【作者】 刘覃；

【导师】 陈晓青；

【作者基本信息】 中南大学 ， 分析化学， 2005， 硕士

【摘要】 本文研究了中药龙葵(Solanum nigrum L.,S.N.L.)中两类活性成分—多糖及总生物碱(Total Steriodal Alkaloids,TSA)的提取、纯化及测定方法。 (1)确定了多糖和TSA的最优提取条件。采用优于传统回流提取的微波辅助提取,以蒸馏水为溶剂,在325W下浸提15min,料液比为1:20,多糖得率达1.94%:并对提取机理进行了探讨。以95%乙醇溶液为溶剂,在455W下浸提8min,料液比为1:20,TSA得率为26.38μg/g;在探究提取机理的同时,采用薄层色谱法研究了微波作用对TSA结构的影响。 (2)分别探讨了多糖、蛋白质、澄清度和TSA的测定方法。 采用苯酚—硫酸法结合DNS法对多糖进行测定,确定了最佳检测条件:总糖含量与吸光度值在18.0～66.0μg/mL范围内线性关系良好;单糖含量与吸光度值在0.40～2.00mg/mL范围内有良好线性关系。 采用改进考马斯亮蓝法分析测定了多糖中蛋白质的含量。线性回归方程A=-0.0604+0.84543x,R=0.9997。 确定了检测样品液澄清度的试验条件参数。以440nm处的吸光度和660nm处的透过率为指标,分析纯化处理前后样品的澄清状况。 采用酸性染料比色法对TSA进行了测定,TSA含量与吸光度值在3.20～16.0μg/mL范围内线性关系良好。 (3)研究了多糖的纯化工艺并对最终产品进行了初步表征。 采用4000r/min下高速离心10min去除不溶性杂质。 采用天然澄清剂ZTCi+1—Ⅱ—NKA-9大孔树脂联用吸附澄清,最佳工艺参数为:澄清剂两组份加入顺序先B后A;加入量B6%(v/v),A3%;澄清温度B80℃,A60℃;提取液处理浓度1:5(g/mL)。树脂洗脱体积3.2mL(每1mL样品液);树脂用量为干树脂质量:提取液上样体积=0.4g/mL。此时样品液在660nm处的透过率从5.40%上升到88.8%,440nm处的吸光度则从2.69下降到0.258。 研究了采用酶解—Sevag法联用去除多糖中的蛋白质,确定了最佳清除条件:酶解温度46℃,酶解时间7.5h,胰蛋白酶用量2%更多还原

【Abstract】 The extraction, purification and analysis methods of the biologically active composition polysaccharides and TSA in S.N.L. were discussed.（1） Technological microwave-assited extraction process of polysaccharides and TSA was experimented, which excel the traditional reflux extraction in many respects. The solvent of distilled water was used to extract from S.N.L under 325W for a duration of 15 min;ratio of feed-stock weight to the volume of solvent was 1:20; the yield of polysaccharides was 1.94%; the extraction mechanism was also investigated. And the yield of TSA was 26.38μg/g when extracted with 1:20 of 95% ethanol aqueous, at 455W for 8 min. The effect of microwave radiation on the structure of TSA was discussed by TLC and the extraction mechanism was investigated as well.（2） Analysis methods of polysaccharides, protein and clarity were discussed as well as that of TSA. The content of polysaccharides was determined by phenol-sulfuric acid method combined with DNS method and the analysis condition was as follows: for the saccharides, the linear determination range is 18.0⁶6.0μg/mL; for the monosaccharides, the linear determination range is 0.40².00mg/mL. The modified CBB method for determinating the content of protein was studied and the calibration curve was A=-0.0604+0.84543x （R=0.9997）. The clarity of extraction solution was analysed by determinating T₆60/% and A₄40 before and after the purification treatment. And the acidic dye colorimetry for determinating the content of TSA was also studied: the linear determination range is 3.20¹6.0μg/mL.（3） Technological process of purification for polysaccharides and elementary characterization on the finally obtained preparation were studied.The unsolvable impurity was removed by high-speed centrifugation at 4000r/min for 10min.Absorption and clarification were completed by the natural ZTC1+1— II clarifier combined with NKA-9 resin and the optimum condition was as follows: the adding sequence is first B and then A; the dosage is 6%（v/v） B and 3% A; the reacting temperature is 80 °C for B while 60°C for A; the concentration of solution was 1:5（g/mL）; the elution volume was 3.2mL for every lmL specimen; the dosage of dry resin was 0.4 g for every lmL specimen. And the result shows T?56o rises from 5.40% to 88.8% while the A440 declines from 2.69 to 0.258.Polysaccharides was then deproteinized by Trypsin hydrolyzation combined with Sevag method and the following was the optimum conditions: the specimen was hydrolyzed at 46°C and 6.0pH for 7.5h after adding 2%（v/v） trypsin before extracting for 7 times in Sevag method. The result shows the content of residual protein is only 0.0940% and the clarifier was high to 99.89%.Technological process of further isolation and purification was completed by chromatographic column. Employed with 16X500 mm column, the installation was simple and the adsorption and elution was easy to operate. In this experiment, four fine fractions SNL1, SNL2, SNL3 and SNL4 were obtained by gradient elution with the PB of 7.6pH and NaCl solution through the DE-52 cellulose column chromatography before dialysis and ethanol precipitation. Their homogeneity was further examined by the Sephadex G-100 column chromatography.Physical&chemical characters and homogeneity of the four fine preparations were analysed as well as the structures was investigated by UV and FT-IR spectra.更多还原