【作者】 王成章；

【导师】 张宗和；

【作者基本信息】 中国林业科学研究院 ， 林产化学加工工程， 2007， 博士

【摘要】 银杏叶聚戊烯醇（GP）是银杏叶中新的有效类脂化合物，与人体多萜醇结构相似，参与糖蛋白的生物合成，目前国内外尚未开发利用。本文以银杏叶为原料，研究GP提取分离机理和纯化工艺；聚戊烯基磷酸酯（GPP）衍生物的合成机理，探讨了GP和GPP保护肝损伤、抗肿瘤和抗病毒药效和药理，为GP保健制剂和新药开发提供基础。首次系统研究GP分析的方法，建立了GP的TLC、RP-TLC定性和HPLC定量分析方法。以C95-prenol聚戊烯醇为外标法，分析不同树龄和采集时期的GP，GP富集在春季最低，在秋季增幅最快，最佳采集时间是9-10月。苗叶经过精制处理，GP精制品不仅得率在0.7％～0.9％间，而且含量在90％左右。首次系统研究不同提取方式对提取GP的影响，超声波提取效果最好，GP提取率达90％左右，纯度达18.6％。索氏抽取法作为工业生产首选，GP提取率高达80％以上，纯度16％-19％。采用皂化反应和溶剂冷冻分离聚戊烯醇不皂化物，皂化剂为5％NaOH-EtOH，石油醚软膏与皂化剂的比例为1：5（g／ml）。溶剂A冷冻温度-10℃至-20℃，溶剂B冷冻温度-20℃以下，溶剂A母液中聚戊烯醇为55.6％，GP回收率为98.5％，溶剂B沉淀物中聚戊烯醇含量64.7％，GP回收率为90％。采用介质分离聚戊烯醇不皂化物，不同介质对GP分离的效果为硅胶＞氧化铝＞NKA-2＞聚酰胺＞D101。首次采用分子短程蒸馏分离聚戊烯醇不皂化物，馏余物中聚戊烯醇由不皂化物中的46.2％提高到83.7％，而且无溶剂残留，是工业化制备高纯度聚戊烯醇理想的分离方法。采用二级分子蒸馏分离和溶剂重结晶甾醇类化合物，银杏叶甾醇收率为0.03-0.08％，纯度大于95％。利用GC-MS分析，其中8个是甾醇类化合物，除β-谷甾醇外，其它银杏叶甾醇是首次报道。首次以GP为原料，分别合成聚戊烯基氯磷酸酯、聚戊烯基磷酸单酯（GPP）和聚戊烯基磷酸二乙酯等衍生物，反应产物收率高于80％。由IR、¹H-NMR、¹³C-NMR和高分辨质谱鉴定其化学结构。首次研究GP保护肝脏、体内抗肿瘤和体外抗病毒的药效学和药理。结果表明GP无毒副作用，对CCl₄、D-Gal和酒精引起急性肝损伤有保护作用，能明显改善CCl₄致大鼠慢性肝损伤的肝功能，对肝组织羟脯氨酸和胶原蛋白含量明显降低，具有抗慢性肝细胞损伤和抗肝纤维化的作用。GPP对移植性Heps、肉瘤S₁₈₀和艾氏癌EC荷瘤鼠的抑制作用明显好于GP。GP能延长EC荷瘤鼠、荷人脑瘤SF763裸鼠和A549人肺腺癌荷瘤裸鼠的延命率，GP与CTX、PDD、ADM联合化疗Heps、S₁₈₀和EC荷瘤小鼠，具有很好的协同作同，明显提高联合化疗率，联合⁶⁰Co放疗Heps荷瘤鼠，其效果高于贞芪冲剂，具有辅助化放疗的作用。GP能明显增加Heps荷瘤鼠胸腺指数，EC荷瘤鼠的脾指数和胸腺指数，GP可明显提高小鼠巨噬细胞的吞噬指数和吞噬功能，具有体液免疫作用；GP能提高S₁₈₀荷瘤小鼠肿瘤细胞凋亡指数（APO）至6.35，促进S₁₈₀荷瘤小鼠肿瘤细胞凋亡，使S₁₈₀荷瘤小鼠CD₄／CD₈比值接近正常小鼠；GP可通过降低端粒酶活性，促进肿瘤细胞凋亡，达到抑制肿瘤细胞增殖的药理作用。GP对HepG 2215细胞和MDCK细胞无毒性，对HepG2215分泌的HBV DNA具有很好的抑制作用，GP为25μg／ml和12.5μg／ml的抑制率分别为75.9％和76.6％；GP对H₃N₂甲型流感病毒没有直接的杀灭作用，但能提高MDCK细胞的存活数量：100μg／mlGP对MDCK细胞的存活率为55.6％，具有明显的保护作用。更多还原

【Abstract】 Polyprenols from ginkgo biloba L （GP） is new effective lipids in ginkgo biloba L, whichhave similar structure as dolichols（DH） in human. GP and DH mainly take part in thebiosynthsis of glycoprotein in vivo, but GP have’nt been developed yet.The extraction and separation of GP have been studied in this dissertation. The synthesiticmechanism of polyprenyl monophosphates （GPP） was investigated from GP also. To develophealthyfood and new drugs of GP, the bioactives and pharmacology of GP and GPP forhepatoprotective properties, antitumor and antivirus were researched.The dissertation investigated systemically the analysitic methodology of GP, andestablished qualitative and quantitative scheme of TLC, RP-TLC and HPLC for GP. The contentand seasonal variation of GP were studied with C₉₅-prenol as external standard, the resultsshowed that the content of GP was lowest in spring and highest in fall, the optimum collect timeis from sept. to oct.. The yield of GP was 0.7%～0.9% with about 90% polyprenol by treatmentwith seedlings.The effection on GP with different extraction styles were systematically researched first.the ultrasonic extraction was best effective with short time, high yield and purity, itstechnological parameter was 40Khz, 150W, solid to liquid ratio （g/ml）1:10,30min,twice, theextract rate of GP was about 90% with purity 18.6%. Zauschneria extract was selected asindustrial production with over 80% yield of GP and 16%-19% polyprenols.Non-saponifialble matter of GP was made and separated by saponification and solventrefrigeration., saponifier 5%NaOH-EtOH, ratio of paste of petroleum to saponifier 1:5 （g/ml）.Solvent A got GP recovery of 98.5% with 55.6% polyprenols in the twice refrigerating fluids at-10℃to -20℃, solvent B got GP revovery of 90% with 64.7% polyprenols at below -20℃inthe twice precipitates while refrigerating.Column chromatography was selected to purify non-saponifialble matter of GP. Theseperating effect was Silica gel＞alumina＞NKA-2＞polyamide＞D101. Molecular shortdistillation was first used to purify non-saponifialble matter of GP, total polyprenols could beraised from 46.2% to 83.7% in distilled remainder and there was no solvent residue, so it wouldbe the best ideal separation in industry.Sterols in lipid of GP could be separated byⅡgrade molecular short distillation andrecrystallizatiion with a yield of 0.3-0.8% and over purity 95%. 8 sterols were identified byGC-MS, besidesβ-sitosterol, the other sterols were first reported in ginkgo biloba L.The derivants of GP were synthesized first by reaction of acylation, hydrolysis and esterifywith phosphorus oxychloride, pyro-phosphoric acid and diethyl chlorophosphate as phosphatein wild alkali, the hydrolysate was purified by silicon gel chromatography and prepared byHPLC,and the yields of monopolyprenyl phosphate and polyprenyl diethyl phosphate andpolyprenylphosphatedichlordate were over 80%, and their structure was identified with IR、 ¹H-NMR、¹³C-NMR and HRMS.The bioactives and pharmacology of GP for hepatoprotective properties showed that GP isinnocuity and has a powerful protective effect on acute hepatic injury induced by carbontetrachloride, D-galactosamine and alcohol; GP and YI Shanfu could significantly decreasedserum levels of AST and ALT and significantly improved degree of proliferation of hepaticfibrous tissue and declined content of hydrop roline and collagen. GP holds significant effectson the treatment of hepatic fibrosis of chronic liver injury induced by CCl₄ in Rats.The bioactives and pharmacology of GP and GPP for antitumor showed that GPP havebetter inhibition than GP on mice transplanted tumor Heps, S₁₈₀ and EC. GP couldsignificantly prolong survival time of mice transphtnted EC tumor, nude bearing SF763 tumorand nude bearing A549 tumor. GP has good synergistic reaction on mice transplanted tumors ofHeps, S₁₈₀ and EC in combination with Fu-5, CTX, PDD and ADM, In particular the lowerdoses of GP with ⁶⁰Co radiotherapy had the best therapeutic effect on mice transplantedHeps, SoGP have distinct subsidiary effect on chemotherapy and radiotherapy and can reducetoxicity.GP had higher the clearance than CTX, and could increase remarkablely the phagocyticfunction of the macrophages, increase the index of the thymus gland in mouse with Heps andindex of the spleen in mouse with EC, The APO level in mouse with S₁₈₀ tumor in 5mg/kg GPwas 6.35, which made the rate of CD₄/CD₈ near to the normal mouse. GP have goodpharmacological functions of humoual immunity, inducing apoptosis and inhibitingproliferation.The bioactives and pharmacology of GP for antivirus in vitro showed GP is no toxicityon cell HepG 2215 and MDCK, and has better inhibition on HBV DNA secreted by cell HepG2215, with inhibition for GP 75.9% at 25μg/ml and 76.6% at 12.5μg/ml; GP had directly nokill on H₃N₂ influenza virus, but GP can raise the survival numbers of cell MDCK, the survivalrate can be raised to 55.6% at GP100μg/ml.更多还原