Study On Preparation And Antioxidant Activity Of Carboxymethyl Poria Cocos Polysaccharides With Different Molecular Weight

Polysaccharide is a kind of polymer with high molecular weight in nature.A large number of studies have shown that polysaccharides exhibited a series of remarkable bioactivities,such as anti-oxidation,anti-tumor and immunoregulation.These biological activities are influenced by many factors,such as molecular weight,monosaccharide composition and glycoside bond connection.At present,most of the polysaccharides are extremely complex in monosaccharide composition and glycoside bond connection mode.The factors affecting the biological activity have changed after degradation.Therefore it is impossible to clearly explain the exact relationship between the change of biological activity and the molecular weight.It has been reported that alkali-soluble Poria cocos polysaccharide is a kind of glucan mainly composed of??1?3?glycoside bond,which can eliminate the influence of glycoside bond and monosaccharide composition on the biological activity of polysaccharide after degradation of polysaccharides.So the alkali-soluble Poria cocos polysaccharide was selected as the research object.In this paper,the alkali-soluble Poria cocos polysaccharide was carboxymethylated and then degraded carboxymethyl Poria Polysaccharide by H₂O₂.The effects of different molecular weights of carboxymethyl Poria cocos polysaccharides on antioxidant activity were studied.The main experimental results are as follows:?1?Alkali-soluble Poria cocos polysaccharide extracted by dilute alkali is white lamellar,and the physicochemical properties and structure of alkali-soluble Poria cocos polysaccharides were analyzed.The extraction rate of Poria cocos polysaccharide was about60%.The results showed that alkali-soluble Poria cocos polysaccharide was a kind of glucan composed of 1?3 glycoside bond,which did not contain protein,nucleic acid,free amino acid,starch and phenolic substances.The total sugar content was 95.14±0.48%,and the uronic acid content was 0.1161±0.036%.The molecular weight of alkali-soluble Poria cocos polysaccharide was estimated to be about 37×10⁴ Da.?2?The carboxymethyl group was modified by secondary addition of alkali to obtain carboxymethyl Poria cocos polysaccharide CMP-1 with a degree of substitution?DS?of0.903±0.007.The results of infrared spectrum analysis of CMP-1 showed that the absorption peaks around 1640 cm^-11 and 1430 cm^-11 were the carboxymethyl characteristic absorption peak.The molecular weight of CMP-1 measured by GPC was 60.9×10⁴ Da and the polydispersity index was 3.56,which means it was a kind of polysaccharide with relatively large molecular weight and wide molecular weight distribution.?3?Carboxymethyl Poria cocos polysaccharides were degraded by hydrogen peroxide and the structure of polysaccharides was analyzed.Controlling of the concentration of H₂O₂ is2%and the reaction temperature is 50?,the carboxymethyl Poria cocos polysaccharide degradation products CMP-1-1,CMP-1-2 and CMP-1-3 were obtained at the reaction time of40 min,80 min and 120 min,which respectively with molecular weight of 10.7×10⁴ Da,3.22×10⁴ Da and 1.09×10⁴ Da.The degree of substitution of the three degraded polysaccharides was basically the same as that of CMP-1.Periodate oxidation and Smith degradation analysis showed the ratios of glycosidic bonds in CMP-1,CMP-1-1,CMP-1-2and CMP-1-3 respectively were in accordance.The Congo Red test proved that CMP-1and its degradation products all contain triple helix structure.?4?The antioxidant activities of carboxymethyl Poria cocos polysaccharides with different molecular weights were studied.The results showed that carboxymethyl Poria cocos polysaccharides with different molecular weights had certain antioxidant activities,including the ability to reduce Fe^3+,scavenge DPPH free radicals and inhibit hydroxyl free radicals.Low molecular weight carboxymethyl Poria cocos polysaccharides showed better antioxidant activity,of which CMP-1-3 had the best antioxidant activity,and the antioxidant effect increased with the increase of polysaccharide concentration.?5?The protective effects of carboxymethyl Poria cocos polysaccharide with different molecular weight on oxidative stress were studied.The results showed that carboxymethyl Poria cocos polysaccharides with different molecular weights could decrease the activity of LDH,decrease the content of MDA,and increase the activity of antioxidant enzymes?SOD and CAT?in oxidative damaged cells.Low molecular weight carboxymethyl Poria cocos polysaccharide showed better antioxidant activity.CMP-1-2 had the best effect on maintaining the stability of cell membrane,inhibiting lipid peroxidation and improving the activity of antioxidant enzymes in cells in the concentration range of 100-1000 ug/mL.In this paper,carboxymethyl Poria cocos polysaccharides were prepared by twice alkalization method and then degraded by H₂O₂ oxidation method.Finally,four polysaccharides with large molecular weight span were obtained.Their molecular weights were 60.9×10⁴ Da,10.7×10⁴ Da,3.22×10⁴ Da and 1.09×10⁴ Da.The experimental results showed that the composition and the structure of these four polysaccharides were similar,but there were only differences in molecular weight.The results of antioxidant study showed that the decrease of molecular weight was beneficial to the improvement of antioxidant activity of polysaccharides,but not the smaller the molecular weight,the better the antioxidant activity.This indicated that the biological activity of polysaccharides might also be affected by some specific spatial structure.The above results could provide relevant basis for further exploring the effect of molecular weight on the spatial structure of polysaccharides.