Study On Chemical Constituents Of Omphalia Lapidescens Schroet. And Their Cytotoxicities

Objective:To investigate the cytotoxicities of the Omphalia lapidescens Schroet.,chemical constituents of EtOAc extracts of Omphalia lapidescens was separated and identified,and their cytotoxic activities was valued.Methods:1.Chromatographic separation techniques such as silica gel chromatography,Rp-18 chromatography and Sephadex LH-20 were used for the isolation and purification.The structures of the chemical constituents were identified on the basis of mass spectrometry,NMR spectroscopy techniques.2.MTT assay was used to detect the inhibitory effect of EtOAc extracts of Omphalia lapidescens Schroet.on different cancer cell lines?HGC-27,A549,SMMC-7721,MCF-7,HCT-116,Hela?and one normal cell line?LO₂?,then calculate the IC₅₀0 filtrating the most sensitive one.3.The cytotoxic activities of all isolates against human gastric cancer cell line HGC-27 were determined by a colorimetric MTT assay;Hoechst 33342 fluorescence staining was used to visualize motphology changes of nuclear DNA in live cells;Western Blot was used to detect the protein expressions of Bax,Bcl-2 and Caspase 3in HGC-27 cells.Results:1.Study on the chemical components of Omphalia lapidescens Schroet.Twenty-seven compounds were isolated from Omphalia lapidescens,including three new steroids?22E,24R?-9?,11?-epoxyergosta-7,22-diene-3?,5?,6?-triol?1^*?,?22E,24R?-9?,15?-dihydroxyergosta-4,6,8?14?,22-tetraene-3-one?2^*?and?22E,24R?-ergosta-7,9?11?,22-triene-3?,5?,6?-triol?3^*?.2.The antitumor activity of EtOAc extract and steroid compounds from Omphalia lapidescens in vitro.The ethyl acetate fraction of Omphalia lapidescens extract can inhibit the proliferation of tumor cell lines HGC-27,A549,SMMC-7721,MCF-7,and HCT-116.The human gastric cancer cell HGC-27 showed the highest sensitivity to drugs in all cells.The IC₅₀0 value was 33.41?g/mL.The inhibitory effect of Hela on human cervical cancer cells was not shown.It also had a certain inhibitory effect on the normal liver cell LO₂.The cytotoxic effects of the isolated steroids were evaluated by an MTT colorimetric assay using the HGC-27 human gastric cancer cell line and A549 human lung cancer cell line.The general structure-cytotoxicity relationships were summarized.It can be deduced that in 3,5,6-trioxygenated?⁷-ergosteroids the orientation and the etherification of OH-6 have a significant impact on cytotoxicity;acetylation of OH-3 can increase the cytotoxic activity.As for the subtype of ergosteroids?3,5,6,7-tetraoxygenated ergosteroids?,the presence of?^8?14?had no significant effect on the cytotoxicity,but epoxidation of?^8?14?resulted in a significant loss of the inhibitory effect.Among all the compounds,compound 10 had the most obvious inhibitory effect on HGC-27 cell line with IC₅₀0 values 4.17±0.48?M.After treated with compound 10for 24 hours,the cells showed morphological changes characteristic of apoptosis,including chromatin condensation,phase bright nuclear fragmentation and apoptotic bodies could be found.Results on Western Blot showed that compound 10dose-dependent dowm-regulated the expression of pro-Caspase 3.It suggested that the inhibitory effect of compound 10 on HGC-27 may be related to the induction of apoptosis.Conclusion:1.The compounds isolated from the ethyl acetate extract of Omphalia lapidescens extract were mainly steriods,including three new steroids.2.The Omphalia lapidescens extract part of ethyl acetate can inhibit the growth of a variety of tumor cell lines with the different intensities,of which the most sensitive to human gastric cancer cells.The cytotoxic activity in vitro of components showed that those compounds have a certain structure-cytotoxic relationship in human caner cell line.3.The compound 10 isolated from Omphalia lapidescens has the most obvious inhibitory effect on HGC-27 cells.The morphology of the cells was observed by fluorescence staining.The results showed that compound 10 could produce apoptotic bodies andinduce apoptosis.Western Blot results showed that it could reduce the protein expression of pro-Caspase 3 in the cells.It may be related to the regulation of Caspase 3,which induced apoptosis.