螳螂内生真菌D.eschscholzii次级代谢产物TL1-1的分离制备工艺及抑菌活性研究

发布时间：2018-07-05 04:20

本文选题：TL1-1 + 硅胶柱层析　；参考：《华东理工大学》2017年硕士论文

【摘要】：5-羟基-2-甲基苯并二氢吡喃酮(5-hydroxy-2-methylchrommanone,TL1-1)是螳螂(Tenodora aridifolia)肠道内内生真菌Daldinia eschscholzii代谢产物中分离到的一种酚类化合物。据文献报道,TL1-1对人类白血病细胞HL-60有显著的细胞毒性,对农业致病菌Micrabotryum violaceuw有显著的抑菌活性。然而,TL1-1的发酵工艺水平低,难以满足活性研究的需求量。且现有的TL1-1分离方法仍局限于TLC法、HPLC法,难以进行放大实验。本论文针对上述问题,在实验室其他同学500 L发酵工艺优化放大的基础上,针对TL1-1的不同分离制备工艺进行考察与比较。本论文建立了硅胶柱层析法分离制备TL1-1的工艺。采用硅胶柱层析对发酵液浸膏进行分离,上样浸膏与硅胶质量比为1:20;洗脱剂为石油醚-乙酸乙酯体系,按照100:1、50:1、20:1、10:1、5:1的顺序进行梯度洗脱,每个梯度的洗脱体积为2 BV,体积流量为1 BV/h;当洗脱条件石油醚:乙酸乙酯=20:1时,富集获得TL1-1组分,纯度为59.75%,得率为68.82%。硅胶柱层析法因得率低、纯度低、难再生且有机溶剂消耗大等问题,难以符合高效的、经济节约的分离制备要求。在前述基础上,本论文又研究了大孔吸附树脂法分离制备TL1-1的工艺。通过吸附量与解吸率的比较,筛选出XAD-16树脂为最佳吸附树脂;通过静态动力学与热力学实验对工艺条件进行优化,发现XAD-16树脂对TL1-1的吸附符合准一级动力学模型、Weber-Morris模型以及Freundlich经验模型;通过动态吸附与解吸附实验,获得XAD-16树脂分离制备TL1-1的最佳工艺条件:上样量为11 BV,初始浓度为0.508 mg/mL,溶液pH值为4.0,上样流速为2 BV/h;洗脱剂为乙醇-水体系,浓度梯度依次为去离子水(4BV)、40%乙醇溶液(4BV)、95%乙醇溶液(4BV),体积流量为2BV/h。检测并收集95%乙醇洗脱液,在洗脱体积范围为1.5-3.5 BV时,富集获得TL1-1,纯度为84.64%,得率为75.06%。本论文还进行了 10倍放大实验,获得TL1-1的纯度为80.33%,得率为70.02%,与未经放大的实验结果相比几乎持平,说明XAD-16树脂在大规模分离制备TL1-1方面具有很好的应用前景。本论文对XAD-16树脂分离所得的TL1-1进行了结构确认。利用HPLC法进行初步定性分析,发现分离所得化合物与标准品的保留时间相一致。利用LC-MS法与1HNMR法分别对TL1-1进行分子量测定与结构确认,获得的TL1-1分子量(178)与结构氢谱数据均与文献报道结果一致,进一步确认化合物为TL1-1。此外,通过抑菌圈实验对分离制备所得TL1-1的抑菌活性进行了初步考察,发现TL1-1对烟草青枯病菌具有较好的抑制作用。综上所述,本论文主要对两种分离制备TL1-1的工艺方法进行了考察。与硅胶柱层析相比,XAD-16大孔吸附树脂在操作性、纯度、得率、可再生、安全性以及放大制备等方面皆具有显著的优势。这说明大孔吸附树脂法在分离制备天然产物方面具有非常好的应用前景与价值。
[Abstract]:5-hydroxy-2-methylchrommannione (TL1-1) is a phenolic compound isolated from the metabolites of endophytic fungus Daldinia eschscholzii of Tenodora aridifolia. According to the literature, TL1-1 has significant cytotoxicity to human leukemia cell HL-60, and significant inhibitory activity to agricultural pathogenic bacteria Micrabotryum violaceuw. However, the fermentation process level of TL 1-1 is low, so it is difficult to meet the demand of activity research. The existing separation methods of TL1-1 are still limited to TLC and HPLC, so it is difficult to carry out amplification experiments. Based on the optimization and amplification of the 500L fermentation process of other students in the laboratory, the different separation and preparation processes of TL1-1 were investigated and compared in this paper. In this paper, the separation and preparation of TL 1-1 by silica gel column chromatography were established. The fermentation liquor extract was separated by silica gel column chromatography. The mass ratio of sample extract to silica gel was 1: 20. The eluent was petroleum ether ethyl acetate system, and the gradient elution was carried out in the order of 100: 1: 50: 1: 1 20: 1 10: 1 1: 1. The elution volume of each gradient is 2 BV and the volume flow rate is 1 BV / h. When the elution condition is petroleum ether: ethyl acetate 20: 1, the TL1-1 component is enriched, the purity is 59.75 and the yield is 68.82%. Due to the problems of low yield, low purity, difficult regeneration and large consumption of organic solvents, silica gel column chromatography is difficult to meet the requirements of efficient and economical separation and preparation. On the basis of the above mentioned, the separation and preparation of TL 1-1 by macroporous adsorption resin were studied. XAD-16 resin was selected as the best adsorption resin by comparison of adsorption capacity and desorption rate, and the process conditions were optimized by static kinetic and thermodynamic experiments. It was found that the adsorption of TL1-1 by XAD-16 resin was in accordance with the quasi-first-order kinetic model Weber-Morris model and Freundlich empirical model. The optimum conditions for the preparation of TL1-1 by XAD-16 resin were obtained: the sample amount was 11 BV, the initial concentration was 0.508 mg / mL, the pH value of solution was 4.0, the flow rate was 2 BV / h, and the eluent was ethanol water system. The concentration gradient was 40% ethanol solution (4BV) and 95% ethanol solution (4BV), and the volume flow rate was 2BV / h. The 95% ethanol eluate was detected and collected. When the elution volume was in the range of 1.5-3.5 BV, TL1-1 was obtained, the purity was 84.644.64, and the yield was 75.06B. The experiment of 10 times amplification showed that the purity of TL1-1 was 80.33, the yield of TL1-1 was 70.022.Compared with the unamplified experimental results, XAD-16 resin had a good application prospect in large-scale separation and preparation of TL1-1. In this paper, the structure of TL 1-1 from XAD- 16 resin was confirmed. A preliminary qualitative analysis by HPLC was carried out. It was found that the retention time of the isolated compounds was consistent with that of the standard compounds. The molecular weight and structure of TL1-1 were determined by LC-MS and 1H NMR, respectively. The obtained molecular weight (178) and structural hydrogen spectrum data of TL1-1 were in agreement with those reported in the literature, and the compound was further confirmed as TL1-1. In addition, the bacteriostatic activity of TL1-1 was preliminarily investigated by bacteriostasis test, and it was found that TL1-1 had a good inhibitory effect on tobacco bacterial wilt. To sum up, two methods of separation and preparation of TL 1-1 were investigated in this paper. Compared with silica gel column chromatography, XAD-16 macroporous adsorption resin has significant advantages in operation, purity, yield, regeneration, safety and amplification. This shows that macroporous adsorption resin method has a very good application prospect and value in separation and preparation of natural products.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ920.1;TQ450.1

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