高纯度二苯乙烯苷制备工艺及其抗衰老机制研究
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~(-1)0%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。
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~(-1)0%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.
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~(-1)0%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。
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~(-1)0%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.
引文
1. Hata K, Kozawa M, Baba K. A new stilbene glucoside from Chinese crude drugHeshouwu. Roots of Polygonum multiflorum [J]. Yakugaku Zasshi,1975,95（2）:211-213.
2.吕丽爽,汤坚.二苯乙烯苷和白藜芦醇抗氧化构效关系研究［J］.食品与机械,2009,25(5):57-58.
3. Wood J.G, Rogina B, Lavu S, Howitz K, Helfand S.L, Tatar M, Sinclair D. Sirtuinactivators mimic caloric restriction and delay ageing in metazoans [J]. Nature,2004,430(7000):686-698.
4. Xin Yu, Guorong Li. Effects of resveratrol on longevity, cognitive ability andaging-related histological markers in the annual fish Nothobranchius guentheri [J].Experimental Gerontology,2012,47(12):940-949.
5. Yee Ting Wong, Jan Gruber, Andrew M. Jenner, Mary Pei-Ern Ng, Runsheng Ruan,Francis Eng Hock Tay. Elevation of oxidative-damage biomarkers during aging in F2hybrid mice: Protection by chronic oral intake of resveratrol [J]. Free Radical Biologyand Medicine,2009,46(6):799-809.
6.边秀娟,包国荣.电子自旋共振法测定何首乌中大黄素、二苯乙烯苷清除超氧阴离子自由基的实验研究[D].福州,福州中医学院,2002,12-13.
7.吕丽爽,汤坚.何首乌中二苯乙烯苷的醇提工艺[J].食品与发酵工业,2004,30(10):132-135.
8.戚爱棣.何首乌中二苯乙烯苷提取工艺优选及炮制对其含量影响[J].中草药,2002,33(7):609-611.
9.王娟,沈平孃,沈永嘉.均匀设计优选微波辅助萃取何首乌中有效成分的研究[J].中草药,2003,34(4):314-317.
10.千梅,何况旻,方红美,陈从贵.何首乌中二苯乙烯苷的超声提取工艺研究[J].农产品加工学刊,2006,(10):115-122.
11.白研,葛发欢,史庆龙.超临界CO2流体萃取制首乌中的有效成分[J].中药材,2005,28(4):290-291.
12.班翊,刘其礼,金悠.二苯乙烯苷的稳定性研究[J].中草药,2004,35(11):1235-1237.
13. Yao S, Li Y, Kong L. Preparative isolation and purification of chemical constituentsfrom the root of polygonum muhiflorumby high-speed counter-currentchromatography [J]. J Chromatogr A,2006,1115(1-2):64-71.
14.张纯,杨少麟,袁海龙.高效液相色谱法测定何首乌中二苯乙烯甙的含量及其稳定性考察[J].中国中药杂志,1999,24(6):357-360.
15.刘振丽,宋志前.不同地区制首乌中二苯乙烯苷含量测定及稳定性考察[J].中成药,2002,24(9):684-685.
16.吕丽爽,汤坚,何其傥.何首乌中二苯乙烯苷的制备及抗氧化机理的研究[D].无锡,江南大学,2006,27-28,46-47,61-66.
17.孙江浩,袁志芳,王春英,许慧君,张兰桐.何首乌中二苯乙烯苷在大鼠体内的药动学[J].中草药,2005,36(3):405-409.
18.王春英,李敏,袁志芳,杨维,解凤立,张兰酮.二苯乙烯苷大鼠在胃肠吸收动力学研究[J].中草药,2008,39(8):1215-1219.
19.王春英,郭栋,袁志芳,冯小龙,张兰桐.二苯乙烯苷在大鼠体内外的代谢研究[J].中国医药工业杂志,2009,40(2):120-123.
20.楼招欢,吕圭源,陈素红,顾慧.首乌苷制剂中二苯乙烯苷含量测定及其在小鼠体内组织分布研究[J].中国现代应用药学,2011,28(2):89-91.
21.孙江浩,张兰桐,王春英,袁志芳,卢兴红.液-质联用法测定小鼠体内二苯乙烯苷代谢物[J].药学学报,2003,38(12):968-970.
22.许爱霞,张振明,葛斌.何首乌多糖对氧自由基及抗氧化酶活性的作用研究[J].中国药师,2005,8(11):900-902.
23.胡存华,赵立波,王晓敏.二苯乙烯苷增强正常血管内皮细胞抗氧化作用研究[J].医药导报,2007,26(2):138-139.
24. Howes R.M. The free radical fantasy: A panoply of paradoxes [J]. Ann N.Y Acad Sci,2006,1067(1):22-26.
25.刘丽,李林,赵玲.二苯乙烯苷对慢性脑缺血大鼠学习记忆的影响[J].中国药理学与毒理学杂志,2008,22(2):108-115.
26.周琳,杨期东,马志健.何首乌对Alzheimer病大鼠学习记忆及乙酰胆碱酯酶的作用[J].中国药师杂志,2004,21(5):394-396.
27. Lan Zhang, Ying Xing, Cui-Fei Ye, Hou-Xi Ai, Hai-Feng Wei, Lin Li.Learning-memory deficit with aging in APP transgenic mice of Alzheimer's diseaseand intervention by using tetrahydroxystilbene glucoside [J]. Behavioural BrainResearch,2006,173(2):246-254.
28. Ting Wang, Yuan-Jian Yang, Peng-Fei Wu, Wei Wang, Zhuang-Li Hu, Li-Hong Long,Na Xie, Hui Fu, Fang Wang, Jian-Guo Chen. Tetrahydroxystilbene glucoside, aplant-derived cognitive enhancer, promotes hippocampal synaptic plasticity [J].European Journal of Pharmacology,2011,650(1):206-214.
29.高瑄,胡英杰,符林春.何首乌二苯乙烯苷的调节血脂作用[J].中国中药杂志,2007,32(4):323-326.
30.韩晓,吴成爱,王伟.何首乌二苯乙烯苷降血脂作用机理研究[J].中华中医药学刊,2008,26(8):1687-1689.
31. Yang P.Y, Almofti M.R, Lu L. Reduction of atherosclerosis in cholesterol-fed rabbitsand decrease ofexpressions of intraccllular adhesion molecule-1andvascjlarendothelial growth factor in foam cells by a water-solutionfraction ofpolygonum multiflorum [J]. J Pharmacol Sci,2005,99(3):294-300.
32. WenJuan Yao, WenJun Fan, Chao Huang, Hui Zhong, XiangFan Chen, Wei Zhang.Proteomic analysis for anti-atherosclerotic effect of tetrahydroxystilbene glucoside inrats. Biomedicine&Pharmacotherapy,2012,67(2):140-145.
33. Xiaomin Wang, Libo Zhao, Tianzhao Han, Shaofei Chen, Jialing Wang. Protectiveeffects of2,3,5,4′-tetrahydroxystilbene-2-O-beta-d-glucoside, an active component ofPolygonum multiflorum Thunb, on experimental colitis in mice [J]. EuropeanJournal of Pharmacology,2008,578(2):339-348.
34. Zequn Jiang, Jimin Xu, Minghai Long, Zhiming Tu, Guangxiao Yang, Guangyuan He.2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (THSG) induces melanogenesis inB16cells by MAP kinase activation and tyrosinase upregulation [J]. Life Sciences,2009,85(9):345-350.
35. Xin Han, Shuang Ling, Woting Gan, Li Sun, Ju Duan, Jin-Wen Xu.2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside ameliorates vascular senescence andimproves blood flow involving a mechanism of p53deacetylation [J]. Atherosclerosis,2012,225(1):76-82.
36. Cairong Li, Fei Cai, Yinqiao Yang, Xinyuan Zhao, Chun Wang, Juan Li, Yanlong Jia,Jing Tang, Qing liu. Tetrahydroxystilbene glucoside ameliorates diabetic nephropathyin rats: Involvement of SIRT1and TGF-β1pathway [J]. European Journal ofPharmacology,2010,649(1):382-389.
37. Rong Qin, Xiaobing Li, Gang Li, Lizhen Tao, Yan Li, Jing Sun, Xiaogang Kang,Jianzong Chen. Protection by tetrahydroxystilbene glucoside against neurotoxicityinduced by MPP+: The involvement of PI3K/Akt pathway activation [J]. ToxicologyLetters,2011,202(1):1-7.
38. Klar A.J.S, Fogel S, MacLeod K. MAR1-A regulator of the HMa and HMα loci inSaccharomyces cerevisiae [J]. Genetics,1979,93(1):37-50.
39. Guarente L. Diverse and dynamic functions of the Sir silencing complex [J]. NatGenet,1999,23(3):281-285.
40. Rine J, Herskowitz I. Four genes responsible for a position effect on expression fromHML and HMR in Saccharomyces cerevisiae [J]. Genetics,1987,116(1):9-22.
41. Gottschling D.E, Aparicio O.M, Billington B.L, Zakian V.A. Position effect at S.cerevisiae telomeres: Reversible repression of Pol II transcription [J]. Cell,1990,63(4):751-762.
42. Landry J, Sutton A, Tafrov S.T, Heller R.C, Stebbins J, Pillus L, Sternglanz R. Thesilencing protein SIR2and its homologs are NAD-dependent protein deacetylases [J].Proc Natl Acad Sci,2000b,97(11):5807-5811.
43. Smith J.S, Brachmann C.B, Celic I, Kenna M.A, Muhammad S, Starai V.J, Avalos J.L,Escalante-Semerena J.C, Grubmeyer C, Wolberger C, Boeke J.D. A phylogeneticallyconserved NAD+-dependent protein deacetylase activity in the Sir2protein family [J].Proc Natl Acad Sci,2000,97(12):6658-6663.
44. Imai S, Armstrong C.M, Kaeberlein M, Guarente L. Transcriptional silencing andlongevity protein Sir2is an NAD-dependent histone deacetylase [J]. Nature,2000a,403(6771):795-800.
45. Tissenbaum H.A, Guarente L. Increased dosage of a sir-2gene extends lifespan inCaenorhabditis elegans [J]. Nature,2001,410(6825):227-230.
46. Rogina B, Helfand S.L. Sir2mediates longevity in the fly through a pathway relatedto calorie restriction [J]. Proc Natl Acad Sci,2004,101(45):15988-16003.
47. Astrom S.U, Cline T.W, Rine J. The Drosophila melanogaster sir2+gene isnonessential and has only minor effects on position-effect variegation [J]. Genetics,2003,163（3）:931-937.
48. Jang M, Cai L, Udeani G.O, Slowing K.V, Thomas C.F, Beecher C.W, Fong H.H,Farnsworth N.R, Kinghorn A.D, Mehta R.G. Cancer chemopreventive activity ofresveratrol, a natural product derved from grapes [J]. Science,1999,275(5297):218-220.
49. Middleton E, Jr, Kandaswami C, Theoharides T.C. The effects of plant flavonoids onmammalian cells: Implications for inflammation, heart disease, and cancer [J].Pharmacol Rev,2000,52(4):673-751.
50. Baur J.A, Sinclair D.A. Therapeutic potential of resveratrol: The in vivo evidence [J].Nat Rev Drug Discov,2006,5(6):493-506.
51. Huige Li, Ning Xia, Ulrich F rstermann. Cardiovascular effects and molecular targetsof resveratrol [J]. Nitric Oxide,2012,26(2):102-110.
52. Sylwia Borska, Magdalena Chmielewska, Teresa Wysocka, MalgorzataDrag-Zalesinska, Maciej Zabel, Piotr Dziegiel. In vitro effect of quercetin on humangastric carcinoma: Targeting cancer cells death and MDR [J]. Food and ChemicalToxicology,2012,50(9):3375-3383.
53. Pravinkumar Bhutada, Yogita Mundhada, Kuldeep Bansod, Chetan Bhutada, SantoshTawari, Pankaj Dixit, Dharmendra Mundhada. Ameliorative effect of quercetin onmemory dysfunction in streptozotocin-induced diabetic rats [J]. Neurobiology ofLearning and Memory,2010,94(3):293-302.
54. Brack C, Bechter-Thuring E, Labuhn. N-acetylcysteine slows down ageing andincreases the life span of Drosophila melanogaster [J]. Cell Mol Life Sci,1997,53(11-12):960-966.
55. Kang H.L, Benzer S, Min K.T. Life extension in Drosophila by feeding a drug [J].Proc Natl Acad Sci,2002,99(2):838-843.
56. Melov S, Wolf N, Strozyk D, Doctrow S.R, Bush A.I. Mice transgenic for Alzheimerdisease beta-amyloid develop lens cataracts that are rescued by antioxidant treatment[J]. Free Radic Biol Med,2005,38(2):258-261.
57. Melov S, Doctrow S.R, Schneider J.A, Haberson J, Patel M, Coskun P.E, Huffman K,Wallace D.C, Malfroy B. Lifespan extension and rescue of spongiformencephalopathy in superoxide dismutase2nullizygous mice treated with superoxidedismutase-catalase mimetics [J]. Neurosci,2001,21(21):8348-8353.
58. Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y,Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-iida T, Nishikawa S,Nagai R, Nabeshima YI. Mutation of the mouse klotho gene leads to a syndromeresembling ageing [J]. Nature,1997,390(6655):45-51.
59. Shiraki-Iida T, Iida A, Nabeshima Y, Anazawa H, Nishikawa S, Noda M, Kuro-o M,Nabeshima Y. Improvement of multiple pathophysiological phenotypes of klotho(kl/kl) mice by adenovirus-mediated expression of the klotho gene [J]. Gene Med,2000,2(4):233-242.
60. Saito Y, Yamagishi T, Nakamura T, Ohyama Y, Aizawa H, Suga T, Matsumura Y,Masuda H, Kurabayashi M, Kuro-o M, Nabeshima Y, Nagai R. Klotho proteinprotects against endothelial dysfunction [J]. Biochem Biophys Res Commun,1998,248(2):324-329.
61. Suga T, Kurabayashi M, Sando Y, Ohyama Y, Maeno T, Maeno Y, Aizawa H,Matsumura Y, Kuwaka T, Kuro-o M, Nabeshima Y, Nagai R. Disruption of the kothogene causes pulmonary emphysema in mice. Defect in maintenance of pulmonaryintegrity during postnatal life [J]. Am J Respir Cell Mol Biol,2000,22(1):26-33.
62. Okada S, Yoshida T, Hong Z, Ishii G, Hatano M, Kuro-o M, Nabeshima Y, NabeshimaY, Tokuhisa T. Impairment of B lymphopoiesis in precocious aging (klotho) mice [J].Int Immunol,2000,12(6):861-871.
63. Utsugi T, Ohno T, Ohyama Y, Uchiyama T, Saito Y, Matsumura Y, Aizawa H, Itoh H,Kurabayashi M, Kawazu S, Tomono S, Oka Y, Suga T, Kuro-o M, Nabeshima Y,Nagai R. Decreased insulin production and increased insulin sensitivity in the klothomutant mouse, a novel animal model for human aging [J]. Metabolism,2000,49(9):1118-1123.
64. Mori K, Yahata K, Mukoyama M, Suganami T, Makino H, Nagae T, Masuzaki H,Ogawa Y, Sugawara A, Nabeshima YM Nakao K. Disruption of klotho gene causesan abnormal energy homeostasis in mice [J]. Biochem Biophys Res Commun,2000,278(3):665-670.
65. Uchida A, Komiya Y, Tashiro T, Yorifuji H, Kishimoto T, Nabeshima Y, Hisanaga S.Neurofilaments of Klotho, the mutant mouse prematurely displaying symptomsresembling guman aging [J]. J Neurosce Res,2001,64(4):364-370.
66. Ohyama Y, Kurabayashi M, Masuda H, Nakamura T, Aihara Y, Kaname T, Suga T,Arai M, Aizawa H, Matsumura Y, Kuro-o M, Nabeshima Y, Nagail R. Molecularcloning of rat klotho cDNA: markedly decreased expression of klotho by acuteinflammatory stress [J]. Biochem Biophys Res Commun,1998,251(3):920-925.
67. Matsumura Y, Aizawa H, Shiraki-Iida T, Nagai R, Kuro-o M, Nabeshima Y.Identification of the human klotho gene and its two transcripts encoding membraneand secreted klotho protein [J]. Biochem Biophys Res Commun,1998,242(3):626-630.
68. Shiraki-Iida T, Azawa H, Matsumura Y, Sekine S, Iida A, A nazawa H, Nagai R,Kuro-o M, Nabeshima Y. Structure of the mouse klotho gene and its two transcriptsencoding membrane and secreted protein [J]. FEBS Lett,1998,424(1):6-10.
69. Imura A, Iwano A, Tohyama O, Tsuji Y, Nozaki K, Hashimoto N, Fujimori T,Nabeshima Y. Secreted Klotho protein in sera and CSF: implication forposttranslational cleavage in release of Klotho protein from cell membrane [J]. FEBSLett,2004,565(1):143-147.
70. Chang Q, Hoefs S, Van der Kemp AW, Topala CN, Bindels RJ, Hoenderop JG. Thebeta-glucuronidase klotho hydrolyzes and activates the TRPV5channel [J]. Science,2005,310(5747):490-493.
71. Wolf I, Levanon-Cohen S, Bose S, Ligumsky HM Sredni B, Kanety H, Kuro-o M,Karlan B, Kaufman B, Koeffler HP, Rubinek T. Klotho: a tumor suppressor and amodulator of the IGF-1and FGF pathways in human breast cancer [J]. Oncogene,2008,27(56):7094-7105.
72. Tatar M, Bartke A, Antebi A. The endocrine regulation of aging by insulin-likesignals [J]. Science,2003,299(5611):1346-1351.
73. Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R. AC. Elegans mutant that livestwice as long as wild type [J]. Nature,1993,366(6454):461-464.
74. Morris JZ, Tissenbaum HA, Ruvkun G. A phosphatidylinositol-3-OH kinase familymember regulating longevity and diapause in Caenorhabditis elegans [J]. Nature,1996,382(6591):536-539.
75. Clancy DJ, Gems D, Harshman LG, Oldham S, Stocker H, Hafen E, Leevers SJ,Partridge L. Extension of life-span by loss of CHICO, a Drosophila insulin receptorsubstrate protein [J]. Science,2001,292(5514):104-106.
76. Bartke A, Brown-Borg H, Mattison J, Kinney B, Hauck S, Wright C. Prolongedlongevity of hypopituitary dwarf mice [J]. Exp Gerontol,2001,36(1):21-28.
77. Taguchi A, Wartschow LM, White MF. Brain IRS2signaling coordinates life spanand mutrient homeostasis [J]. Science,2007,317(5836):369-372.
78. Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum HA, Ruvkun G. TheFork head transcription factor DAF-16transduces insulin-like metabolic andlongevity signals in C. elegans [J]. Nature,1997,389(6654):994-999.
79. Lin K, Hsin H, Libina N, Kenyon C. Regulation of the Caenorhabditis eleganslongevity protein DAF-16by insulin/IGF-1and germline signaling [J]. Nat Genet,2001,28(2):139-145.
80. Henderson ST, Johnson TE. Daf-16integrates developmental and environmentalinputs to mediate aging in the nematode Caenorhabditis elegans [J]. Curr Biol,2001,11(24):1975-1980.
81. Giannakou ME, Goss M, Junger MA, Hafen E, Leevers SJ, Patridge L. Long-livedDrosophila with overexpressed dFOXO in adult fat body [J]. Science,2004,305(5682):361.
82. Hwangbo DS, Gershman B, Tu MP, Palmer M, Tatar M. Drosophila dFOXO controlslifespan and regulates insulin signaling in brain and fat body [J]. Nature,2004,429(6991):562-566.
83. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC,Blenis JM Greenberg ME. Akt promotes cell survival by phosphorylating andinhibiting a forhead transcription factor [J]. Cell,1999,96(6):857-868.
84. Kops GJ, Dansen TB, Polderman PEM Saarloos I, Wirtz KW, Coffer PJ, Huang TT,Bos JL, Medema RH, Burgering BM. Forkhead transcription factor FOXO3a protectsquiescent cells from oxidative stress [J]. Nature,2002,419(6904):316-321.
85. Yamamoto M. Clark JD, Pastor JV, Gurnani P, Nandi A, Kurosu HM Miyoshi M,Ogawa Y, Castrillon DH, Rosenblatt KP, Kuro-o M. Regulation of oxidative stress bythe anti-aging hormone klotho [J]. J Biol Chem,2005,280(45):38029-38034.
86. Chateau MT, Araiz C, Descamps S, Galas S. Klotho interferes with a novelFGF-signalling pathway and insulin/Igf-like signaling to improve longevity and stressresistance in Caenrhabditis elegans [J]. Aging (Albany NY),2010,2(9):567-581.
87. Lee J, Jeong DJ, Kim J, Lee S, Park JH, Chang B, Jung SI, Yi L, Han YM Yang Y,Kim KI, Lim JS, Yang I, Jeon S, Bae DH, Kim CJ, Lee MS. The anti-aging geneKLOTHO is a novel target for epigenetic silencing in human cervical carcinoma [J].Mol Cancer,2010,9(1):109.
88. Wolf I, Laitman Y, Rubinek T, Abramovitz L, Novikov I, Beeri R, Kuro-o M,Koeffler HP, Catane R, Freedman LS, Levy-Lahad E, Karlan BY, Friedman E,Kaufman B. Functional variant of KLOTHO: a breast cancer risk modifier amongBRCA1mutation carriers of Ashkenazi origin [J]. Oncogene,2010,29(1):26-33.
89. Bartucci M, Morelli C, Mauro L, Ando S, Surmacz E. Differential insulin-like growthfactor I receptor signaling and function in estrogen receptor (ER)-positive MCF-7andER-negative MDA-MB-231breast cancer cells [J]. Cancer Res,2001,61(18):6747-6754.
90. Chen B, Wang X, Zhao W, Wu J. Klotho inhibits growth and promotes apoptosis inhuman lung cancer cell line A549[J]. J Exp Clin Cancer Res,2010,29(1):99.
91. Lu L, Katsaros D, Wiley A, De la Longrais IA, Puopolo M, Yu H. Klotho expressionin epithelial ovarian cancer and its association with insulin-like growth factors anddisease progression [J]. Cancer Invest,2008,26(2):185:192.
92. Michio Imai, Kazuhiko Ishikawa, Naomichi Matsukawa, Iwao Kida, Junsuke Ohta,Masashi Ikushima, Yukana Chihara, Xu Rui, Hiromi Rakugi MD, PhD, ToshioOgihara. Klotho protein activates the PKC pathway in the kidney and testis andsuppresses25-hydroxyvitamin D3lalpha-hydroxylase gene expression [J]. Endocrine,2004,25(3):229-234.
93. Hiroshi Kurosu, Yasushi Ogawa, Masayoshi Miyoshi, Masaya Yamamoto, AnimeshNandi, Kevin P. Rosenblatt, Michel G. Baum, Susan Schiavi, Ming-Chang Hu, OrsonW. Moe, and Makoto Kuro-o. Regulation of fibroblast growth factor-23signaling byklotho [J]. J Biol Chem,2006,281(10):6120-6123.
94. Negri AL. The klotho gene: a gene predominantly expressed in the kidney is afundamental regulator of aging and calcium/phosphorus metabolism [J]. J Nephrol,2005,18(6):654-658.
95. Weindruch R, Walford R. The retardation of aging and disease by dietary restriction[M]. C.C. Thomas, Springfield, Illinois,1988.
96. Mosoro E.J. Overview of caloric restriction and aging [J]. Mech Aging Dev,2005,126(9):913-922.
97. Min K.J, Tatar M. Restriction of amino acids extends lifespan in Drosophilamelanogaster [J]. Mech Aging Dev,2006,127(7):643-646.
98. Kealy R.D, Lawler D.F, Ballam J.M, Mantz S.L, Biery D.N, Greeley E.H, Segre M,Smit G.K, Stowe H.D. Effects of diet restriction on lifespan and agerelated changes indogs [J]. Am Vet Med Assoc,2002,220(9):1315-1320.
99. Bordone L, Guarente L. Calorie restriction, SIRT1and metabolism: Understandinglongevity. Nat Rev Mol Cell Biol,2005,6(4):298-305.
100. Nisoli E, Tonello C, Cardile A, Cozzi V, Bracale R, Tedesco L, Falcone S, Valerio A,Cantoni O, Clementi E. Calorie restriction promotes mitochondrial biogenesis byinducing the expression of eNOS [J]. Science,310(5746):314-317.
101. Lavrovsky Y, Chatterjee B, Clark R.A, Roy A.K. Role of redox-regulatedtranscription factors in inflammation, aging and age-related diseases [J]. ExpGerontol,2000,35(5):521-532.
102. Merry B.J. Oxidative stress and mitochondrial function with aging—The effects ofcalorie restriction [J]. Aging Cell,2004,3(1):7-12.
103. Reiling J.H, Sabatini D.M. Stress and mTORture signaling [J]. Oncogene,2006,25(48):6373-6383.
104. Kim J.H, Chu S.C, Gramlich J.L, Pride Y.B, Babendreier E, Chauhan D, Salgia R,Podar K, Griffin J.D, Sattler M. Activation of the PI13K/mTOR pathway byBVRABL contributes to increased production of reactive oxygen species [J]. Blood,2005,105(4):1717-1723.
105. Kopchick J.J, Andry J.M. Growth hormone (GH), GH receptor, and signaltransduction [J]. Mol Genet Metab,2000,71(1-2):293-314.
106. Cohen P. Overview of the IGF-I system [J]. Hormone Research in Paediatrics,2006,65(Suppl.1):3-8.
107. Giustina A, Velduis J.D. Pathophysiology of the neuroregulation of growth hormonesecretion in experimental animals and the human [J]. Endocr Rev,1998,19(6):717-797.
108. Silvia G, Emanuele M, Stephen E. Borst, Christiaan Leeuwenburgh. Modulation ofGH/IGF-1axis: Potential strategies to counteract sarcopenia in older adults OriginalResearch Article [J]. Mechanisms of Ageing and Development,2008,129(10):593-601.
109. Oguszewski C.L, Meister L.H, Zaninelli D.C, Radominski R.B. One year of GHreplacement therapy with a fixed low-dose regimen improves body composition, bonemineral density and lipid profile of GH-deficient adults [J]. Eur J Endocrinol,2005,152(1):67-75.
110. Blackman M.R, Sorkin J.D, Munzer T, Bellantoni M.F, Busby-Whitehead J, StevensT.E, Jayme J, O’Connor K.G, Christmas C, Tobin J.D. Growth hormone and sexsteroid administration in healthy aged women and men: A randomized controlled trial[J]. J Am Med Assoc,2002,288(18):2282-2292.
111. Guarente L, Kenyon C. Genetic pathways that regulate aging in model organisms [J].Nature,2000,408(6809):255-262.
112. Greer E.L, Brunet A. FOXO transcription factors at the interface between longevityand tumor suppression [J]. Oncogene,2005,24(50):7410-7425.
113. Masoro E.J, Iwasaki K, Gleiser C.A, Mcmahan C.A, Seo E.J, Yu B.P. Dietarymodulation of the progression of nephropathy in aging rats: An evaluation of theimportance of protein [J]. Am J Clin Nutr,1989,49(6):1217-1227.
114. Masoro E.J, McCaRTER r.j, Katz M.S, McMahan C.A. Dietary restriction alterscharacteristics of glucose fuel use [J]. J Gerontol,1992,47(6): B202-B208.
115. Kenyon C. A conserved regulatory system for aging [J]. Cell,2001,105(2):165-168.
116. Paul C. Henning, Dennis E. Scofield, Kevin R. Rarick, Joseph R. Pierce, Jeffery S.Staab, Harris R. Lieberman, Bradley C. Nindl. Effects of acute caloric restrictioncompared to caloric balance on the temporal response of the IGF-I system [J].Metabolism,2013,62(2):179-187.
117. Vladimir N. Anisimov, Andrzej Bartk. The key role of growth hormone–insulin–IGF-1signaling in aging and cancer [J]. Critical Reviews in Oncology/Hematology,2013, In Press.
118. Achille Pich, Elena Margaria, Luigi Chiusa, Renata Ponti, Massimo Geuna. DNAploidy and p53expression correlate with survival and cell proliferative activity inmale breast carcinoma [J]. Human Pathology,1996,27(7):676-682.
119. Gerald P. Holmquist, Shuwei Gao. Somatic mutation theory, DNA repair rates, andthe molecular epidemiology of p53mutations [J]. Mutation Research/Reviews inMutation Research,1997,386(1):69-101.
120. Jirouta Kitagaki, Yili Yang. DNA intercalator korkormicin A preferentially kills tumorcells expressing wild type p53[J]. Biochemical and Biophysical ResearchCommunications,2011,414(1):186-191.
121. Levine A.J. p53, the cellular gatekeeper for growth and division [J]. Cell,1997,88:323-331.
122. Levine A.J, Hu W, Feng Z. The p53pathway: what questions remain to be explored?[J]. Cell Death Differ,2006a,13:1027-1036.
123. Vosden K.H, Lu X. Live or let die: The cell’s response to p53[J]. Nat Rev Cancer,2002,2:594-604.
124. Levine A.J, Feng Z, Mak T.W, You H, Jin S. Coordination and communicationbetween the p53and IGF-1-AKT-TOR signal transduction pathways [J]. Genes Dev,2006b,20:267-275.
125. Feng Z, Zhang H, Levine A.J, Jin S. The coordinate regulation of the p53and mTORpathways in cells [J]. Proc Natl Acad Sci,2005,102(23):8204-8209.
126. Jones R.G, Plas D.R, Kubek S, Buzzai M, Mu J, Xu Y, Birnbaum M.J, Thompson C.B.AMP-activated protein kinase induces a p53-dependent metabolic checkpoint [J]. MolCell,2005,18(3):283-293.
127. Schmitt C.A, Fridman J.S, Yang M, Lee S, Baranov E, Hoffman R.M, Lowe S.W. Asenescence program controlled by p53and p16INK4a contributes to the outcome ofcancer therapy [J]. Cell,2002,109(3):335-346.
128. Maier B, Gluba W, Bernier B, Turner T, Mohammad K, Guise T, Sutherland A,Thorner M, Scrable H. Modulation of mammalian life span by the short isoform ofp53[J]. Genes Dev,2004,18:306-319.
129. Barton A. Some aspects of cell division in Saccharomyces cerevisiae [J]. J GenMicrobiol,1950,4(1):84-86.
130. Longo V.D, Fabrizio P. Regulation of longevity and stress resistance: A molecularstrategy conserved from yeast to humans?[J]. Cell Mol Life Sci,2002,59(6):903-908.
131. Muller I. Parental age and the life-span of zygotes of Saccharomyces cerevisiae [J].Antonie van Leeuwenhoek,1985,51:1-10.
132. Brenner S. The genetics of Caenorhabditis elegans [J]. Genetics,1974,77(1):71-94.
133. Ellis R.E, Yuan J.Y, Horvitz H.R. Mechanisms and functions of cell death [J]. AnnuRev Cell Biol,1991,7:663-698.
134. Coulson A, Sulston J, Brenner S, Karn J. Toward a physical map of the genome of thenematode Caenorhabditis elegans [J]. Proc Natl Acad Sci,1986,83(20):7821-7825.
135. C.elegans Sequencing Consortium. Genome sequence of the nematode C.elegans: Aplatform for investigating biology [J]. Science,1998,282(34):2012-2018.
136. Friedman D.B, and Johnson T.E. A mutation in the age-1gene in Caenorhabditiselegans lengthens life and reduces hermaphrodite fertility [J]. Genetics,1988a,118(38):75-86.
137. Friedman D.B, and Johnson T.E. Three mutants that extend both mean and maximumlife span of the nematode, Caenorhabditis elegans, define the age-1gene [J]. Gerontol,1988b,43(4): B102-B109.
138. Morris J.Z, Tissenbaum H.A, Ruvkun G. A phosphatidylinositol-3-OH kinase familymember regulating longevity and diapause in Caenorhabditis elegans [J]. Nature,1996,382:536-539.
139. Kohler R.E. Lords of the fly: Drosophila genetics and the experimental life [M].University of Chicaago Press, Chicago, Illinois,1994.
140. Collins B, Mazzoni E.O, Stanewsky R, Blau J. Drosophila CRYPTOCHROME is acircadian transcriptional repressor [J]. Curr Biol,2006,16(5):441-449.
141. Gotter A.L. A Timeless debate: Resolving TIM’s noncircadian roles with possibleclock function [J]. Neuroreport,2006,17(12):1229-1233.
142. Shaw p.j, Tononi G, Greenspan R.J, Robinson D.F. Stress response genes protectagainst lethal effects of sleep deprivation in Drosophila [J]. Nature,2002,417:287-291.
143. Koh K, Evans J.M, Hendricks J.C, Sehgal A. A Drosophila model for age-associatedchanges in sleep: wake cycles [J]. Proc Natl Acad Sci,2006,103(37):13843-13847.
144. Walker D.W, Benzer S. Mitochondrial “swirls” induced by oxygen stress and in theDrosophila mutant hyperswirl [J]. Proc Natl Acad Sci,2004,101(28):10290-10295.
145. Kujoth G.C, Hiona A, Pugh T.D, Someya S, Panzer K, Wohlgemuth S.E, Hofer T, SeoA.Y, Sullivan R, Jobling W.A, Morrow J.D, Van Remmen H, Sedivy J.M, Yamasoba T,Tanokura M, Weindruch R, Leeuwenburgh C, Prolla T.A. Mitochondrial DNAmutations, oxidative stress, and apoptosis in mammalian aging [J]. Science,2005,309(5733):481-484.
146. Landis G.N, Abdueva D, Skvortsov D, Yang J, Rabin B.E, Carrick J, Tavare S, TowerJ. Similar gene expression patterns characterize aging and oxidative stress inDrosophila melanogaster [J]. Proc Natl Acad Sci,2004,101(20):7663-7668.
147. Crother D.C, Kingborn K.J, Page R, Lomas D.A. Therapeutic targets from aDrosophila model of Alzheimer’s disease [J]. Curr Opin Pharmacol,2004,4(5):513-516.
148. Chee F, Mudher A, Newman T.A, Cuttle M, Lovestone S, Shepherd D.Overexpression of tau results in defective synaptic transmission in Drosophilaneuromuscular junctions [J]. Biochem Soc Trans,2006,34:88-90.
149. Feany M, Bender W. A Drosophila model of Parkinson’s disease [J]. Nature,2000,404:394-398.
150. Whitworth A.J, Wes P.D, Pallanck L.J. Drosophila models pioneer a new approach todrug discovery for Parkinson’s disease [J]. Drug Discov Today,2006,11(3-4):119-126.
151. Marsh J.L, Pallos J, Thompson L.M. Fly models of Huntington’s disease [J]. HumMol Genet (spec),2003,12(2): R187-193.
152. Long T.A, Montgomerie R, Chippindale A.K. Quantifying the gender load: Canpopulation crosses reveal interlocus sexual conflict?[J]. Philos Trans R Soc Lond BBiol Sci,2006,361(1466):363-374.
153. Osiewacz H.D, Scheckhuber C.Q. Impact of ROS on ageing of two fungal modelsystems: Saccharomyces cerevisiae and Podospora anserine [J]. Free Radic Res,2006,40(8):1350-1358.
154. Wessells R.J, Fitzgerald E, Cypser J.R, Tatar M, Bodmer R. Insulin regulation ofgeart function in aging fruit flies [J]. Nat Genet,2004,36:1275-1281.
155. Zhou Y, Xu B.C, Maheshwari H.G, He L, Reed M, Lozykowski M, Okada S, CataldoL. Coschigamo K, Wagner T.E. A mammalian model for Laron syndrome producedby targeted disruption of the mouse growth hormone receptor/binding protein gene(the Laron mouse)[J]. Proc Natl Acad Sci,1997,94(24):13215-13220.
156. Kurosu H, Yamamoto M, Clark J.D, Pastor J.V, Nandi A, Gurnani P, McGuinness O.P,Chikuda H, Yamamoto M, Kawaguchi H. Suppression of aging in mice by thehormone Klotho [J]. Science,2005,309(5742):1829-1833.
157. Minako K, Noboru M, Kohji K. Abnormal accumulation of luteal bodies in ovaries ofthe senescence accelerated mouse (SAM)[J]. The Journal of Reproduction andDevelopment,2001,47(3):153-164．
158.周惠嘉．制备衰老动物模型的一种可控臭氧装置[J]．中国药理学通报,1996,12(3):285-286．
159. Gong G, Xu F. Study of aging model in mice [J]. Journal of China PharmaceuticalUniversity,1991,21(2):101-103.
160. Peng C, Chan H.Y, Huang Y, Yu H, Chen Z.Y. Apple polyphenols extend the meanlifespan of Drosophila melanogaster [J]. Agric Food Chem,2011,59:2097-2106.
161. Shizhen Li. Polygonum multiflorum [M]. Compendium of Materia Medica,1590,Nanjing, China: P910.
162. Wei Wang, Jinye Wang, Tiren Shi. The effect on lifespan and lipid metabolism ofaging quail of Polygonum multiflorum [J]. Journal of Integrative Medicine,1988,4(8):35-37.
163. Dong Sai, Qing Yang. The effect on drosophila’s growth and lifespan of Polygonummultiflorum [J]. Chinese Journal of Gerontology,1989,9(S1):1-2.
164. Houchun Liu, Wansheng Chen. Polygonum multiflorum water-soluble components of2,3,5,4'-tetrahydroxy stilbene-2-O-beta-D glucoside in vitro antioxidant effects [J].Journal of Pharmaceutical Practice,2000,18(4):232-234.
165. LiShuang Lv, XiaoHong Gu, Jian Tang, Chi-Tang Ho. Antioxidant activity of stilbeneglycoside from Polygonum multiflorum Thunb in vivo [J]. Food Chemistry,2007,104(4):1678-1681.
166. P. Enes, J., Sanchez-Gurmaches, I. Navarro, J., Gutiérrez, A., Oliva-Teles. Role ofinsulin and IGF-I on the regulation of glucose metabolism in European sea bass(Dicentrarchus labrax) fed with different dietary carbohydrate levels [J]. ComparativeBiochemistry and Physiology Part A: Molecular&Integrative Physiology,2010,157(4):346-353.
167. Andrzej Bartke. Long-lived Klotho mice: new insights into the roles of IGF-1andinsulin in aging [J]. Trends in Endocrinology&Metabolism,2006,17(2):33-35.
168. Massimiliano Bonafè, Fabiola Olivieri. Genetic polymorphism in long-lived people:Cues for the presence of an insulin/IGF-pathway-dependent network affecting humanlongevity [J]. Molecular and Cellular Endocrinology,2009,299(1):118-123.
169. Guiyuan Lv, Zhaohuan Lou, Suhong Chen, Hui Gu, Letian Shan.Pharmacokinetics and tissue distribution of2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside from traditional Chinese medicine Polygonum multiflorumfollowing oral administration to rats [J]. Journal of Ethnopharmacology,2011,137(1):449-456.
170. Hiroshi Kurosu, Masaya Yamamoto, Jeremy D. Clark, Johanne V. Pastor, AnimeshNandi, Prem Gurnani, Owen P. McGuinness, Hirotaka Chikuda, MasayukiYamaguchi, Hiroshi Kawaguchi, Iichiro Shimomura, Yoshiharu Takayama, JoachimHerz, C. Ronald Kahn, Kevin P. Rosenblatt, Makoto Kuro-o. Suppression of aging inmice by the hormone Klotho [J]. Science,2005,309(5742):1829-1833.
171. Holzenberger M. The GH/IGF-1axis and longevity [J]. Eur. J. Endocrinol.(suppl.1),2004,151:23-27.
172. David J. Clancy, David Gems, Lawrence G. Harshman, Sean Oldham, Hugo Stocker,Ernst Hafen, Sally J. Leevers, Linda Partridge. Extension of life-span by loss ofCHICO, a drosophila insulin receptor substrate protein [J]. Science,2001,292(5514):104-106.
173. M. Tatar, A. Kopelman, D. Epstein, M.-P. Tu, C.-M. Yin, R. S. Garofalo. A mutantDrosophila insulin receptor homolog that extends life-span and impairsneuroendocrine function [J]. Science,2001,292(5514):107-110.
174. Li Liu, Lin Li, Ling Zhao,2,3,5,4’-tetrahydroxyl diphenylethylene-2-o-β-D-glucosideon learning and memory abilities of rats with chronic cerebral ischemia.[J] Chin JPharmacia Topical,2008,22(2):108-115.
175. Lin Zhou, Qidong Yang, Zhijian Ma, Polygonum multiflorum on learning, memoryand acetylcholinesterase of Alzheimer disease rats.[J] Journal of Apoplexy andNervous Diseases,2004,6(5):394-396.
176. A.M Matsumoto, B.T Marck, D.A Gruenewald, T Wolden-Hanson, M.A Naai, Agingand the neuroendocrine regulation of reproduction and body weight. ExperimentalGerontology,2000,12(35):1251-1265.
177. Zhou Yang, David Bishai, Jeffrey Harman, Convergence of body mass with aging:The longitudinal interrelationship of health, weight, and survival. Economics&Human Biology,2008,12(6): Pages469-481.
178. James M Harper, Andrzej T Galecki, David T Burke, Richard A Miller, Body weight,hormones and T cell subsets as predictors of life span in genetically heterogeneousmice Original Research Article, Mechanisms of Ageing and Development,2004,5(125):381-390.
179. Aziz A. Khazaeli, Wayne Van Voorhies, James W. Curtsinger, The relationshipbetween life span and adult body size is highly strain-specific in Drosophilamelanogaster Original Research Article. Experimental Gerontology,2005,5(40):377-385.
2.吕丽爽,汤坚.二苯乙烯苷和白藜芦醇抗氧化构效关系研究［J］.食品与机械,2009,25(5):57-58.
3. Wood J.G, Rogina B, Lavu S, Howitz K, Helfand S.L, Tatar M, Sinclair D. Sirtuinactivators mimic caloric restriction and delay ageing in metazoans [J]. Nature,2004,430(7000):686-698.
4. Xin Yu, Guorong Li. Effects of resveratrol on longevity, cognitive ability andaging-related histological markers in the annual fish Nothobranchius guentheri [J].Experimental Gerontology,2012,47(12):940-949.
5. Yee Ting Wong, Jan Gruber, Andrew M. Jenner, Mary Pei-Ern Ng, Runsheng Ruan,Francis Eng Hock Tay. Elevation of oxidative-damage biomarkers during aging in F2hybrid mice: Protection by chronic oral intake of resveratrol [J]. Free Radical Biologyand Medicine,2009,46(6):799-809.
6.边秀娟,包国荣.电子自旋共振法测定何首乌中大黄素、二苯乙烯苷清除超氧阴离子自由基的实验研究[D].福州,福州中医学院,2002,12-13.
7.吕丽爽,汤坚.何首乌中二苯乙烯苷的醇提工艺[J].食品与发酵工业,2004,30(10):132-135.
8.戚爱棣.何首乌中二苯乙烯苷提取工艺优选及炮制对其含量影响[J].中草药,2002,33(7):609-611.
9.王娟,沈平孃,沈永嘉.均匀设计优选微波辅助萃取何首乌中有效成分的研究[J].中草药,2003,34(4):314-317.
10.千梅,何况旻,方红美,陈从贵.何首乌中二苯乙烯苷的超声提取工艺研究[J].农产品加工学刊,2006,(10):115-122.
11.白研,葛发欢,史庆龙.超临界CO2流体萃取制首乌中的有效成分[J].中药材,2005,28(4):290-291.
12.班翊,刘其礼,金悠.二苯乙烯苷的稳定性研究[J].中草药,2004,35(11):1235-1237.
13. Yao S, Li Y, Kong L. Preparative isolation and purification of chemical constituentsfrom the root of polygonum muhiflorumby high-speed counter-currentchromatography [J]. J Chromatogr A,2006,1115(1-2):64-71.
14.张纯,杨少麟,袁海龙.高效液相色谱法测定何首乌中二苯乙烯甙的含量及其稳定性考察[J].中国中药杂志,1999,24(6):357-360.
15.刘振丽,宋志前.不同地区制首乌中二苯乙烯苷含量测定及稳定性考察[J].中成药,2002,24(9):684-685.
16.吕丽爽,汤坚,何其傥.何首乌中二苯乙烯苷的制备及抗氧化机理的研究[D].无锡,江南大学,2006,27-28,46-47,61-66.
17.孙江浩,袁志芳,王春英,许慧君,张兰桐.何首乌中二苯乙烯苷在大鼠体内的药动学[J].中草药,2005,36(3):405-409.
18.王春英,李敏,袁志芳,杨维,解凤立,张兰酮.二苯乙烯苷大鼠在胃肠吸收动力学研究[J].中草药,2008,39(8):1215-1219.
19.王春英,郭栋,袁志芳,冯小龙,张兰桐.二苯乙烯苷在大鼠体内外的代谢研究[J].中国医药工业杂志,2009,40(2):120-123.
20.楼招欢,吕圭源,陈素红,顾慧.首乌苷制剂中二苯乙烯苷含量测定及其在小鼠体内组织分布研究[J].中国现代应用药学,2011,28(2):89-91.
21.孙江浩,张兰桐,王春英,袁志芳,卢兴红.液-质联用法测定小鼠体内二苯乙烯苷代谢物[J].药学学报,2003,38(12):968-970.
22.许爱霞,张振明,葛斌.何首乌多糖对氧自由基及抗氧化酶活性的作用研究[J].中国药师,2005,8(11):900-902.
23.胡存华,赵立波,王晓敏.二苯乙烯苷增强正常血管内皮细胞抗氧化作用研究[J].医药导报,2007,26(2):138-139.
24. Howes R.M. The free radical fantasy: A panoply of paradoxes [J]. Ann N.Y Acad Sci,2006,1067(1):22-26.
25.刘丽,李林,赵玲.二苯乙烯苷对慢性脑缺血大鼠学习记忆的影响[J].中国药理学与毒理学杂志,2008,22(2):108-115.
26.周琳,杨期东,马志健.何首乌对Alzheimer病大鼠学习记忆及乙酰胆碱酯酶的作用[J].中国药师杂志,2004,21(5):394-396.
27. Lan Zhang, Ying Xing, Cui-Fei Ye, Hou-Xi Ai, Hai-Feng Wei, Lin Li.Learning-memory deficit with aging in APP transgenic mice of Alzheimer's diseaseand intervention by using tetrahydroxystilbene glucoside [J]. Behavioural BrainResearch,2006,173(2):246-254.
28. Ting Wang, Yuan-Jian Yang, Peng-Fei Wu, Wei Wang, Zhuang-Li Hu, Li-Hong Long,Na Xie, Hui Fu, Fang Wang, Jian-Guo Chen. Tetrahydroxystilbene glucoside, aplant-derived cognitive enhancer, promotes hippocampal synaptic plasticity [J].European Journal of Pharmacology,2011,650(1):206-214.
29.高瑄,胡英杰,符林春.何首乌二苯乙烯苷的调节血脂作用[J].中国中药杂志,2007,32(4):323-326.
30.韩晓,吴成爱,王伟.何首乌二苯乙烯苷降血脂作用机理研究[J].中华中医药学刊,2008,26(8):1687-1689.
31. Yang P.Y, Almofti M.R, Lu L. Reduction of atherosclerosis in cholesterol-fed rabbitsand decrease ofexpressions of intraccllular adhesion molecule-1andvascjlarendothelial growth factor in foam cells by a water-solutionfraction ofpolygonum multiflorum [J]. J Pharmacol Sci,2005,99(3):294-300.
32. WenJuan Yao, WenJun Fan, Chao Huang, Hui Zhong, XiangFan Chen, Wei Zhang.Proteomic analysis for anti-atherosclerotic effect of tetrahydroxystilbene glucoside inrats. Biomedicine&Pharmacotherapy,2012,67(2):140-145.
33. Xiaomin Wang, Libo Zhao, Tianzhao Han, Shaofei Chen, Jialing Wang. Protectiveeffects of2,3,5,4′-tetrahydroxystilbene-2-O-beta-d-glucoside, an active component ofPolygonum multiflorum Thunb, on experimental colitis in mice [J]. EuropeanJournal of Pharmacology,2008,578(2):339-348.
34. Zequn Jiang, Jimin Xu, Minghai Long, Zhiming Tu, Guangxiao Yang, Guangyuan He.2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (THSG) induces melanogenesis inB16cells by MAP kinase activation and tyrosinase upregulation [J]. Life Sciences,2009,85(9):345-350.
35. Xin Han, Shuang Ling, Woting Gan, Li Sun, Ju Duan, Jin-Wen Xu.2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside ameliorates vascular senescence andimproves blood flow involving a mechanism of p53deacetylation [J]. Atherosclerosis,2012,225(1):76-82.
36. Cairong Li, Fei Cai, Yinqiao Yang, Xinyuan Zhao, Chun Wang, Juan Li, Yanlong Jia,Jing Tang, Qing liu. Tetrahydroxystilbene glucoside ameliorates diabetic nephropathyin rats: Involvement of SIRT1and TGF-β1pathway [J]. European Journal ofPharmacology,2010,649(1):382-389.
37. Rong Qin, Xiaobing Li, Gang Li, Lizhen Tao, Yan Li, Jing Sun, Xiaogang Kang,Jianzong Chen. Protection by tetrahydroxystilbene glucoside against neurotoxicityinduced by MPP+: The involvement of PI3K/Akt pathway activation [J]. ToxicologyLetters,2011,202(1):1-7.
38. Klar A.J.S, Fogel S, MacLeod K. MAR1-A regulator of the HMa and HMα loci inSaccharomyces cerevisiae [J]. Genetics,1979,93(1):37-50.
39. Guarente L. Diverse and dynamic functions of the Sir silencing complex [J]. NatGenet,1999,23(3):281-285.
40. Rine J, Herskowitz I. Four genes responsible for a position effect on expression fromHML and HMR in Saccharomyces cerevisiae [J]. Genetics,1987,116(1):9-22.
41. Gottschling D.E, Aparicio O.M, Billington B.L, Zakian V.A. Position effect at S.cerevisiae telomeres: Reversible repression of Pol II transcription [J]. Cell,1990,63(4):751-762.
42. Landry J, Sutton A, Tafrov S.T, Heller R.C, Stebbins J, Pillus L, Sternglanz R. Thesilencing protein SIR2and its homologs are NAD-dependent protein deacetylases [J].Proc Natl Acad Sci,2000b,97(11):5807-5811.
43. Smith J.S, Brachmann C.B, Celic I, Kenna M.A, Muhammad S, Starai V.J, Avalos J.L,Escalante-Semerena J.C, Grubmeyer C, Wolberger C, Boeke J.D. A phylogeneticallyconserved NAD+-dependent protein deacetylase activity in the Sir2protein family [J].Proc Natl Acad Sci,2000,97(12):6658-6663.
44. Imai S, Armstrong C.M, Kaeberlein M, Guarente L. Transcriptional silencing andlongevity protein Sir2is an NAD-dependent histone deacetylase [J]. Nature,2000a,403(6771):795-800.
45. Tissenbaum H.A, Guarente L. Increased dosage of a sir-2gene extends lifespan inCaenorhabditis elegans [J]. Nature,2001,410(6825):227-230.
46. Rogina B, Helfand S.L. Sir2mediates longevity in the fly through a pathway relatedto calorie restriction [J]. Proc Natl Acad Sci,2004,101(45):15988-16003.
47. Astrom S.U, Cline T.W, Rine J. The Drosophila melanogaster sir2+gene isnonessential and has only minor effects on position-effect variegation [J]. Genetics,2003,163（3）:931-937.
48. Jang M, Cai L, Udeani G.O, Slowing K.V, Thomas C.F, Beecher C.W, Fong H.H,Farnsworth N.R, Kinghorn A.D, Mehta R.G. Cancer chemopreventive activity ofresveratrol, a natural product derved from grapes [J]. Science,1999,275(5297):218-220.
49. Middleton E, Jr, Kandaswami C, Theoharides T.C. The effects of plant flavonoids onmammalian cells: Implications for inflammation, heart disease, and cancer [J].Pharmacol Rev,2000,52(4):673-751.
50. Baur J.A, Sinclair D.A. Therapeutic potential of resveratrol: The in vivo evidence [J].Nat Rev Drug Discov,2006,5(6):493-506.
51. Huige Li, Ning Xia, Ulrich F rstermann. Cardiovascular effects and molecular targetsof resveratrol [J]. Nitric Oxide,2012,26(2):102-110.
52. Sylwia Borska, Magdalena Chmielewska, Teresa Wysocka, MalgorzataDrag-Zalesinska, Maciej Zabel, Piotr Dziegiel. In vitro effect of quercetin on humangastric carcinoma: Targeting cancer cells death and MDR [J]. Food and ChemicalToxicology,2012,50(9):3375-3383.
53. Pravinkumar Bhutada, Yogita Mundhada, Kuldeep Bansod, Chetan Bhutada, SantoshTawari, Pankaj Dixit, Dharmendra Mundhada. Ameliorative effect of quercetin onmemory dysfunction in streptozotocin-induced diabetic rats [J]. Neurobiology ofLearning and Memory,2010,94(3):293-302.
54. Brack C, Bechter-Thuring E, Labuhn. N-acetylcysteine slows down ageing andincreases the life span of Drosophila melanogaster [J]. Cell Mol Life Sci,1997,53(11-12):960-966.
55. Kang H.L, Benzer S, Min K.T. Life extension in Drosophila by feeding a drug [J].Proc Natl Acad Sci,2002,99(2):838-843.
56. Melov S, Wolf N, Strozyk D, Doctrow S.R, Bush A.I. Mice transgenic for Alzheimerdisease beta-amyloid develop lens cataracts that are rescued by antioxidant treatment[J]. Free Radic Biol Med,2005,38(2):258-261.
57. Melov S, Doctrow S.R, Schneider J.A, Haberson J, Patel M, Coskun P.E, Huffman K,Wallace D.C, Malfroy B. Lifespan extension and rescue of spongiformencephalopathy in superoxide dismutase2nullizygous mice treated with superoxidedismutase-catalase mimetics [J]. Neurosci,2001,21(21):8348-8353.
58. Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y,Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-iida T, Nishikawa S,Nagai R, Nabeshima YI. Mutation of the mouse klotho gene leads to a syndromeresembling ageing [J]. Nature,1997,390(6655):45-51.
59. Shiraki-Iida T, Iida A, Nabeshima Y, Anazawa H, Nishikawa S, Noda M, Kuro-o M,Nabeshima Y. Improvement of multiple pathophysiological phenotypes of klotho(kl/kl) mice by adenovirus-mediated expression of the klotho gene [J]. Gene Med,2000,2(4):233-242.
60. Saito Y, Yamagishi T, Nakamura T, Ohyama Y, Aizawa H, Suga T, Matsumura Y,Masuda H, Kurabayashi M, Kuro-o M, Nabeshima Y, Nagai R. Klotho proteinprotects against endothelial dysfunction [J]. Biochem Biophys Res Commun,1998,248(2):324-329.
61. Suga T, Kurabayashi M, Sando Y, Ohyama Y, Maeno T, Maeno Y, Aizawa H,Matsumura Y, Kuwaka T, Kuro-o M, Nabeshima Y, Nagai R. Disruption of the kothogene causes pulmonary emphysema in mice. Defect in maintenance of pulmonaryintegrity during postnatal life [J]. Am J Respir Cell Mol Biol,2000,22(1):26-33.
62. Okada S, Yoshida T, Hong Z, Ishii G, Hatano M, Kuro-o M, Nabeshima Y, NabeshimaY, Tokuhisa T. Impairment of B lymphopoiesis in precocious aging (klotho) mice [J].Int Immunol,2000,12(6):861-871.
63. Utsugi T, Ohno T, Ohyama Y, Uchiyama T, Saito Y, Matsumura Y, Aizawa H, Itoh H,Kurabayashi M, Kawazu S, Tomono S, Oka Y, Suga T, Kuro-o M, Nabeshima Y,Nagai R. Decreased insulin production and increased insulin sensitivity in the klothomutant mouse, a novel animal model for human aging [J]. Metabolism,2000,49(9):1118-1123.
64. Mori K, Yahata K, Mukoyama M, Suganami T, Makino H, Nagae T, Masuzaki H,Ogawa Y, Sugawara A, Nabeshima YM Nakao K. Disruption of klotho gene causesan abnormal energy homeostasis in mice [J]. Biochem Biophys Res Commun,2000,278(3):665-670.
65. Uchida A, Komiya Y, Tashiro T, Yorifuji H, Kishimoto T, Nabeshima Y, Hisanaga S.Neurofilaments of Klotho, the mutant mouse prematurely displaying symptomsresembling guman aging [J]. J Neurosce Res,2001,64(4):364-370.
66. Ohyama Y, Kurabayashi M, Masuda H, Nakamura T, Aihara Y, Kaname T, Suga T,Arai M, Aizawa H, Matsumura Y, Kuro-o M, Nabeshima Y, Nagail R. Molecularcloning of rat klotho cDNA: markedly decreased expression of klotho by acuteinflammatory stress [J]. Biochem Biophys Res Commun,1998,251(3):920-925.
67. Matsumura Y, Aizawa H, Shiraki-Iida T, Nagai R, Kuro-o M, Nabeshima Y.Identification of the human klotho gene and its two transcripts encoding membraneand secreted klotho protein [J]. Biochem Biophys Res Commun,1998,242(3):626-630.
68. Shiraki-Iida T, Azawa H, Matsumura Y, Sekine S, Iida A, A nazawa H, Nagai R,Kuro-o M, Nabeshima Y. Structure of the mouse klotho gene and its two transcriptsencoding membrane and secreted protein [J]. FEBS Lett,1998,424(1):6-10.
69. Imura A, Iwano A, Tohyama O, Tsuji Y, Nozaki K, Hashimoto N, Fujimori T,Nabeshima Y. Secreted Klotho protein in sera and CSF: implication forposttranslational cleavage in release of Klotho protein from cell membrane [J]. FEBSLett,2004,565(1):143-147.
70. Chang Q, Hoefs S, Van der Kemp AW, Topala CN, Bindels RJ, Hoenderop JG. Thebeta-glucuronidase klotho hydrolyzes and activates the TRPV5channel [J]. Science,2005,310(5747):490-493.
71. Wolf I, Levanon-Cohen S, Bose S, Ligumsky HM Sredni B, Kanety H, Kuro-o M,Karlan B, Kaufman B, Koeffler HP, Rubinek T. Klotho: a tumor suppressor and amodulator of the IGF-1and FGF pathways in human breast cancer [J]. Oncogene,2008,27(56):7094-7105.
72. Tatar M, Bartke A, Antebi A. The endocrine regulation of aging by insulin-likesignals [J]. Science,2003,299(5611):1346-1351.
73. Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R. AC. Elegans mutant that livestwice as long as wild type [J]. Nature,1993,366(6454):461-464.
74. Morris JZ, Tissenbaum HA, Ruvkun G. A phosphatidylinositol-3-OH kinase familymember regulating longevity and diapause in Caenorhabditis elegans [J]. Nature,1996,382(6591):536-539.
75. Clancy DJ, Gems D, Harshman LG, Oldham S, Stocker H, Hafen E, Leevers SJ,Partridge L. Extension of life-span by loss of CHICO, a Drosophila insulin receptorsubstrate protein [J]. Science,2001,292(5514):104-106.
76. Bartke A, Brown-Borg H, Mattison J, Kinney B, Hauck S, Wright C. Prolongedlongevity of hypopituitary dwarf mice [J]. Exp Gerontol,2001,36(1):21-28.
77. Taguchi A, Wartschow LM, White MF. Brain IRS2signaling coordinates life spanand mutrient homeostasis [J]. Science,2007,317(5836):369-372.
78. Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum HA, Ruvkun G. TheFork head transcription factor DAF-16transduces insulin-like metabolic andlongevity signals in C. elegans [J]. Nature,1997,389(6654):994-999.
79. Lin K, Hsin H, Libina N, Kenyon C. Regulation of the Caenorhabditis eleganslongevity protein DAF-16by insulin/IGF-1and germline signaling [J]. Nat Genet,2001,28(2):139-145.
80. Henderson ST, Johnson TE. Daf-16integrates developmental and environmentalinputs to mediate aging in the nematode Caenorhabditis elegans [J]. Curr Biol,2001,11(24):1975-1980.
81. Giannakou ME, Goss M, Junger MA, Hafen E, Leevers SJ, Patridge L. Long-livedDrosophila with overexpressed dFOXO in adult fat body [J]. Science,2004,305(5682):361.
82. Hwangbo DS, Gershman B, Tu MP, Palmer M, Tatar M. Drosophila dFOXO controlslifespan and regulates insulin signaling in brain and fat body [J]. Nature,2004,429(6991):562-566.
83. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC,Blenis JM Greenberg ME. Akt promotes cell survival by phosphorylating andinhibiting a forhead transcription factor [J]. Cell,1999,96(6):857-868.
84. Kops GJ, Dansen TB, Polderman PEM Saarloos I, Wirtz KW, Coffer PJ, Huang TT,Bos JL, Medema RH, Burgering BM. Forkhead transcription factor FOXO3a protectsquiescent cells from oxidative stress [J]. Nature,2002,419(6904):316-321.
85. Yamamoto M. Clark JD, Pastor JV, Gurnani P, Nandi A, Kurosu HM Miyoshi M,Ogawa Y, Castrillon DH, Rosenblatt KP, Kuro-o M. Regulation of oxidative stress bythe anti-aging hormone klotho [J]. J Biol Chem,2005,280(45):38029-38034.
86. Chateau MT, Araiz C, Descamps S, Galas S. Klotho interferes with a novelFGF-signalling pathway and insulin/Igf-like signaling to improve longevity and stressresistance in Caenrhabditis elegans [J]. Aging (Albany NY),2010,2(9):567-581.
87. Lee J, Jeong DJ, Kim J, Lee S, Park JH, Chang B, Jung SI, Yi L, Han YM Yang Y,Kim KI, Lim JS, Yang I, Jeon S, Bae DH, Kim CJ, Lee MS. The anti-aging geneKLOTHO is a novel target for epigenetic silencing in human cervical carcinoma [J].Mol Cancer,2010,9(1):109.
88. Wolf I, Laitman Y, Rubinek T, Abramovitz L, Novikov I, Beeri R, Kuro-o M,Koeffler HP, Catane R, Freedman LS, Levy-Lahad E, Karlan BY, Friedman E,Kaufman B. Functional variant of KLOTHO: a breast cancer risk modifier amongBRCA1mutation carriers of Ashkenazi origin [J]. Oncogene,2010,29(1):26-33.
89. Bartucci M, Morelli C, Mauro L, Ando S, Surmacz E. Differential insulin-like growthfactor I receptor signaling and function in estrogen receptor (ER)-positive MCF-7andER-negative MDA-MB-231breast cancer cells [J]. Cancer Res,2001,61(18):6747-6754.
90. Chen B, Wang X, Zhao W, Wu J. Klotho inhibits growth and promotes apoptosis inhuman lung cancer cell line A549[J]. J Exp Clin Cancer Res,2010,29(1):99.
91. Lu L, Katsaros D, Wiley A, De la Longrais IA, Puopolo M, Yu H. Klotho expressionin epithelial ovarian cancer and its association with insulin-like growth factors anddisease progression [J]. Cancer Invest,2008,26(2):185:192.
92. Michio Imai, Kazuhiko Ishikawa, Naomichi Matsukawa, Iwao Kida, Junsuke Ohta,Masashi Ikushima, Yukana Chihara, Xu Rui, Hiromi Rakugi MD, PhD, ToshioOgihara. Klotho protein activates the PKC pathway in the kidney and testis andsuppresses25-hydroxyvitamin D3lalpha-hydroxylase gene expression [J]. Endocrine,2004,25(3):229-234.
93. Hiroshi Kurosu, Yasushi Ogawa, Masayoshi Miyoshi, Masaya Yamamoto, AnimeshNandi, Kevin P. Rosenblatt, Michel G. Baum, Susan Schiavi, Ming-Chang Hu, OrsonW. Moe, and Makoto Kuro-o. Regulation of fibroblast growth factor-23signaling byklotho [J]. J Biol Chem,2006,281(10):6120-6123.
94. Negri AL. The klotho gene: a gene predominantly expressed in the kidney is afundamental regulator of aging and calcium/phosphorus metabolism [J]. J Nephrol,2005,18(6):654-658.
95. Weindruch R, Walford R. The retardation of aging and disease by dietary restriction[M]. C.C. Thomas, Springfield, Illinois,1988.
96. Mosoro E.J. Overview of caloric restriction and aging [J]. Mech Aging Dev,2005,126(9):913-922.
97. Min K.J, Tatar M. Restriction of amino acids extends lifespan in Drosophilamelanogaster [J]. Mech Aging Dev,2006,127(7):643-646.
98. Kealy R.D, Lawler D.F, Ballam J.M, Mantz S.L, Biery D.N, Greeley E.H, Segre M,Smit G.K, Stowe H.D. Effects of diet restriction on lifespan and agerelated changes indogs [J]. Am Vet Med Assoc,2002,220(9):1315-1320.
99. Bordone L, Guarente L. Calorie restriction, SIRT1and metabolism: Understandinglongevity. Nat Rev Mol Cell Biol,2005,6(4):298-305.
100. Nisoli E, Tonello C, Cardile A, Cozzi V, Bracale R, Tedesco L, Falcone S, Valerio A,Cantoni O, Clementi E. Calorie restriction promotes mitochondrial biogenesis byinducing the expression of eNOS [J]. Science,310(5746):314-317.
101. Lavrovsky Y, Chatterjee B, Clark R.A, Roy A.K. Role of redox-regulatedtranscription factors in inflammation, aging and age-related diseases [J]. ExpGerontol,2000,35(5):521-532.
102. Merry B.J. Oxidative stress and mitochondrial function with aging—The effects ofcalorie restriction [J]. Aging Cell,2004,3(1):7-12.
103. Reiling J.H, Sabatini D.M. Stress and mTORture signaling [J]. Oncogene,2006,25(48):6373-6383.
104. Kim J.H, Chu S.C, Gramlich J.L, Pride Y.B, Babendreier E, Chauhan D, Salgia R,Podar K, Griffin J.D, Sattler M. Activation of the PI13K/mTOR pathway byBVRABL contributes to increased production of reactive oxygen species [J]. Blood,2005,105(4):1717-1723.
105. Kopchick J.J, Andry J.M. Growth hormone (GH), GH receptor, and signaltransduction [J]. Mol Genet Metab,2000,71(1-2):293-314.
106. Cohen P. Overview of the IGF-I system [J]. Hormone Research in Paediatrics,2006,65(Suppl.1):3-8.
107. Giustina A, Velduis J.D. Pathophysiology of the neuroregulation of growth hormonesecretion in experimental animals and the human [J]. Endocr Rev,1998,19(6):717-797.
108. Silvia G, Emanuele M, Stephen E. Borst, Christiaan Leeuwenburgh. Modulation ofGH/IGF-1axis: Potential strategies to counteract sarcopenia in older adults OriginalResearch Article [J]. Mechanisms of Ageing and Development,2008,129(10):593-601.
109. Oguszewski C.L, Meister L.H, Zaninelli D.C, Radominski R.B. One year of GHreplacement therapy with a fixed low-dose regimen improves body composition, bonemineral density and lipid profile of GH-deficient adults [J]. Eur J Endocrinol,2005,152(1):67-75.
110. Blackman M.R, Sorkin J.D, Munzer T, Bellantoni M.F, Busby-Whitehead J, StevensT.E, Jayme J, O’Connor K.G, Christmas C, Tobin J.D. Growth hormone and sexsteroid administration in healthy aged women and men: A randomized controlled trial[J]. J Am Med Assoc,2002,288(18):2282-2292.
111. Guarente L, Kenyon C. Genetic pathways that regulate aging in model organisms [J].Nature,2000,408(6809):255-262.
112. Greer E.L, Brunet A. FOXO transcription factors at the interface between longevityand tumor suppression [J]. Oncogene,2005,24(50):7410-7425.
113. Masoro E.J, Iwasaki K, Gleiser C.A, Mcmahan C.A, Seo E.J, Yu B.P. Dietarymodulation of the progression of nephropathy in aging rats: An evaluation of theimportance of protein [J]. Am J Clin Nutr,1989,49(6):1217-1227.
114. Masoro E.J, McCaRTER r.j, Katz M.S, McMahan C.A. Dietary restriction alterscharacteristics of glucose fuel use [J]. J Gerontol,1992,47(6): B202-B208.
115. Kenyon C. A conserved regulatory system for aging [J]. Cell,2001,105(2):165-168.
116. Paul C. Henning, Dennis E. Scofield, Kevin R. Rarick, Joseph R. Pierce, Jeffery S.Staab, Harris R. Lieberman, Bradley C. Nindl. Effects of acute caloric restrictioncompared to caloric balance on the temporal response of the IGF-I system [J].Metabolism,2013,62(2):179-187.
117. Vladimir N. Anisimov, Andrzej Bartk. The key role of growth hormone–insulin–IGF-1signaling in aging and cancer [J]. Critical Reviews in Oncology/Hematology,2013, In Press.
118. Achille Pich, Elena Margaria, Luigi Chiusa, Renata Ponti, Massimo Geuna. DNAploidy and p53expression correlate with survival and cell proliferative activity inmale breast carcinoma [J]. Human Pathology,1996,27(7):676-682.
119. Gerald P. Holmquist, Shuwei Gao. Somatic mutation theory, DNA repair rates, andthe molecular epidemiology of p53mutations [J]. Mutation Research/Reviews inMutation Research,1997,386(1):69-101.
120. Jirouta Kitagaki, Yili Yang. DNA intercalator korkormicin A preferentially kills tumorcells expressing wild type p53[J]. Biochemical and Biophysical ResearchCommunications,2011,414(1):186-191.
121. Levine A.J. p53, the cellular gatekeeper for growth and division [J]. Cell,1997,88:323-331.
122. Levine A.J, Hu W, Feng Z. The p53pathway: what questions remain to be explored?[J]. Cell Death Differ,2006a,13:1027-1036.
123. Vosden K.H, Lu X. Live or let die: The cell’s response to p53[J]. Nat Rev Cancer,2002,2:594-604.
124. Levine A.J, Feng Z, Mak T.W, You H, Jin S. Coordination and communicationbetween the p53and IGF-1-AKT-TOR signal transduction pathways [J]. Genes Dev,2006b,20:267-275.
125. Feng Z, Zhang H, Levine A.J, Jin S. The coordinate regulation of the p53and mTORpathways in cells [J]. Proc Natl Acad Sci,2005,102(23):8204-8209.
126. Jones R.G, Plas D.R, Kubek S, Buzzai M, Mu J, Xu Y, Birnbaum M.J, Thompson C.B.AMP-activated protein kinase induces a p53-dependent metabolic checkpoint [J]. MolCell,2005,18(3):283-293.
127. Schmitt C.A, Fridman J.S, Yang M, Lee S, Baranov E, Hoffman R.M, Lowe S.W. Asenescence program controlled by p53and p16INK4a contributes to the outcome ofcancer therapy [J]. Cell,2002,109(3):335-346.
128. Maier B, Gluba W, Bernier B, Turner T, Mohammad K, Guise T, Sutherland A,Thorner M, Scrable H. Modulation of mammalian life span by the short isoform ofp53[J]. Genes Dev,2004,18:306-319.
129. Barton A. Some aspects of cell division in Saccharomyces cerevisiae [J]. J GenMicrobiol,1950,4(1):84-86.
130. Longo V.D, Fabrizio P. Regulation of longevity and stress resistance: A molecularstrategy conserved from yeast to humans?[J]. Cell Mol Life Sci,2002,59(6):903-908.
131. Muller I. Parental age and the life-span of zygotes of Saccharomyces cerevisiae [J].Antonie van Leeuwenhoek,1985,51:1-10.
132. Brenner S. The genetics of Caenorhabditis elegans [J]. Genetics,1974,77(1):71-94.
133. Ellis R.E, Yuan J.Y, Horvitz H.R. Mechanisms and functions of cell death [J]. AnnuRev Cell Biol,1991,7:663-698.
134. Coulson A, Sulston J, Brenner S, Karn J. Toward a physical map of the genome of thenematode Caenorhabditis elegans [J]. Proc Natl Acad Sci,1986,83(20):7821-7825.
135. C.elegans Sequencing Consortium. Genome sequence of the nematode C.elegans: Aplatform for investigating biology [J]. Science,1998,282(34):2012-2018.
136. Friedman D.B, and Johnson T.E. A mutation in the age-1gene in Caenorhabditiselegans lengthens life and reduces hermaphrodite fertility [J]. Genetics,1988a,118(38):75-86.
137. Friedman D.B, and Johnson T.E. Three mutants that extend both mean and maximumlife span of the nematode, Caenorhabditis elegans, define the age-1gene [J]. Gerontol,1988b,43(4): B102-B109.
138. Morris J.Z, Tissenbaum H.A, Ruvkun G. A phosphatidylinositol-3-OH kinase familymember regulating longevity and diapause in Caenorhabditis elegans [J]. Nature,1996,382:536-539.
139. Kohler R.E. Lords of the fly: Drosophila genetics and the experimental life [M].University of Chicaago Press, Chicago, Illinois,1994.
140. Collins B, Mazzoni E.O, Stanewsky R, Blau J. Drosophila CRYPTOCHROME is acircadian transcriptional repressor [J]. Curr Biol,2006,16(5):441-449.
141. Gotter A.L. A Timeless debate: Resolving TIM’s noncircadian roles with possibleclock function [J]. Neuroreport,2006,17(12):1229-1233.
142. Shaw p.j, Tononi G, Greenspan R.J, Robinson D.F. Stress response genes protectagainst lethal effects of sleep deprivation in Drosophila [J]. Nature,2002,417:287-291.
143. Koh K, Evans J.M, Hendricks J.C, Sehgal A. A Drosophila model for age-associatedchanges in sleep: wake cycles [J]. Proc Natl Acad Sci,2006,103(37):13843-13847.
144. Walker D.W, Benzer S. Mitochondrial “swirls” induced by oxygen stress and in theDrosophila mutant hyperswirl [J]. Proc Natl Acad Sci,2004,101(28):10290-10295.
145. Kujoth G.C, Hiona A, Pugh T.D, Someya S, Panzer K, Wohlgemuth S.E, Hofer T, SeoA.Y, Sullivan R, Jobling W.A, Morrow J.D, Van Remmen H, Sedivy J.M, Yamasoba T,Tanokura M, Weindruch R, Leeuwenburgh C, Prolla T.A. Mitochondrial DNAmutations, oxidative stress, and apoptosis in mammalian aging [J]. Science,2005,309(5733):481-484.
146. Landis G.N, Abdueva D, Skvortsov D, Yang J, Rabin B.E, Carrick J, Tavare S, TowerJ. Similar gene expression patterns characterize aging and oxidative stress inDrosophila melanogaster [J]. Proc Natl Acad Sci,2004,101(20):7663-7668.
147. Crother D.C, Kingborn K.J, Page R, Lomas D.A. Therapeutic targets from aDrosophila model of Alzheimer’s disease [J]. Curr Opin Pharmacol,2004,4(5):513-516.
148. Chee F, Mudher A, Newman T.A, Cuttle M, Lovestone S, Shepherd D.Overexpression of tau results in defective synaptic transmission in Drosophilaneuromuscular junctions [J]. Biochem Soc Trans,2006,34:88-90.
149. Feany M, Bender W. A Drosophila model of Parkinson’s disease [J]. Nature,2000,404:394-398.
150. Whitworth A.J, Wes P.D, Pallanck L.J. Drosophila models pioneer a new approach todrug discovery for Parkinson’s disease [J]. Drug Discov Today,2006,11(3-4):119-126.
151. Marsh J.L, Pallos J, Thompson L.M. Fly models of Huntington’s disease [J]. HumMol Genet (spec),2003,12(2): R187-193.
152. Long T.A, Montgomerie R, Chippindale A.K. Quantifying the gender load: Canpopulation crosses reveal interlocus sexual conflict?[J]. Philos Trans R Soc Lond BBiol Sci,2006,361(1466):363-374.
153. Osiewacz H.D, Scheckhuber C.Q. Impact of ROS on ageing of two fungal modelsystems: Saccharomyces cerevisiae and Podospora anserine [J]. Free Radic Res,2006,40(8):1350-1358.
154. Wessells R.J, Fitzgerald E, Cypser J.R, Tatar M, Bodmer R. Insulin regulation ofgeart function in aging fruit flies [J]. Nat Genet,2004,36:1275-1281.
155. Zhou Y, Xu B.C, Maheshwari H.G, He L, Reed M, Lozykowski M, Okada S, CataldoL. Coschigamo K, Wagner T.E. A mammalian model for Laron syndrome producedby targeted disruption of the mouse growth hormone receptor/binding protein gene(the Laron mouse)[J]. Proc Natl Acad Sci,1997,94(24):13215-13220.
156. Kurosu H, Yamamoto M, Clark J.D, Pastor J.V, Nandi A, Gurnani P, McGuinness O.P,Chikuda H, Yamamoto M, Kawaguchi H. Suppression of aging in mice by thehormone Klotho [J]. Science,2005,309(5742):1829-1833.
157. Minako K, Noboru M, Kohji K. Abnormal accumulation of luteal bodies in ovaries ofthe senescence accelerated mouse (SAM)[J]. The Journal of Reproduction andDevelopment,2001,47(3):153-164．
158.周惠嘉．制备衰老动物模型的一种可控臭氧装置[J]．中国药理学通报,1996,12(3):285-286．
159. Gong G, Xu F. Study of aging model in mice [J]. Journal of China PharmaceuticalUniversity,1991,21(2):101-103.
160. Peng C, Chan H.Y, Huang Y, Yu H, Chen Z.Y. Apple polyphenols extend the meanlifespan of Drosophila melanogaster [J]. Agric Food Chem,2011,59:2097-2106.
161. Shizhen Li. Polygonum multiflorum [M]. Compendium of Materia Medica,1590,Nanjing, China: P910.
162. Wei Wang, Jinye Wang, Tiren Shi. The effect on lifespan and lipid metabolism ofaging quail of Polygonum multiflorum [J]. Journal of Integrative Medicine,1988,4(8):35-37.
163. Dong Sai, Qing Yang. The effect on drosophila’s growth and lifespan of Polygonummultiflorum [J]. Chinese Journal of Gerontology,1989,9(S1):1-2.
164. Houchun Liu, Wansheng Chen. Polygonum multiflorum water-soluble components of2,3,5,4'-tetrahydroxy stilbene-2-O-beta-D glucoside in vitro antioxidant effects [J].Journal of Pharmaceutical Practice,2000,18(4):232-234.
165. LiShuang Lv, XiaoHong Gu, Jian Tang, Chi-Tang Ho. Antioxidant activity of stilbeneglycoside from Polygonum multiflorum Thunb in vivo [J]. Food Chemistry,2007,104(4):1678-1681.
166. P. Enes, J., Sanchez-Gurmaches, I. Navarro, J., Gutiérrez, A., Oliva-Teles. Role ofinsulin and IGF-I on the regulation of glucose metabolism in European sea bass(Dicentrarchus labrax) fed with different dietary carbohydrate levels [J]. ComparativeBiochemistry and Physiology Part A: Molecular&Integrative Physiology,2010,157(4):346-353.
167. Andrzej Bartke. Long-lived Klotho mice: new insights into the roles of IGF-1andinsulin in aging [J]. Trends in Endocrinology&Metabolism,2006,17(2):33-35.
168. Massimiliano Bonafè, Fabiola Olivieri. Genetic polymorphism in long-lived people:Cues for the presence of an insulin/IGF-pathway-dependent network affecting humanlongevity [J]. Molecular and Cellular Endocrinology,2009,299(1):118-123.
169. Guiyuan Lv, Zhaohuan Lou, Suhong Chen, Hui Gu, Letian Shan.Pharmacokinetics and tissue distribution of2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside from traditional Chinese medicine Polygonum multiflorumfollowing oral administration to rats [J]. Journal of Ethnopharmacology,2011,137(1):449-456.
170. Hiroshi Kurosu, Masaya Yamamoto, Jeremy D. Clark, Johanne V. Pastor, AnimeshNandi, Prem Gurnani, Owen P. McGuinness, Hirotaka Chikuda, MasayukiYamaguchi, Hiroshi Kawaguchi, Iichiro Shimomura, Yoshiharu Takayama, JoachimHerz, C. Ronald Kahn, Kevin P. Rosenblatt, Makoto Kuro-o. Suppression of aging inmice by the hormone Klotho [J]. Science,2005,309(5742):1829-1833.
171. Holzenberger M. The GH/IGF-1axis and longevity [J]. Eur. J. Endocrinol.(suppl.1),2004,151:23-27.
172. David J. Clancy, David Gems, Lawrence G. Harshman, Sean Oldham, Hugo Stocker,Ernst Hafen, Sally J. Leevers, Linda Partridge. Extension of life-span by loss ofCHICO, a drosophila insulin receptor substrate protein [J]. Science,2001,292(5514):104-106.
173. M. Tatar, A. Kopelman, D. Epstein, M.-P. Tu, C.-M. Yin, R. S. Garofalo. A mutantDrosophila insulin receptor homolog that extends life-span and impairsneuroendocrine function [J]. Science,2001,292(5514):107-110.
174. Li Liu, Lin Li, Ling Zhao,2,3,5,4’-tetrahydroxyl diphenylethylene-2-o-β-D-glucosideon learning and memory abilities of rats with chronic cerebral ischemia.[J] Chin JPharmacia Topical,2008,22(2):108-115.
175. Lin Zhou, Qidong Yang, Zhijian Ma, Polygonum multiflorum on learning, memoryand acetylcholinesterase of Alzheimer disease rats.[J] Journal of Apoplexy andNervous Diseases,2004,6(5):394-396.
176. A.M Matsumoto, B.T Marck, D.A Gruenewald, T Wolden-Hanson, M.A Naai, Agingand the neuroendocrine regulation of reproduction and body weight. ExperimentalGerontology,2000,12(35):1251-1265.
177. Zhou Yang, David Bishai, Jeffrey Harman, Convergence of body mass with aging:The longitudinal interrelationship of health, weight, and survival. Economics&Human Biology,2008,12(6): Pages469-481.
178. James M Harper, Andrzej T Galecki, David T Burke, Richard A Miller, Body weight,hormones and T cell subsets as predictors of life span in genetically heterogeneousmice Original Research Article, Mechanisms of Ageing and Development,2004,5(125):381-390.
179. Aziz A. Khazaeli, Wayne Van Voorhies, James W. Curtsinger, The relationshipbetween life span and adult body size is highly strain-specific in Drosophilamelanogaster Original Research Article. Experimental Gerontology,2005,5(40):377-385.