| Sanghuangporus baumii is a kind of rare fungus that has a very important medicinal value.In recent years,S.baumii was found to have many medicinal functions,such as antitumor,enhancing the immunity of organism,anti-inflammatory,anti-aging,lowering blood glucose,preventing the liver damage and repairing damage.The current researches found that the pharmacological active substances in S.baumii were polysaccharide,flavonoids,pyrone,steroids,and triterpenoids compounds.Tumor is one kind of diseases that seriously affect the health,even lead to death,and it is also the disease that can not be cured.At present,Chinese herbal medicine is becoming the new materials that find new antitumor drugs and the development and utilization of Chinese herbal medicine's antitumor activity is becoming a hotspot of antitumor research.In this study,Total triterpenoids was separated and purificated from S.baumii,and the antitumor activity of total triterpenoids and its mechanism was studied with four kinds of tumor cell(human breast cancer cells MCF-7,human lung cancer cell A549,human colon cancer cell Caco-2 and human cervical cancer cell Hela).Some of the experimental datas and theoretical basis about the research of screening new antitumor drugs from Chinese herbal medicine and the antitumor mechanism of Chinese herbal medicine were provided by the study.The main content and results were as follows:1.Total triterpenoids was extracted from S.baumii with the method of extraction and ultrasound.The extraction process was optimized by Response surface method(RSM).The optimized extracting conditions were as follows:the volume fraction of ethanol was 70%,the temperature and time of ultrasonic was 60oC and 40min,the liquid to solid was 20:1,the ultrasonic power was 240w,respectively.The yield of total triterpenoids extracted with these conditions was 9.83 mg/g.2.The purification of total triterpenoids extracted was completed with macroporous adsorption resin.Four types of adsorption resin(HPD-100,AB-8,X-5 and D-101)were used in the experiment and the conditions of static adsorption and elution were optimized.The optimized purification conditions were as follows:the macroporous adsorption resin was D101.In the dynamic adsorption:the pH,concentration,flow rate and volume of the sample were 6.0,1.0 mg/mL,3BV/h and 4BV,respectively.The eluent were distilled water(3BV),20%of ethanol(2BV)and 70%of ethanol(4BV)in turn,the flow rate of each eluent was 3BV/h,and last eluent(the 70%of ethanol)was collected.The purity of purified total triterpenoids was 22.49%and nearly five times higher than that before purified.3.The inhibition of total triterpenoids on the proliferation of these four kinds of tumor cell was tested with MTT method,CASY cell analyzer and flow cytometry analyzer.The result showed that the proliferation of these cells was significantly inhibited by total triterpenoids.The minimum inhibition concentration of total triterpenoids for Hela cell and Caco-2 cell was 0.2mg/mL,for MCF-7 cell and A549 cell was 0.3mg/mL,and for these four kinds of cells,the minimum treated time was 48h.The concentration of total triterpenoids is higher,the inhibition on the proliferation of four kinds of tumor cell is stronger.4.The effect of total triterpenoids on the morphology of these four kinds of tumour cell was detected by optical microscope,laser confocal microscope and electron microscope,the result showed that cells treated with total triterpenoids were appeared abnormal morphology,such as growth abnormalities,irregular shape,abnormal proliferation,appeared white spot in the cells;cell volume smaller,cytoplasm condensed;nuclei chromatin coiling appeared many cavity structure called cavitation in the cells,appeared cavity like apoptosis body in some cells,part of the chromatin condensed state,highly condensed chromatin,marginalization;the nuclei were corrugated or in creasing,and appeared a few apoptotic body;The effect of total triterpenoids on the ectropion of cell membrane and membrane potential energy of the mitochondrial membrane in these cells was detected with phosphatidylserine valgus mitochondrial membrane potential detection kit,respectively,the results showed that the phosphatidylserine was turned to outside and the membrane potential energy of mitochondrial membrane was reduced in cells treated with total triterpenoids.The effect of total triterpenoids on the integrity of DNA in these cells was detected with DNA Ladder detection kit,the results showed that the integrity of DNA was destroyed in cells.Comprehensive results showed that the apoptosis of these cells was induced by total triterpenoids.5.The effect of total triterpenoids on the mitochondrial apoptotic pathways and endoplasmic reticulum mediated apoptosis pathway of these tumor cells was detected.The expression,subcellular localization and interaction of proteins related mitochondrial apoptotic pathways and endoplasmic reticulum mediated apoptosis pathway were detected by quantitative real-time fluorescent PCR and protein immunoblot,protein subcellular localization,and protein immune coprecipitation,respectively.The results showed that on the one hand,the expression of the promote apoptosis gene related mitochondrial apoptotic pathways(p53,Bax,Cytc,Apaf1,caspase-3 and caspase-9)was significant increased;the apoptosis suppression gene related mitochondrial apoptotic pathways(Bc1-2)was significant reducted in cells treated with total triterpenoids.In addition,the aoptosis process(The Cytc was released from the double membrane of mitochondria into the cytoplasm,combined with Apaf-1 and formed an aoptosis complex contains Apaf-1,Cytc,caspase-9,activated caspase-3.)was significant increased in cells treated with total triterpenoids.On the other hand,the expression of the gene related endoplasmic reticulum mediated apoptosis pathway(Grp78,Ire1-?,TRAF-2 and caspase-12)was significant increased.In addition,the aoptosis process(The Ire1-? separated with Grp78,and then combined with and activated TRAF-2,the activated TRAF-2 enabled a large number of caspase-12 and formed an aoptosis complex.)was significant increased in cells treated with total triterpenoids.These results suggested that total triterpenoids could induce the apoptosis of these tumor cells by activating both mitochondrial apoptosis pathway and endoplasmic reticulum mediated apoptosis pathway. |
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