中华鲎活性物质的分离纯化、结构鉴定及活性研究

发布时间：2018-04-12 06:51

本文选题：中华鲎 + 液相色谱分析　；参考：《集美大学》2017年硕士论文

【摘要】：丰富的海洋生物是新型抗肿瘤活性物质的重要来源。近年来,已从海洋生物中分离和鉴定出了许多具有抗肿瘤活性的天然物质,显示出诱人的研究开发前景。鲎是一种古老的海洋动物,生活了4亿多年,被称作是“活化石”,其体内含有特殊的活性物质。世界上仅存四种鲎,美洲鲎、蝎鲎、巨鲎与中华鲎。分布在我国海域的鲎称作中华鲎。我国中华鲎资源丰富,占世界上鲎资源的98%。本研究对中华鲎进行活性物质的分离纯化、鉴定及活性分析,其主要的研究结果如下:(1)对中华鲎进行部位分割,分为鲎壳、鲎脚、鲎内脏、鲎尾4个部分。分别用甲醇进行萃取,得到四个部位的粗提物,采用高效液相色谱技术(HPLC),对这四个部位进行化学成分的分析。确定最佳色谱条件为紫外检测波长220 nm,上样浓度为1 mg/mL,流速为0.2 mL/min,流动相为乙腈-0.1%甲酸超纯水溶液。相似度分析显示,鲎尾与鲎壳、鲎脚、鲎内脏的化学成分相似度在60%以下。在流动相为60%的乙腈水-95%的乙腈水条件下,鲎尾与其它部位存在大量色谱重合,但流动相为100%的乙腈时,鲎尾与其它部位的色谱差异很大,存在很多其它部位没有的色谱峰。因此,以化学成分差异最大的鲎尾作为实验材料,进行下一步的分离纯化。(2)通过凝胶柱层析、中压反相C18柱层析以及正相硅胶柱层析技术,从鲎尾的甲醇粗提物中分离纯化得到13种化合物。采用NMR核磁共振技术、ESI-MS电喷雾质谱技术,通过对一维和二维核磁谱谱图的解析,鉴定化合物最终的结构。鉴定出11种化合物分别为:胆甾醇、亚油酸甲酯、犬尿酸、矿油精、亚油酸、邻苯二甲酸二辛酯、2-十五烷酮、胆甾醇乙基碳酸酯、甲基丙烯酸月桂酯、胆甾醇苯乙酸酯、N-Cbz-甘氨酸乙酯。(3)对分离得到的11种化合物进行MTT细胞体外活性增殖试验。在24 h细胞毒性增殖试验中犬尿酸、亚油酸、2-十五烷酮、甲基丙烯酸月桂酯对人的肝癌HepG2细胞有较好的抑制活性,其中犬尿酸、亚油酸、2-十五烷酮对人的正常LO2细胞无细胞毒作用而甲基丙烯酸月桂酯对人的正常LO2细胞有较强的杀伤作用。对犬尿酸、2-十五烷酮和顺铂进行72 h细胞毒性增殖实验,犬尿酸、2-十五烷酮和顺铂对HepG2细胞的IC50分别是75.50μg/m L、48.65μg/mL、19.39μg/mL,对LO2细胞的IC50分别是280.23μg/m L、250.61μg/m L、118.53μg/m L。结果表明,无论在24 h和72 h条件下,犬尿酸、2-十五烷酮对人的肝癌HepG2细胞都有较好的抑制活性,并且对人的正常LO2细胞无细胞毒作用,甲基丙烯酸月桂酯对人的正常LO2细胞有杀伤作用。
[Abstract]:Rich marine life is an important source of new anti-tumor active substances.In recent years, many natural substances with anti-tumor activity have been isolated and identified from marine organisms, showing attractive research and development prospects.Limulus, an ancient marine animal that has lived for more than 400 million years, is called a "living fossil" and contains special active substances.Only four species of Limulus, American Limulus, Scorpius, Giant Limulus and Chinese Limulus exist in the world.The Limulus Limulus which distributes in our country sea area is called the Chinese Limulus.China is rich in Limulus resources, accounting for 98 percent of the total resources in the world.In this study, the active substances were isolated, identified and analyzed. The main results were as follows: 1. The main results of this study were as follows: 1) the Chinese Limulus was divided into four parts: horseshoe crab shell, horseshoe crab foot, viscera and tail of TAL.The crude extracts of four parts were obtained by methanol extraction. The chemical constituents of the four parts were analyzed by high performance liquid chromatography (HPLC).The optimum chromatographic conditions were determined as follows: UV detection wavelength 220 nm, sample concentration 1 mg / mL, flow rate 0.2 mL / min, and mobile phase was acetonitrile-0.1% formic acid ultrapure water solution.The similarity analysis showed that the similarity of chemical components between the tail of Limulus and the shell, foot and viscera of Limulus amebocyte lysate was less than 60%.Under the condition of 60% acetonitrile water and 95% acetonitrile water as mobile phase, there is a large amount of chromatographic overlap between Limulus tachypleus tachypleus tail and other parts, but when the mobile phase is 100% acetonitrile, the chromatogram of Limulus tachypleus tachypleus tail is very different from that of other parts, and there are many chromatographic peaks that do not exist in other parts.Therefore, the tachypleus tachypleus tail, which has the greatest difference in chemical composition, was used as the experimental material for the next step of separation and purification. The gel column chromatography, the medium pressure reverse phase C18 column chromatography and the normal phase silica gel column chromatography were used.13 compounds were isolated and purified from crude methanol extract from Limulus tachypleus.NMR NMR technique and ESI-MS electrospray mass spectrometry (ESI-MS) were used to identify the final structures of the compounds by analyzing the 1D and 2-D NMR spectra.Eleven compounds were identified as follows: cholesterol, methyl linoleate, canine uric acid, mineral oil, linoleic acid, dioctadecyl phthalate 2-pentadecanone, cholesterol ethyl carbonate, lauryl methacrylate.N-Cbz-glycine ethyl ester (N-Cbz-glycine ethyl ester) was used to study the activity of 11 compounds isolated from MTT cells in vitro.Canine uric acid, linoleic acid 2-pentadecanone and lauryl methacrylate had a good inhibitory activity on human hepatocellular carcinoma HepG2 cells in 24 h cytotoxic proliferation test.Linoleic acid 2-pentadecanone has no cytotoxic effect on human normal LO2 cells, while lauryl methacrylate has a strong killing effect on human normal LO2 cells.The results showed that canine uric acid 2-pentadecanone had a good inhibitory activity on human hepatoma HepG2 cells at 24 h and 72 h, and had no cytotoxic effect on human normal LO2 cells.Lauryl methacrylate can kill human normal LO2 cells.
【学位授予单位】：集美大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

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