| Objective:To explore the extraction process,monosaccharide composition and in vitro antioxidant capacity of Euonymus fortunei var.radicans Sieb polysaccharide by using modern Chinese medicine research methods,high-performance liquid phase,in vitro cell culture and flow cytometry and other molecular biology experimental techniques.The influence of human ovarian adenocarcinoma cells(SKOV3)provides a theoretical basis for the further development of new anti-oxidant and anti-human ovarian adenocarcinoma-related disease drugs with a clear mechanism of action.Methods:(1)Through the single factor test,the best material-liquid ratio,the best extraction temperature,the best ultrasonic power,and the best extraction time of Euonymus fortunei var.radicans Sieb.polysaccharide extraction were obtained,on this basis,the extraction rate of polysaccharides from Euonymus fortunei var.radicans Sieb.was evaluated,and the influencing factors were the material-liquid ratio,extraction temperature,ultrasonic power and extraction time.The orthogonal test L9(3~4)method was used to optimize the extraction process.Under these conditions,the Euonymus fortunei var.radicans Sieb.polysaccharide was purified with AB-8 macroporous resin.(2)Passed the precision,repeatability,stability and recovery test,investigate the analytical method of high performance liquid chromatography before derivatization of the column PMP,and use this method to analyze the composition of polysaccharides of Euonymus fortunei var.radicans Sieb.(3)By examining DPPH free radical scavenging rate,superoxide anion free radical inhibition rate,reducing power,and hydroxyl radical inhibition rate of Euonymus fortunei var.radicans Sieb.polysaccharide,and comparing with VC,BHT,Lycium barbarum polysaccharide and Astragalus polysaccharide.(4)The effect of Euonymus fortunei var.radicans Sieb.polysaccharide on the proliferation of human ovarian adenocarcinoma(SKOV3)cells was detected by CCK-8,and the flow cytometry technique was used to detect the cycle effect of Euonymus fortunei var.radicans Sieb.polysaccharide on SKOV3 cells.Result:(1)The optimum extraction process conditions for Euonymus fortunei var.radicans Sieb.polysaccharide are:extraction temperature 80℃,ultrasonic power 144W,material-liquid ratio 1:60(g/m L),extraction time 75min,and extraction times 2 times.Under the optimal extraction conditions,the extraction rate of polysaccharide from Euonymus fortunei var.radicans Sieb.was 2.627%.Polysaccharide purified by AB-8 macroporous resin is 53.47%.(2)After investigation,it was found that the precision,repeatability,stability and recovery of HPLC analysis method of PMP pre-column derivatization were high.According to the analysis of this method,it is known that Euonymus fortunei var.radicans Sieb.polysaccharide is composed of D-galactose,D-glucose,D-arabinose,D-mannose,L-fucose,and L-rhamnose.39.460,21.938,5.085,3.486,and 0.610mg/g,of which the content of D-glucose is the highest(39.460mg/g),and the content of L-rhamnose is the lowest(0.610mg/g).The content of the monosaccharide in Euonymus fortunei var.radicans Sieb.polysaccharide was converted into a molar ratio of D-galactose:D-glucose:D-arabinose:D-mannose:L-fucose:L-rhamnose was21.82:64.41:42.97:8.29:6.24:1.(3)Through in vitro antioxidant test,it was found that in the concentration range of 0.1～1mg/m L,DPPH free radical inhibition rate comparison,Lycium barbarum polysaccharide>Astragalus polysaccharide>Euonymus fortunei var.radicans Sieb.polysaccharide≈BHT;superoxide anion free radical inhibition rate comparison,VC>Euonymus fortunei var.radicans Sieb.polysaccharide>Lycium barbarum polysaccharide≈Astragalus polysaccharide;reducing power comparison,BHT>Euonymus fortunei var.radicans Sieb.polysaccharide>Astragalus polysaccharide≈Lycium barbarum polysaccharide.In the range of the solution concentration of 0.05～0.4mg/m L,the hydroxyl radical inhibition rate was compared,VC>Euonymus fortunei var.radicans Sieb.polysaccharide>Astragalus polysaccharide≈Lycium barbarum polysaccharide.polysaccharide has high DPPH free radical scavenging activity(47.12%,1mg/m L)and superoxide anion free radical scavenging activity(29.59%,1mg/m L),reducing power(absorbance OD value A700nmis 1.081,1mg/m L)and hydroxyl radical scavenging activity(59.38%,0.4mg/m L;IC50:0.3204mg/m L).(4)According to the CCK-8 detection technology,Euonymus fortunei var.radicans Sieb.polysaccharide can inhibit the increase of SKOV3 cells,and It is positively correlated with polysaccharide concentration and incubation time.When SKOV3 cells were cultured in 1000μg/m L Euonymus fortunei var.radicans Sieb.polysaccharide culture medium for 96 h,the highest value-added inhibition rate was 72.61%.The effects of polysaccharides on the cycle of SKOV3 cells were detected by flow cytometry.SKOV3 cells were cultured with polysaccharides for 72 h,which significantly affected the cell cycle.Compared with the control group,the number of G0/G1 cells increased significantly,and the difference was statistically significant(P<0.05).Conclusion:Under the optimal extraction process conditions of extraction temperature 80℃,ultrasonic power 144 W,material-liquid ratio 1:60(g/m L),extraction time 75 min,extraction number 2 times,the extraction rate of Euonymus fortunei var.radicans Sieb polysaccharide is 2.627%,after AB-8macroporous resin Purification purity is 53.47%;the Euonymus fortunei var.radicans Sieb polysaccharide contains 6 monosaccharides of D-galactose,D-glucose,D-arabinose,D-mannose,L-fucose and L-rhamnose,molar ratio It is21.82:64.41:42.97:8.29:6.24:1;after purification,the Euonymus fortunei var.radicans Sieb polysaccharide has strong antioxidant capacity in vitro,which can inhibit the proliferation of SKOV3 cells and increase the number of G0/G1cells,it shows the inhibitory effect on the proliferation of human ovarian adenocarcinoma cell line SKOV3,and can block the division of human ovarian adenocarcinoma cell line SKOV3 in the G0/G1 phase. |
PDF Full Text Request