Study On Extraction And Bioactivity Of Black Ginseng Saponins

Black ginseng is a new processed ginseng product after red ginseng,which is processed by nine steaming and nine sunning processes.Ginsenoside is the main component of black ginseng,which has high biological activities,such as antioxidant,anti-tumor,hypoglycemic and so on.In order to explore the biological activity of black ginseng saponins,black ginseng was prepared by freeze-thaw technology and fermentation at different stages.Ethanol ultrasonic extraction method was used to extract black ginseng saponins,and response surface analysis was used to determine the optimal extraction process.Saponins were purified by macroporous resin.Liquid mass spectrometry was used to compare the constituents of panax ginseng and black ginseng.Then,the antioxidant activities of ginsenosides and black ginseng saponins,as well as the inhibition and apoptosis mechanism of gastric cancer MGC-803 cells were compared.The black ginseng prepared by this method has black color and higher Rg₃content than the traditional method,which can be used as a new preparation method.Ultrasonic assisted extraction of black ginseng saponins with ethanol as solvent.According to the optimization results of response surface experiment,the optimal extraction conditions of black ginseng saponins were determined as follows:solid-liquid ratio 1:39,ultrasonic time78 min,ethanol concentration 71%,under which the yield was 11.73%.The refined black ginseng saponins were purified by D101 macroporous resin,and the content was 89.02%.Fourteen monomer saponins were detected by LC-MS,including Rg₃,Rg₅and Compound K rare saponins.It is speculated that it was formed by the transformation of common ginsenosides during the preparation of black ginseng.In order to explore the antioxidant capacity of ginsenosides and black ginseng saponins,the effects of ginsenosides and black ginseng saponins on DPPH free radical scavenging capacity were studied.The DPPH free radical scavenging rate of black ginseng saponins and black ginseng saponins reached 86.01%and 50.63%at 4 mg/m L.In terms of hydroxyl radical scavenging rate,black ginseng saponin and ginsenoside reached 77.9%and 44.8%at 4 mg/m L.It is speculated that the antioxidant capacity of black ginseng saponins is stronger than that of ginseng.In order to investigate the effect of black ginseng saponins on apoptosis of 803 cells,MTT,morphological observation,flow cytometry and WB experiments were carried out.MTT assay showed that the IC₅₀of ginsenoside at 24 h was 296.64μg/m L,that of 48 h was266.46μg/m L,that of 72 h was 188.35μg/m L,that of black ginseng at 24 h was 141.25μg/m L.The IC₅₀of 48 h and 72 h were 120.23μg/m L and 95.5μg/m L respectively.It shows that black ginseng has stronger anti-tumor effect than ginseng.The apoptosis of gastric cancer MGC-803 cells treated with black ginseng saponin was observed by inverted microscope and fluorescence microscope.Flow cytometry showed that the total apoptosis rate increased from 0.23%to 66.96%.Cell cycle results showed that the ratio of G0/G1phase changed from 62.51%to 96.19%,S phase decreased from 37%to 3.14%,and there was no significant change in G2/M cells.so it could be inferred that cells were blocked at G0/G1 and cell division was inhibited.Mitochondrial membrane potential decreased to23.45%and ROS level increased to 74.65%.WB showed that the expression of Bax,Cyt-C,Caspase3 and Caspase9 increased by 1.18,1.08,1.16,1.32 times,and the expression of Bcl-2 decreased by 1.03 times.The apoptosis mechanism is mainly through the change of the expression of Bax,Bcl-2 and other proteins,leading to the decrease of mitochondrial membrane potential,activation of Cyt-C,Caspase3,Caspase9 and lysis,and the apoptosis of gastric cancer MGC-803 cells.