本文選題：中華鱟 + 液相色譜分析�。� 參考：《集美大學》2017年碩士論文

【摘要】：豐富的海洋生物是新型抗腫瘤活性物質的重要來源。近年來,已從海洋生物中分離和鑒定出了許多具有抗腫瘤活性的天然物質,顯示出誘人的研究開發(fā)前景。鱟是一種古老的海洋動物,生活了4億多年,被稱作是“活化石”,其體內含有特殊的活性物質。世界上僅存四種鱟,美洲鱟、蝎鱟、巨鱟與中華鱟。分布在我國海域的鱟稱作中華鱟。我國中華鱟資源豐富,占世界上鱟資源的98%。本研究對中華鱟進行活性物質的分離純化、鑒定及活性分析,其主要的研究結果如下:(1)對中華鱟進行部位分割,分為鱟殼、鱟腳、鱟內臟、鱟尾4個部分。分別用甲醇進行萃取,得到四個部位的粗提物,采用高效液相色譜技術(HPLC),對這四個部位進行化學成分的分析。確定最佳色譜條件為紫外檢測波長220 nm,上樣濃度為1 mg/mL,流速為0.2 mL/min,流動相為乙腈-0.1%甲酸超純水溶液。相似度分析顯示,鱟尾與鱟殼、鱟腳、鱟內臟的化學成分相似度在60%以下。在流動相為60%的乙腈水-95%的乙腈水條件下,鱟尾與其它部位存在大量色譜重合,但流動相為100%的乙腈時,鱟尾與其它部位的色譜差異很大,存在很多其它部位沒有的色譜峰。因此,以化學成分差異最大的鱟尾作為實驗材料,進行下一步的分離純化。(2)通過凝膠柱層析、中壓反相C18柱層析以及正相硅膠柱層析技術,從鱟尾的甲醇粗提物中分離純化得到13種化合物。采用NMR核磁共振技術、ESI-MS電噴霧質譜技術,通過對一維和二維核磁譜譜圖的解析,鑒定化合物最終的結構。鑒定出11種化合物分別為:膽甾醇、亞油酸甲酯、犬尿酸、礦油精、亞油酸、鄰苯二甲酸二辛酯、2-十五烷酮、膽甾醇乙基碳酸酯、甲基丙烯酸月桂酯、膽甾醇苯乙酸酯、N-Cbz-甘氨酸乙酯。(3)對分離得到的11種化合物進行MTT細胞體外活性增殖試驗。在24 h細胞毒性增殖試驗中犬尿酸、亞油酸、2-十五烷酮、甲基丙烯酸月桂酯對人的肝癌HepG2細胞有較好的抑制活性,其中犬尿酸、亞油酸、2-十五烷酮對人的正常LO2細胞無細胞毒作用而甲基丙烯酸月桂酯對人的正常LO2細胞有較強的殺傷作用。對犬尿酸、2-十五烷酮和順鉑進行72 h細胞毒性增殖實驗,犬尿酸、2-十五烷酮和順鉑對HepG2細胞的IC50分別是75.50μg/m L、48.65μg/mL、19.39μg/mL,對LO2細胞的IC50分別是280.23μg/m L、250.61μg/m L、118.53μg/m L。結果表明,無論在24 h和72 h條件下,犬尿酸、2-十五烷酮對人的肝癌HepG2細胞都有較好的抑制活性,并且對人的正常LO2細胞無細胞毒作用,甲基丙烯酸月桂酯對人的正常LO2細胞有殺傷作用。
[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.
【學位授予單位】：集美大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R915

【參考文獻】

相關期刊論文 前10條

1 李艷青;陸園園;邢瑩瑩;奚濤;;海洋動物來源活性物質的研究新進展[J];藥學進展;2015年12期

2 張燕婷;周申生;鄧春梅;康信煌;;海洋微生物代謝產物分離純化及其生物活性研究進展[J];廣東化工;2015年14期

3 賈秀娟;劉有軍;楊煥;周義發(fā);;利用質譜技術解析糖類結構的研究進展[J];質譜學報;2014年05期

4 張亞;蘇品;廖曉蘭;黃璜;;多肽的分離純化技術研究進展[J];微生物學雜志;2013年05期

5 羅曉航;余鑫;盧曉燕;方婷;陳錦權;;鮑魚臟器粗多糖體外抗氧化活性研究[J];中國海洋藥物;2012年06期

6 王桂芳;馬廷燦;劉買利;;核磁共振波譜在分析化學領域應用的新進展[J];化學學報;2012年19期

7 方紹慶;尹偉力;耿金培;許紅巖;劉寧;陸承平;;中國鱟物種特異性鑒定的熒光PCR方法研究[J];動物醫(yī)學進展;2011年11期

8 張月紅;姜波;劉長建;王麗娜;謝宇;周琪;張麟;;鮑魚多糖提取工藝的研究[J];中央民族大學學報(自然科學版);2011年03期

9 ;Ds-echinoside A,a new triterpene glycoside derived from sea cucumber,exhibits antimetastatic activity via the inhibition of NF-κB-dependent MMP-9 and VEGF expressions[J];Journal of Zhejiang University-Science B(Biomedicine & Biotechnology);2011年07期

10 傅小偉;金益英;周石磊;崔礫礫;黃斌;;蛋白質分離純化技術研究進展[J];廣東化工;2011年04期

相關碩士學位論文 前1條

1 張曉梅;降血壓和降膽固醇大豆肽的分離純化[D];江南大學;2006年

，

本文編號：1738629