Isolation And Chemical Composition And Protective Effect On Acute Alcoholic Liver Injury Of Atractylodes Macrocephala Polysaccharide

The research object of this paper is the crude polysaccharide of Atractylodes macrocephala(BZCP)produced by Shaanxi Laisente Biotechnology Co.,Ltd.according to the process of water extraction and alcohol precipitation.In this paper,the BZCP was decolorized,anion exchange chromatography and dialysis to obtain the refined polysaccharide of Atractylodes macrocephala Koidz(BZJP)with the content of 97.12%.The structure of BZJP was analyzed and then the effect of polysaccharide from Atractylodes macrocephala Koidz on acute alcoholic liver injury was studied from animal level and cell level.Finally,the possible mechanism was discussed by molecular docking technology.Following parts were mainly included:1.Isolation and purification of BZCPThe optimal decolorization process of macroporous resin was determined by single factor investigation and orthogonal test.The optimal decolorization process was as follows:the type of macroporous resin was HPD600,the concentration of sample was 25 mg/mL,the pH of sample was 6,the decolorization temperature was 75℃ and the decolorization time was 6 h.Under these conditions,the decolorization rate and recovery rate can reach 79.75%and 80.31%respectively.The purified polysaccharide BZJP was obtained by DEAE cellulose-52 ion exchange column chromatography and dialysis,which contains almost no protein and 97.12%polysaccharide.2.Chemical composition of BZJPThe content of BZJP polysaccharide and protein were determined by phenol sulfuric acid method and Coomassie blue staining method respectively.The results showed that the content of BZJP polysaccharide was 97.12%,and the content of protein was almost zero(0.08%).The structure of BZJP was analyzed by high performance gel permeation chromatography(HPGPC),amino column method,HPLC analysis with pre-column PMP derivatization,gas chromatography,IR spectroscopy,methylation analysis and NMR spectroscopy.BZJP was a homogeneous polysaccharide,and its relative molecular weight was 5465 Da by HPGPC.BZJP was mainly composed of glucose(Glc),mannose(Man)and xylose(Xyl)by the combination of amino column method,PMP derivatization and gas chromatography,and their molar ratio was determined by gas chromatography,Glc:Man:Xyl=6.8:3.0:1.0.IR spectroscopy confirmed that BZJP was a polysaccharide with D-glucopyran ring and β-terminal epimerism.Methylation analysis showed that BZJP was composed of five residues,namely→4)-αGlcp(1→,→6)-αGlcp(1→,→2)-aManp(1→,βXy1p(1→and→3)-βXylp(1→It was inferred that BZJP was a linear polysaccharide with a terminal sugar group of βXylp(1→.It further inferred that the primary structural formula of BZJP was βXylp-[(1→3)-βXy1p]6-(1→6)-αGlcp-[(1→4)-aGlcp]40-[(1→2)-aManp]12 by NMR.3.Protective effect of Atractylodes macrocephala polysaccharide on acute alcoholic liver injury in miceWe investigate the protective effect of Atractylodes macrocephala polysaccharides on acute alcoholic liver injury in mice.The serum alanine aminotransferase(ALT),aspartate aminotransferase(AST)and triglyceride(TG)were determined in the supernatant,and the contents of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)were measured.The results showed that Atractylodes macrocephala polysaccharide could significantly inhibit the increase of serum AST,ALT and TG activities caused by ethanol induced acute liver injury,effectively inhibit the increase of liver index,the increase of MDA content and the decrease of SOD and GSH-Px activities caused by liver injury,and further alleviate the liver injury of mice.Conclusion:Atractylodes macrocephala polysaccharide has better protective effect on ethanol induced acute liver injury in mice.4.Protective effect of BZJP on alcohol induced injury of L-02(1)The concentration and time of alcohol modeling were determined by MTT method.The time and concentration of alcohol modeling were determined as 100 mmol/L and 6 h,respectively.(2)The intervention concentration of BZJP was determined by MTT method.The final concentration of BZJP in 0～800 μg/mL for 24 h had no cytotoxicity on L-02 cells.Combined with the concentration range of polysaccharide in other literatures,three concentrations of 50,100 and 200 μg/mL were selected for further cell experimental study.(3)Objective to investigate the protective effect of BZJP on ethanol induced L-02 hepatocyte injury.After different concentrations of BZJP were pre incubated for different time,the model was established.The activities of ALT and AST in the cell culture supernatant and the oxidation antioxidant indexes MDA,SOD and GSH-Px of hepatocytes were detected to observe the protective effect of BZJP on the oxidative damage of hepatocytes by ethanol.The results showed that BZJP could significantly improve the above indexes caused by liver injury,and the effect of high-dose BZJP pre incubation for 1 h was better.5.The molecular docking technique was used to explore the protective effect of Atractylodes macrocephala polysaccharide against alcoholic liver injury.Molecular docking of the possible acid hydrolysate/microbial degradation products of BZJP with the specific receptor ASGPR on liver parenchymal cells and MR on liver Kupffer cells revealed that these oligosaccharides can bind to the two receptors well.Therefore,one of the mechanisms of BZJP’s anti-alcoholic liver injury is likely to be:BZJP acid hydrolysate/microbial degradation products combine with ASGPR and/or MR to enter the liver and play a role in liver protection.