【作者】 刘晓丽；

【导师】 赵谋明； 宁正祥；

【作者基本信息】 华南理工大学 ， 食品科学， 2007， 博士

【摘要】 余甘子（Phyllanthus emblica L.）是一种具有较高食用和药用价值的植物果实,含有多种对人体有益的活性物质,包括多酚类物质、生物碱、活性多糖、维生素、氨基酸以及微量元素。余甘子具有抗氧化、抗癌、抗突变、降血压、降血脂等保健作用,是一种极具潜力的食品资源。余甘子在我国栽培历史悠久、资源丰富。截止2002年,我国的野生及栽培余甘林面积达6万hm²以上,产量达10万t。由于余甘子口感酸涩,作为食品原料加工适应性差,开发利用率很低,造成很大的资源浪费。因此,大宗野生余甘子资源的深度开发,对高效利用余甘子资源和促进山区经济发展具有重要意义。本课题首先对六个产地的野生余甘子进行筛选,测定其多酚类物质含量、水溶性多糖含量及各提取物的体外抗氧化活性,结果表明,惠州产地余甘子的多酚类物质含量及抗氧化活性相对较高,其总多酚含量为220.9mg/g,其多酚提取物清除DPPH自由基、羟基自由基、超氧阴离子自由基的EC50值分别为11.2μg/mL、64.6μg/mL及12.1μg/mL,还原力达到0.5的提取物浓度为84.5μg/mL,与对照芦丁的抗氧化活性相近,是分离、提取天然抗氧化剂的优质原料,因此,选取惠州野生余甘子进行后续试验。以总多酚提取率和抗氧化活性为指标,通过单因素实验及响应面分析法,确定了对总多酚提取率和抗氧化活性影响显著的四个因素（浸提时间、浸提温度、pH和乙醇浓度）,建立了该指标与四个因素间的数学模型,并得出余甘子多酚最佳提取工艺参数为:乙醇浓度为60%,浸提温度50℃,浸提时间3.9h,pH3.50。在此优化工艺下余甘子多酚提取率为217.9±0.9mg/g,还原力为1.203±0.013。采用有机溶剂分级、聚酰胺柱层析和制备HPLC等分离方法,以自由基清除活性作为检测指标,从余甘子多酚提取物中分离得到三种化合物,利用紫外可见光扫描光谱、质谱和核磁共振波谱等方法分别鉴定为异鞣云实素、槲皮素和山奈酚。其中,异鞣云实素和山奈酚首次在余甘子果实中分离得到。采用体外实验方法,对余甘子中多酚化合物在DPPH自由基、羟基自由基和超氧阴离子自由基体系,亚油酸体系、鼠肝匀浆体系和血清脂蛋白体系的脂质过氧化反应和过渡金属离子螯合方面的抗氧化活性进行研究。结果表明,三种化合物都有较强的自由基清除活性,其中异鞣云实素的自由基清除能力最强。各多酚化合物均可抑制Fe²⁺引发的亚油酸过氧化和小鼠肝脂质过氧化反应,其中异鞣云实素的抑制能力最强。余甘子多酚化合物可以螯合Fe²⁺,抑制脂质过氧化能力与螯合Fe²⁺能力一致。各多酚化合物均可抑制Cu²⁺诱导的低密度脂蛋白（LDL）氧化,提示具有潜在的预防动脉粥样硬化的功能。通过紫外可见光谱分析,推断多酚化合物可以通过螯合Cu²⁺抑制LDL氧化。对余甘子多酚提取物中促进免疫细胞增殖的活性成分进行了研究,采用硅胶柱层析和反相高效液相色谱柱分离,从正丁醇相得到化合物CI、CII和CIII,利用紫外可见光扫描光谱、质谱分析和核磁共振波谱等方法分别鉴定槲皮素-3-O-β-D-葡萄糖苷、老鹳草素和人参皂苷Rb1。人参皂苷Rb1为首次在余甘子果实中分离得到。通过体外抗氧化试验、免疫细胞增殖试验与大鼠动脉环收缩试验研究表明,三种化合物都具有显著的自由基清除作用。老鹳草素在10⁷5μg/mL浓度范围内对ConA诱导的淋巴细胞增殖都有促进作用。人参皂苷Rb1在低浓度时对ConA诱导的淋巴细胞增殖有抑制作用,当浓度超过25μg/mL时表现出促增殖作用,继续升高浓度又呈抑制作用,表现出双向调节作用。槲皮素-3-O-β-D-葡萄糖苷可以浓度依赖性的抑制ConA诱导的淋巴细胞增殖。三种化合物在10¹60μmol/L浓度范围内对离体大鼠胸主动脉环均具有浓度依赖性舒张作用,其中老鹳草素的血管舒张活性最强。更多还原

【Abstract】 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）.更多还原