The interactions of GBE and other drugs may occur in all probability in the clinic. So the main purpose of the present study is to prepare SEDDS for improving oral absorption of GBE and to investigate the interactions of GBE and drugs. The results will provide theory bases for clinical administration and prevention of side effects.In the pre-formulation study, the solubility of GBE in 1,2-propanediol and glycerol was higher than other chose vehicles at 25℃. The methods of content and dissolution determination for the active components of GBE were developed and validated. The flavonol glycosides were determined after hydrolyzing by HPLC-UV for content and dissolution. The terpene lactones were determined by HPLC-ELSD and HPLC-ESI-MS for the contents and dissolutions, respectively. Pseudo-ternary phase diagrams were constructed to identify the efficient self-emulsifying region using Miglyol 812 and ethyl oleate as oils, 1, 2-propanediol as cosolvent, Tween 80 and T₅₀C₅₀（50％Tween 80-50％Cremophor EL35） as emulsifiers. For both systems, the self-enulsifying region was larger when ethyl oleate as oil than Miglyol 812. The mixed surfactant T₅₀C₅₀ increased the self-emulsifying region slightly comparing with Tween 80 at the same weight level. We investigated the effects of temperature, flow shape, pH and volume of media on the self-emulsifying efficiency. At last, the conditions of 37℃, low speed and diluting with 500-fold volume of the distilled water were used for evaluating the efficiency of self-emulsifying. GBE did not affect the self-emulsifying region, but prolonged self-emulsifying time and increased the droplet size comparing with the blank vehicles. Optimum formulation for in vitro dissolution and bioavailability assessment consisted of 45％T₅₀C₅₀, 45％ethyl oleate and 10％1, 2-propanediol. The content of GBE was fixed at 12.5％w/w of the vehicle. The in vitro dissolution rates of the active components of GBE from optimum SEDDS were faster than those of the GBE tablets. The dissolution of SEDDS at 20 rain was more than 90％, and the dissolution of tablet at 20 rain was less than 40％. The stability of GBE SEDDS was good in the accelerated test and under room temperature.A sensitive and selective method using HPLC-ESI-MS was developed for the quantification of bilobalide and ginkgolides in canine plasma. The lower limit of quantification （LLOQ） of the method was 2.5 ng/ml for ginkgolide B and 10.0 ng/ml for bilabolide, ginkgolide A and ginkgolide C. The accuracy of the method was within 15％of the actual values over a wide range of plasma concentrations. The intra-day and inter-day precision was better than 15％（R.S.D.）. The plasma profiles of ginkgolides and bilobalide in dogs following oral administration of tablets and self-emulsifying formulation of GBE at a single dose of 800 mg (3BE were investigated. The short T_max values of SEDDS for all three tested components showed the fairly rapid onset compared to the conventional tablets. The marked differences were also observed for the C_max. The C_max of SEDDS increased 44.8％, 52.4％and 44.4％for bilobalide gikgolide A and B compared to the conventional tablets, respectively. And the AUC_0-10h of SEDDS was increased significantly comparing with the conventional tablets. The relative bioavailability of bilobalide, ginkgolide A and ginkgolide B was 162.1±42.5％, 154.6±65.3％and 155.8±26.8％, respectively. These data clearly demonstrate the utility of SEDDS in improving the rate and extent of oral absorption of GBE. The analysis variance, two-one sided tests and （1-2α） confidence interval analysis of main pharmacokinetic parameters showed that the absorptions of bilobalide, ginkgolide A and B for SEDDS and tablets were not bioequivalence.This study attempted to investigate the effect of GBE on the pharmacokinetics of theophylline, a cytochrome P450 （CYP） 1A2 substrate and an important therapeutic agent with narrow therapeutic window （5～20μg/ml） used for the treatment of asthma. GBE （10 or 100 mg/kg, p.o.） or water （control group） was given to rats （6 rats for each group） for 5 consecutive days and on the sixth day theophylline （10 mg/kg） was administered either orally or intravenously. The results showed that pretreatment of rats with GBE resulted in an increase in the total clearance of theophylline of about 30 percent （GBE 10 mg/kg, p＜0.05） and 70 percent （GBE 100 mg/kg, p＜0.01） compared with the control group after intravenous administration of theophylline （10 mg/kg）. After pretreatment with GBE （100 mg/kg）, the AUC_0-24h of theophylline was reduced by 40％following intravenous administration and by 38％following oral administration. These results demonstrated that GBE pretreatment increased CYP1A2 metabolic activity and the clearance of theophylline in rats.The aim of this study is to investigate the effects of GBE and the active components of GBE on the transport activity of P-glycoprotein （P-gp） in the rat small intestine. The efflux of P-gp substrates from rat everted sac in the absence or presence Of verapamil, GBE, rutin, quercetin or terpene lactones was measured. Fexofenadine and celiprolol were used as P-gp substrates. Verapamil and the quercetin inhibited the efflux from the intestine of the two drugs tested. GBE, rutin and terpene lactones did not affect the efflux of fexofenadine and celiprolol in the intestine.更多还原