| Leaves of Alstonia scholaris(DTY),the dry leaves of Apocynaceae Alstonia trees(Alstonia scholaris R.Br.),mainly contains alkaloids,flavonoids,triterpenoids,and its property are sweet,short,flat.In recent years,DTY is observed to possess anti-tumor,anti-inflammatory,antibacterial,anti-diabetic and hypolipidemic activities.We selected the DTY as the object of research,and got the active components by using modern extraction and separation.We filtered the active components of anti-lung cancer in vivo and vitro and the anti-lung cancer mechanism and the Biological agents properties of the active component were studyed preliminarily.We did the study to promote the further development and utilization of DTY,and to provide a scientific basis for the creating of anti-lung cancer traditional Chinese medicine.This thesis first summarized the research progress on chemical composition,pharmacological effects of DTY.The experiment parts of the research are as follows:1.System composition pre-test and the determination of the representative composition for DTYThis experiment first did the system composition pre-test for DTY and established a method for the determination of the representative composition.The results of experiment showed that:DTY mainly contains alkaloids,flavonoids,and triterpenoids.The content of total alkaloids,total triterpenoids,picrinine,oleanolic acid and ursolic acid was 2.13%,2.45%,0.085%,0.55%,1.80%,respectively.2.Evaluation of the anti-lung cancer activity of different solvent extracts in DTYFirst two cell modes of human lung adenocarcinoma cell line A549 and SPC-A-1 were established,used to evaluate the activity of the different solvent extracts and different solvent parts extracted by using different solvents follow by of DTY on the prevention for lung cancer.The anti-lung cancer activity of 85%ethanol extract was the best,with an IC50 of A549 and SPC-A-1 cells was 275.48μg·mL-1 and 297.03μg·mL-1,less than the international standards of evaluation of drug antitumor activity.Therefore,we selected 85%ethanol as extraction solvent for anti-lung cancer activity screening of DTY.3.Anti-lung cancer in vitro activity screening for the effective components of DTY.85%ethanol extract of DTY was added into macroporous resin column and washed follow by using water,10%ethanol,20%ethanol,30%ethanol,40%ethanol,50%ethanol,60%ethanol,70%ethanol,80%ethanol,95%ethanol.They were named as DTY-water part,DTY-10%ethanol part,DTY-20%ethanol part,DTY-30%ethanol part,DTY-40%ethanol part,DTY-50%ethanol part,DTY-60%ethanol part,DTY-70%ethanol part,DTY-80%ethanol part,DTY-95%ethanol part,respectively.85%ethanol extract of DTY was separated into 10 parts,and the human lung adenocarcinoma cell line A549 and SPC-A-1 were used to do anti-lung cancer in vitro activity screening for the effective components systematically by MTT assay.The result is that DTY-50%ethanol part,DTY-60%ethanol part,DTY-70%ethanol part,DTY-80%ethanol part,and DTY-95%ethanol part had activity.The five parts of A549 cells with the IC50 of the extract is 438.93,58.94,81.70,35.10,89.00 μg·mL-1,respectively;the five parts of SPC-A-1 cells with the IC50 of the extract is 492.34,61.33,87.65,35.51,95.04 μg·mL-1,respectively.Chromogenic reaction,thin layer,UV,and HPLC were used to analyze the composition in various parts.The result was that DTY-50%ethanol part,DTY-60%ethanol part mainly contained alkaloids and DTY-70%ethanol part,DTY-80%ethanol part,DTY-95%ethanol part mainly contained triterpenoids,moreover,the content of alkaloids and triterpenoids in the effective parts has larger correlation with anti-lung cancer activity.4.Preparation and macroporous resin screening of the anti-lung cancer active components in DTYFirstly,we filtered 5 type of macroporous resin such as HPD400A,NKA-9,D101,DA201,and AB-8.Integrated the static adsorption and desorption results of two types of components,we decided to choose the AB-8 macroporous resin as the best macroporous resin.Secondly,combined with the results of in vitro activity screening and component analysis,60%ethanol can separate alkaloids and triterpenoids,so 85%ethanol extract of DTY was added into AB-8 macroporous resin,and the impuritie was washed aways by 30%ethanol.Then the total alkaloids were eluted by 60%ethanol,and finally total triterpenes were eluted by 80%ethanol.5.Study of anti-tumor activity of active components and their compatibility in DTYStudy of anti-tumor activity of total alkaloids,total triterpenes and their compatibility on mice bearing lewis lung-tumor model in vivo and a combination of compatibility and evaluation on the immune response,and cyclophosphamide(20mg/kg/d)was used as a positive control.The inhibition rate of total alkaloids,total triterpenes and their compatibility of high-dose group(15g·kg-1·d-1)was 45.33±9.78%,46.00±15.11%,51.11±12.00%,respectively.The results showed that the two components have good antitumor effect on mice with lewis lung cancer,and the antitumor effect of their compatibility was better than the separate components.Compared with the cyclophosphamide treatment group,the thymus index,spleen index,live index,the TNF-a and IL-6 of total alkaloids,total triterpenoids and their compatibility in high and low dose treatment groups had increased,which indicated that the component of the alkaloids and triterpene components could inhance immune function in mice and have a synergistic effect.6.Preliminary study on the mechanism of the anti-lung cancer active component in DTYThe total alkaloid component(5 mg·mL-1),the total triterpene components(5 mg·mL-1)and the total alkaloids+total triterpenoid components(2.8 mg·mL-1)acted on A549 cell for 48h,and the flow test results were that the apoptosis rate was 30.17%,32.22%,34.45%,respectively.The total alkaloids composition,the total triterpene composition and total alkaloids+total triterpenoid component made the cell cycle arrest at G2/M period,and the blocking percentage is 4.48%,8.28%,10.56%,respectively.The results showed that the alkaloids and triterpene components had same mechanism to the cell cycle,and the two components may have synergistic anti-tumor effect.7.Preliminary study on the biopharmaceutical properties of the active components in DTYThe apparent solubility range of the total alkaloids composition and the total triterpene composition in the pH 2.5～8.0 buffer was 69.49～238.64μg·mL-land 20.76～137.09 μg·mL-1,which showed that the two compositions belong to very slightly soluble or almost insoluble substances.The oil-water partition coefficient logP range of the total alkaloids composition and the total triterpene composition in the pH1.2～8.0 was 1.06～2.11 and 1.78～2.86.logP were all between the range0～3,which speculated that the two compositions had good intestinal absorption.The rat intestinal perfusion model was carried out to study the absorption of picrinine.There was no significant difference(P>0.05)on the intestinal absorption parameter of picrinine based at the concentration of 10～30μmol·L-1,which showed that the intestinal absorption of picrinine was not affected by its concentration,but mainly acted as a passtive diffusion mechanism.The Peff*of picrinine in the total alkaloids composition gradually decreased by the order of duodenum,colon,jejunum,ileum,which is 4.50±0.85,3.69±0.92,2.67±0.64,1.51±0.62,respectively.The Peff*picrinine in total alkaloids and monomer picrinine were compared,and the absorption of picrinine in total alkaloids is slightly better than the monomer picrinine,but it had no significant difference(P>0.05). |
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