| At present,Research on the isolation,purification,analysis and chemical properties,and pharmacological effects of Ganoderma lucidum (GL) polysaccharides has been done very systematically and deeply. But very few studies on GL peptides and their bioactivities have been found reported. By adopting the sample's . OH inhibitory activities as the trace-checking index,the anti-oxidant peptides and their bioactivities in the fermented GL powder were studied systematically and deeply through several modern technologies,such as ultrafiltration,gel permeate chromatography,amino acids analysis,RP-HPLC and Capillary electrophoresis (CE). The main results are shown as follows:1. Defatting pretreatment of fermented GL powder could increase the . OH inhibitory activities of its water extraction(GLW). Also the extracting ratio of proteins and peptides increase dramatically,and the total sugar content increased a little too. Experiment result showed:under the conditions of pH5.5,temp. 4C,solvent to material ratio 15:3(V/W),the -OH inhibitory activities of GL water extraction and the content of peptides reached maximum values both. GLW was further separated by a 10,OOODa ultrafiltrate membrane into 70.67% GLL (filtrate) and 29.33% GLH (retentate). By using ligand exchange chromatography,or a two-step gel chromatogram method respectively,two peptides,GLP- I and GLP- II,were separated from GLL. Amino acids analysis showed that the purity of GLP- I is 91.53%,much higher than that of GLP-II (48.55%). For furtherresearch,the peptides GLP- I was separated into 8 chromatogram peaks by RP-HPLC. And then by using CE,the peptides GLP- I was separated into 13 chromatogram peaks. In peptides GLP-1,the amount of acidic amino acids,alkaline amino acids,essential amino acids were 29.83%,6.82%,36.67% respectively. And the amount of Glu,which has a good-to-brain activity,was 6.82%. The F value (ratio of branched amino acids to aromatic amino acids) of peptides GLP- I was 3.2. Measured by gel permeation chromatography,the peptides GLP- I 's molecular weight was 2362Da.2. Experiments about the inhibitory effect of the peptides GLP- I on lipoxygenase,the auto-oxidizing of lipid,free radicals and the antioxidant system of mice were performed. It turned out that the peptides GLP- I had significant antioxidant activities: The peptides GLP-1 could inhibit the oxidizing of oil significantly and it had a better effect in soybean oil system than in lard system. Also GLP- I could inhibit the lipoxygenase activities significantly,the IC.so value was 1.69X 10-2mg/ml. When the concentration of GLP- I reached 0.5mg/ml,the lipoxygenase activity almost lost totally.(2) In different free radical generating systems,GLL,GLP- I and GLP-II all showed very strong free radical inhibitory abilities. The GLP- I could inhibit the . OH effectively with an IC50 value of 2.5 X 10"2mg/ml.(3) The peptides GLP-1 could prevent the oxidation of mice liver tissues (dose:3.5mg/ml,inhibitory ratio 66%;compared to control group:p<0.01). And the peptides GLP- I could significantly inhibit the lipids peroxidation which were catalyzed by adding deduced reagents Fe2+,or H2O2.(4) The peptides GLP- I could protect the membrane of mice livermitochondria by inhibiting the lipids peroxidation. With a concentration of 5mg/ml,the inhibitory effect on mitochondria swelling reached 79.1%. Experiment also showed that GLP- I could inhibit the MDA formation of mice liver mitochondria effectively with an apparent concentration-effect relationship.(5) The auto-hemolysis of mice RBC could be inhibited by the peptides GLP- I significantly. The inhibitory ration reached beyond 90% with a 2.5 X 10-'mg/ml concentration of the peptides GLP- I .3. The peptides GLP- II had a good and durative therapeutic effect on Alloxan-induced hyperglycemic mice.4. It was found that GLL(Po. 800mg/kg.dX 12d) could significantly increase the reservoir of mice hepatin in muscle and liver. Also GLL could reduce the body lipid index of normal mice. The two parts separated by ultrafil... |
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