Study Of High Purety Tetrahydroxystilbene Glucoside’s Anti-aging Effect And The Possible Mechanism

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.