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ObjectiveTo observe the effects of ultrafiltration extract from Angelica Sinensis Radix and Hedysari Radix on PC12 cell apoptosis induced by H2O2; To discuss its mechanism of action.Methods H2O2 was used in the incubation of PC12 cells to establish the oxidative damage nerve cell apoptosis model. The experiment was divided into normal control group, model group, and three different dosages (0.375, 0.75, 1.5 g/L) of ultra-filtration extracted from Angelica Sinensis Radix and Hedysari Radix after modeling for interference. Rate of cell apoptosis was detected by flow cytometry; mitochondrial membrane potential was measured by laser scanning confocal microscopy; the protein expressions of cleaved Caspase-3, Bax and Bcl-2 were detected by Western blot.Results Compared with the model group, rates of cell apoptosis in the different dosage groups decreased significantly; membrane potential of mitochondria increased; the protein expressions of cleaved Caspase-3 and Bax decreased; the expression of Bcl-2 increased; the ratio of Bcl-2 and Bax increased (P<0.05).Conclusion The ultra-filtration extracted from Angelica Sinensis Radix and Hedysari Radix can inhibit PC12 cell apoptosis induced by H2O2.
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Protein is one of the three major food groups needed for proper nutrition. Proteolytic enzymes or proteinases are the group of enzymes whose catalytic function is to hydrolyze (breakdown) proteins. Production and partial purification of protease enzyme by Bacillus subtilis was the aim of this study. Bacillus subtilis was allowed to grow in shake flask broth culture, 3.5L and 7L fermenters for purpose of inducing protease enzyme. We are finding out the effect of minerals which are useful for the production of protease by Plackett Burman design. Minerals such as magnesium sulphate, potassium dihydrogen phosphate and manganese sulphate were showing the results for the production of protease. The protease enzyme was purified by ultra filtration, ammonium sulphate precipitation, dialysis, and lyophilization.The activity of protease was increased as there was increase in the enzyme concentration. Purified protease enzyme had a maximum activity at pH 9.0 of carbonate buffer and the optimum incubation time was 48hr. The protease assay is done for the crude enzyme at different temperature. It showed greater activity at 50 °C but after that it started decreasing the activity so, we had selected the temperature at 40 ℃ for the good activity..
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Objective To investigate whether the administration of the ultra-filtration extract mixture from Angelica sinensis and Hedysarum polybotrys is able to protect cardiomyocytes from oxidative injury of rats induced by H2O2 and its potential mechanism. Methods Myocardial cells from 2—3 d neonatal rats were cultured in DF medium and the cellular injury was induced by H2O2. The ultra-filtration extract mixture from A. sinensis and H. polybotrys was given in three doses of 3.75,7.5,and 15 mg/mL. Morphological changes of cardiomyocytes were observed by microscope. Survival rate of myocardial cells was assessed using MTT. The cardiomyocyte damages were estimated by detecting lactate dehydrogenase (LDH) and creatine kinase (CK) releases in the medium,superoxide dismutase (SOD) activities and intracellular malondialdehyde (MDA),and myeloperoxidase (MPO) contents. The levels of caspase-3 and hsp70 expression in cardiomyocytes were measured by RT-PCR. Results The ultra-filtration extract mixture could protect the cardiomyocytes from H2O2 injury in a dose-dependent manner (3.75,7.5,and 15 mg/mL). The ultra-filtration extract mixture could significantly decrease LDH and CK leakages and intracellular MDA and MPO contents,increase SOD activity,upregulate hsp70 expression,and downregulate caspase-3 expression. Conclusion The ultra-filtration extract mixture has protection on cardiomyocytes injured by H2O2 through improving cell antioxidant ability,upregulating hsp70 expression,and inhibiting caspase-3 activity.