油茶枯饼中天然抗氧化剂提取和筛选.pdf

I摘 要我国南方地区包括江西省盛产油茶，油茶经榨取油料后的枯饼中尚存在很多活性物质，如茶皂素，多糖，黄酮类物质等等，但由于长期得不到合理和充分的利用，导致资源浪费，甚至对环境也造成了一定的污染对油茶枯饼这种废弃资源的回收利用不仅有助于缓解环境压力，而且对提高油茶副产品的经济价值和社会价值有着非常重要的意义本论文以油茶枯饼中的茶皂素、多糖和黄酮类物质为研究对象，应用微波萃取装置分别对其中的茶皂素和多糖进行微波萃取研究讨论了乙醇浓度、提取时间、固液比和提取温度（功率）对提取率的影响通过正交试验，得到微波提取茶皂素的最佳工艺条件为萃取剂 90％乙醇-水溶液、固液比 1:20（w/v） 、萃取时间 9 min 和萃取温度 90 ℃；提取茶多糖的最佳工艺条件为纯水提取、提取时间为 16 min、固液比 1:20（w/v） ，功率为 800 W并将微波提取的粗多糖经通过乙醇醇析和大孔树脂分离纯化，得到不含茶皂素、黄酮及蛋白质组分的多糖产品用硅胶柱层析法，以氯仿-甲醇-水（70:30:5，v/v/v）为洗脱液从茶皂素的微波提取物中分离得到两种主要黄酮，经 UV, ESI-MS 数据解析和相关文献对比， 对其结构进行了确证， 这两种黄酮分别为山奈酚3-O-[2-O-β-D-木糖-6-O-α-L-鼠李糖]-β-D-葡萄糖苷(Ⅰ)和山奈酚 3-O-[2-O-β-D-半乳糖-6-O-α-L-鼠李糖]-β-D-葡萄糖苷(Ⅱ)，其分子量为分别为 726 和 756。

以两种黄酮苷为对照品，建立了测定油茶枯饼提取物中两种主要黄酮的高效液相色谱方法利用本文建立的毛细管电泳法、HPLC 法，和参考文献的苯酚-硫酸法分别测定了油茶枯饼中茶皂素，黄酮和多糖的含量，为茶枯饼活性资源的开发利用提供理论基础，经测定，以上三种成分在茶枯饼中的含量分别为 16.96％、4.92％和 11.25％通过显色反应、光谱扫描和 HPLC-MS 分析对大孔树脂乙醇梯度洗脱物中的茶皂素，黄酮进行了纯度鉴定，结果表明，60%乙醇洗脱组分为茶皂素，其分子量分布范围为 1190-1332Da，10％、20％乙醇洗脱组分为两种黄酮的混合物，可用于抗氧化活性研究采用化学发光法，利用发光体系 CuSO4-phen-VitC-H2O2-DNA，分别考察了油茶皂素，多糖和黄酮类物质对羟基自由基引发 DNA 损伤的保护作用，以半抑制率浓度大小反映这三种物质的抗氧化能力强弱，并且与芦丁、槲皮素和绿原II酸等公认的强抗氧化剂进行了比较结果表明油茶皂素和黄酮具有较显著的抗氧化能力，而油茶多糖的抗氧化能力不明显；油茶皂素较油茶黄酮、芦丁、槲皮素和绿原酸四种物质的抗氧化活性要弱；油茶黄酮抗氧化活性较芦丁、槲皮素的抗氧化活性弱，但略强于绿原酸。

关键词：油茶枯饼，茶皂素，黄酮，高效液相色谱，化学发光，抗氧化活性IIIABSTRACTIt is rich in Camellia Oleifera in South china, including Jiang Xi province. Thereare many active substances in the oiltea seed cakes after squeezing out the fuel oils，such as： tea saponin， polysaccharides, flavonoids， and so on. As the oil-tea seed cakeshave not been fully usd for a long time, the resources are be wasted and cause muchpollution to the environment. So it is very important to develop the resources in theoil-tea cakes for relieving pressure of environment and improving the economic andsocial value of the By-Products from Camellia Oleifera.Tea saponin, polysaccharides，flavonoids in the Camellia Oleifera were bestudied in the article. Extraction of tea saponin and poysaccharides were done withmicrowave technique by discussing the influence of ethyl alcohol density, extractiontime, liquid-solid and extraction temperature (power) to the extraction rate. Theoptimum conditions for saponin extract was as follows: 90% ethyl alcohol, solid-liquid ratio was 1:20, extraction time was 9min with temperature 90℃; while aquapure extract, solid-liquid ratio was 1:20, extraction time16min with power 800W forpolysaccharides. The microwave extract of polysaccharides were then be purified byprecipitating with 75% ethanol and separating with macroporos resin. the saponin andflavonoids were removed and no protein in the polysaccharides extract.Two flavonoids were gotten from microwave extracts of saponin by silica gelcolumn chromatography with chloroform-methanol-water （70:30:5， v/v/v） for eluent.The two flavonoids had been identified respectively as kaemferol-3-0-[2-O-β-D-xylose-6-0-α-L-rhamnose]-β-D-glucoside(Ⅰ)和 kaemferol-3-0-[2-O-β-D- galactose-6-0-α-L-rhamnose]-β-D-glucoside (Ⅱ) by UV, ESI-MS analysis and literaturereference. The molecular weight were respectively 726 and 756. HPLC method wasestablished for the determination of the two flavorids in the oil-tea cakes.The contents of the three substances in oil-tea cakes — saponin 、polysaccharides、 flavonoids were detemined repectively as 6.8%, 4.8% and 12.5%with capillaryelectrophoresis (established before), HPLC and colorimetry. Which provided the basictheories for the development of the oil-tea cakes. The saponin and flavonoids in theIVeluates were analyized by color reaction, spectrum scan and HPLC-MS, the resultshowed that the eluate with 60% ethanol were saponin while those with 10% and 20%ethanol were mixture of the two flavonoids.The effect of the saponin ，polysaccharides and flavonoids in the oil-tea cakes toprotect DNA from been damaged by hydroxyl radicals were respectively studied withchmeiluminescence system CuSO4-phen-VitC-H2O2-DNA. And were compared withthree other antioxidants—rutin、quercetin and chlorogenic acid. Antioxidation abilitywere reflected by half inhibiting concentration. The result showed that saponin andpolysaccharides have both abbility of antioxidation, however, the antioxidant capacityof saponin was not as strong as the other four substances, and the antioxidant activityof flavonoids was lower than rutin and quercetin, but not worse than chlorogenic acid.Keywords : oil-tea cakes, tea saponin, flavonoids, high-performance liquidchromatography, chemiluminescence, antioxidant activity南昌航空大学硕士学位论文原创性声明本人郑重声明：所呈交的硕士学位论文，是我个人在导师指导 下，在南昌航空大学攻读硕士学位期间独立进行研究工作所取得的 成果。

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