Study On The Biological-activity And Structure-activity Relationship Of Phytosterols

Phytosterol exhibits anti-tumor,anti-inflammatory,anti-oxidation effects and can significantly improve body cholesterol levels and regulate immune activity.However,at present,the research on the biological activity of sterols is a single compound as target,and few reports have compared the sterols with different structures.In addition,no research was report in terms of the mechanisms of anti-tumor,anti-inflammatory,cholesterol-lowering and other biological activities of phytosterol.Therefore,in our research,the anti-tumor,anti-inflammatory,cholesterol-lowering and other biological activities of six kinds of common phytosterols(ergosterol,ergosterol ester,?-sitosterol,stigmasterol,campesterol and stellasterol)and its possible molecular mechanism of biological activities was explored.Firstly,the relationship between structure of phytosterol and anti-inflammatory activity was investigated.The inflammatory response model of mouse macrophage RAW264.7 was stimulated by lipopolysaccharide(LPS)and phytosterol was added for pretreatment.The results showed that phytosterols can decrease the inflammatory response symptoms of LPS-induced macrophage model,inhibit cell proliferation activity and reduce phagocytosis of RAW264.7 and the produce of NO and TNF-? in the inflammatory model.Sterol esters can significantly reduce of NO production in the cell supernatant compared to sterol at same concentration,but in phagocytosis activity and TNF-? production,sterols have a stronger effect,which may contribute to the structure of sterols in the C-3 with the hydroxyl and ester groups.In addition,we found that the reduction of NO and phagocytosis are relevant to number of double bond and branched chain configuration,while the inhibition of the secretion of TNF-?is related to the molecular weight of sterols.Lastly the effect of different concentrations of different phytosterols shows that the anti-inflammatory activity of phytosterols is not worked by one factor,it is a complex regulatory process,which may be related to their structure.Then the relationship between structure of phytosterol and antitumor activities was studied.In this research,CCK-8 experiments were performed on several cancer cells(Caco-2,HT-29,SW480,and MCF-7)to screen phytosterols that can effectively inhibit the proliferation of cancer cells,which can provide the theory basis for subsequent experiments.The results showed that the inhibition of 6 kinds of phytosterols were significantly different from the normol group.Ergosterol and ergosterol esters on HT-29 cells and MCF-7 cells had inhibitory effects(P<0.01,P<0.05),indicating that sterols and esters had inhibitory effects on the same cancer cells and the inhibitory effect of sterols on cancer cells was higher than that of esters,which may contribute to the hydroxyl activity of C-3 makes the sterols have better antitumor activity.Inhibitory activity on Caco-2 and MCF-7: ?-sitosterol(one double bond,one alcoholic hydroxyl group)> campesterol(one double bond)> stigmasterol(two double bonds,multiple branches)> stellasterol(two double bonds).Inhibitory activity on HT-29 cells: stellasterol(two double bonds)> stigmasterol(two double bonds,multiple branches)> campesterol(one double bond)> ?-sitosterol(one double bond,an alcoholic hydroxyl group).These results suggested that the number of branched and double bonds of sterols have different roles on inhibiting cancer cells.Lastly,the relation between structure of phytosterol and cholesterol absorption in Caco-2 cells was investigated.The monolayer model of intestinal epithelium was constructed using polarized Caco-2 human colon cancer epithelial cells and the effects of various phytosterols on the absorption of cholesterol in Caco-2 cells were investigated by fluorescence microscope and microplate reader.Furthermore,the effect of phytosterols on the expression of cholesterol absorption transporter NPC1L1 studied by Western Blot experiment,for which to better understanding of the mechanism of action of phytosterols on cholesterol absorption.The results demonstrated that all 6 kinds of phytosterols could significantly reduce the absorption of cholesterol in a concentration-dependent manner.The effect of ergosterol ester was better than that of ergosterol.And the effect of five kinds of sterols on the cholesterol absorption was based on the molecular weight: stellasterol(398)> campesterol(400)> stigmasterol(412)> ?-sitosterol(414),which indicated that the size of molecular weight affects the chosterol-lowering effect of sterols.The effect of NPC1L1 protein was most significant at 200 ?M,and the effect was: ergosterol> ergosterol acetate > Ezetimibe;stigmasterol> ?-sitosterol> Ezetimibe> campesterol> stellasterol,which probably due to the number and position of double bonds of sterols.In this study,the anti-inflammatory and anti-tumor activities,and cholesterol absorption in Caco-2 cells of different phytosterols were investigated and compared.Results showed that the bioactivities of phytosterols were related to their structure and molecular weight.It is a study of plant sterol bioactivity and as a health food additive.It provides a theoretical basis for the development of health foods and drugs,and also provides new perspectives for the construction of small molecule drugs.