| Objective:To study the extraction process, oxidation resistance, anti-ulcer activity and mechanisms of collagen from Rana chensinensis skin (CRCS) on absolute ethanol induced gastric ulcer in mice.Methods:The content of collagen in Rana chensinensis skin was determined by the method of Hydroxyproline in this study. Papain, pepsin and trypsin were respectively used to extract CRCS and the extraction effects were compared to select the best enzyme. The best extraction process was determined according to single factor and orthogonal experiments. The molecular weight was determined by the method of sephadex gel chromatog raphy.The oxidation resistance (reducing power and effects on scavenging of hydroxyl radicals) was determined and compared with L-ascorbic acid (Vc).We designed 4 groups of 20 mice each, namely control (C), absolute ethanol (ABE), cimetidine (CIM,52.48 mg/kg), and CRCS (60.58mg/kg) in order to demonstrate the anti-ulcer of CRCS in absolute ethanol induced gastric ulcer. The mice were respectively administrated medicine and the groups of C and ABE were administrated saline solution by the oral rout for 10 days and then all of them were administrated 0.2 mL absolute ethanol except C by the same rout. The half of each group was used to determine UI and the others were used to determine biochemical changes including malondialdehyde (MDA), glutathione peroxidase (GSH-Px), nitric oxide (NO), prostaglandin E2 (PGE2), and non-protein sulfhydryl (NP-SH). The similar experiment was carried and the mice were pretreatment with L-NAME (100 mg/kg) or indomethacin (14.4 mg/kg) before absolute ethanol. The UI was determined in the same way.Results:The best extraction process:reaction time was 36h, temperature was room temperature, pH was 5.5, enzymic concentration was 1%, the ratio of solid to liquid was1:20. The extraction rate of CRCS is 24.71% and the purity is 72.03%. All of the molecular weight of CRCS is less than 30000 Da and the minimum is 200 Da. CRCS is weak that reducing capacity and antioxidant activity on scavenging hydroxyl free radical effects compared with L-ascorbic acid (Vc).In pharmacological experiments, the UI of ABE group was significantly increased compared with C (64.36±4.2 vs 0 mm2,p< 0.01). The UI of CRCS and CIM was significantly reduced compared with absolute ethanol group (11.36±1.20,15.03±1.78 vs 64.36±.20 mm2, p< 0.01). The results clearly demonstrated the gastricprotective effect of CRCS. In the experiment of biochemical changes, CRCS significantly increased the level of NO (7.31±0.092 vs 4.37±.068,p<0.05) and decrease the level of MDA (0.71±.032 vs 1.72±0.038, p< 0.01) compared with ABE group. The levels of GSH-Px and PGE2 in CIM and CRCS groups were not significantly different but it was growth trend compared with ABE group. In the experiment of mechanisms, the UI of CRCS and CAR were decreased significantly compared with the respective group with L-NAME (13.3±1.415 mm2 vs 51.8±.068,20.6±.048 mm2vs 60±9.41 mm2,p < 0.01). Thus NO is involved in the gastricprotection elicited by both CRCS and carbenoxolone. The results show that in the presence of indomethacin the gastroprotective effect of carbenoxulone was inhibited(43±.23 vs 20.6±0.5 mm2, p< 0.05), but it was not significantly different (13±.04 vs 13.3±.42,p>0.05) of CRCS. Thus PGE2is not involved in the gastroprotection by CRCS.Conclusion:The extraction rate and purity of CRCS were 24.71% and 72.03%. The study demonstrated that CRCS is weak that reducing capacity and antioxidant activity on scavenging hydroxyl free radical effects and clearly protective effect against absolute ethanol induced gastric ulcer and the mechanism is related to up-regulation of NO. |
PDF Full Text Request