【作者】 倪学文；

【导师】 吴谋成；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2004， 博士

【摘要】 银杏酚酸是银杏叶提取物中的限量因子，在银杏外种皮中含量最高，其独特的药理和毒理活性日益受到人们的广泛关注，但与银杏叶相比，银杏酚酸的研究还很少。本文旨在利用现代色谱分离理论和分析鉴定技术，建立银杏酚酸高效准确的定量和分析方法；对从银杏外种皮中分离纯化银杏酚酸的工艺条件进行系统的研究，并对其结构进行鉴定：探讨银杏酚酸对细菌和病原真菌的抑制作用；利用免疫学和细胞生物学的实验方法，从免疫调节、细胞凋亡等角度探讨银杏酚酸抗肿瘤的机理。其主要研究结果如下： 1 银杏酚酸的反相高效液相色谱测定方法研究 根据银杏酚酸结构和性质的差异，利用反相高效液相色谱（RP-HPLC）分离银杏酚酸，对流动相组成、pH值和柱温各种影响因素进行了研究，得到银杏酚酸的RP-HPLC分析条件为：色谱柱为SUPELCOSIL^TMLC-18（15cm×4.6mm，5μm）；流动相为90％甲醇，乙酸调pH值为3.15；紫外检测波长λ=310nm；流速为0.7ml／min；柱温为30℃；灵敏度AUFS=0.02。在确定的色谱条件下，银杏酚酸的峰高分离度R_h均大于1，银杏酚酸各组分的容量因子均在3～8之间。 在银杏酚酸高效液相色谱分离理论的基础上，建立了银杏酚酸HPLC的定量测定方法，运用二极管阵列检测器分析了样品中银杏酚酸色谱峰和对照品的色谱峰，结果表明二者各对应色谱峰的紫外光谱完全相同，定量分析中所确定的色谱峰是正确的。样品用甲醇浸提、过滤后可直接进行HPLC分析，平均回收率为97.02％，相对标准偏差RSD（n=5）为1.28％，线性范围在0.498～8.879μg，线性相关系数为0.9998，最低检测限为7.2ng。银杏酚酸的高效液相色谱定量测定方法简便、快速、灵敏、准确，可用于银杏产品的质量监控。 2 银杏酚酸的提取工艺、纯化和结构鉴定 对从银杏外种皮中提取银杏酚酸的工艺进行优化，得到银杏酚酸的优化提取条件为：以80％乙醇为提取溶剂，采用1：7（W／V）的料液比，在60℃条件下回流提取3次，每次4h，此条件下银杏酚酸浸出率为8.021％。利用大孔吸附树脂进行分离富集银杏酚酸，对银杏酚酸在树脂上的吸附、解吸特性和吸附动力学行为进行了研究。得到银杏酚酸分离纯化的工艺流程为：银杏外种皮采用1：7的料液比，用80％乙醇在60℃条件下回流提取3次，每次4h，提取液经抽滤浓缩后，经过D4020吸附树脂纯化（水，65％，90％乙醇梯度洗脱），洗脱液浓缩后用S₁溶剂萃取，再浓缩，冷冻干燥，再经AB-8吸附树脂纯化（75％乙醇和95％乙醇梯度洗脱），冷冻干燥后得银杏酚酸的分离纯化及其生物活性研究燥后得到淡黄色银杏酚酸，产物纯度为95%，得率为0.89%。 通过对从银杏外种皮中纯化得到的银杏酚酸进行定性实验，紫外、红外光谱分析，二极管阵列检测器检测和反相高效液相色谱一电喷雾电离质谱伍SI一Ms)联用分析，证实了纯化得到的银杏酚酸为C13:0，C15:1，C17:2，C15:O和C17:1，其相对百分组成为12.0:41.77:4.89:3.52:37.83。 3银杏酚酸的抑菌活性研究 采用常量肉汤稀释法，研究了银杏酚酸对细菌和病原真菌的抑制作用。银杏外种皮水提取物、乙醇提取物和纯化的银杏酚酸都有很好的抑制细菌和植物病原菌的活性，且随着银杏酚酸含量增加，抑菌活性增强，可见银杏酚酸是其中主要的抑菌活性物质。银杏酚酸主要表现为抑制革兰氏阳性菌，对蜡状芽抱杆菌、枯草芽抱杆菌和金黄色葡萄球菌等有很好的抑制作用，而对大肠杆菌等革兰氏阴性菌几乎没有抑制作用，同时表明银杏酚酸的烷基侧链在抑菌中起了关键作用. 4银杏酚酸抗肿瘤作用及机理研究 银杏酚酸浓度为10，20，40m叭gd时，对小鼠S:。。肉瘤的抑制率分别为38，93%，42.90%和62.94%，40m叭gd的抑瘤率超过了阳性对照药物cy。银杏酚酸使荷瘤小鼠的胸腺重量减轻，但无明显差异，中低剂盘组能显著提高荷瘤小鼠脾脏的重量，但高剂量组却减轻脾脏重量;银杏酚酸能明显增强DNFB诱导的迟发型超敏反应，显著增强荷瘤小鼠脾细胞抗体生成能力，且能明显提高荷瘤小鼠血清lgG溶血素的含量。说明银杏酚酸对荷瘤小鼠有免疫调节的作用，提高荷瘤小鼠的细胞免疫和体液免疫能力，对免疫功能的影响有一个最佳剂量范围，可通过提高机体免疫功能来抑制肿瘤。 银杏酚酸能降低荷瘤小鼠血清乳酸脱氢酶的活性，但只有高剂量组表现出显著性差异;能显著提高荷瘤小鼠红细胞过氧化氢酶活性，提示银杏酚酸的体内抗移植性肿瘤的活性可能是通过改变一些酶的活性来实现的。 5银杏酚酸诱导宫颈癌Hela细胞凋亡的研究 银杏酚酸能剂量依赖性地显著抑制人宫颈癌hela细胞增殖，经银杏酚酸作用后的hela细胞在倒置和荧光显微镜下可见凋亡的形态学特征改变，DNA琼脂糖凝胶电泳可见明显的DNA梯状条带，单细胞电泳可见明显DNA断片拖尾，原位末端标记检测为阳性，流式细胞仪分析图上出现典型的凋亡细胞峰，细胞周期分析表明银杏酚酸主要是影响hela细胞S期向GZ舰期的转变过程，将细胞阻滞在S期，减少进入GZ八吐期的细胞数量，减少其有丝分裂，引起细胞凋亡。当银杏酚酸浓度为1.5、6、12、24mg/L时，细胞凋亡率分别为10.07%、30.69%、49.64%和5更多还原

【Abstract】 Ginkgolic acids are claimed to be harmful compounds and strictly limited in the extract of Ginkgo biloba leaves (EGb). Contents of Ginkgolic acids are the highest in Ginkgo biloba exopleuras and their special pharmacological and poisonous activities have increasingly caused the extensive concern of people. But compared with Ginkgo biloba leaves, the research of Ginkgolic acids is still few. This paper is aimed to establish an efficiently quantitative and analytical method of Ginkgolic acids by using modern chromatographic separation theory and identification technique. The technological conditions of isolation and purification Ginkgolic acids from Ginkgo biloba exopleuras were studied systematically and their structures were identified. The antibacterial activity of Ginkgolic acids was also discussed and their antitumor mechanism was studied by immune adjustment and apoptosis. The main results are as follows:1. Determination of Ginkgolic acids by reversed-phase high performance liquid chromatography (RP-HPLC)According to the difference of Ginkgolic acids in structure and character, an RP-HPLC analytical method was developed for separation of Ginkgolic acids. Effects of mobile phase constitute, pH and temperature on retention behaviors of Ginkgolic acids in column were discussed. The optimal chromatographic conditions of RP-HPLC were as follows: chromatographic column was SUPELCOSIL?LC-18(15cm X4.6mm,5um); mobile phase was MethanoI/H2O(90:10,V/V), adding acetic acid and making pH become 3.15; UV detection wavelength was at 310nm; flow rate was 0.7ml/min and AUFS was 0.02; column temperature was 30*C. Under the above conditions, the peaking high resolutions (Rj,) between different Ginkgolic acids were all over 1, and the capacity factors (k) of Ginkgolic acids were between 3 and 8.The quantitative determination method of Ginkgolic acids was established based on RP-HPLC separation theory. The UV spectrums of Ginkgolic acids were identified by HPLC-DAD, the results showed that the spectrums of standard Ginkgolic acids were identical to that of Ginkgolic acids from samples. The sample could be analyzed directly by HPLC after extracting with methanol and filtration. The average recoveries of the method were 97.02% and the coefficients of variation were 1.28%(n=5). The linear range was from 0.498ug to 8.879g, correlation coefficient was 0.9998. The minimum thatcould be determined was 7.2ng. This simple, rapid, sensitive and accurate method could be used in quantitative determination of Ginkgolic acids in ginkgo products.2. Extracting technology, purification and structure identification of Ginkgolic acidsThe technological conditions of extracting Ginkgolic acids from Ginkgo biloba exopleuras were optimized, and the adsorbing and desorbing properties of Ginkgolic acids on different macroreticular adsorbents were studied. The optimal purification conditions were as follows: Ginkgo biloba exopleuras was extracted with 7 volumes of 80% methanol by refluxing at 60 for 3 times and 4h for each time. The extracts were filtrated, concentrated and purified on D4020 resin (HaO, 65%, 90% ethanol grads desorbing). The desorbing solution was concentrated and extracted with solvent S|. Then, Ginkgolic acids were further purified on AB-8 resin (75% and 95% ethanol grads desorbing). The obtained Ginkgolic acids product had 95% purity and 0.89% yield.UV, IR, HPLC-DAD and LC/ESI/MS were applied to identify the structure of Ginkgolic acids from Ginkgo biloba exopleuras. Five Ginkgolic acids C13:0, C15:l, C17:2, C15:0 and C17:l were confirmed, their proportion relative were!2.0: 41.77: 4.89: 3.52: 37.83.3. Antibacterial activity of Ginkgolic acidsThe antibacterial activity of Ginkgolic acids was discussed by the dilution broth method. Water extraction and ethanol extraction of Ginkgo biloba exopleuras, purified Ginkgolic acids all showed strong antibacterial and antifungal activities. As contents of Ginkgolic acids increasing, the antibacterial activity became stronger. Ginkgolic acids were the main inhibitor. Ginkgolic acid更多还原