Effects Of The Extract Of Gynostemma Pentaphyllum Makino On The Anti-oxidation Activity And DNA Damage In Aged Rat

Background: There are 11 categories of Gynostemma Pentaphyllum Makino(GP) in China, which are rich in bioactive elements. It was reported that GP has rich Ginsenosid such as Rb₁, Rb₃, Rd, F₂, K, Rg₃, essential amino acids, germanium, selenium, zinc, etc., which have the biological functions of anti-aging, anti-ulcer, restraining the proliferation of cancer cell and improving the immunity of the body. Nevertheless, the various elements and potential bioactivities of GP and its extracts are deserved to be detected, and in particular, effects of the extract of GP on the anti-oxidation activity and DNA damage in the aged rats are still unclear.Objective: To examine the effects of the different doses of GP's extract on the anti-oxidation, anti-aging activity, DNA damage and apoptosis, so as to explore the mechanism of GP's extract and its benefits to human being and to provide methodology reference.Method: 94 adult Wistar rats, weighed from 190g to 245g, and aged from 3 to 4 months were randomized into 6 groups with 14-16 rats in each group, Which were a general control group, an aged control group, a vitamin EC group, GP group 1, GP group 2 and GP group 3. Except for the general control, all of the rats in the other 5 groups were subcutaneously injected with D-galactose at the dose of 100mg/kg BW to induce the aging animal model, and the rats in the general control were injected with the same dose of saline. The rats in the general control and the aged control were fed with common feeding stuff, and the rats in the vitamin EC group were treated with VE 200mg/kg and VC 1000mg/kg, while the rats of the GP groups 1,2 and 3 were supplemented with 200 mg/kg, 800 mg/kg and 4000 mg/kg of GP extract respectively. The trial last 8 week. Food and water were free to supply for the rats. The samples of blood and urine were taken for measurements, which were included in anti-oxidative activities, such as SOD andGSH-Px activities and plasma MDA level with commercial kits, and detecting the membrane fluidity of erythrocyte by fluorescence polarization method, DNA spontaneous or oxidized damage by SCGE, the content of O -MeG in the urine by the capillary electrophoresis, and the Bel-2 and P53 protein expression with immunocytochemistry. Result:1. The results of anti-oxidation activities in rats showed that: ?activities and content of SOD, GSH-Px and MDA in the aged control were 128.70NU/ml, 338.42 U (activity unit) and 6.55 nmol/ml respectively. Compared with that in the general control, SOD and GSH-Px activities decreased 20.3% and 19.9% respectively (PO.01) in the aged control, while the content of MDA increased 16.1%, and the activity of GSH-Px in GP group 1 decreased 14.5%, MDA level in GP group 1 and 2 increased 12.4% and 10.3%(P<0.05) respectively. Compared with that in the aged control, the activities of SOD and GSH-Px in vitamin EC group increased, the level of MDA decreased (PO.05), and the GSH-Px and SOD in GP group 3 increased to 412.00 U(PO.Ol) and 152.43 NU/ml(P<0.05) respectively, the level of MDA decreased to 5.91 nmol/ml (P<0.05);?the membrane fluidity of erythrocyte was significantly improved compared with the control group, the P values of the aged control group, GP group 1 and 2 were 0.323,0.319 and 0.317 respectively, and the n values were 4.813,4.616 and 4.512, which significantly increased after the trial (P<0.05). Compared with the aged control group, the P and n values in vitamin EC group are 0.307 and 4.173, while the P and n values in GP group 3 decreased (PO.05);and compared between GP group 3 and 1, the P and n values also decreased (PO.05), which means that the P and n values have the trend of decrease with the increase of the dose of GP extract.2. The results of the DNA damage and apoptosis showed that: ?There was no significant difference in DNA spontaneous damage in all the groups of lymphocytes by SCGE. The DNA oxidative damage in the aged control are 179.38, 219.88 and 262.43AU(PO.05) with the oxidation of 5, 10 and 25 u mol/L H2O2 respectively. Compared with that, the DNA damage in GP group 1,2,3 and vitamin EC groupsdecreased significantly (PO.05), but no significant difference of the DNA damage in GP group 2 and 3 was found compared with that of the general control and vitamin EC group;?The content of O6-MeG in urine was higher in the aged control than the others, but there was no significant difference (PO.05);?The mean optical density values of the P53 protein expression of liver and brain was 0.207740 and 0.184997 in the aged control, 0.175305 and 0.156304 in GP group 1, and the value of brain in the vitamin EC group increased clearly (PO.05), the mean optical density values of brain and liver in the aged control significantly increased (PO.01), which means that the P53 protein expression was stronger than that of other groups. Compared with the general control and the vitamin EC group, the mean optical density values of brain and liver in GP group 1 and 3 were increased (PO.05), but was lower than the aged control, which means the P53 protein expression in this group was in a medium level;?Compared with the general control, the mean optical density values of liver and brain of Be 1-2 protein expression were 0.357382 and 0.332599 in the aged control group, 0.412761 and 0.357589 in the vitamin EC group, 0.385556 and 0.344915 in GP group 1, and the mean optical density value of liver in GP group 2 was 0.416365. The data showed that the Bcl-2 protein expression in general control group was stronger than the others. Compared with the aged group, the mean optical density values in GP group 1 was lower than the general control group, the vitamin EC group, GP group 2 and 3 (PO.05), which showed that the Bcl-2 protein expression in GP group 1 was in a medium level. Conclusion:1. the GP extract could enhance anti-oxidative activities, such as strength the activities of anti-oxidative enzyme SOD and GSH-Px, and lower the level of MDA and improve the membrane fluidity of erythrocytes. The DNA damage or apoptosis were decreased after the treatment of GP extract. The more the amount of GP extract was treated, the stronger the above activities were.2. SCGE, O6-MeG and P53 protein expression detection methods are often used in the detection of DNA damage. From the comparison, the advantages of SCGE were: lessamount of sample, convenience, short experimental period, low expense and high delicacy. It was a kind of simple and delicate DNA damage detection techniques.