Study On Hepatoprotective Effects And Struture Of Ganoderma Lucidum Peptides

There are many research reports about polysacchrides and triterpene from Ganoderma lucidum(GLPS and GLT) but Ganoderma lucidum peptides(GLP) at present.Previously we have reported that GLP has been separated and it possess potent antioxidative capacity and hepatoprotective effects against CCl₄-induced liver injury in mice.In order to research the hepatoprotective effects and relationship between its structure and effect,material was selected and desalination was done,and the hepatoprotective effects of GLP were studied against D-galactosamine(D-GalN) /alcohol/Bacille-Calmette-Gu'erin and lipopolysaccharide(BCG+LPS) induced liver damage in mice.The hepatoprotective effects and antioxidative capacities of GLP, GLPS and GLT were explored.GLP were separated by Sephadex G-15 and the distribution profiles of GLP were studied.The amino acid sequence of a peptide was analyzed by RP-HPLC and HPLC-MS/MS.1.Selection of material and extraction/desalination of GLP(1) The content of crude protein and ratio of inhibitory activities on -OH of water-solution from five material of Ganoderma lucidum(GL) were determined.The result showed that the content of crude protein and the ratio of inhibitory activities on·OH of GL fruit body and GL mycelium one(GLM1) were equivalent.The ratio of inhibitory activities on·OH of GLM1 was close to GLM2,though the content of crude protein of GLM1 were inferior to GLM2.Finally,GLM1 was selected to be material in the experiment,because it is a product by industry.(2) Two fractions-peptides were separated by Cu²⁺-Sephadex G-25 ligand chromatogram method.(3) Desalination of GLP by six kinds of macroporous adsorption resins was investigated.The result revealed that desorption rate and desalination rate of S-8 were both relatively highest with 70%ethanol as desorpting agent,but the best resin to desalt GLP was DA201-C,and absorption rate and desorption rate of it was approached to S-8.According to the static adsorption,the DA201-C resin was chosen to do the dynamic adsorption tests,and the optimum adsorbing condition was 1.6 mL/min of flowing velocity,5.5 of pH and 2.0mg/mL of concentration.Compared with Cation and Anion exchange resin treatment and S-8 resin,DA201-C resin treatment was the best desalt method,the ratio of inhibitory activities on·OH of GLP and desalting rate of which were highest except that its nitrogen recovery ratio was not better than S-8.2.The hepatoprotective effects of GLP against D-GalN/alcohol/BCG+LPS induced liver damage in mice(1) Compared to mice of D-GalN-induced hepatic damage,pretreatment of mice with GLP at doses of 120 mg/kg-bw and 180mg/kg·bw were manifested by a significant decrease in the activities of marker enzymes(AST,ALT) in serum and MDA level in liver(p＜0.01),and by a significant increase in SOD activity and GSH level in liver (p＜0.01).The biochemical results were supplemented by histopathological examination of liver sections.Specially,at the dose of 180mg/kg·bw GLP,the biochemical parameters and the liver histopathological characters were close to normal mice.(2) The mice'index markedly changed except liver histopathological characters with alcohol treatment.Mice intoxicated with alcohol alone developed extremely evident hepatocellular damage from an extremely significant change in activities of AST, ALT and SOD and content of TG,GSH and MDA,when compared with normal control group.Compared to mice of alcohol-induced hepatic damage,pretreatment of mice with GLP at doses of 60 mg/kg·bw,120 mg/kg·bw and 180mg/kg·bw were manifested by a significant decrease in the activities of marker enzymes(AST,ALT) and content of TG in serum and MDA level in liver(p＜0.01),and by a significant increase in activity of SOD and GSH level in liver(p＜0.01).The elevated liver index and spleen index was markedly reduced by GLP treatment(P＜0.01).At the dose of 180mg/kg·bw GLP,the liver histopathological characters were close to normal mice. At the dose of 120 mg/kg·bw GLP,the effect was equivalent to mice pretreated with 50 mg/kg·bw tiopronin.(3) Compared to mice of BCG+LPS-induced hepatic damage,pretreatment of mice with GLP at doses of 120 mg/kg·bw and 180 mg/kg·bw were manifested by a significant decrease in the activities of marker enzymes(AST,ALT) in serum and liver index and spleen index(p＜0.05,p＜0.01),and by a significant increase in activities of SOD and GSH level and a significant decrease in content of MDA and NO in liver(p＜0.01).The biochemical results were supplemented by histopathological examination of liver sections.Specially,at the dose of 180 mg/kg·bw GLP,the biochemical parameters and the liver histopathological characters were close to normal mice,and some were better than normal mice.In conclusion,pretreatment of mice with GLP at doses of 180 mg/kg·bw afforded a significant protection in the alleviation of D-GalN/Alcohol/BCG+LPS-induced hepatocellular injury.3.Study on antioxidant activity of GLP and GLPS and hepatoprotective effects of GLP,GLPS and GLT(1) The ratio of inhibitory activities on·OH of GLP(7.5mg/mL)+GLPS(25mg/mL) was higher than the same concentration GLP or GLPS,but was not remarkable.(2) The inhibitory effect on liver MDA of GLP+GLPS was excelled to the same concentration GLP or GLPS in vitro.It showed prominent effect when GLPS (12.5mg/mL) was compounded with GLP at each concentration.(3) Compared to mice of D-GalN-induced hepatic damage,pretreatment of mice with GLP(150mg/kg·bw),GLPS(800mg/kg·bw),GLT(100mg/kg·bw) and combination of them were manifested by a significant decrease in the activities of marker enzymes (AST,ALT) in serum and MDA level in liver(p＜0.01),and by a significant increase in activity of SOD/GSH-Px and GSH level in liver(p＜0.01).The change of every index was not evident except GSH in mice treated with combination of GLP,GLPS and GLT.The liver histopathological characters of mice treated with combination of GLP,GLPS and GLT markedly got better and recovered,better than the same concentration GLP or GLPS or GLT. 4.Study on distribution profiles and structure of GLPGLP were separated by Sephadex G-15 column and finally divided into two peaks, one distributed between 5398.8 to 1148.9Da in molecular weight,and the other distributed between 1148.9 to 52.0Da,the main of which was between 530.0 to 244.5Da.By means of HPLC-MS,MS/MS,the amino acid sequence of a peptide, with M/Z 365.1 from peak I in HPLC,was determined,which was Ser-Asp-Gly-Ser.