Isolation And Identification Of Polyphenols From Emblica (Phyllanthus Emblica L.) Fruit And Their Biological Activities

Emblica (Phyllanthus emblica L.) is a kind of fruit containing much phenolics, alkaloids, polysaccharoses, vitamin, amino acids and trace elements. Emblica fruit is reported to have hypolipidemic, antimicrobial, antitumor, hepatoprotective and hypoglycemic activities. It has been regarded as a potential resource of food. But raw fruits, being highly acidic and astringent in taste, are unacceptable to consumers. Therefore, they are generally processed to sugar-rich products such as preserves, candies, etc. and utilised for preparation of some pharmaceutical products. Further development of this resources means much for increasing its added value of emblica fruit and facitilating the development of agro-industrial economics.The contents of various phenolics of methanolic extracts of emblica fruit from six regions in China were measured. The antioxidant activities of these extracts were also evaluated. Among all the methanolic extracts analysed, the Huizhou sample exhibited a significantly (P < 0.05) higher phenolic contents than other samples with the value of 220.9mg/g. The Huizhou sample was found to have the strongest antioxidant activities in scavenging DPPH radicals, superoxide anion radicals, and had the highest reducing power. Thus, Huizhou sample was selected for further experiments.In the experiments of developing the extraction program of phenolics from emblica fruit, the effects of extraction time, extraction temperature, ethanol solution concentration and extraction times on extraction program were studied. Response surface methodology was further taken based on the single factor experiments. The mathematic model was built between the response, total phenolics yield, antioxidant activity and four factors. Optimal conditions for obtaining the highest total phenolics yield and antioxidant activity were 3.9hr of extraction time, 50℃of extraction temperature, pH 3.50 and 60% of ethanol solution concentration. Under this condition, the yield of total phenolics and the reducing power were 217.9±0.9mg/g and 1.203±0.013, respectively.An activity-directed fractionation and purification process was used to identify the antioxidative components of emblica fruit. The ethyl acetate fraction was subjected to separation and purification using polyamide chromatography and HPLC. Three compounds were identified to be isocorilagin, quercetin, and kaempferol, respectively, by spectral methods, 1H and 13C nuclear magnetic resonnance spectroscopy, ultraviolet-visible spectrophotometry and mass spectroscopy and comparison with literatures. Compounds isocorilagin and kaempferol were identified from emblica fruit for the first time. The antioxidant activities of purified compounds were were evaluated by in vitro experiments using scavenging assays of DPPH radicals, hydroxyl radicals, and superoxide anion radicals, chelating ability of ferrous ion, reducing power, inhibition capability of Fe (II)-induced lipid peroxidation and low density lipoprotein (LDL), respectively. All the purified compounds showed strong radical scavenging activity. Amongst, isocorilagin showed the highest antioxidant activity than other purified compounds. The EC50 values for DPPH radicals, hydroxyl radicals and superoxide anion radicals were 3.9, 7.2 and 5.4μg/mL, respectively. These three compounds could inhibit linoleic acid peroxidation and microsomal lipid peroxidation induced by Fe²⁺. Isocorilagin showed significant (P < 0.05) potency to chelate Fe²⁺ with the EC50 value of 0.33mmol/L. All the compounds were effect in peotecting LDL against oxidation in a concentration-dependent manner. The data suggested that phenolics from emblica fruit may have a preventive effect against atherosclerosis by inhibiting LDL oxidation. According to the differential UV-visible spectrum of samples in the presence of Cu²⁺, all the purified compounds could chelate copper ions.The components with immunomodulatory properties were investigated. These active components were prepared with silica column chromatography and reverse-HPLC. The three compounds were identified to be quercetin 3-β-D-glucopyranoside, geraniin, and ginsenoside Rb1, respectively, by spectral methods, 1H and 13C NMR spectroscopy, UV-visible spectrophotometry, MS and comparison with literatures. Geraniin was identified from emblica fruit for the first time.In vitro determination of antioxidant activities, immune cell proliferation and contractile response of isolated aortic rings induced by phenylephrine were made. The results showed these three compounds had strong radical-scavenging activities. Amongst, geraniin showed the highest antioxidant activity than other purified compounds. Geraniin can significantly stimulate the proliferation of lymphocyte cells (P < 0.05). Ginsenoside Rb1 at low concentrations inhibited ConA-induced proliferation of lymphocytes, but enhanced proliferation at higher concentrations. However, lymphocyte proliferation was been inhibited again at much higher concentrations. Quercetin 3-β-D-glucopyranoside appear to inhibit lymphocyte proliferation in a concentration-dependent manner. All the compounds relaxed precontracted rat aortic rings in a concentration-dependent manner. Geraniin showed significantly higher vasodilation capacity compared to other purified compounds (P < 0.05).