| Prostate cancer is one of the malignant tumors that are seriously endangering the world's male health,the incidence of it increases with age,but there is no effective treatment for androgen independent prostate cancer at present.The sea buckthorn is Elaeagnus acid thorn shrubs or small trees,it is widely used as a dual-purpose of drug and food in our country.In this paper,we use the seabuckthorn fruit residue,optimizing the total flavonoids extraction from residue by response surface,and purifying the crude extracts,then screening out the purified samples which have inhibitory effect on the proliferation of human prostate cancer PC-3 cells in vitor by MTT method,and elaborating the possible mechanism at last.The research conclusions are as follows:(1)With the extraction rate of total flavonoids as index,extraction process of total flavonoids from residue was analysised by response surface,The final optimization conditions is:The extraction temperature is 60?,the ratio of material to liquid is 1:16,and the volume fraction of ethanol is 65%,the extraction rate of total flavonoids reach 111.75%±2.64%under this condition.(2)The crude extracts of flavonoids from seabuckthorn residue were purified by AB-8 macroporous resin,the total flavonoids,quercetin,isorhamnetin,kaempferol content were determined by colorimetry and HPLC methods,the results suggest that AB-8 can enrich the flavones effectively,the content of total flavonoids in S30 samples was the highest,up to 47.04%,and the S50 samples were 25.41%.The main composition of flavonoids from seabuckthorn residue are quercetin,isorhamnetin and kaempferol flavonoid glycosides,these three flavonoid aglycones content increased significantly after hydrolyzed,among them,the content of quercetin and isorhamnetin in SH30 hydrolysis sample were 2.98%,3.90%,and in SH50 quercetin,isorhamnetin and kaempferol content were 5.03%,19.54%,0.43%respectively.(3)The PC-3 cell model of androgen independent prostate cancer in vitro was used to screen samples of proliferation inhibition of PC-3 cells by MTT,the results suggest that the effect of S10,S30,S50 and SH30 on the inhibition of PC-3 cells in vitro are poor and are not ideal active samples.SH50 sample had strong inhibitory effect on PC-3 cell proliferation in vitro,and the half inhibitory concentrations of PC-3 cells at 48 h and 72 h were 43.09 ?g/mL and 33.16 ?g/mL respectively.(4)It suggests that quercetin and isorhamnetin are the main factors of SH50 in inhibiting the effect of PC-3 cells in vitro compared these three samples' inhibitory effects.(5)Cell scratch and cell morphology tests suggest that the lateral migration ability of cells can be significantly reduced after the action of 25 ?g/mL SH50 on PC-3 cells 24 h,and the cells can show obvious characteristics of apoptosis after the action of SH50.(6)The effects of SH50 on apoptosis,cell cycle and expression of related proteins in PC-3 cells are tested by flow cytometry and Western blot,the results show that SH50 could effectively increase the apoptosis rate of PC-3 cells,and have a strong effect on inducing cell apoptosis.And the cell cycle can be blocked at G2/M stage,which affect DNA synthesis and delay the cell cycle,thus inhibiting the proliferation of PC-3 cells.At the same time,SH50 can inhibit the proliferation of PC-3 cells by up regulating the expression of Bax protein and down regulating the expression of Bcl-2 protein.To sum up,the SH50 sample obtained from the crude extracts of flavonoids from Seabuckthorn residue has a strong inhibitory effect on the proliferation of PC-3 cells in vitro,it is presumed that the possible mechanism of action is to induce apoptosis,block cell cycle in G2/M stage and regulate the expression of Bax and Bcl-2 protein.This provides a scientific basis and theoretical guidance for improving the comprehensive utilization of Seabuckthom fruit residue and developing the anti prostate cancer products of Seabuckthorn. |
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