Study On Isolation, Purification, Anti-aging Activity Of Huai Yam Polysaccharide And Its Regulation On Klotho Gene Expression

Huai Yam is one of the famous four Traditional Chinese Medicines in Henan province.Polysaccharide is the mainly active ingredient with a variety of pharmacological effects in Huai yam.Then Huai yam polysaccharide has become a hot spot in the research of chemical constituents and functional components.However,at present,most of the anti-aging researches of Huai yam polysaccharide were focused on the detection of biological activity,but the molecular mechanism of its anti-aging action,especially the researches on the regulation of the anti-aging gene have not paid enough attention.In this study,Huai yam polysaccharide was used as this research object.The polysaccharide was separated by water extraction and was purified by alcohol precipitation.The anti-aging activity of Huai yam polysaccharide and its regulation on anti-aging gene Klotho were systematically studied through anti-aging experiments in vitro and vivo.The anti-aging and longevity effect of Huai yam polysaccharide provide a scientific basis for the further research.This research is mainly divided into four parts:1.Extraction and separation of polysaccharide from Huai yam:The polysaccharide from Huai yam was extracted by water extraction and alcohol precipitation.After deproteinization by Sevage method,SDS-PAGE,Molish reaction and infrared spectroscopy were used to identify the extract.The content of polysaccharide determined by phenol-sulfuric acid method is 91.77%.2.Anti-aging activity of Huai Yam polysaccharide in vitro:CCK-8 method and?-galactosidase staining method were used to detect the effect of Huai yam polysaccharide on NRK-52E cells and TCMK-1 cells.The survival rate of normal cells has shown that Huai yam polysaccharide has no obvious inhibitory and side effects.Meanwhile,it has obvious protective effect on cellular oxidative stress damage caused by H₂O₂,indicating that Huai yam polysaccharide has a significant anti-aging activity.3.Anti-aging activity of Huai Yam polysaccharide in vivo:Aging model of mice were induced by D-galactose,then different doses of Huai yam polysaccharide were administrated orally.Anti-aging activity of Huai yam polysaccharide was studied in vivo.The results showed that Huai yam polysaccharide can obviously improve the abilities of learning and the spatial recognition of mice.And there was a significant dose-effect relationship with the anti-aging effect.At the same time,histopathological sections of hippocampus showed that Huai yam polysaccharide can significantly improve the degradation and deformation of the neuronal cell in aging mice,and repair the damaged neuronal cells in mice hippocampal tissue.In addition,Huai Yam polysaccharide can significantly improve the pathological state of liver and kidney tissues of aging mice,and repair the reserve capacity of liver and kidney.Thereby it can also improve the activities of glutathione peroxidase?GSH-Px?,superoxide dismutase?SOD?,catalase?CAT?and malondialdehyde?MDA?in aging mice.4.Study on the regulation of Klotho gene expression by Huai yam polysaccharide:The RNA was extracted from mice brain and kidney tissues,then the mRNA was reversed transcription into cDNA.The expression level of anti-aging gene Klotho in mice brain and kidney tissues was detected by traditional PCR and real-time PCR.And the results showed that the Huai yam polysaccharide could significantly increase the expression level of Klotho gene in mice brain and kidney tissues.The above results showed that Huai yam polysaccharide has the obvious anti-aging activity.It is a new type of drug and functional food for further study.Its anti-aging mechanism may be to eliminate the harmful free radicals,effectively improve the physiological state of the organs,repair the functions of various tissues of the body.The anti-aging effect can be achieved by regulating the expression level of the anti-aging gene Klotho in vivo.And the anti-aging effect can provide experimental basis and scientific basis for the"Kidney Deficiency Theory"and"Genetic Program Theory".