Extraction And Purification Of Polysaccharides From Finger Citron And Their Hepatoprotective Effect On Alcohol Induced Hepatocyte Injury

Finger citron[Citrus medica L.var.sarcodactylis(Noot.)Swingle],belongs to the family Rutaceae.Traditional Chinese medical books reported that finger citron had the function of anti-alcohol and hepatoprotective activity.The main functional components in finger citron were polysaccharides.Based on the effect of different extraction methods on characteristics of finger citron polysaccharides(FCP),FCP were prepared by ultrasonic-microwave synergistic extraction(UMSE)using the response surface optimization method and were graded into different fractions.Finally,the protective effect on alcohol induced hepatocyte injury was investigated.The main contents and results were as follows:(1)Effects of different extraction methods on characteristics and alcohol dehydrogenase(ADH)activity of FCP were investigated,including traditional water extraction(TWE),ultrasonic assisted extraction(UE),ultrasonic-microwave synergistic extraction(UMSE),microwave assisted extraction(ME)and compound enzyme assisted extraction(CEE).The results showed that the average yield of FCP extracted by UMSE was as high as 4.15%.There were differences in molecular weight of FCP prepared by different extraction methods.Five polysaccharides had typical absorption for pyran polysaccharides and?-glycosidic bond.Monosaccharide composition mainly included arabinose,galactose and glucose.The polysaccharides extracted by UMSE had the highest activating effect on ADH,with the percentage activation of 46.58%,but polysaccharides extracted by TWE inhibited the activity of ADH.Hence,UMSE was determined to extract polysaccharides from finger citron.(2)In order to improve the efficiency of UMSE,the effects of microwave power,material-liquid ratio and extraction time on the extraction yield of FCP were studied.The results of Box-Behnken response surface showed that the optimum conditions were as follows:microwave power of 700 W,ratio of water volumn to raw material weight of 57(m L/g)and extraction time of 910 s.Under these conditions,the extraction yield of FCP could be up to5.68%,which was increased by 51.06%and 37.86%compared to microwave extraction and traditional water extraction,respectively.(3)After the deproteinization and decolorization,FCP were separated into three fractions of FCP-1,FCP-2 and FCP-3 by Cellulose DE-52.FCP-2 and FCP-3 were separated into FCP-2A and FCP-3A by Sephadex G-200,respectively.FCP-1,FCP-2A and FCP-3A were homogeneous polysaccharides.The purity of FCP-1,FCP-2A and FCP-3A were 82.7%,86.5%and 88.4%determined by phenol-sulfuric acid method,respectively.After ultrafiltration centrifugation,the purity of three polysaccharides were 88.6%,91.7%and 93.6%,respectively.HPGFC,HPAEC,IR and uronic acid were used to analyse the homogeneous polysaccharides.The average molecular weight of FCP-1,FCP-2A and FCP-3A were1.96×10~4 Da,1.47×10~5 Da and 2.06×10~5 Da,respectively.FCP-2A and FCP-3A were composed of arabia sugar,galactose and glucose,and FCP-1 was composed of arabia sugar,galactose,glucose and mannose.IR spectra showed that FCP-1 mainly contained?-D-galactose,FCP-2A contained?-D-glucose and?-D-glucose,FCP-3A mainly contained?-D-glucose.All of three polysaccharides were pyranoses.The contents of uronic acid in FCP-1,FCP-2A and FCP-3A were 5.20%,23.01%and 29.54%,respectively.(4)In order to investigate hepatoprotective activity of FCP and fractions from FCP,antioxidant activity in vitro,effect on ADH activity and protective effect on alcohol induced hepatocyte injury were studied.The antioxidant capacity of FCP in vitro was determined by DPPH free radical scavenging activity,ABTS radical scavenging activity and reducing power.The sequence of antioxidant capacity was as follows:FCP-3A>FCP-3>FCP>FCP-1>FCP-2A>FCP-2.When the concentration of polysaccharides was 1.0 mg/mL~10.0 mg/mL,FCP and five fractions can activate ADH activity.The sequence of activation capacity was as follows:FCP-2A>FCP-2>FCP>FCP-3A>FCP-3>FCP-1.With the 20%survival rate of alcohol induced HepG2 as a model group,it was found that when the concentration of polysaccharides was 37.5?g/mL~150.0?g/m L,FCP and fractions can protect the alcohol induced HepG2 injury by viability of HepG2 cells and indexs(AST,GSH,MDA and SOD).FCP-3 and FCP-3A had the strongest hepatoprotective activity among the samples.