【作者】 何静仁；

【导师】 谢笔钧；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2003， 博士

【摘要】 银杏的药用价值早在几百年前已有记载，银杏提取物现已成为世界上公认的预防和治疗心脑血管疾病的有效植物药之一，然而对其引起不良反应的有害成分和／或银杏叶提取物中的限量因子银杏酸的安全性研究鲜见报道。本文旨在尝试利用现代色谱分离的理论和近代分析鉴定技术，在已建立银杏酸高效、准确的分析方法的基础上探讨其分离机制，掌握银杏酸的分布模式及变化规律；同时利用现代免疫学和药理实验方法，从整体动物水平、细胞水平以及分子水平全面了解银杏酸引起的不良反应及早期指标，着重揭示其引起过敏反应的变应原作用，并研究银杏酸致过敏作用的免疫发病机理，此外，还对具有显著抗过敏作用的物质OPC对银杏酸引起的致敏反应的抗过敏作用机制进行了探讨。 主要研究结果： 1 次级化学平衡反相高效液相色谱分离银杏酸及其机制的研究 对已建立的反相银化高效液相色谱（PR-AHPLC）分离银杏酸的方法的机制进行了初步探讨。结果表明PR-AHPLC方法能很好分离银杏酸同系物，分离因子和分离度均显著提高，达到1.3和1.6以上。色谱分离的热力学和动力学表明，配位化合物的分配模型是反相银化液相色谱分离行为的主要保留机制；次级化学平衡的参与显著改变了溶质物理分配平衡的热力学参数，是反相银化液相色谱有效提高银杏酸同系物分离选择性的机制，由此说明次级化学平衡是液相色谱中分离同系物和异构体的理想方法。将该方法应用于脂肪酸同系物也同样得到了很好的分离。 2 银杏酸的分布及含量变化规律的研究 通过LC／（-）ESI／MS、FTICR-SIMS及PR-AHPLC等分析鉴定了银杏不同部位的银杏酸种类、组成、存在形式、含量分布以及变化规律与影响因素。结果表明银杏叶、外种皮、果仁均含有C_13：0、C_15：1、C_15：0、C_17：2及C_17：15种银杏酸，并以C₁₃～C₁₇银杏酸同系物及其相应的类似长链酚的形式存在，结构上具有相似性和规律性，但同系物的含量及比例差别极大，且与对应长链酚的比例并不对应。银杏中的主要银杏酸是以单烯链的C_15：1、C_17：1及饱和侧链的C_13：0形式存在，约占95％以上。银杏的品种、产地、树龄及生长期等均影响银杏酸的比例及含量变化，与脂肪酸相关性的研究进一步证实了漆树酸生物合成的研究中认为脂肪酸是漆树酸侧链烃基的前体的假设。 3 银杏酸的纯化及免疫原GA-BSA的构象特征 采用含相调节剂的一定极性溶剂系统浸提银杏酸，树脂法分离及重结晶纯化后，经LC／ESI／MS联用分析，鉴定银杏叶和外种皮中分离的银杏酸均为GA_13：0，GA_15：1，GAIS:。和GA，7:!，反相银化高效液相色谱测定结果表明银杏叶中分离的银杏酸含量为91.2%，外种皮中分离的银杏酸纯度为97.7%。 圆二色谱和原子力显微镜分析和观察了银杏酸免疫原GA一BSA的构象与分子形貌，表明半抗原银杏酸GA与BSA结合的祸联物较好地具备了完全抗原的一定结构，既具有大分子物质BSA的部分构象，但同时受半抗原的结构影响，并具有与半抗原相似结构的吸收峰。BSA与GA藕联后，原有序的分子簇集变为相对无序，但仍保留BSA部分Q一螺旋结构。 4银杏酸的急性毒性和一般药理作用 雄性小鼠灌胃和腹腔注射银杏酸的!J氏。分别为2706mg/kg和1650 mg/kg;雌性小鼠灌胃和腹腔注射银杏酸的LDS。分别为2429mg/kg和1324mg/kg。一般药理实验研究表明，银杏酸对小鼠一般活动及中枢神经系统无显著影响;银杏酸对大鼠的呼吸幅度无明显影响，但高剂量的银杏酸在给药后Zh左右能引起大鼠呼吸频率的显著增快;银杏酸对大鼠心率的影响较大，低剂量的银杳酸在给药后30min开始引起心率显著下降，特别是在45一105min引起心率的过性降低，而高剂量的银杏酸在给药后15min即引起大鼠心率的下降，在75一90min内亦引起过性的降低;银杏酸对大白鼠SP、MP血压的影响较大，对DP血压的影响极小，高剂量的银杏酸在给药后15min起引起SP、MP血压的显著下降，特别在75一gomin引起血压的过性降低。 银杏酸引起的不良反应与细胞免疫及体液免疫状态有关。大鼠经致敏方式接触银杏酸后，在不同时间的细胞免疫水平与体液免疫水平的研究结果表明，大鼠CD3+及CD4+T淋巴细胞的表达增加，而CDS+T淋巴细胞的表达受到明显抑制，同时参与体液免疫的B淋巴细胞百分率呈增高趋势，提示银杏酸可能作为变应原使机体敏感性增强，诱导T细胞分裂增殖，增强细胞免疫应答，同时促进B细胞增殖分化成工gE抗体产生细胞。 5银杏酸变应原的致过敏作用 豚鼠全身过敏反应试验、皮肤过敏反应试验，以及在上述实验结果基础上建立银杏酸变应原的致过敏性变态反应的实验动物模型，通过被动皮肤过敏反应试验、主动肥大细胞脱颗粒实验、致敏豚鼠腹腔肥大细胞组胺释放试验等证实了银杏酸引起过敏反应的变应原作用。银杏酸作为过敏原能引起过敏反应，具有变应原性，但抗原性较弱，属于半抗原;病理观察表明GA一BSA银杏酸全抗原所致过敏性变态反应主要表现为炎细胞浸润和血管炎，同时具有毛细管扩张和通透性增加所致组织水肿等变态反应的基本病理更多还原

【Abstract】 Ginkgo biloba L., which was a traditional Chinese medicinal plant, has been recorded for several hundred years. And now it is approbated in the world for prevention and treatment of cardiovascular diseases. Bad-effect caused by extract of Ginkgo biloba leaves (EGb) have been reported. Ginkgolic acids are claimed to be allergenic compounds in Ginkgo biloba leaves, extrocarp and nut, but little reports were published on the safety of ginkgolic acids and the harmful compounds resulting in bad-effect in the usage of EGb. This paper is aimed to establish an efficient analytical method, probe its separation mechanism and find out the distribution and variation characteristic of ginkgolic acids by using modern chromatographic separation theory and identification technique. On the other hand, the bad-effect caused by ginkgolic acids and its early-hint were also studied systematically at animals level, cell level and molecular level. Emphases were put to reveal the allergenicity and immune sensitization mechanism of ginkgolic acids anaphylactogen. Anti-allergic mechanism of OPC, which was effective in preventing allergy caused by ginkgolic acids, was also discussed. Main research results:1 Separation of ginkgolic acids by second chemical equilibrium-high performance liquid chromatography (HPLC) and its mechanismAccording to the difference of ginkgolic acids in structure and character, separation mechanism of reversed-phase argentatin high performance liquid chromatography (RP-AHPLC) analytical method, which has been developed to separate ginkgolic acids in our laboratory, was studied utilizing basic theory and method of complexation chemistry in second chemical equilibrium. Ginkgolic acids homologues were separated efficiently by RP-AHPLC. Separation factor and resolution were up to 1.3 and 1.6 respectively. The results of chromatographic thermodynamics and kinetics showed that the partition model of complexation compound was the main retention mechanism of reversed-phase argentatin high performance liquid chromatographic behavior; the thermodynamic parameters of solute in physical partition equilibrium were changed markedly duo to the participation of second chemical equilibrium, which was the mechanism that the separation selectivity of ginkgolic acids homologues was increased by RP-AHPLC. The results revealed that second chemical equilibrium was a perfect method for the separation of homologues and isomers in HPLC. This method was also applied to separation of fatty acids homologues successfully.2 Distribution and contents variation characteristics of ginkgolic acidsThe kinds, composition, form, content distribution, variation rule and affecting factors of ginkgolic acids from different parts of Ginkgo biloba were studied by LC/(-)ESI/MS, FTICR-SIMS, RP-AHPLC and GC. The results showed that five kinds of ginkgolic acids C13: 0, C13: 1, C15:0, C17: 2 and C17:1 were present in Ginkgo biloba leaves, extrocarp and nuts, together with long chain phenols, the homophylic derivatives of ginkgolic acids. The contents and proportions of ginkgolic acids homologues were different evidently. The main ginkgolic acids in Ginkgo biloba were C15: 1, C17:1,which has unsaturated side chain, and C13: 0, which has saturated chain, and were up to 95% in sum. The variety, areas, age and growth period has different effect on the contents and proportions of ginkgolic acids. The pertinence between ginkgolic acids and fatty acids confirmed further the hypothesis that fatty acids were precursors of alkyl chain of anacardic acids.3 Purification of ginkgolic acids and conformation characteristic of ginkgolic acids complete antigen (GA-BSA)Ginkgolic acids were extracted by moderate polarity solvents containing phase-regulators, and separated on different resins, then purified by re-crystalization. LC/(-) ESI/MS was applied to identify the samples. Four ginkgolic acids Ci3,o, Cis, i, C^, o, and Cn, i from Ginkgo biloba leaves and extrocarp were confirmed. The contents of ginkgolic acids from Ginkgo biloba leaves and ext更多还原