高速逆流色谱法分离纯化黄顶菊及芒果花中活性成分及活性研究

发布时间：2018-06-07 07:55

本文选题：逆流色谱 + 黄酮　；参考：《北京化工大学》2017年博士论文

【摘要】：入侵植物可以产生有各种医药功能的代谢产物。我们实验室曾经研究建立了简单而成功的分析方法来鉴定有用的目标黄酮类成分,这是获得入侵植物的提取物中有效成分的重要一步。建立了制备分离十种总黄酮的方法。选择D4020树脂,因为它具有高吸附和解吸能力。根据以下优化的参数进行动态吸附/解吸过程:黄顶菊提取物浓度为2.72mg/mL, pH 4.50,处理量16BV,流速2.12mL/min,水(4BV)和乙醇:水溶液(90:10, v/v) 5BV。根据上述条件,提取物中总黄酮含量从4.30%到30%,提高了 7倍,使用D4020树脂的回收率为90%。此外,对于分离3-硫酸异鼠李素和黄芪苷,通过使用树脂D4020研究其动态吸附和解吸过程。3-硫酸异鼠李素的含量8.7%,回收率74.15%,黄芪苷的含量30.8%,回收率92.2%。此外,采用制备型HSCCC分离提纯,得到96.48%的3-硫酸异鼠李素4.5mg和24.4mg纯度为98.46%的黄芪苷。在第二个部分中,建立了选定的溶剂体系正相与溶剂洗脱-挤出操作的两步HSCCC方法。第一步中,通过使用正己烷:乙酸乙酯:甲醇:水(4:6:4:6, v/v)的溶剂体系得到3.7mg没食子酸(98.87%) , 3.9mg没食子酸乙酯(99.55%),以及鞣花酸、乙基没食子酸酯的混合物。在第二步中,通过洗脱和挤出高速逆流色谱操作后,使用二氯甲烷-甲醇-水(4: 3: 2, v/v)进行混合物的进一步纯化。分离出3.8mg纯度为98.68%乙基没食子酸酯的和5.7mg纯度为99.7%的鞣花酸。通过高效液相色谱,超高压液相色谱-四极杆-飞行时间串联质谱法,核磁碳谱和核磁氢谱分析确认分离的酚类化合物的结构。在第三部分研究中,研究了分离的化合物的DPPH抗氧化以及粗提物的抗菌敏感性实验。研究表明没食子酸乙酯的抗氧化能力好,芒果花的醇提取物可抑制多种病原菌的生长。在浓度为20mg/mL的大肠杆菌,克雷伯菌和金黄色葡萄球菌的保留带分别为13.20mm, 19.80mm和23.00mm,而仙人掌显示出最显着的保留带26.OOmm。因此,观察到每个测试生物体的最小抑制区(MIC)从1.00至2.50mg/mL变化。MIC值不同的原因可能是提取物中发现的各类具有生物活性的化感物质的反应机理不一样,而合成抗生素的抑制机制具有特异性和单一性。从研究结果中可以得到结论,上述生物活性成分的分离方法可以扩展到其他天然产物中活性物质的分离。
[Abstract]:Invasive plants can produce metabolic products with various medical functions. Our laboratory has studied and established a simple and successful analytical method to identify useful target flavonoids. This is an important step in obtaining effective components from the extracts of invasive plants. A method for preparing ten kinds of total flavonoids was established. Because it has the ability of high adsorption and desorption, the dynamic adsorption / desorption process based on the following optimized parameters: the concentration of the extract of anathea is 2.72mg/mL, pH 4.50, the amount of treatment 16BV, the flow rate 2.12mL/min, water (4BV) and ethanol: the aqueous solution (90:10, v/v) 5BV. based on the above conditions, the content of total flavonoids in the extract is from 4.30% to 30%, which has increased by 7 times, so that the content of total flavonoids in the extract is increased by 7 times. The recovery rate of D4020 resin was 90%.. For the separation of 3- and astragaloside, the content of.3- sulfate was 8.7%, the recovery rate was 74.15%, the content of astragaloside was 30.8%, the recovery rate was 92.2%., and 96.48% 3- sulphuric acid was obtained by using the prepared HSCCC. ISO rhamnin 4.5mg and 24.4mg were 98.46% of astragaloside. In second parts, the selected solvent system positive phase and the two step HSCCC method of solvent elution and extrusion operation were established. In the first step, 3.7mg gallic acid (98.87%), 3.9mg gallic acid was obtained by using hexane: ethyl acetate: methanol: water (4:6:4:6, v/v) solution system. Ethyl (99.55%), as well as a mixture of ellagic acid, ethyl gallate. In the second step, the mixture was further purified using dichloromethane methanol water (4: 3: 2, v/v) by elution and extrusion high speed counter flow chromatography. The purity of 3.8mg was 98.68% ethyl gallate and 5.7mg was 99.7% tannic acid. High performance liquid chromatography, high pressure liquid chromatography - quadrupole flight time tandem mass spectrometry, nuclear magnetic carbon and nuclear magnetic hydrogen spectrum analysis confirmed the structure of separated phenolic compounds. In the third part, the antioxidation of the separated compounds and the antimicrobial susceptibility test of the crude extracts were studied in the third part of the study. The study showed the resistance of ethyl gallate. The alcohol extract of the mango flower could inhibit the growth of various pathogens. In the Escherichia coli with a concentration of 20mg/mL, the retention zones of Klebsiella and Staphylococcus aureus were 13.20mm, 19.80mm and 23.00mm, respectively, while the cactus showed the most significant reservation zone of 26.OOmm., which observed the minimum inhibitory area of each test organism. MIC) the variation of.MIC values from 1 to 2.50mg/mL may be due to the different mechanisms of the bioactive allelochemicals found in the extracts, while the inhibition mechanism of the synthetic antibiotics is specific and unitary. The separation of active substances from other natural products.
【学位授予单位】：北京化工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O652.6;TQ28

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