Studies On The Antitumor Effects And Molecular Mechanism Of Triterpene Acids From Fomes Officinalis Ames

Fomes Officinals Ames is a kind of commonly used drugs by Xinjiang Uygurdoctors as a non-prescription for medical treatment.Uygur doctors consider thatFomes Officinals Ames is dry and hot when used as drug，which could warm thestomach，depress gas and relieve asthma， dispelwind and eliminate dampness，diminish swelling，have diuretic effect and solute snake vonem. According to somestudies， it was found that Fomes Officinals Ames could enhance their strength，extend the time of anti-fatigue and hypoxia tolerance，and could enhance the ability ofbody response to stress. Further analyzing， processing， and purifying FomesOfficinals Ames as well as the in-depth study of the pharmacological activity of activeingredients helped to expand the drug development and utilization prospects.The main ingredients of Fomes Officinals Ames include polysaccharides，saponins and terpenoids，among which triterpenoids content accounts for as high as16%. Former studies mostly focus on the polysaccharides of Fomes Officinals Ames，finding that it can enhance imunity and eliminate oxygen free radicals. However，there are few reports about the pharmacological activity of the triterpene acids fromFomes officinalis Ames both at home and abroad.Firstly，the extrating process of the triterpene acids from Fomes officinalis wasstudied，during which the response surface design method was adopted to optimizethe extracting conditions. The results showed that in the condition of ultrasonic power200W， ultrasonic time30min，ultrasonic temperature40℃， the total triterpeneacids from Fomes officinalis Ames got the highest yield. After that， the responsesurface design method was adopted to optimize the extracting conditions， and theoptimum processing was concluded： ultrasonic power245.2W，extraction time34.5min， extraction temperature50℃. This model predicted total flavonoids was14.89%. Verified tests showed that in the three parallel experiments using the setexperimental conditions， the average yield was14.88%， and had a good fit with thetheoretical predictions，so this model was stable and effective， which had a certainpractical significance. Then silica-gel column chromatography was used to do gradient elution fordiferent ratio of solvent system to purify triterpene acids from Fomes officinalisAmes， and also the qulification test would be conducted to the triterpene acidscompounds with HPLC-MS. Fomes officinalis Ames crude extractings with silica gelsample were eluted with different polar solvent to get Chloroform elution content.Then silica gel column chromatography was employed to different gradient ofChloroform and methanol(V/V100:0；98:2；95:5；9:1；8:2)with TLC to monitor，and integrate the triterpene acid compounds. This method showed that the samplepurity was as high as74.14%，5.13times of that before purified， and productrecovery rate was70.26%. The purified products were analyzed with HPLC-MS，andcomparison of HPLC and MS was made according to related literature. It wascondluded that the three triterpene acids include： Dehydrosulphurenic acid，3-keto-dehydrosulfurenic acid，Fomitopsins C，Fomefficinic acid F，Fomefficinic acidG，dehydroeburicoic acid，Dehydroebu-riconic acid.The third part is to explore the antitumor activity of Fomes Officinals Ames.Total triterpene acids from Fomes officinalis Ames acted on A549cell in nude mice，and it was found that total triterpene acids from Fomes officinalis Ames functionedwell enough to inhibit the growth of tumor. In vitro， A549cell、Hela cell、U937celland HepG2cell were selected respectively， and MTT was adopted to detect theinhibition of total triterpene acids from Fomes officinalis Ames to theabove-mentioned cells. After the result was the IC50was determined: A54929.91μg/mL，U93771.51μg/mL，Hela56.87μg/mL，HepG242.98μg/mL. A549cell，sensitive to drug， was chosen. After total triterpene acids from Fomes officinalisAmes acted on A549cells， the cells would apoptosis with the use of microscope.With FCM， it was also observed that the drug can induce the apoptosis of A549cells.The fourth part of the study is to investigate the molecular mechanism ofanti-tumor effect of total triterpene acids from Fomes officinalis Ames. Whenstimulated by total triterpene acids from Fomes officinalis Ames， human A549cellsproduced reactive oxygen species ROS and superoxide dismutase SOD， in order to determine the role of oxidative stress in tumor cells，the researchers found that thetotal triterpene acids from Fomes officinalis Ames could increase ROS level in humanA549cells， and reduce the content of SOD by means of concentration-dependent.Then it was observed that changes of total triterpene acids from Fomes officinalisAmes in mitochondrial membrane potential and FITC-Annexin V/PI double stainingmethod. It was found that the total triterpene acids from Fomes officinalis Ames couldelevate concentration-dependent mitochondrial membrane of A549cells and induceapoptosis. Using western blot method，it was observed the effect of total triterpeneacids from Fomes officinalis Ames to mitochondrial pathway mediated apoptosis andrelated proteins (Bcl-2，Bax，Cyto C，Caspase-3and activation the expression ofCaspase-3，Caspase-9and activation of Caspase-9) expression，determine the specificmolecular mechanisms of its anti tumor effects.The results showed that totaltriterpene acids from Fomes officinalis Ames could raise the Bax/Bcl-2the proportionby concentration-dependent manner， induced mitochondrial release of cytochrome Cand activation of Caspase-3and caspase-9to induce apoptosis in A549cells. Hence，on the one hand，total triterpene acids from Fomes officinalis Ames inhibited growthof human A549cells was to increase cellular oxidative stress injury，on the other handwas to activate the mitochondrial pathway mediated apoptosis.