Structures Of Ganoderma Lucidum Endogenesis Peptides And Intervention To Liver Injury

Ganoderma (G.) lucidum is pharmaceutical-edible mushroom with high medicinal value, which is widely used to tumors and liver disease prevention and treatment. It is generally considered that the active factors are polysaccharides and triterpenes, and the research in G. lucidum (endogenesis) peptides (GLP) is rare to be reported. The GLP was studied systematically first time in the paper. The structures of GLP, hepatoprotective effects and their mechanism of GLP, structure-activity relationship were investigated using the membrane separation technology and RP-HPLC, capillary electrophoresis, HPLC-ESI-MS/MS and other modern analysis method. Mostly Results are as follows:1. Separation and purification of GLPUsing the scavenging activities of hydroxyl radicals (·OH) as tracking index, the activity of the G. lucidum mycelium powder water extract and its various components were compared, and the optimization extraction conditions of GLP were identified. The results showed that GLP was potent scavenging agents of·OH and its radical scavenging capacity was much better than G. lucidum polysaccharide. Cu2+-Sephadax G-25 coordination chromatography could effectively separate the small peptides and amino acid from G. lucidum, the purity of GLP is up to 91.53%and the yield rate is 9.66%. GLP were separated by RP-HPLC and capillary electrophoresis, which eight chromatogram peaks and 13 peaks occurred respectively, and they were inferred to be peptide peaks preliminarily.2. Antioxidant activity and mechanism of GLPFor antioxidant is one of the important mechanisms of hepatoprotection, GLP antioxidant activity was systematically evaluated using many methods in lipid system, water-solubility system and animal in vitro experiment. In the above three kinds of system experiments, GLP all exhibited potent antioxidant activity. The oxidation mechanism involved:(1) Scavenging free radicals; (2) Inhibit lecithin oxidation: Protecting the biological membrane and integrity of structure and function; (3) Potent reducing ability; Protect glutathione and-SH from oxidation; (4) Chelating nonheme iron and other metal ions which promote lipid oxidation; Inhibit lipoxidase activity and lipid peroxidation; (5) The peptides have synergistic contributions to the oxidative stress release among them.3. Hepatoprotective effects and mechanism of GLPFour kinds of common liver damage animal models were used to study hepatoprotective effect and its mechanism of GLP by not only the determination of biochemical parameters, but also observations of liver histopathological examination. GLP showed good hepatoprotective effects in the four acute liver damage model, which were induced by CC14, D-Gal, alcohol, BCG+LPS, especially when GLP administration at the dosage of 180 mg/kg-bw, all biochemical parameters determined and pathology changes were close to normal controls. It is indicated that ganoderma lucidum hepatoprotective active factors included GLP, except for polysaccharide and triterpene. Its hepatoprotective mechanism involved:(1) Good antioxidant activity:The ability of scavenging reactive oxygen free radicals/reducing and resisting phospholipids oxidation; (2) Positive immune adjustment effect; (3) Good amino acid composition.4. Apoptosis of human hepatoma HepG2 cells induced by GLPOn the basis of the above work, the inhibition effect of GLP on human hepatoma HepG2 cells was investigated for the first time. The results showed that in vitro, GLP could restrain the proliferation of human liver HepG2 cell in a dose and time dependent manner. GLP could cause G0/G1 cell cycle arrest in HepG2 cells to induce the apoptosis, and there were certain dose dependent between GLP concentration and apoptosis rate. The HepG2 apoptosis induced by GLP was due to Ca2+overloading in cells, the downregulation of the expression levels of Bcl-2 and survivin which were apoptosis inhibiting gene, the upregulation of p53 which was the apoptosis improving gene, the activation of Caspase-3 activity. The effects were in a dose dependent manner.5. Comparison of antioxidant activities and hepatoprotective effects among GLP, G lucidum polysaccharides and G. lucidum triterpeneAntioxidant activities and hepatoprotective effects of GLP, G. lucidum polysaccharides (GLPS), G. lucidum triterpene (GLT) and their combination were compared. The inhibitory effects of GLP (3.75mg/ml)+GLPS (12.5mg/ml) on liver MDA in vitro were synergistic, whether they have free radicals inducer or not. Compared to the hepatic damage mice induced by D-Gal, 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 activities of SOD/GPX and level of GSH in liver (P<0.01). However the change of each index was not evident except GSH among treatment groups namely groups of GLP, GLPS, GLT and their combination. The liver tissue pathology photos showed that the histopathological characters of mice treated with combination of GLP, GLPS and GLT exhibited markedly recovery and it was better than the same concentration GLP or GLPS or GLT treatment.Antioxidant activities and hepatoprotective activities of GLP were excelled to GLPS, but inferior to GLT.6. The primary structure and structure-activity relationship of GLPThe GLP were separated and identified by HPLC-ESI-MS/MS online, and the structures of three kinds of oligopeptides were analyzed. One peptide with m/z 365.1 was Ser-Asp-Gly-Ser, the second peptide with m/z 566.8 was Ala-His-Leu(Ile)-Leu(Ile)-Leu(Ile), the third peptide with m/z 814.5 was Leu(Ile)-Leu(Ile)-Leu(Ile)-Thr-Phe-His-Ala.All amino acids of the tetrapeptides were with antioxidant effect, the pentapeptide and heptapeptide were all high F value oligopeptides.