Purification,Structure Characterization,Liver Protecting Activity And Effect On CYP2E1 Enzyme Of Poria Cocos Polysaccharide

Poria cocos（Schw.）Wolf,the national medicinal and edible Chinese herbology variety,is mainly used for the treatment of spleen deficiency,lack of food,restlessness,liver fatigue and other diseases.Polysaccharide is an active chemical component in the Poria cocos sclerotium,which has many pharmacological activities such as anti-inflammatory,anti-oxidation and immune regulation.Due to the problems of low yield and difficult separation and purification of natural active polysaccharide from Poria cocos,the current research reports mainly focus on its crude extracts or derivatives,but the fine structure of its homogeneous and many biological activities remain to be explored.Objective:In this study,different fractions of Poria cocos polysaccharide were isolated and purified from Poria cocos by water extraction and alcohol precipitation combined with anion exchange resin and gel column chromatography.And their hepatoprotective activities were compared in vitro.The homogenous component with the highest activity was screened,and its structural characteristics were analyzed by physical and chemical methods.Then,the regulation mechanism of its liver protection effect was explored through the liver injury model in vivo.In order to clarify the structural characteristics and action mechanism of Poria cocos hepatoprotective polysaccharide,explore the relationship between its structure and function.Methods:（1）Acute liver injury model was induced by CCl₄ in mice.Through the general state of mice,organ indexes,liver tissue pathological changes,liver marker enzyme changes,oxidative stress and inflammation factors,a comprehensive evaluation of the liver protection of alcohol extracts,water extracts and polysaccharides of Poria cocos;（2）Poria cocos polysaccharides were fractionally purified by DEAE-52cellulose resin and Sephadex S-500 gel column chromatography.Using CCl₄ to induce the liver injury model in vitro,screening the homogeneous fraction of Poria cocos polysaccharides that exert hepatoprotective activity based on cell viability and liver marker enzyme change levels;（3）Combined with physical and chemical methods,the structural characteristics of PCP-1C were identified through FT-IR,IC,SEC-RI-MALLS,methylation and 1D and 2D NMR analysis;（4）The model of acute liver injury induced by CCl₄ in mice was used to further verify the liver-protecting activity of the Poria cocos homogeneous polysaccharides in vivo.Combining with H&E staining,biochemical index determination and ELISA analysis,the effect of the purified homogeneous polysaccharides on the prevention of liver injury was comprehensively evaluated.Use Western-blotting to explore its possible mechanism of action to explore the possible connection between its structure and functionResults:（1）Pretreatment with water extract,alcohol extract and crude polysaccharide of Poria cocos have varying degrees of protective effect against CCl₄-induced liver injury in mice.Liver pathological injury was alleviated.Compared with the model group,each administration group had significant reductions in ALT,AST,MDA,IL-1β,IL-6,TNF-αand a significant increase in SOD activity,with dose-dependence effects observed in the polysaccharide groups,among which the high dose polysaccharide group had the closest results to the positive drug group;（2）Five different polysaccharide fractions（PCP1,PCP2,PCP-1A,PCP-1B and PCP-1C）were isolated from Poria cocos polysaccharide（PCP）by DEAE-cellulose and Sephadex gel.The results of physical and chemical properties showed that the different fractions were well enriched and separated.In vitro hepatoprotective activity test indicated that PCP-1C was the main hepatoprotective active component,and showed well homogeneity;（3）The molecular weight of PCP-1C was 17 k Da.It was composed of galactose,glucose,mannose and fucose in a molar percentage of 43.5:24.4:17.4:14.6.Structural characterization showed that PCP-1C has a backbone consisted of 1,6-α-D-Galp,which branches composed of T-α-D-Manp,1,3-β-D-Glcp,1,4-β-D-Glcp,1,6-β-D-Glcp,T-β-D-Glcp,T-α-L-Fucp and 1,3-α-L-Fucp;（4）The evaluation results of the liver-protecting activity of PCP-1C in vivo showed that PCP-1C exhibited superior liver-protecting activity.It can effectively reduce the elevation of liver injury characteristic transaminase in serum,increased the activity of liver antioxidant enzymes SOD and GSH-Px,and reduced the production of MDA and inflammatory factors.In addition,PCP-1C also alleviated liver injury in mice by inhibiting the expression of CAR and interfering with CCl₄-induced activation of cytochrome P4502E1 enzyme.Conclusion:PCP is the main active part of Poria cocos to protect the liver.Based on the results of in vitro experiments,the main active homogeneous component PCP-1C was further separated and enriched.PCP-1C had a molecular weight of 17 k Da and was composed of fucose,galactose,glucose and mannose.It was mainly composed of1,6-α-D-galactose andα-D-mannose as its repeating unit.Pharmacological experiments showed that the polysaccharide could regulate the expression of CAR protein,inhibit the activity of CYP2E1 enzyme,and reduce the production of toxic CCl₄ metabolites.These provide a new prospect for the application of polysaccharides in liver protection.