The Inhibitory Effect On Laryngeal Cancer In Vitro And Flavonoids Study Of Ethyl Acetate Extracts From Selaginella Doederleinii Hieron

Selaginellae Doederleinii Herba,originated from the dried whole plant of Selaginella doederleinii Hieron.(Selaginellaecae),is a commonly used Chinese herbal medicine and an ethnic drug in many minority areas of the southern China,and is recorded in Chinese material medica standards of some provinces in China.It has the functions of clearing heat,detoxication and anticancer,and is also widely used for the treatment of sore throat,respiratory inflammation,jaundice and the different cancers such as lung cancer,nasopharyngeal carcinoma and liver cancer.The previous work of the research team demonstrated that Selaginellae Doederleinii Herba showed significant activity in anti-laryngocarcinoma,and its ethyl acetate extract(SDO-Et OAC)had stronger effectiveness than the other extract portions.In this thesis,we studied the inhibitory effect of SDO-Et OAC in vitro against laryngeal carcinoma,investigated its antitumor mechanism as well as elucidate its antitumor substances.Furthermore,the contents of total flavonoids and amentoflavone in Selaginellae Doederleinii Herba and five other species of Selaginella plant drugs were determined by UV-Vis spectrophotometry and High Performance Liquid Chromatography(HPLC)method respectively.This provided the reliable methods for evaluating and controlling the quality of S.doederleinii and other species of genus Selaginella.And the appropriate harvest periods of the five plant drugs have been confirmed.In this study,the following methods were applied to evaluate in vitro antitumor activity of SDO-Et OAC against laryngeal carcinoma and clarify the possible action mechanism.The MTT method was used to test inhibitory effect of proliferation of Hep-2 cells treated with different concentrations of SDO-Et OAC at different time;an inverted microscopy was used to observe morphologic changes of Hep-2 cells by the treatment of the drug;flow cytometry method(FCM)was applied to analyze change of cell apoptosis and the cell cycle distribution after them were treated with various dose of SDO-Et OAC;western blot assay was used to measure the protein expression.The results showed that SDO-Et OAC inhibited growth of human laryngeal carcinoma cell line Hep-2 in a dose-dependent and time-dependent manner.The morphological changes reflecting the cell apoptosis could be observed in a reserved microscopy,for example,edge of the cell was irregular,and the number of adherence cell decreased and chromatin was condensation.Flow cytometric analysis presented that SDO-Et OAC could arrest the cells in S phase and induce the cell apoptosis,and apoptosis rate increased with increasing dose.The results of western blot suggested that the mechanisms of Hep-2 cell apoptosis induced by SDO-Et OAC may be associated with down-regulating Bcl-2,up-regulating Bax,activating Caspase-3 and Caspase-9.Nine principles were isolated and purified from Selaginella doederleinii Hieron.by chromatographic methods.Their structures were identified on the basis of spectral data and physicochemical properties.Six of them were elucidated as 7,4′,7′′,4′′′-tetra-O-methylamentoflavone(1),heveaflavone(2),2′′,3′′-dihydroochnaflavone(3),chrysocauloflavone I(6),amentoflavone(7)and delicaflavone(9).The compound(3)was isolated from this plant for the first time.UV-Vis method and HPLC method were applied to detect the contents of the total flavonoids and amentoflavone in Selaginellae Doederleinii Herba and five other Selaginella plant drugs.The results of this experiment showed that the contents were significantly different in the six plant drugs.And the variation tendencies of the total flavonoids and amentoflavone contents from July to October indicated the diversity and complexity of the accumulation of the active ingredients in plants from the same genus.Therefore,the author put forward that it was appropriate to collect S.moellendorffii,S.uncinata and S.remotifolia in summer or in autumn,while S.delicatula and S.heterostachys were suggested to be harvested only in autumn.In conclusion,the research clarified the possible molecular mechanism of the antitumor effect of SDO-Et OAC in human laryngocarcinoma Hep-2 cell in vitro.The results also laid a foundation for further studies in pharmacological activities of biflavonoids,and provided valuable references for controlling the quality of Selaginella plant drugs and exploring or developing novel and efficient medicinal resources.