Evaluation Of Activities In Vitro And Synthesis Of Hydroxytyrosol Prodrug

Objectives The prodrugs of hydroxytyrosol were designed, synthetized and studied on the antineoplastic activity and the induction of apoptosis of tumor cells. We also investigated whether the prodrug can release the active drug throughout the incubation under the effect of tumor cells, providing the scientific evidence for vivo study in the future.Methods 1 The prodrug of hydroxytyrosol was completed with hydroxytyrosol as the raw material, after esterification, reacted with p-toluenesulfonyl chloride,methanesulfonyl chloride and nitrophenyl sulfonyl chloride. respectively 2 The effectiveness of different prodrugs inhibiting glioma cells C6, lung cancer cells A549 and colon tumor cells HT-29 was detected by CCK-8. 3 With different concentration of the selected prodrug B1 and B2 on C6 cells for 24 hours, the change of morphologic was observed by invert microscope. 4 After incubating of B1 and B2 on C6 cells for 24 hours,using hoechst33342-PI double staining to observe the change of morphologic by inverted fluorescence microscope. 5 After incubating of B1 and B2 on C6 cells for 24 hours, the changes of cell cycle were analyzed by flow cytometry. 6 After C6 cells were treated with prodrug B1 and B2, the cells cultured medium and cells was collected and extracted by ethyl acetate, then the extracted solutions were analyzed by HPLC.Results 1 The preparation of target compounds was completed and it was demonstrated by light spectrum according to a predetermined synthetic methods. 2 The CCK-8 results showed that the anti-tumor effect of the prodrug B1 and B2 is the best among the 8 kinds of prodrug on glioma cells C6, lung cancer cells A549 and colon tumor cells HT-29.Compared with HT, B1 and B2 also has better inhibition rate. In three kinds of tumor cells, C6 cells were most sensitive to B1 and B2, and the effect of anti-tumor is dosedependent. However, the inhibition rate of B2 reached more than 80% at the concentration of 250umol/L, and there was no significant change on the inhibition rate with the increase dose of B2, When the concentration at 1000umol/L, the inhibition rate was decreased modestly. 3 After different concentration of the prodrug B1 and B2 on C6 cells for 24 hours, the invert microscop results showed that the cells of experimental group became round, more and more cells died. With the increasing dose, the cells became sparse and appeared cell debris. 4 Under the inverted fluorescence microscope,early apoptotic cell with bright blue fluorescence were observed in experimental groups.With the increasing dose, experimental group showed necrotic and late apoptotic cells with red fluorescence. 5 B1 and B2 arrested C6 cells in G1 phase to induced apoptosis by flow cytometry. 6 The HPLC assay showed that hydroxytyrosol prodrug of B1 and B2 on C6 cells after 24 h can release active drug HT. But the amount was very small, as well as the proportion of HT accounted for B1 and B2.Conclusions With hydroxytyrosol as the active drug, through the esterification,research work in this thesis designed and synthetized three series total 8 componds of hydroxytyrosol prodrug. Then in vitro tumor cell proliferation inhibition test selected two kinds of prodrug B1 and B2, which have strongest anti-tumor activity. And through arresting C6 cells in G1 phase to induced apoptosis. Finally, the HPLC assay showed that hydroxytyrosol prodrug of B1 and B2 can release active drug HT, provides a reference for further study in vivo.