本文選題：TL1-1 + 硅膠柱層析��； 參考：《華東理工大學(xué)》2017年碩士論文

【摘要】：5-羥基-2-甲基苯并二氫吡喃酮(5-hydroxy-2-methylchrommanone,TL1-1)是螳螂(Tenodora aridifolia)腸道內(nèi)內(nèi)生真菌Daldinia eschscholzii代謝產(chǎn)物中分離到的一種酚類化合物。據(jù)文獻(xiàn)報(bào)道,TL1-1對(duì)人類白血病細(xì)胞HL-60有顯著的細(xì)胞毒性,對(duì)農(nóng)業(yè)致病菌Micrabotryum violaceuw有顯著的抑菌活性。然而,TL1-1的發(fā)酵工藝水平低,難以滿足活性研究的需求量。且現(xiàn)有的TL1-1分離方法仍局限于TLC法、HPLC法,難以進(jìn)行放大實(shí)驗(yàn)。本論文針對(duì)上述問(wèn)題,在實(shí)驗(yàn)室其他同學(xué)500 L發(fā)酵工藝優(yōu)化放大的基礎(chǔ)上,針對(duì)TL1-1的不同分離制備工藝進(jìn)行考察與比較。本論文建立了硅膠柱層析法分離制備TL1-1的工藝。采用硅膠柱層析對(duì)發(fā)酵液浸膏進(jìn)行分離,上樣浸膏與硅膠質(zhì)量比為1:20;洗脫劑為石油醚-乙酸乙酯體系,按照100:1、50:1、20:1、10:1、5:1的順序進(jìn)行梯度洗脫,每個(gè)梯度的洗脫體積為2 BV,體積流量為1 BV/h;當(dāng)洗脫條件石油醚:乙酸乙酯=20:1時(shí),富集獲得TL1-1組分,純度為59.75%,得率為68.82%。硅膠柱層析法因得率低、純度低、難再生且有機(jī)溶劑消耗大等問(wèn)題,難以符合高效的、經(jīng)濟(jì)節(jié)約的分離制備要求。在前述基礎(chǔ)上,本論文又研究了大孔吸附樹(shù)脂法分離制備TL1-1的工藝。通過(guò)吸附量與解吸率的比較,篩選出XAD-16樹(shù)脂為最佳吸附樹(shù)脂;通過(guò)靜態(tài)動(dòng)力學(xué)與熱力學(xué)實(shí)驗(yàn)對(duì)工藝條件進(jìn)行優(yōu)化,發(fā)現(xiàn)XAD-16樹(shù)脂對(duì)TL1-1的吸附符合準(zhǔn)一級(jí)動(dòng)力學(xué)模型、Weber-Morris模型以及Freundlich經(jīng)驗(yàn)?zāi)Ｐ?通過(guò)動(dòng)態(tài)吸附與解吸附實(shí)驗(yàn),獲得XAD-16樹(shù)脂分離制備TL1-1的最佳工藝條件:上樣量為11 BV,初始濃度為0.508 mg/mL,溶液pH值為4.0,上樣流速為2 BV/h;洗脫劑為乙醇-水體系,濃度梯度依次為去離子水(4BV)、40%乙醇溶液(4BV)、95%乙醇溶液(4BV),體積流量為2BV/h。檢測(cè)并收集95%乙醇洗脫液,在洗脫體積范圍為1.5-3.5 BV時(shí),富集獲得TL1-1,純度為84.64%,得率為75.06%。本論文還進(jìn)行了 10倍放大實(shí)驗(yàn),獲得TL1-1的純度為80.33%,得率為70.02%,與未經(jīng)放大的實(shí)驗(yàn)結(jié)果相比幾乎持平,說(shuō)明XAD-16樹(shù)脂在大規(guī)模分離制備TL1-1方面具有很好的應(yīng)用前景。本論文對(duì)XAD-16樹(shù)脂分離所得的TL1-1進(jìn)行了結(jié)構(gòu)確認(rèn)。利用HPLC法進(jìn)行初步定性分析,發(fā)現(xiàn)分離所得化合物與標(biāo)準(zhǔn)品的保留時(shí)間相一致。利用LC-MS法與1HNMR法分別對(duì)TL1-1進(jìn)行分子量測(cè)定與結(jié)構(gòu)確認(rèn),獲得的TL1-1分子量(178)與結(jié)構(gòu)氫譜數(shù)據(jù)均與文獻(xiàn)報(bào)道結(jié)果一致,進(jìn)一步確認(rèn)化合物為T(mén)L1-1。此外,通過(guò)抑菌圈實(shí)驗(yàn)對(duì)分離制備所得TL1-1的抑菌活性進(jìn)行了初步考察,發(fā)現(xiàn)TL1-1對(duì)煙草青枯病菌具有較好的抑制作用。綜上所述,本論文主要對(duì)兩種分離制備TL1-1的工藝方法進(jìn)行了考察。與硅膠柱層析相比,XAD-16大孔吸附樹(shù)脂在操作性、純度、得率、可再生、安全性以及放大制備等方面皆具有顯著的優(yōu)勢(shì)。這說(shuō)明大孔吸附樹(shù)脂法在分離制備天然產(chǎn)物方面具有非常好的應(yīng)用前景與價(jià)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.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ920.1;TQ450.1

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