Studies On The Antioxidant Activity And Immune Function Of Total Flavones From Red Clover

Red clover is a perennial herb fabaceous plant native to the Mediterranean that is found widespread throughout China. It contains flavonoids,protein,amino acids,carbohydrate and vitamin, etc.The total weight of flavonoids extracted from the Red clover showed an approximate 2% compared with the dry weight of the Red clover.Isoflavone from red clover mainly contain Biochanin A,Formononetin,Genistein,Daidzein etc.The total flavonoids of red clover have various physiology, as shown by animal research and from human clinical medicine.its have weakly estrogenic activity,promote animal growth,inhibit the growth of cancer cells and improve ductility of arterial blood vessel etc,but the study was less focus on the anti-oxidative activity and immune function of isoflavone from red clover.Therefore,the aim of the experiment is to evaluate the effects of the total flavonoids from red clover on the anti-oxidative activity and immune function.Total flavonoids of red clover was extracted by microwave and ultrasonic wave syner-gistic extraction technology.Free radical models were used to study the effects of total flavonoids from red clover scavenging free radical in vitro.Spectrophotometry was applied to determine erythrocyte hemolysis induced by hydrogen peroxide; To study the effect of antioxygen on total flavonoids from red clover in vitro,the malondialdehyde(MDA) producted by red cell membranes lipid peroxidation and mouse liver homogenate lipid peroxidation was determined with the method of thiobarbituric acid(TBA) colorimetry.To evaluate the effect of total flavonoids from red clover on the immune function,the tech-nology of the carbo clearance,delayed type hypersensitivity and organ index was util-ized.The experiment results were shown as the following:1.The result showed that total flavonoids of red clover (6.25～200 mg/mL) in vitro had scavenging activity on hydroxyl radical and superoxide radical by the autoxidation of py-rogallol production at the concentration of 6.25～100 mg/mL with dose dependent mode.Linear regression equation are Y=18.699+0.395X(r2=0.843) and Y=13.82+0.663X(r2=0.997) respectively,the 50% inhibiting concentration(IC50) values of scavenge hydroxyl radical and superoxide radical are 44.06 mg/mL and 54.57 mg/mL respectively.2.The results indicated that total flavonoids from red clover significantly inhibited the erythrocytic membranous lipid peroxidation and erythrocytic hemolysis induced by hy- drogen peroxide at the concentration of 0.025～0.2 mg/mL with dose dependent mode.The IC50 values of inhibit erythrocytic hemolysis and the erythrocytic membranous lipid per-oxidation were 0.0910 and 0.0905 mg/mL respectively.Total flavonoids of red clover significantly inhibited MDA mouse liver homogenate lipid peroxidation formation at the concentration of 0.10～0.78 mg/mL,the IC50 value for nhibited mouse liver homogenate lipid peroxidation formation was 0.174 mg/mL.The results showed that total flavonoids of red clover have antioxidation effect within a range of certain concentration in vitro.3.To evaluate the effect of total flavonoids from red clover on the immune function,the technology of the carbo clearance,delayed type hypersensitivity and organ index was utilized.The experimental results indicate that as the increase of total flavonoids concen-tration from red clover,the thickening in mouses metatarsus was increased,significant increase ocured in the group of 50 mg/mL compared with placebo(p < 0.05).Total flavonoids from the red clover can enhanced the clearance index(K) and phagocytic index(α) value for clearance of carbon particles in mice,The experiment demonstrates that it can enhance the function of macrophage system in mice at the concentration of 50 mg/mL and 20 mg/mL(P < 0.05).significant differences were not found on the immune organs index of mice in all test groups(P < 0.05),but spleen index and thymus index have the trend of increase as the increase of red clover total flavonoids concentration.it indicate that total flavonoids from the red clover can enhance mouse function in some scope of concentra-tions.