Extraction And Separation Of Total Saponins From Dioscorea And Its Mechanism Study Of Antioxidant Action In Podocytes

Objective:To extract total saponins from Dioscorea by ultrasonic-assisted method,optimize the extraction process of total saponins from Dioscorea combined with Box-Behnken response surface methodology,and determine the best extraction method.The MPC-5 cell injury model induced by adriamycin was used to investigate the protective effect of total saponins from Pangolin on adriamycin-induced MPC-5 cell injury.mechanism of apoptosis.Methods: 1.The total saponins of Pangolin were extracted by ultrasonic-assisted method.The effects of ultrasonic time,extraction temperature,ethanol concentration,extraction time and other factors on the yield of total saponins of Pangolin were investigated.2.AB-8 macroporous adsorption resin was used to purify the above-mentioned total saponins,with ascorbic acid as a positive control,DPPH and ABTS free radicals were selected to compare the in vitro antioxidant activities of saponins before and after purification.3.MTT assay was used to detect cell viability,to verify the establishment of adriamycin injury model on MPC-5 cells,and to study the protective effect of total saponins of Pangolin on MPC-5 cells.4.Using western blot and other techniques to study the protective effect of total saponins from doxorubicin on the oxidative stress of MPC-5 cells induced by doxorubicin,and to study the oxidative stress of total saponins from doxorubicin-induced chronic kidney disease through Nrf2 signaling pathway.Molecular mechanisms of stimulation and apoptosis.Results: 1.The optimal extraction process of total saponins from Pangolin by ultrasonic-assisted method is: Under the conditions of 80% ethanol concentration,80 min extraction time,78 ℃ extraction temperature,25 min ultrasonic time,the content of total saponins was 16.24%,the experimental data was stable.2.The results showed that the total saponins of Pangolin had certain scavenging ability on DPPH and ABTS free radicals before and after purification,but the scavenging ability was weaker than VC.The antioxidant ability of saponins after purification was higher than that of unpurified.The antioxidant activity of unpurified saponins and purified saponins to DPPH free radical was 4.9 mg/ m L and 3.8 mg/ m L,respectively,and the antioxidant activity to ABTS free radical was 2.8 mg/ m L and 2.2 mg/ m L,respectively.The total saponins of Pangolin can inhibit the generation of free radicals.3.From the perspective of oxidative stress,the protective effect of total saponins of Pangolin on adriamycin-induced MPC-5 cell damage model was studied.MTT assay was used to detect the cell viability of MSC-5 cells.The results showed that adriamycin at 0.25μg/ m L could cause 50-60% damage of podocytes,resulting in decreased cell viability.When the concentration of total saponins in Pangolin was 0-20 μg/ m L,adriamycin could reduce the damage of MSC-5 cells and play a protective role on cells.It was found that adriamycin can increase the levels of MDA and ROS in cells and reduce the activities of SOD and GSH-Px,thus increasing cell apoptosis and causing oxidative stress in cells.In this experiment,the concentrations of total saponins of Pangolin were 5 μg/ m L,10 μg/ m L and 15 μg/ml,respectively,to study the protective effect of oxidative stress on MPC-5 cells.It was found that total saponins of Pangolin could reduce the levels of MDA and ROS in MPC-5 cells,increase the activities of SOD and GSH-Px,and reduce the cell apoptosis rate.Furthermore,oxidative stress levels induced by adriamycin were inhibited in a dose-dependent manner in MPC-5 cells.After adriamycin treatment,the expression of Nrf2 and HO-1,Bax and Bcl-2 in cytoplasm,nucleus and total protein of Mc C-5 cells were significantly increased,while the expression of Bcl-2 was significantly decreased.In the positive control group and the total saponins administration group,compared with the model group,the expression levels of Nrf2 and HO-1 in cytoplasm,nucleus and total protein in the administration group were significantly decreased,the expression levels of Bax in total protein were significantly decreased,and the expression levels of Bcl-2 were significantly increased.However,the effect of total saponins in pangolin was significantly weaker than that in the positive dexamethasone group.Through analysis and discussion,it was concluded that adriamycin caused oxidative stress reaction in cells and activated Nrf2,thus protecting cells.Pangolin total saponins could activate Nrf2 oxidative stress pathway,increase the expression of downstream protein HO-1,induce the transcription of protective cells and genes,and reduce MDA and ROS levels in MPC-5 cells.Increasing the levels of SOD and GSH-Px can reduce cell apoptosis,reduce the oxidative stress response of MPC-5cells,and then protect the oxidative stress damage of MPC-5 cells induced by adriamycin.By setting the concentration gradient,it was found that there was a certain dose dependence.When the concentration of total saponins in Pangolin was 15 μg/ m L,The protective effect of MPC-5 cells was strongest.Conclusions:(1)The best extraction process of pangolin total saponin: ethanol concentration 80%,extraction time 80 min,extraction temperature 78 °C,ultrasound time25 min,under this condition,the content of total saponin is 16.24%.And pangolin total saponins have a certain scavenging ability to DPPH and ABTS free radicals.(2)Pangolin total saponin has a protective effect on the oxidative stress produced by doxorubicin-induced MPC-5 cells,and its mechanism of action is closely related to the regulation of the Nrf2 signaling pathway.