Effective Chemical Constituents And Antitumor Effect Of Leaves Of Phyllanthus Emblica L

1. Objective:Phyllanthus emblica L, one species of Phyllanthus, which belong to Family Euphorbiaceae, is a traditional herbal medicine which is widely distributed in Guangxi, Yunnan, Fujian, Guizhou ang Guangdong province et al. being widely used as a folk TCM for 35 kinds of diseases in the world. Literatures survey indicated that there are 17 countries and nations of the world using various parts of Phyllanthus emblica L in their medical treatment. It was collected in several editions of Chinese Pharmacopoeia. Leaves of Phyllanthus emblica L, sweet and little bitter in flavor, cool in nature, it has various functions of clearing heat, detoxifying, damp elimination detumescence, diuresis, anti-inflammatory,et al. In folk clinic, Leaves of Phyllanthus emblica L were used for the treatment of oral carity ulcer, scrotum eczema, furuncle, dermatitis, anorectal diseases hypertension, viperbite and Dieda Sunshang as well. However, Literatures survey indicated that a little systemic chemical constituents, pharmacology effects and quality control research on leaves of Phyllanthus emblica L were reported. On the basis of the previous studies of the program of Guangxi Natural Science Foundation about leaves of Phyllanthus emblica L, we had accomplished relevent chemical constituents, pharmacology effects and the content analysis research on leaves of Phyllanthus emblica L from Guangxi province as follows. To clarify the active constituents and pharmacological effects of its and establish the quality control method of its, To provided a scientific foundation for the research development of leaves of Phylanthus emblica L. 2. Method:Our studies included four parts as follows:(1) On the basis of Literatures survey, recent research development of leaves of Phylanthus emblica L were described in the paper. (2) According to the result of activity sifting, we made a systemic extraction and separation study on the chemical constituents of its. Soaking and percolating with 95% and 50% ethanol in order, recycle ethanol, extracts mixed with silica.gel, refluxed and extracted with Petrol ether, ethyl acetate and n-butanol, isolated chemical constituents from these extractions with silica.gel column, polymide column, Sephadex LH-20 column, prepared HLPC and recrys. etc. and lastly identify them with physical and chemical property and UV, IR,1H-NMR, 13C-NMR, MS. (3) Research the methods of qualitative and quantitative analysis on leaves of Phyllanthus Embllca L by TLC and HPLC. TLC was used to distinguish the quality of Phyllanthus Embllca L; The external standard method was adopted to determine gallic acid content in leaves of Phyllanthus Embllca L. The HPLC chromatographic conditions were as follows:Reliasil C18 column (5μm,250 mm×4.6 mm), MeOH:0.2% H3PO4 (3:97) as mobile phase, at a flow rate of 1.0 ml.min-1 and detective wavelength 269 nm. The qualitative and quantitative methods were established by studying on leaves of Phyllanthus Embllca L, which provided a reliable technique for the establishment of the quality standard of its. (4) According to the result of previous activity sifting, we study the effect of gallic acid on the proliferation and apoptosis of a human hepatocellular carcinoma Bele7404 cell lines in vitro, which extracted and isolated from leaves of Phyllanthus emblica L. The effect of gallic acid on proliferation and cell cycle of hepatocellular carcinoma Bele7404 cell lines was determined by MTT assay method A common light microscope, inverted phase contrast microscopy and fluorescence microscopy were used to observe the morphological changes of apoptosis of human hepatocellular carcinoma Bele7404 cell lines in vitro induced by different concentrations of gallic acid. The analysis of early apoptosis phase of human hepatocellular carcinoma Bele7404 cell lines induced by gallic acid in vitro was determined by Flow Cytometry using Annexin V-FITC/PI method. Induction apoptotic rate of Bele7404 cell was determined by TUNEL method.3. Result: On the basis of Literatures survey, recent research development of its chemical constituents, pharmacological effects and its healthy functions both as food and medicine were described in the paper. By means of those research works, 11 compounds were isolated from Petrol ether extract, ethyl acetate extract and n-butanol extract, which were defined by chemical analysis and spectrum analysis:β-sitosterol(Ⅰ),β-Carotene(Ⅱ), Avicularin(Ⅲ), Kaemferol(Ⅳ), Quercetin(Ⅴ), kaempferol-7-methylether (Ⅵ), ellagic acid (Ⅶ), gallic acid (Ⅷ), ethyl gallate(Ⅸ), tetratriacontanoic acid (X),1-Triacontanol(Ⅺ), Among them, compoundsⅢ,Ⅵ,Ⅸ,Ⅹ,Ⅺwere discovered in it for the first time, Furthermore, take gallic acid as an index, the gallic acid content research of leaves of Phyllanthus emblica L has been done by HPLC chromatographic conditions throung the external standard method, which comes from different habitats in different seasons. Study result showed that processing method obviously and regularly affects its content, the gallic acid content in Jiangzhou District of Chongzuo City and at Qinzhou city LingShan Municipal Luwu mountain are higher by contrast. Furthermore, in-vitro activity experiment showed that compoundⅧcan obviously restrain the Bele7404 cell lines proliferation in vary degree, show the typical signals of the early apoptosis. Typical morphology changes was found in apoptotic cells by MTT assay and microscope. The amount and the rate of early apoptosis cell increased significantly in a dose-and time-dependent manner. which were observed by Flow Cytometry Using Annexin V-FITC/PI and by TUNEL method.4. Conclusion:This dissertation was established in traditional Chinese medical science, on the basis of its flavor, nature, efficacy, therapeutic action and clinical application, the advance activity sifting were studied on leaves of Phyllanthus Embllca L. According to the result of activity sifting, we made a systemic extraction and isolation study on the chemical constituents of its, compoundⅢ,Ⅵ,Ⅸ,Ⅹ,Ⅺwere discovered in it for the first time. For the first time, take gallic acid as an index component. studied on the qualititave and quantitave analysis of gallic acid in leaves of Phyllanthus Embllca L by TLC and HPLC. Furthermore, analyzed comparatively the content of gallic acid in different habitats and different process methods, which provided a reliable technique for the quality moniter and control of its. For the first time, studied on the antitumor effects of gallic acid isolated from leaves of Phyllanthus emblica L in-vitro. Activity experiment showed that gallic acid can obviously restrain the Bele7404 cell lines proliferation in vary degree, showed the typical signals of the early apoptosis induced. The rate of early apoptosis cell significantly increased in a dose-and time-dependent manner. Results showed that gallic acid is one of the important chemical constituent which have antitumor effect by apoptosis induced, which provided a scientific foundation for evaluating antitumor pharmacological effect and elucidating potential action mechanisms of leaves of Phyllanthus emblica L; provided a scientific evidence for exploiting the traditional Chinese medicine.