| This thesis is divided into five chapters.Chapter Ⅰ Literature review.Triphala is made of Terminalia chebula Retz.,Terminalia bellirica(Gaertn.)Roxb.and Phyllanthus emblica L.The three fruits are often used together,or they are used as main drugs in SanguoTang powder,SanguoTang and other forms.So they are often called Da Sanguo.Thiphala was introduced from India to the Sui and Tang dynasties,the first absence was in the"four medical",attending the plague,disorder fever,old and new fever,prompting hot forming and in 1995 it was contained in Tibetan medicine ministerial standard.It has clear effects on heat,harmonizing qi and blood,dissolving the bad blood,clinically being used to treat sore throats,cough,asthma,indigestion,anemia,liver dysfunction and cardiovascular disease and used as the basis of many prescriptions in Tibetan medicine.In traditional Indian medicine,the proportion of prescription compatibility and dosage of triphala was determined as 1:1:1.While the proportion in the Tibetan medicine was 300 g of Terminalia chebula Retz.,200 g of Terminalia bellirica(Gaertn.)Roxb.and 240 g of Phyllanthus emblica L.and specially emphasized the nuclear processing.According to the results of chemical composition analysis,triphala contains tannins,organic acids,triterpene and other kinds of chemical compositions.The pharmacological action and clinical application research showed that the triphala had the effects of scavenging free radicals,antioxidation,anti-inflammatory,immune regulation,stimulating appetite,pain,antibacterial,anti mutagenesis,guaiac,antitumor,anti-stress,falling blood sugar,liver protection,chemical protection,radiation protection,promoing the effects of chemotherapy.Among them its anti-tumor effect is excelent,which can be divided into antitumor activity in vivo and in vitro,chemotherapy protection,radiation protection,and chemical prevention and so on.Its anticancer mechanism is associated with inducing apoptosis of tumor cells,activating p53 and ERK pathway,and inhibiting DNA synthesis of cancer cells.It was reported that the tannin component as the main chemical composition of triphala has good anti-tumor effects in vivo.For example,Corilagin can inhibite the growth of ovarian cancer cell(A2780),nasopharyngeal carcinoma cell(KB),osteosarcoma cells(OS-732)and human colon cancer cells(HCT-8)in vitro.The IC50 values were 0.25 μg/ml、1.2 μg/ml、7.42 μg/ml and 9.08 μg/ml.It also showed a significant inhibitory effect on the tumor cell lines in transplanted mice and nude mice.The Chebulinic acid also showed good inhibition of proliferation in vitro on human breast cancer cells(hct-15,COLO-205),human breast cancer cells(mda-mb-231),human prostate cancer cells(du-145)and human leukemia cells(K562).The IC50 values were 20.3 μM,18 μM,26.2 μM,28.54 μM,30.66 μM.Because of the good anticancer effects of the tannin in triphala,we enriched the tannins parts to conduct anti-tumor research in vitro.In the early stage of the research group,the tannin preparation technology and anticancer activity of the three single fruit were studied.The purpose of this paper is to establish the preparation process,quality control method and the screening of anticancer activity of triphala and provide experimental basis for its research and development.Chapter Ⅱ studies on the the extraction and purification technology of the Triphala tannins partsThe extraction process of triphala was established.Firstly,the extraction rate of tannin was used as the index and the effect of particle size on the extraction effect was investigated by single factor.After L9(34)orthogonal test,total tannins,Ellagic acid,Gallic acid and the extraction of the Corilagin were used as test indexs.Through the extraction solvent,solvent dosage,extraction time and extraction times of inspection,the best extraction process of tannin was set up,namely A2%ethanol extract D3 times,each time using C1 h,dosage of solvent was B2.Then the process was verified and the experimental results showed that the result was stable and the extraction rate was higher and the process was economical and easy to operate.The purification process of triphala using macroporous resin had been established.Firstly,adsorption capacity and adsorption of total tannins and major components were servrd as indexes to decided the best type of macroporous resin.Then used the transfer rate of total tannins,the single component,content and adsorption-resolution rate as the main indexes to investigate the maximum resin,the sample liquid concentration,diameter ratio,adsorption velocity,the mixed solvent,the complex velocity and elution solvent,amount of elution times and elution velocity parameters.Determined the optimal purification process as followed:The extraction solution was prepared by the best extraction process of the tannin,then enriched to the concentration to I2 g/mL.Centrifuged it at 3000 r/min the for 30 min.According to the volume ratio as G2,it was put on the B-type macroporous resin and the resin column diameter/high was R1 with dynamic adsorption and the adsorption velocity was J2 Bv/h.When the adsorption was completed,K2%ethanol was used to elucidate L2 BV,and the flow rate was P2 BV/h.Then M2%ethanol was used to wash off N2BV with the rate of S3 BV/h.Finaly,M2%ethanol elution fluid had been collected and dried under reduced pressure to obtain tannins parts.According to the above method,the total tannin content of the triphala tannins parts was above 55%.The transfer rate was about 62%.The results of amplification showed that the process was stable and feasible,meetting the demand of industrial production.Chapter Ⅲ Study on the quality control methods of the medicinal herbs and tannins parts.Based on the Chinese pharmacopoeia of 2015,a spectrophotometric method was established for the determination of total tannin content in the medicinal herbs and tannins parts.Taken Gallic acid as control,The content of total tannin was determined by using phospho-molybdenum-tungstic acid colorimetry,and the regression equation of Gallic acid was obtained:Y=116.53+0.0309 X,r=0.9991(n=6).The results showed that Gallic acid was linear in the range of 1.008～10.08 μg·mL-1.The content can be calculated by the external standard.Through calculation,the total tannin content of 3 batches of tannins parts(medicinal materials produced in Nepal)were 55.33%,55.05%,55.63%,and RSD were 0.51%,1.22%,0.69%(n=3).In addition,the total tannin content of 3 batches of herbs(Nepal)of origin were 24.16%,24.25%,24.12%,and RSD were 0.71%,1.58%,1.35%(n=3).That indicated that the method was stable and feasible.The method of HPLC had been established to simultaneously determine the content of Gallic acid,Corilagin,Chebulinic acid and Ellagic acid in the tannins parts and triphala.The regression equations were as followed:Y=2815.5X-0.231,r=0.9999;Y=1688.8X-3.3186,r=0.9999;Y=3400.6X-7.7667,r=0.9996;Y=8586.5X-155.56,r=0.9998.The results showed that there was a good linear relationship between the two groups,namely,0.91～4.55 μg,0.274～1.368 μg,0.022～0.175 μg and 0.329～2.634 μg,respectively.The content can be calculated by the external standard.It was determined that the content of the three batches of effective parts(medicinal materials from Nepal)and medicinal materials(from Nepal)were stable,indicating that the method was stable and feasible.Chapter Ⅳ Study on the antitumor activity of Triphala herbs and tannins parts in vitro.Comparative study on the antitumor activity of triphala in various proportions and single drug extracts in vitro.MTT method was used to investigate the effect of ethanol extracts in various proportions of triphala and the three single drugs on the extracorporeal inhibition of tumor cells.Through screening,it can be concluded that the M2%ethanol extracts of the above could all inhibit proliferation on A549,MCF-7,HepG2 and HELA in a dose-dependent manner.Among them,the effect of triphala on A549 and HepG2 was most obvious.The IC50 values of the two kinds of cell proliferation inhibition were 17.02 μg/ml and 15.81 μg/ml,respectively.The corresponding IC50 values of Tibetan propotion were 18.48μg/ml and 17.5 μg/ml.It can be concluded that the inhibition effect of Indian propotion triphala is slightly better than that of the Tibetan one,while the value added inhibition of the three drugs is weaker than that of the compound,which reflecting the advantages of the compound.In addition,the extracts of Indian propotion triphala and its tannin compositions had shown great basis for the study of anti-cancer in the literature review.So the preparation process and quality control method of the tannins parts in Indian propotion were selected,and the anti-cancer activity screening in vitro was conducted.Comparative study on the antitumor activity in vitro of crude extracts and tannins parts of triphala.MTT assay was used to explore the effects on the proliferation inhibition of four kinds of tumor cells:HepG2,A549,McF-7 and HELA.Both of them had inhibitory effect increased with the drug concentration,and showed a dose-dependent relationship.The IC50 values of crude extracts for the proliferation inhibition of the above tumor cells were 24.90μg/ml,21.59 μg/m,67.90 μg/ml and 38.48 μg/ml,respectively.The corresponding IC50 values of the tannins parts were 20.87 μg/ml,15.85 μg/ml,54.72μg/ml and 28.99μg/ml.It can be concluded from the data that both of them have the most obvious effect on the proliferation inhibition of A549.And the effect of tannins parts was better than that of crude extracts.It is suggested that tannin was the effective part of the anti-tumor effect.Chapter Ⅴ Summary and discussion1.The enrichment and purification process for the preparation of tannins in triphala by macroporous adsorption resin had been established,and the final total tannin content could be 55%and the transfer rate was 62%.Verifying results showed that the prepared process was stable and feasible,meetting the needs of industrial production.2.A spectrophotometric method had been established for the determination of total tannin content.The total tannin content of triphala was 24%,and the total tannin content of tannins parts was 55%.Three batches of medicinal materials and tannins parts were determined respectively,and the content was stable.3.A method of HPLC method was established to simultaneously determine the content of Gallic acid,Corilagin,Chebulinic acid and Ellagic acid of triphala as long as the method to simultaneously determine the content of Gallic acid,Corilagin and Ellagic acid of the tannins parts.The content of the four compounds above were 1.69,0.58%,0.99%and 0.044%.As Chebulinic acid in tannins fraction could not be tested,the content of the other three were 0.3%,1.7%and 1.4%.4.Conducted contrasting study to investigate the antitumor activity of the extractions of different proportion of triphala and its single drugs,triphala and its tannins parts on A549,MCF-7,HepG2 and HELA cell lines by MTT assay.Different proportions of triphala and every single drug extracts in vitro antitumor activity comparison results showed that triphala with equal proportion has a slightly better inhibition ratio than Tibetan medicine proportion,and inhibition ratio of the three single drugs was weaker than the compound as a whole.The comparison of the anticancer activity of the crude extracts and tannins parts showed the most obvious effect on the proliferation inhibition of A549.And the inhibition ratio of tannins parts was better than that of crude extracts,suggesting tannin was an effective part of triphala.Innovation1.Macroporous resin was used to enrich the tannins parts of triphala,and the content determination method of total tannin content and 3～4 single component content of the raw materials and tannins fraction were established.2.The results showed that the equal proportion triphala(India)in vitro antitumor activity was better than that of different proportion(Tibetan medicine),and the tannins parts were better than the crude extracts. |
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