Isolation And Identification Of Polysaccharide From Tetrastigma Hemsleyanum Vines And Mitigation Effects Of TVP On EC-induced Toxicity

Ethyl Carbamate(EC)has a wide range of commercial uses in life.It is found in many fermented foods as well as alcoholic beverages and has potential carcinogenic effects.Studies have shown that the toxicity induced by EC is often accompanied by the overproduction of reactive oxygen species(ROS),which leads to oxidative stress in the organism.Polysaccharides,as a natural biologically active compounds widely found in plants,animals and microorganism,has been verified to possess antioxidant capacity for the past years.They are widely present in plants,animals and microorganisms.More and more studies have shown that polysaccharides have better biological activities to relieve oxidative damage.Tetrastigma hemsleyanum Diels et Gilg,belonging to the grape family Vitaceae,a rare herbal plant possessing high medicinal value native to China,is distributed in the eastern,central,southern and south-western provinces of China such as Zhejiang Province and Yunnan Province.Nowadays T.hemsleyanum has passed safety evaluation and could be expected to be used as new food raw materials.However,most studies have focused on the composition and biological activity of the tubers of T.hemsleyanum whereas the vines as a waste have attracted little attention.Therefore,research on the active substances in T.hemsleyanum vines is of great significance to improve the resource utilization of T.hemsleyanum.In this study,we isolated and purified the polysaccharides of the T.hemsleyanum vines and characterized the structures by multiple analytical methods.Next,we aimed to investigate the potential protective effects of T.hemsleyanum vines polysaccharides(TVP)against ECinduced oxidative stress in Caco-2 cells and C.elegans including N2 nematodes,daf-2(-)mutants as well as daf-16(-)mutants and then preliminarily explored the antioxidative mechanism.The main results are as follows:1.Results of characterization analysis manifested that the average molecular weight polysaccharide extracted and purified from T.hemsleyanum(TVP)was 64.89 k Da and TVP was composed of mannose,rhamnose,glucuronic acid,glucose,galactose and arabinose.The TG-DSC results showed that TVP possessed excellent thermal stability.The identification results of Fourier transform infrared spectroscopy(FT-IR)and nuclear magnetic resonance(NMR)indicated that TVP possessed specific functional groups of polysaccharides.2.In vitro,the protective effects of TVP on EC-induced cytotoxicity was studied using Caco-2 cells.The results showed that the intervention of TVP could alleviate ECinduced cytotoxicity,mitochondrial dysfunction and oxidative damage in Caco-2 cells.WB results indicated that TVP could inhibit the apoptosis of Caco-2 cells via Aktm TOR and Bcl-2 signaling pathways.Meanwhile TVP could attenuate oxidative damage in Caco-2 cells via Nrf2-Keap1 and Sirt1-Fox O1 signaling pathways.3.In vivo,the protective effects of TVP on EC-induced toxicity was studied using C.elegans.The results showed that the treatment of TVP alleviated the decrease of survival rate,athletic ability and oxidative damage of N2 nematodes induced by EC.TVP exerted the same mitigation effects in the daf-2(-)mutants.However,TVP did not mitigate EC-induced oxidative damage in daf-16(-)mutants,suggesting that TVP could mitigate oxidative damage in C.elegans by activating DAF-16 /FOXO.