Extraction And Purification Of Total Saponins From Ganoderma Lucidun And Their Antioxidative And Tumor Suppressor Activities

Ganoderma lucidum is an ancient precious medicinal material in my country,which has the functions of calming the mind,strengthening the spleen and stomach,nourishing qi and blood,preventing and treating heart palpitations,diabetes and cancer.As one of the important functional components of Ganoderma lucidum,Ganoderma lucidum saponins have the functions of protecting the liver and kidney,inhibiting tumors,scavenging free radicals,and inhibiting bacteria.However,as secondary metabolites,saponins are difficult to separate and purify,so there are relatively few studies on them.Therefore,this study set up two groups of experimental interventions before and after purification,and compared the active effect of the two groups of interventions,and preliminarily studied the antioxidant effect of Ganoderma lucidum total saponins and the effect of inhibiting the proliferation of colon cancer cells.Firstly,the total saponins of Ganoderma lucidum were extracted by ultrasonic-assisted method,and the optimal extraction conditions of total saponins from Ganoderma lucidum were determined by single factor and response surface methodology,that is,the ratio of solid to liquid was 1:54.9 g/m L,the concentration of ethanol was 80.5%,and the ultrasonic time was 62 min extraction conditions,the average Ganoderma lucidum total saponins extraction amount was 7.252 mg/g.The extract was concentrated by rotary evaporation and freeze-dried into powder.At this time,the total saponin content of Ganoderma lucidum before purification was 9.28%.Secondly,the D101 macroporous adsorption resin was used to purify the Ganoderma lucidum total saponins extract,and the optimal purification conditions were determined by dynamic adsorption experiments(sample flow rate: 3 BV/h,sample concentration0.438mg/m L,sample volume 2.5 BV,washing the dehydration concentration is 90%,the eluent flow rate is 3 BV/h,and the eluent dose is 4BV).After the Ganoderma lucidum total saponin extract was purified under optimal conditions,its total saponin concentration increased from 9.28% to 39.91%.Through the ultra-high performance liquid chromatography analysis of the two interventions before and after purification,the proportion of saponins in the total saponin extract of Ganoderma lucidum after purification was significantly higher than that before purification,and other active components were reduced compared with those before purification.Thirdly,by measuring and comparing the in vitro antioxidant activity of Ganoderma lucidum total saponin extract before and after purification,it was found that Ganoderma lucidum total saponin extract had better DPPH free radical scavenging rate,ABTS free radical scavenging rate,hydroxyl scavenging rate and reducing power.And the effect after purification is better than the effect before purification,indicating that Ganoderma lucidum total saponins are antioxidant active substances worthy of development.Finally,the inhibitory effect of Ganoderma lucidum total saponin extract on colon cancer HCT116 cells was determined by CCK8 method,and flow cytometry was used to detect cell apoptosis and cycle,and fluorescence spectrophotometer was used to detect intracellular mitochondrial membrane potential and intracellular reactive oxygen species content,The expressions of apoptosis-related genes p53,noax,bax,bcl-2,caspase-9 and caspase-3 were detected by real-time fluorescence quantitative PCR.The results showed that Ganoderma lucidum total saponin extract could significantly inhibit the proliferation of HCT116 cells,and the inhibitory effect was positively correlated with time.After the intervention of Ganoderma lucidum total saponins,the apoptosis rate was significantly increased,the cell cycle was arrested in G0 phase,the mitochondrial membrane potential in the cell decreased,the content of reactive oxygen species in the cell increased,and the pro-apoptotic genes p53,noax,bax,caspase-9 and the expression of caspase-3 increased,while the expression of anti-apoptotic gene bcl-2 decreased.Therefore,Ganoderma lucidum total saponins have a significant inhibitory effect on colon cancer cells,and the inhibitory effect can be achieved through the mitochondrial apoptosis pathway.To sum up,the author compared and analyzed the in vitro activities of Ganoderma lucidum alcohol-extracted total saponins by setting two groups of experimental interventions before and after purification.Antioxidants and colon cancer suppressor drug candidates or food additives for further study.