The Preparation Of EGCG From Tea And Studies On Its Effect And Mechanism On Tumors

In this study,we prepare epigallocatechin-3-gallate(EGCG)from green tea,eval uate the antitumor activity of EGCG in vitro and in vivo,and find out the target and effective proteins related to the effects of EGCG and explore its antitumor mechanism.In order to prepare EGCG from green tea by caffeine precipitation and solvent partition,the experiment of EGCG-preparation optimization from tea was carried out with the design of the rotation-regression-orthogonal combination.The parameter models with the purity of EGCG as target functions were established.The optimization of the preparing parameters of EGCG from tea in caffeine precipitation and solvent partition system were as follows:water temperature,caffeine concentration,the number of partition with chloroform,with ethyl hexanoate,and with propyl acetate.Additionally,the mathematic model was well established to estimate the purity of EGCG within the investigated range.By the preparing parameters,the EGCG production system was obtained,followed by further separation and purification.The product was gained and then measured by HPLC.In order to evaluate the antitumor activity of EGCG in vitro and in vivo,MTT assay was used to assess the proliferation of S 180 and HUH-7 cells in vitro treated with EGCG.The mouse tumor model was utilized to investigate the antineoplastic effect of EGCG,and the inhibitory effects of EGCG on S180 and U14 tumors were observed in mouse.In order to find out the target and effective proteins related to the effects of EGCG and explore its antitumor mechanism,the mouse tumor model with cell line S180 and U14 xenograft were treated with or without EGCG,and then the total proteins were extracted from xenografts,analysis the proteins by two-dimensional gel electrophoresis(2-DE).The 2-DE gel was stained with Coomassie Brilliant Blue G-250.The differential proteins between experimental and control groups were analyzed using PDQuest image analysis.The peptide mass fingerprints of differentially expressed proteins were detected based on matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF/TOF MS)and the Swiss-Port database to identify the proteins.The results indicated that the best EGCG preparing parameters is water temperature, 80℃;caffeine concentration,30mmol/L;the number of partition with chloroform, 5;with ethyl hexanoate,3;and with propyl acetate,3.Under these preparing parameters,the product was obtained and though further sublimate,finally its purity was more than 98%,as judged by HPLC.the results indicated that the EGCG significantly inhibited the proliferation of S180 cells and U14 cells in a dose-dependent in vitro.In the tumor-bearing mouse,EGCG inhibited the development of S180 and U14 tumor.The tumor suppressed rate on S180 and U14 was 51.19%and 45.78%,respectively.The PDQuest image analysis displayed about 1000 protein spots in each gel.In S180,twenty-one differentially expressed protein spots were found between two groups,of which 16 spots decreased and 5 increased in the expression levels in the experimental group compared to the normal group.In U14,twenty differentially expressed protein spots were found between two groups,of which 13 spots decreased and 7 increased in the expression levels in the experimental group compared to the normal group.Ten peptide mass fingerprinting maps(PMF)were obtained,and eight proteins including annexin A2,transferrin,alpha-enolase,et al,were primarily identified after database searching,most of which were involved in tumor cell proliferation,invasion and apoptosis.The caffeine precipitation and solvent partition method has been developed that enables the easy and inexpensive preparation of EGCG from tea.And EGCG possesses antitumor effects both in vitro and in vivo.We find out a series of proteins related to EGCG.It exerts its inhibiting effects on tumors through regulating multiple proteins expression directly or indirectly,such as annexin A2,alpha-enolase, ribosomal protein.Those proteins were likely the novel therapeutic targets for EGCG on the treatment of malignant tumors.