Study On The Physicochemical Properties And Anti-liver Injury Activity Of Cat's Claw Polysaccharide

ObjectionThe purpose of this paper was to systematically isolate subgroups of polysaccharide components and homogeneous polysaccharides from the crude polysaccharide of Radix Ranuncoli Ternati,and thereafter,to determine the physicochemical properties of the isolated products.Meanwhile,the anti-hepatic injury and immuno-regulation effects of the crude polysaccharides,the subgroups and the homogeneous polysaccharides were investigated in vivo and in vitro,in order to provide experimental basis for further studies and development of the polysaccharides of R.Ranuncoli,and to provide a theoretical basis for the development of"Lingmao Fang".MethodThe crude polysaccharides?RTP-60,RTP-60-100?of R.Ranuncoli were prepared successively by extraction with water bath or boiling water,precipitation with ethanol and dialysis.RTP-60 was subjected to DEAE-cellulose as well as Sephadex G-100 or Sephadex G-50 column chromatography to afford the polysaccharides RTP-60-I and RTP-60-II.The physicochemical properties of these polysaccharides were determined by mearuring their sugar content,glucuronic acid content,distribution of molecular weight and monosaccharide composition.The structural features of RTP-60-I and RTP-60-II were elucidated by infrared radiation?IR?and nuclear magnetic resonance?NMR?spectra.The hepatitis model was established by repeatedly injecting ConA into mice and used to evaluate the hepato-protective activities of RTP-60 and RTP-60-100.Human L-O₂Cells were used in the in vivo evaluation of the effects of promoting liver cell proliferation and attenuating the ConA or H₂O₂ induced liver cell injury.The inhibitive activities of polysaccharides on the secretion of HBsAg and HBeAg were evaluated by ELISA using HepG2.2.15 cells.The effects of these polysaccharides on the proliferation and cytokine secretion of native spleen cells were estimated in mice.Results?1?By extracting with water or boiling,ethanol precipitating,dissolving,dialyzing and freezing-drying,the yield of the crude polysaccharides of RTP-60 and RTP-60-100were 3.84%and 0.43%,and the sugar content of their were 84.05%and 64.90%respectively.The peak molecular weight of RTP-60 were 2297Da and 639Da.The monosaccharide molar ration of RTP-60 was Ara:Glu:Gal:Man=1.00:7.05:0.94:0.15;The peak molecular weight of RTP-60-100 were 8.67×10⁴Da and 1.30×10⁴Da,The monosaccharide molar ration of RTP-60-100 was Xyl:Ara:Glu:Rha:Gal:Man=0.06:1.00:8.00:0.03:0.28:0.07.?2?The crude polysaccharide?RTP-60?was separated into two fractions?RTP-60-A,RTP-60-B?with DEAE-cellulose column and their yield were 33.91%and 4.89%,and sugar content were 96.98%and 54.28%respectively.The peak molecular weight of RTP-60-A was 2660Da,While the peak molecular weight of RTP-60-B were 2577Da,1.48×10⁴Da and 6.91×10⁴Da.The RTP-60-A was consisted of glucose,while the monosaccharide molar ratio of RTP-60-B was Xyl:Ara:Glu:Gal:Man=0.03:1.00:2.04:0.62:0.04.?3?The crude polysaccharide?RTP-60-100?was separated into two fractions?RTP-60-100-A,RTP-60-100-B?with DEAE-cellulose column and their yield were 19.7%and 4.27%,and sugar content were 99.00%and 53.22%respectively.The glucuronic acid content of RTP-60-100-B was 13.40%.The peak molecular weight of RTP-60-100-A were 3091Da and 822Da,while the peak molecular weight of RTP-60-100-B were 8.23×10⁴Da and 3859Da.The monosaccharide molar ratio of RTP-60-100-A was Xyl:Ara:Glu:Gal=0.03:1.00:5.63:0.07,the ration of RTP-60-100-B was Xyl:Ara:Glu:Rha:Gal:Man:GluA=0.01:1.00:0.25:0.02:0.17:0.01:0.02.?4?RTP-60-A and RTP-60-B were further separated and purified by Sephadex G-100 or Sephadex G-50 column to obtain two homogeneous polysaccharides RTP-60-I and RTP-60-II respectively.The peak molecular weight of RTP-60-I was 2780 Da,while RTP-60-II was 4802Da.RTP-60-I was mainly?-glucan,and the possible linking residues of RTP-60-I were?1??,?1?6?,?1?4?,?1?4,6?.RTP-60-II contained?and?-configuration and the monosaccharide molar ratio of RTP-60-II was Ara:Glu:Gal=1.00:23.57:0.86.?5?Polysaccharides?RTP-60,RTP-60-100?could protect the liver from the injury induced by ConA?this model was similar to that of hepatitis B virus infection?.in addition,it could obviously improve the survival rate of mice with liver injury and reduce the necrotic area of mouse hepatocytes.it could also reduce the inflammatory damage of hepatocytes and promote the proliferation of hepatocytes.the effect of RTP-60 was better than that of RTP-60-100.?6?Crude polysaccharides?RTP-60,RTP-60-100?polysaccharide components?RTP-60-A,RTP-60-100-B?and polysaccharide subcomponents?RTP-60-A-a,RTP-60-A-b,and RTP-60-B-a?could promote the proliferation of human L-O₂hepatocytes at the dose from 50?g/mL to 250?g/mL.RTP-60,RTP-60-100,RTP-60-A,RTP-60-100-B and RTP-60-B-b could inhibit the injury and death of hepatocytes induced by high concentration of ConA while their were at dose of 250?g/mL.homogeneous polysaccharide?RTP-60-II?could promote the proliferation of human L-O₂ hepatocytes at the dose of 250?g/mL and it could inhibit the injury and daeth of hepatocytes induced by high concentration of ConA at the dose of 250?g/mL.?7?Crude polysaccharide?RTP-60?could inhibit injury and death of hepatocytes induced by high concentration of H₂O₂,and the effect of it was pretty good.while the RTP-60-100 was only valid at the dose of 250?g/mL.both RTP-60 and RTP-60-B-b could inhibit injury and death of hepatocytes induced by high concentration of H₂O₂ at the dose from 100?g/mL to 250?g/mL.?8?RTP-60 inhibited remarkedly the secretion of HBsAg and HBeAg by HepG2.2.15cells while the RTP-60-100 only inhibited the secretion of HBeAg at the dose of500?g/mL.?9?RTP-60,RTP-60-100,RTP-60-A,RTP-60-B and RTP-60-100-B could increase the proliferation of spleen cells.?10?Both of RTP-60 and RTP-60-100 could improve the ability of spleen cells to secret IFN-?in normal mice,and the best effects were observed at the dose of 250?g/mL.RTP-60 could improve the secretion of TNF-?by peritoneal macrophages at the dose of 10?g/mL.ConclusionThese results suggested that the polysaccharides from R.Ternatus possess significant hepato-protective and immune-regulative effects and may possibly be used in the treatment of immune-mediated liver disease in the future.