Optimization of extraction process for total saponins from fibrous root of Anemarrhena asphodeloides and its protective effect on PC12 cells induced by oxygen-glucose deprivation / 中草药

ABSTRACT

Objective: To optimize the extraction of total saponins from fibrous root of Anemarrhena asphodeloides (TSFAA) and explore its protective effect on PC12 cells induced by oxygen-glucose deprivation (OGD). Methods: Single factor experiment and Box-Benhken response surface method were used to select the best extraction technology. The model of PC12 cells induced by OGD was established and treated with the total concentration of 20, 40, and 80 mg/L TSFAA. Inverted microscope was used to observe the morphology of PC12 cells, and cell viability was measured by MTT assay. Fluorescence probe was used to detect the intracellular oxygen free radical (ROS), and Annexin V/PI double staining method was performed to measure the apoptotic rate. The apoptotic protein Bcl-2 and Bax expression were detected by Western blotting. Results: The optimum conditions were as follows: The concentration of solvent was 72.22%; The ratio of material to liquid was 1:11; And the extraction time was 73.33 min. Under this condition, the theoretical calculated extraction rate was 8.38% and the measured value was 8.33%. PC12 cells viability was significantly decreased after OGD injury for 4 h. However, TGA showed a dose-dependent protective effect on OGD-induced cell damage; Flow cytometry analysis showed that TSFAA significantly reduced the content of ROS and apoptotic rate of PC12 cells. Also, Western blotting showed that TSFAA up-regulated the expression of Bcl-2 and down-regulated Bax expression. Conclusion: The extraction process for TSFAA optimized by response surface method has high yield and good extraction effect. TSFAA has protective effect on PC12 cells injured by OGD. The mechanism may be related to the decrease in the content of ROS in PC12 cells induced by OGD and the inhibition of mitochondrial apoptosis pathway.