Study On Effective Substance Basis And Action Mechanism Of Tripterygium Hypoglaucum (Levl.) Hutch Based On Activity Of Anti-multidrug Resistance Cancer

The work of this dissertation is to select Chinese medicinal active compounds which have the structure of unsaturated lactone ring or α,β-unsaturated ketone,and to measure the anti-multidrug resistance tumor activity for the selected compounds.According to the activity screening result,to choose Tripterygium hypoglaucum as the subject of studies on therapeutic material basis.This further study are investigate the mechanism of triptolide against multidrug resistance lung cancer cells in vitro and finish the isolation and identification of ingredients from Tripterygium hypoglaucum.This dissertation is divided into four chapter and the contents are summarized as follows:Chapter 1.literature researchThe relevant literature was summarized,and a systematic review was given about advance in studies on the activity reverse or against MDR cancer of chemical ingredients from Chinese herb medicine and research progress on chemical constituents,pharmacological effects,and clinical application of Tripterygium hypoglaucum.Chapter 2.Activity screening of active ingredients in traditional Chinese medicine against multidrug resistance cancer in vitroTo select 3 MDR cancer cells(MCF-7/Adr、Bel7402/5-Fu、A549/Taxol)and 13 Chinese medicinal active compounds(Puerarin,scutellarin,Osthole,Psoralen,Digoxin,Ginsenoside Rg3,Emodin,Curcumin,Triotolide,10-Hydroxy camptothecin,Podophyllotoxin,Oxymatrine,Annonaceous acetogenins)and then MTT method is applied to test anti-MDR cancer activity in vitro.Result revealed that 10-Hydroxy camptothecin and triptolide exhibit significant inhibitory activity for 3 MDR cancer cells,Digoxin and Annonaceous acetogenins also exhibit good activity but for human lung MDR cancer cell,and activity of others are not very obvious.Chapter 3.The action mechanism of Triptolide’s inhibitory effect against A549/TaxolPrevious research showed that triptolide exhibits good inhibitory activity against human lung multi drug resistance cancer cell A549/Taxol,research on the action mechanism of it has not been reported,therefore the preliminary study on its mechanism will provide a reference for development and utilization of triptolide in the future.Flow cytometry was used to determine the cell apoptosis and the cell cycle distribution,and the expression of cleaved-Caspase-3,cleaved-Caspase-9,Bcl-2,Bax were also determined after treatment with triptolide.To investigate the effect of JNK,p38 MAPK,ERK1/2 and AKT on triptolide inhibits A549/Taxol cells proliferation after co-treatment with inhibitors of MAPKs kinases,and the expression of p-ERK1/2c,p-JNKc,p-P38 MAPKc,p-AKT,and p-GSK 3 p were also determined by Western blot assay,then make sure that MAPKs pathway whether involved in the course of triptolide induces apoptosis on A549/Taxol.The expression of P-gp,MRP and LRP were also detected to figure out whether triptolide could inhibit its activity,and then to exhibit reversal activity.Results revealed that ERK and JNK pathway maybe involved in cell apoptosis and cell cycle arrest induced by triptolide on A549/Taxol.Triptolide was found to be able to inhibit proliferation on A549/Taxol cells in dose-dependent manner,with an IC50 of 46.47±0.31nM after 48 h treatment with triptolide,so we chose these two concentrations(0.025,0.05μM)to study in next researches.The result of MTT assay found that SB202190 exerted little effects on the result,while SP600125 and U0126 significantly reinforced the inhibitory effects of triptolide.Since there was no significant effect observed by the addition of SB202190,it was suggested that the modulation of p38 contributed little to the inhibitory effect displayed by TPL on A549/Taxol cells.SP600125 exhibited synergistic effects with TPL,while antagonism caused by U0126 was also noticed.These findings suggested that TPL may exert its inhibitory effects by regulating JNK and ERK signaling pathways.The result of cell phase distribution by flow cytometry revealed that treatment with triptolide induced a strong S-phase arrest.A549/Taxol cells’ apoptosis rate were 8.50%and 24.45%,respectively after treatment with triptolide in concentration of 0.025、0.05μM,but 3.48%in control group.We also found that triptolide could significantly increase the expressions of cleaved-caspase-3,-9,and the up-regulation of the ratio of bax/bal-2 after TPL treatment was observed,which supported our previous claim concerning the role of pro-apoptosis.Meanwhile,the key proteins of MAPKs and AKT pathway were also determined by WB assay,the results indicated that triptolide induces A549/Taxol cells apoptosis maybe through up-regulationg of ERK pathway and down-regulation of JNK and AKT pathway.Chapter 4.Basic material research of Tripterygium hypoglaucumTo choose triptolide as the target compound which has best anti-MDR cancer activity,according to the preliminary study of activity screening.Since content of triptolide is high in Tripterygium hypoglaucum and few related researchs about it,it was selected to chemical ingredients isolation in next research.The roots of Tripterygium hypoglaucum were extracted with 950 ethanol,and the ethanol extract was fractionated by dichloromethane,ethyl acetate and methanol,and the activity of 3 fractions were tested on human lung cancer cells A549/Taxol,and then the active fraction was determined.Silica gel column chromatography,Sephadex LH-20 column chromatography and reversed-phase chromatography technology were used to chemical separation and purification of Tripterygium hypoglaucum’s active fraction,and structures of compound were identified by their physicochemical properties and spectral data.Activity screening results indicated that the cytotoxicity of dichloromethane,ethyl acetate and methanol fraction against A549/Taxol cells,with IC50 of 16.02±0.75μg/ml,72.60±2.51μg/ml and 245.8±10.12μg/ml,respectively.The dichloromethane fraction which is major active fraction was choosed to subsequent isolation.6 chemical constituents were obtained and identified as β-sitosterol,3’-geranyl-5,7,2’,5’-tetrahydroxyisoflavone,3-(2’-methoxyl-4’,5’-dyhydroxy)-phenyl-5,7-dimethoxy-6-(3-methyl-2-butenyl)-benzopyrans,β-sitosteryl palmitate,Triptolide,docosanoic acid 2,3-dihydroxypropyl ester and 2-(5’,7’-diethoxy-2’,2’-dimethyl-2H-benzopyran-6’-)-3-aldehyde-5,6-dimethoxy-benzofuran.