Studies On The Chemistry And Antineoplastic Activities Of Stetoidal Glycosides From The Root Of Cynanchum Auriculatum

Objective Purified the steroidal glycoside constituents of anti-tumor effect of CGA,analyzed its chemical constitution,which provided a sicentific basis for developing of anti-tumor drugs.Methods①Purification of acid hydrolysis production the use of weak acid hydrolyzes CGA, Further purified constituents by silica gel column chromatography , then Isolated from production of the acid hydrolysis by HPLC.②Anti-tumor effects of acid hydrolysis production in vitro, MTT determination of acid hydrolysis products on PC3 (prostate cancer), SGC-7901 (human gastric cancer cell), Hela (human cervical carcinoma cells), NB4 (human leukemia cells), HEPG-2(human hepatocellular carcinoma cells) and B16 (mouse melanoma cells) had cytotoxic. Evaluate acid hydrolysis production on the toxicity of different tumor cell selectivit. Using cell growth curve observe inhibition of hydrolysates on tumor cells. Preliminary judgment hydrolyzate is inhibition or cytotoxicity mainly antitumor. Use Morphological observe cytotoxicity ingredients on tumor cells. Detection of apoptosis of tumor cells by Flow cytometry and Fluorescence cytometry, determine the mechanism of apoptosis of cancer cells initially.③Identify structure of acid hydrolysis productions. Understand zhe Chemical Structure of acid hydrolysis productions byIR, HR-ESI-MS.1H NMR,13C NMR,DERT,HMBC,MQC.Results1. Obtained two hydrolysates by purified acid hydrolysis production of CGA, Compounds landⅡ.2. The results indicated by MTT method:CompoundsⅠandⅡon the six tumor cells have different levels of inhibition, And a dose dependence, CompoundsⅠIC50 of six tumor cells were: PC3:8.5μg/ml,SGC-7901:29.4μg/ml,Hela:27.7μg/ml,NB4:8.9μg/ml,HEPG:17.7μg/ml,B16:15.8μg/ml. CompoundsⅡIC50 of six tumor cells were: PC-3:11.7μg/ml,SGC-7901:28.5μg/ml,Hela:16.75μg/ml,NB4:10.7μg/ml,HEPG-2:19. μg/ml,B 16:9.1μg/ml.3. Cell growth curve show:the different concentrations of compoundⅠandⅡwere treated tumor cells were inhibited, And the concentration and time-dependent rendering.For PC3, CompoundsⅠ,Ⅱconcentration was 20.0μg/ml,52,76,100,124 hours on the inhibitory rates were:20.7%,28.6%,41.7%,58.9% and 22.7%,32.6%, 45.4%,54.9%.For SGC-7901, CompoundsⅠ,Ⅱconcentration was 20.0μg/ml, 52,76,100,124 hours on the inhibitory rates were:21.5%,30.8%,40.6%,57.2% and 10.82%,21.16%,36.9%,44.2%.For Hela, CompoundsⅠ,Ⅱconcentration was 20.0μg/ml,52,76,100,124 hours on the inhibitory rates were:18.3%,25.7%,38.5%, 48.3% and 16.9%,28.8%,42.4%. For HEPG-2,CompoundsⅠ,Ⅱconcentration was 20.0μg/ml,52,76,100,124 hours on the inhibitory rates were:20.3%,29.5%,40.6%, 52.7% and 24.7%,32.6%,48.9%,65.8%.4. Morphological observations:After compoundⅠ,Ⅱfor 3 days at a certain concentration of tumor cells and tumor cells compared with normal group, Cell density decreased significantly more,adherent cells rounded smaller,part of the suspension, cell shrinkage, cell debris visible under the microscope, also increased.5. Fluorescent color display:under the microscope,can see the control cell membrane integrity and nuclear morphology are the side together, uniform nuclear staining.Treatment PC3 with a certain concentration of compoundsⅠandⅡ, PC3 volume reduce, membrane shrinkage, nuclear condensation smaller, darker staining, chromatin uptake, enhanced fluorescence, broken into fragments of different sizes, and changes in cells under different concentrations under the same conditions showing a different state.6. Flow cytometry:compared with normal cells, zhe apoptosis rate with a certain concentration of compoundsⅠandⅡwas significantly higher than normal cells, Apoptosis according to the different concentrations of different changes, within acertain time, the apoptosis rate increases with increased concentration gradient.7. The chemical structure of CompoundsⅠandⅡwere:caudatin, caudatin3-O-β-D-cymarapyranosyl-(1→4)-β-D-cymarapyranosyl-(1→4)-β-D-cymarapyranoside.Conclusion:1. Hydrolyzate of CGA have cytotoxicity for a variety of tumor cells in vitro.2. Understand compoundsⅠ,Ⅱmechanism of inhibition of tumor cell proliferation. 3. CompoundⅡwhich use of acid hydrolysis were first isolated from the plant...