| ObjectionIn the previous study of this thesis,crude polysaccharides from roots of Ranunculus ternatus are found to have a significant protective effect on immune hepatic damage and improve liver function in mice induced by Con A.In this paper,we propose to prepare crude polysaccharides RTP-60 and RTP-60-100 at different temperatures,chemically isolate and purify crude polysaccharide RTP-60 to obtain different polysaccharide sites,polysaccharide fractions and homogeneous polysaccharide components.At the same time,normal human LO2 cell models are used to evaluate the protective effects of crude polysaccharides and their sites,fractions and components on hepatic damage induced by 7 drugs with significant hepatotoxicity,laying the experimental foundation for further new drug development.Method(1)Two kinds of crude polysaccharides are obtained by water extraction,ethanol precipitation,water dissolution,dialysis and freeze-drying methods.(2)The crude polysaccharides are precipitated using different concentrations of ethanol to prepare the polysaccharide sites.(3)The polysaccharide sites are chromatographed using DEAE-cellulose column to obtain polysaccharide fractions.(4)Polysaccharide fractions are obtained by gel column chromatography using Sephadex G-50 column or Sephadex G-100 column to obtain molecular weight homogeneous polysaccharide components.(5)The polysaccharide content is analyzed by phenol-sulfuric acid method.(6)The polysaccharide molecular weight distribution or molecular weight is determined by gel permeation filtration(HP-GPC).(7)The monosaccharide composition of polysaccharides is analyzed by PMP derivatization and capillary electrophoresis(CE).(8)Analysis of the products of periodate oxidation,Smith degradation and methylation reactions as well as infrared spectroscopy(IR)and nuclear magnetic resonance spectroscopy(NMR)are used to resolve and infer the structural information of polysaccharides.(9)The effects of crude polysaccharides,polysaccharide sites,polysaccharide fractions and homogeneous polysaccharide components on the proliferation of LO2 cells is determined by MTT method.(10)Acetaminophen,cisplatin,entecavir,atorvastatin,dexamethasone,azithromycin,paclitaxel and hydrogen peroxide(H2O2)hepatic injury cell models are established respectively,and the protective effect of polysaccharide on drug hepatic injury is determined by MTT method.Result1.The powder of roots of Ranunculus ternatus is extracted with water at 60℃ and 100℃continuously to obtain two kinds of aqueous extracts.The water extract is precipitated by ethanol,dissolved in water,dialyzed and freeze-dried to prepare two crude polysaccharides,RTP-60 and RTP-60-100,respectively.RTP-60 and RTP-60-100 are graded using different concentrations of ethanol,and five polysaccharide sites are obtained for RTP-60-1V,RTP-60-3V,RTP-60-100-1V,RTP-60-100-2V and RTP-60-100-3V,respectively.RTP-60-3V is separated by DEAE-cellulose column chromatography,and two polysaccharide fractions,RTP-60-3V-A and RTP-60-3V-B,are obtained.RTP-60-3V-A and RTP-60-3V-B are chromatographed on Sephadex G-50 and Sephadex G-100 gel columns,respectively,and three polysaccharide components with uniform molecular weights,RTP-60-3V-Ⅰ,RTP-60-3V-Ⅱ and RTP-60-3V-Ⅲ,are obtained.2.The polysaccharide components RTP-60-3V-I,RTP-60-3V-Ⅱ and RTP-60-3V-Ⅲ are all glucans with Mp of 5153 Da,7741 Da and 4628 Da,Mw of 6068 Da,9002 Da and 5464 Da,and Mn of 3972 Da,5999 Da and 3575 Da,respectively.These components all containe four linkage fragments,α-D-Glcp-(1→4)-α-D-Glcp-(1→,→6)-α-D-Glcp-(1→and→4,6)-α-D-Glcp-(1→,but the molar ratios of the four fragments are different.After the structural analysis,RTP-60-3V-Ⅰcontains the following structural fragments①—④:RTP-60-3V-Ⅱ contains the following structural fragments ①～⑤;RTP-60-3V-Ⅲ contains the following structural fragments①③④⑥⑦⑧.3.The results of activity evaluation show that:(1)The crude polysaccharides RTP-60 and RTP-60-100 and their polysaccharide sites,fractions and components(except RTP-60-3V-A and RTP-60-3V-Ⅰ)show significant proliferative activity on LO2 cells.RTP-60-3V-Ⅲ activity is the strongest among RTP-60(P<0.001).RTP-60-100 has the best activity among RTP-60-100 and it’s sites(P<0.001).(2)The crude polysaccharides RTP-60 and RTP-60-100 and their polysaccharide sites,fractions and components(except RTP-60-3V-A and RTP-60-3V-Ⅰ)show significant inhibitory effects on LO2 cells injury caused by acetaminophen,entecavir,atorvastatin,dexamethasone,azithromycin and paclitaxel.However,for the damage caused by cisplatin,only RTP-60-1V show inhibitory activity(P<0.01).For the damage caused by acetaminophen and entecavir RTP-60-3V-B-c has the best effect among these samples(P<0.01).For the damage caused by atorvastatin RTP-60-1V has the best effect among these samples(P<0.001).For the damage caused by dexamethasone,azithromycin and paclitaxel RTP-60-3V-Ⅲ has the best effect among these samples(P<0.001).(3)The crude polysaccharides RTP-60 and RTP-60-100 and their polysaccharide sites,fractions and components(except RTP-60-3V-A and RTP-60-3V-Ⅰ)also show good inhibitory effects on hydrogen peroxide-induced oxidative damage in LO2 cells.It is suggested that antioxidant effect might be one of the possible mechanisms of action against drug-induced liver injury.ConclusionThe crude polysaccharide RTP-60 contains a variety of dextran components with different linkage patterns.RTP-60 and RTP-60-100 and their polysaccharide sites,fractions and components(except for RTP-60-3V-A and RTP-60-3V-Ⅰ)show significant protective effects against acetaminophen,entecavir,atorvastatin,dexamethasone,azithromycin and paclitaxel induced liver injury. |
PDF Full Text Request