【作者】 周暄宣；

【导师】 王四旺；

【作者基本信息】 第四军医大学 ， 药理学， 2013， 博士

【摘要】 衰老，本世纪生物学研究重点课题，通常定义为正常状况下生物发育成熟后，随年龄增加，自身机能减退，内环境稳定能力与应激能力下降，细胞结构、功能逐步退行性变异，趋向死亡，不可逆转的现象，分为生理性衰老（正常衰老）、病理性衰老（异常衰老）两种类型。寻找可延缓衰老进程的活性成分不仅是全世界人们的期望，更是医药研究者们的责任和义务。二苯乙烯苷，源自素有抗衰老灵药之称的蓼科植物何首乌Polygonum multiflorumThunb，其化学名为2，3，5，4’-四羟基二苯乙烯-2-O-β-D-葡萄糖苷；目前国内外尚未见高纯度二苯乙烯苷（含量＞95.0%）规模生产工艺的报道。既往研究表明，二苯乙烯苷对多种衰老相关疾病如动脉粥样硬化、癌症等病症具有明显治疗作用，可清除体内过量自由基，可能通过作用Sirtuin蛋白家族延缓衰老进程。藉此提示我们，二苯乙烯苷具有明确的抗衰老作用，但其抗衰老的作用靶点及其机制尚未阐明。Klotho基因及分泌型Klotho蛋白，表达于远端肾小管及脑脉络丛，通过对胰岛素/IGF-1信号通路及p53基因的调控影响衰老进程，是衰老机制研究的热门靶点。本课题综合运用药物化学、植物化学、药理学、免疫组织化学、分子生物学等实验技术及方法，在优化二苯乙烯苷提取分离纯化工艺的基础上，研究二苯乙烯苷抗衰老新靶点及其机制，为进一步研制抗衰老药物提供新思路。1二苯乙烯苷的提纯工艺研究1.1二苯乙烯苷提取：取何首乌饮片，用1^-10%甘草-乙醇溶液喷洒或稍浸，润透，照《中国药典》（2010年版一部，附录ⅡD）蒸法，蒸至内外均一色，晒干，粉碎成粗粉（10-40目），上样于超临界CO2萃取仪的萃取釜中萃取，浓缩，萃取压力30-40Mpa，萃取温度60℃,萃取1-3小时，必要时可采用70-90%乙醇作为夹带剂，用量为200-400ml/小时，得萃取液，浓缩。浓缩液上样于硅胶柱层析（200-300目）纯化，用20-80%乙醇梯度洗脱，流速为1.0ml/分钟，收集洗脱液，蒸除乙醇，得二苯乙烯苷粗品。1.2二苯乙烯苷纯化及含量测定方法：纯化方法一，以乙酸乙酯萃取后反复重结晶为主要手段，获得高纯度二苯乙烯苷；方法二，通过大孔吸附树脂吸附，获得高纯度二苯乙烯苷。含量测定采用HPLC法，以乙腈和水为流动相，以稀乙醇定容待测样品，建立的标准曲线y=32899840x+123968，r=0.9994，精密度RSD＝0.29%，重复性RSD＝0.26%，稳定性RSD＝1.03%等。1.3二苯乙烯苷中试研究：二苯乙烯苷的提取率为2.65%（2.21-3.34%/8批次）；按照方法一纯化，二苯乙烯苷纯度97.3%（95.6-98.7%/4批次），二苯乙烯苷平均产率80.4%，RSD=2.03%；方法二纯化，二苯乙烯苷纯度97.5%（96.2-98.3%/4批次），平均产率为85.0%，RSD=3.81%。2二苯乙烯苷抗衰老作用靶点及其机制研究2.1对果蝇生命周期的影响：以倍比稀释法获得二苯乙烯苷水溶液(2mg·ml^-1、20mg·ml^-1、200mg·ml^-1)，加入果蝇培养基，以何首乌提取物加入果蝇培养基做参照，观察各组50%果蝇存活时间、5%幸存果蝇存活时间、果蝇平均生命周期，表明二苯乙烯苷可提高果蝇攀爬能力，延长果蝇生命周期（雄14.3%、雌29.2%，p＜0.01，vs.空白组），证实二苯乙烯苷具抗衰老作用。2.2对病理性衰老小鼠的作用：用D-半乳糖皮下注射的方法建立小鼠病理性衰老模型，48天后采用水迷宫法检测小鼠记忆功能，确定造模成功后，以二苯乙烯苷(含量≥70.0%，42mg·kg^-1、84mg·kg^-1、168mg·kg^-1)生理盐水溶液或等量生理盐水给小鼠灌胃，每日一次，于给药第29天及给药第57天上午，麻醉采血后处死小鼠，制备血清，大脑、心脏、肾脏、睾丸、附睾组织匀浆和组织蜡块备用。以水迷宫实验配合ELISA试剂盒、荧光实时定量PCR、免疫组化、HE染色法及病理切片进行测定，结果：①二苯乙烯苷可改善小鼠因病理性衰老引起的记忆衰退并调节体重至健康范围；②减少小鼠血清中自由基、一氧化氮、IGF-1含量（p＜0.01，vs.空白组）；③提高血清中超氧化物歧化酶、钙离子、Klotho蛋白水平（p＜0.01，vs.空白组）；④促进大脑及肾脏中Klotho基因表达（p＜0.01，vs.空白组）；⑤增加Klotho蛋白在大脑、心脏、肾脏、睾丸、附睾中表达（p＜0.01，vs.空白组），且8周药效优于4周，168mg·kg^-1优于其它两剂量组。说明二苯乙烯苷能调控病理性衰老小鼠体内Klotho基因及蛋白表达，其抗衰老机制与Klotho基因及蛋白相关。2.3对生理性衰老小鼠的作用：选用18月龄、雄性、自然衰老小鼠，灌胃给予二苯乙烯苷(含量≥95.0%，124mg·kg^-1)生理盐水溶液，对照组给予何首乌提取物(二苯乙烯苷含量≥70.0%，168mg·kg^-1)生理盐水溶液，空白组给予等量生理盐水，每日一次，给药8周后，第57天上午，麻醉采血后处死小鼠，制备血清，大脑、心脏、肾脏、皮肤组织匀浆和组织蜡块备用。采用ELISA试剂盒配合荧光实时定量PCR、免疫组化、HE染色法、western blot及病理切片，发现：①二苯乙烯苷可减轻老年小鼠体重，减少老年小鼠皮下脂肪细胞数量、厚度及面积（p＜0.01，vs.空白组），促进Klotho蛋白在肾脏、大脑表达（p＜0.01，vs.空白组），降低大脑及肾脏中胰岛素、胰岛素受体、IGF-1水平（p＜0.01，vs.空白组），降低肾脏及心脏中IGF-1受体水平（p＜0.01，vs.空白组）；②何首乌提取物可降低肾脏中胰岛素、IGF-1水平（p＜0.01，vs.空白组），降低肾脏及心脏中胰岛素受体水平（p＜0.01，vs.空白组），升高大脑及心脏中胰岛素水平（p＜0.01，vs.空白组），升高心脏中IGF-1水平（p＜0.01，vs.空白组），升高肾脏及大脑中IGF-1受体水平（p＜0.01，vs.空白组）。综上，以何首乌为原料，优化并制定了二苯乙烯苷提取和纯化制备工艺，以“一种二苯乙烯苷注射剂及其制备工艺”申报中国发明专利（申请号201110196199.6），该工艺技术为规模化原料药生产提供了方法保障。研究证实，二苯乙烯苷对果蝇、病理型衰老小鼠及自然衰老小鼠具有显著抗衰老作用，发现该抗衰老的作用靶点主要是小鼠体内Klotho蛋白，其可能的作用机制与调控Klotho蛋白含量及胰岛素/IGF-1通路信号因子水平相关。本研究结果为揭示二苯乙烯苷抗衰老确切机制和进一步研制抗衰老新药奠定了重要基础。更多还原

【Abstract】 Aging, a major biological research this century, defined as an irreversible processwhich companied with decreased homeostatic capacity and stress, progressive anddegenerative changes in the structure, function. There were two kinds of aging here, onewas induced with age （natural aging） and the other was induced with diseases （abnormalaging）. Looking for active ingredients that could delay aging process is a tirelessly subjectfor the whole world, and also an urgent duty for the pharmaceutical researchers.The2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside was found in Polygonummultiflorum Thunb which was considered as a magical medicine for anti-aging, andabbreviated as tetrahydroxystilbene glucoside （TSG）. The scale process to get high puretyof TSG （TSG＞95%） has not been report yet. Previous research showed that: TSG wasuseful in courses in a variety of aging related diseases, such as atherosclerosis, cancer, alsoclearing the body’s excess free radicals away, and delaying the aging process witheffecting on Sirtuin protein family. The aforementioned conclusion provided us theoreticaland experimental evidence, and the scientific dispute about the anti-aging effects ofSirtuin protein family pressed us to look for a new target and mechanism to explain the anti-aging activity of TSG.Klotho gene and protein expressed in distal renal tubular and brain choroid plexus arepotential anti-aging target which worked as a regulator on insulin/IGF-1signal pathwayand p53gene.The present study demonstrated a new drug-interfered anti-aging idea through theresearch on TSG’s anti-aging activity and the related mechanism based on aforementionedevidence, and experimental techniques （medicinal chemistry, phytochemistry,pharmacology, immunohistochemistry, and molecular biology）.Section1: Optimization of TSG’s purification process（1） Extraction of TSG: Spray the Polygonum multiflorum Pieces with1^-10%licorice-ethanol solution until the solution run through the pieces. According to the"Chinese Pharmacopoeia"（2010a, Appendix II D） to steam aforementioned pieces till theinside and outside were the same color. Season these pieces before crush them into acoarse powder （10-40mesh）. Extract the powder with Supercritical CO2extractioninstrument for1-3hours, extraction pressure:30-40Mpa, temperature:60℃.70-90%ethanol can be used as an entrainer necessary and the dosage was200-400ml/h.Concentrate the extracts then purify it by silica gel column chromatography （200-300mesh）, gradient elution:20-80%ethanol, flow rate:1.0ml/min. Distill the ethanol off fromaforementioned collection to get crude TSG.（2） Purification and determination of TSG: method1obtained high purity TSGwith ethyl acetate and repeated recrystallization. Method2obtained high purity TSG withmacroporous resin. The content of TSG was measured by HPLC, mobile phase:acetonitrile and water, volume samples with dilute ethanol, standard curve:y=32899840x+123968, r=0.9994, precision: RSD=0.29%, repeatability: RSD=0.26%,stability: RSD=1.03%.（3） Pilot of TSG: TSG’s extraction rate was2.65%（2.31-3.34%/8batches）. Thepurity of TSG was97.3%（95.6-98.7%/4batches）, and the average yield of TSG was80.4%in method1, RSD=2.03%. The purity of TSG was97.5%（96.2-98.3%/4batches）, and the average yield of TSG was85.0%in method2, RSD=3.81%.Section2: the research of TSG’s anti-aging targets and its mechanism（1） Protect effect on drosophila’s lifespan of TSG: To observe the50%survival day,average of the last5%of surviving drosophila, and the mean lifespan in order. Comparedthe TSG (2mg·L^-1,20mg·L^-1,200mg·L^-1) groups and PME group (31mg·L^-1) with controlgroup. Conclusion: TSG and PME were protective in drosophila challenged with controlgroups not only climbing ability but also lifespan （male:14.3%、female:29.2%，p＜0.01，vs. control group）.（2） Effect on pathological aging mice of TSG: The pathological aging mice wasinduced with D-galactose for48days and tested with mirrors. Then intragastricadministered on mice with TSG (≥70%，42mg·kg^-1、84mg·kg^-1、168mg·kg^-1), and thecontrol group was administered only the vehicle. We employed mirrors, ELISA kits, HEstaining, immunofluorescence chemistry and quantitative PCR to get the following results:①long-term administration of TSG improved the memory, and regulated the body weightof aging mice induced by D-galactose.②Reduced the levels of ROS, NO and IGF-1in theserum （p＜0.01，vs. control group）.③Increased the levels of SOD, Ca2+and Klothoprotein in the serum （p＜0.01，vs. control group）.④Increased the levels of Klotho genein the cerebrum and kidney （p＜0.01，vs. control group）.⑤Increased the levels of Klothoprotein in cerebrum, heart, kidney, testis, and epididymis （p＜0.01，vs. control group）.Conclusion: the role of TSG, played in pathological aging mice induced by D-galactose,demonstrated its anti-aging ability and depended on the effect of Klotho protein. Theaforementioned results proofed that TSG regulated the levels of klotho gene and protein inpathological aging mice induced by D-galactose, and klotho gene and protein may be thekey for TSG’s anti-aging mechanism.（3） Effect on natural aging mice of TSG: Compared the18months aging miceintragastric administered with TSG (≥95%，124mg·kg^-1) and PME (TSG≥70%，168mg·kg^-1) with the mice intragastric administered with saline for8weeks. Results: TSGand PME reduced the body weight and subcutaneous fat content of mice, increased the levels of klotho protein in kidney and brain.①TSG significantly reduced the levels ofinsulin, insulin-R, and IGF-1in kidney, brain and heart （p＜0.01，vs. control group）. Italso reduced the levels of IGF-1R in kidney and heart （p＜0.01，vs. control group）, butincreased it in brain （p＜0.01，vs. control group）.②PME reduced the levels of insulinand IGF-1in kidney （p＜0.01，vs. control group）, insulin-R in kidney and heart （p＜0.01，vs. control group）, but increased the levels of insulin in brain and heart （p＜0.01，vs. control group）, IGF-1in heart （p＜0.01，vs. control group）, and IGF-1R in kidney andbrain （p＜0.01，vs. control group）. Conclusion: These results ensured the anti-aging effectof TSG and provided a new mechanism related to klotho protein and insulin/IGF-1signalpathway.In summary, the study used polygonum multiflorum as raw materials to optimize anddevelop TSG’s extract and purify process. A Chinese invention patent about TSG injectionand its preparation process has been declared （Application NO.201110196199.6） whichprovided a method for TSG’s large-scale manufacture. This study proofed that TSG had asignificant anti-aging activity in drosophila, pathological aging mice and natural agingmice. The targets of TSG maybe klotho protein and its mechanism may relate to theeffects of klotho on insulin/IGF-1signal pathway. These results revealed TSG’s anti-agingmechanism and laid an important foundation for anti-aging medicine’s furtherdevelopment.更多还原