铁皮石斛原球茎培养物化学成分的分离与鉴定

发布时间：2018-06-30 00:41

本文选题：铁皮石斛原球茎培养物 + 高速逆流色谱　；参考：《山东师范大学》2017年硕士论文

【摘要】：铁皮石斛是我国一种传统的名贵中草药,它主要的药用部分是其新鲜或者干燥的茎。铁皮石斛具有滋阴补阳,健胃补脾,润肺止咳的功效,还可以抗癌防癌,延年益寿。面对巨大的社会需求量,导致了铁皮石斛资源短缺而且价格昂贵,但是,铁皮石斛原球茎培养物的出现,可以让目前市场上铁皮石斛缺乏以及供不应求的现状得到一些缓解,通过研究铁皮石斛原球茎培养物的化学成分,我们可以更好地了解人工栽培的铁皮石斛原球茎培养物与纯野外生长的铁皮石斛之间的区别,从而使铁皮石斛原球茎培养物更好地更加充分地得以利用。本论文的主要研究内容有如下四点:1.研究了通过高速逆流色谱技术从铁皮石斛原球茎培养物中分离制备出芥子酸,此举为石斛属内首次分离。分离采用的溶剂体系为石油醚-乙酸乙酯-甲醇-水(4:6:3:7,v/v),固定相流速为12 mL/min,流动相流速为2 mL/min,单次进样200 mg,转速800 rpm,检测波长280 nm,固定相保留率46.67%,一步分离得到纯度为93.11%的芥子酸。体外抗氧化实验结果显示,芥子酸具有较强的DPPH·自由基清除能力,计算可得其IC50值为0.046mg/m L。2.利用高速逆流色谱法进行分离,优化两相溶剂系统后采用叔丁基甲醚-正丁醇-乙腈-水(5:1:2:6,v/v)的两相溶剂体系,一步分离得到三种化合物,固定相流速为15 mL/min,流动相流速为1 mL/min,单次进样150 mg,转速800 rpm,检测波长280 nm,固定相保留率41.67%化合物I是β-D-glucopyranose 1-[(E)-3-(4-hydroxyphenyl)-2-propenoat,纯度为97.8%;化合物II是β-D-glucopyranose 1-[(E)-3-(3,4-dihydroxyphenyl)-2-propenoat,纯度为98.4%;化合物III是1-O-sinapoyl glucopyranoside,纯度为98.6%。通过HPLC测定三种化合物的纯度,运用ESI-MS,1 H NMR和13 C NMR鉴定这三种化合物的结构。实验结果表明,三种化合物对DPPH·具有清除作用,在浓度为0.01mg/m L-0.2 mg/mL的时候,清除率随着浓度的增加而增加,其中化合物I的效果较差,化合物II的效果最好,经计算得化合物I,II和III清除DPPH·自由基的IC50值分别为0.6219 mg/m L,0.0497 mg/mL和0.1111mg/m L。3.优化了铁皮石斛原球茎培养物中多糖提取工艺及不同脱蛋白方法的比较。以提取温度,提取时间,料液比三因素进行正交试验得到多糖最佳提取工艺为提取温度90℃、提取时间60 min、料液比1:40 g/mL,多糖提取率最高为78.51%。通过一系列单因素实验和正交优化实验,分析比较Sevage法、盐酸法、三氯乙酸法和木瓜蛋白酶法四种脱蛋白工艺。结果显示,木瓜蛋白酶法反应时间90 min,温度70℃、酶含量30 U时,多糖含量85.01%,蛋白含量为5.89%,效果最为理想。Sevage溶剂脱蛋白中随着洗脱次数的增加,多糖含量一直在减少,损失严重;盐酸法中HCl浓度为25%时效果最好,多糖含量52.17%,蛋白含量为4.82%;三氯乙酸法中TCA浓度为0.03 g/m L时,多糖含量最高35.50%,蛋白含量7.34%。4.铁皮石斛原球茎培养物提取多糖脱蛋白后经过DEAE-52阴离子交换柱层析纯化,得到DOKM-A、DOKM-B、DOKM-C、DOKM-D四个洗脱峰。DOKM-A洗脱峰经多次重复实验进行收集,通过Sephadex G-75柱层析纯化,得到DOKM-A-1,DOKM-A-2两个洗脱峰,经过岛津高速液相检测显示DOKM-A-1相比DOKM-A-2纯度较高,它的分子量是12 KDa。单糖组成分别为甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖,各单糖摩尔比为1:1.821:0.367:1.269:0.355:0.323:1.024。将得到的各部分多糖分别进行DPPH·的清除作用实验,铁皮石斛原球茎培养物总多糖对DPPH·的清除作用较好,经过DEAE-52阴离子交换柱层析纯化所得的DOKM-A、DOKM-B、DOKM-C、DOKM-D这四种多糖对DPPH·的清除作用相对较差,DOKM-ADOKM-DDOKM-BDOKM-C,而多糖DOKM-A再次经Sephadex G-75柱层析纯化所得的DOKM-A-1对DPPH·的清除作用相对DOKM-A较差。
[Abstract]:Dendrobium officinale is a traditional Chinese herbal medicine, its main medicinal part is its fresh or dry stem. Dendrobium officinale has the effect of Nourishing Yin, invigorating the spleen, moistening the lung and relieving cough. It also can prevent cancer and prolong life. Facing the huge social demand, it leads to the shortage and high price of Dendrobium officinale, but it is expensive, but, however, The emergence of the protocorm culture of Dendrobium officinale can relieve the current situation of the shortage of Dendrobium officinale and the current situation of supply shortage. By studying the chemical composition of the protocorm culture of Dendrobium candidum, we can better understand the cultured protocorm culture of Dendrobium officinale and the pure wild Dendrobium. The main research contents of this paper are as follows: 1. the preparation of erucic acid from the protocorm culture of Dendrobium officinale by high speed counter flow chromatography is the first separation. This is the first separation of Dendrobium in the genus Dendrobium. The solvent system used for separation of Dendrobium is the first time. Petroleum ether ethyl acetate methanol water (4:6:3:7, v/v), fixed phase velocity of 12 mL/min, flow velocity of 2 mL/min, single injection 200 mg, rotational speed 800 rpm, detection wavelength 280 nm, fixed phase retention rate 46.67%, one step separation of the purity of 93.11% erucic acid. In vitro anti oxidation experimental results show that erucic acid has a strong DPPH free radical. The IC50 value of 0.046mg/m L.2. was calculated to be separated by high speed counter current chromatography. After optimizing the two phase solvent system, the two-phase solvent system of tert butyl ether - n-butanol acetonitrile water (5:1:2:6, v/v) was used. Three compounds were separated in one step, the velocity of the stationary phase was 15 mL/min, the flow velocity was 1 mL/min, and the single injection was 15. 0 mg, rotation speed 800 rpm, detection wavelength 280 nm, fixed phase retention rate 41.67% compound I is beta -D-glucopyranose 1-[(E) -3- (4-hydroxyphenyl) -2-propenoat, purity of 97.8%; compound II is beta -D-glucopyranose. The purity of the three compounds was determined by HPLC. The structure of the three compounds was identified by ESI-MS, 1 H NMR and 13 C NMR. The results showed that the three compounds had scavenging effect on DPPH. When the concentration was 0.01mg/m L-0.2 mg/mL, the clearance rate increased with the increase of concentration, and the effect of compound I was poor and the effect of compound II. The best result is that the IC50 values of the compounds I, II and III for removing DPPH free radicals are 0.6219 mg/m L, 0.0497 mg/mL and 0.1111mg/m L.3., respectively, to optimize the extraction process of Polysaccharide from the protocorm culture of Dendrobium officinale and the comparison of the different deproteinized methods. The optimum extraction temperature, extraction time, and the ratio of material to liquid are obtained by orthogonal test. The extraction process was 90 C, the extraction time was 60 min, the ratio of material to liquid was g/mL at 1:40, and the highest extraction rate of polysaccharide was 78.51%. through a series of single factor experiments and orthogonal optimization experiments. Sevage, hydrochloric acid, three chloroacetic acid method and papain method were compared. The results showed that the reaction time of papain was 90 min, When the temperature was 70, the content of the enzyme was 30 U, the content of polysaccharide was 85.01% and the protein content was 5.89%. The most ideal.Sevage solvent deproteinized with the increase of the times of elution, the content of polysaccharide had been reduced and the loss was serious. When the concentration of HCl was 25% in hydrochloric acid, the content of polysaccharide was 52.17%, the protein content was 4.82%, and the concentration of TCA in the chloroacetic acid method was 0.03. The concentration of TCA was 0.03. At the time of g/m L, the content of polysaccharide was 35.50%. The protein content of the protocorm culture of Dendrobium officinale 7.34%.4. was purified by DEAE-52 anion exchange column chromatography. The.DOKM-A elution peak of four elution peaks of DOKM-A, DOKM-B, DOKM-C and DOKM-D was collected by repeated experiments and purified by Sephadex G-75 column chromatography. DOKM-A-2 two elution peaks, after SHIMADZU high speed liquid phase detection, showed that DOKM-A-1 had higher purity than DOKM-A-2, and its molecular weight was 12 KDa. monosaccharides, which were mannose, rhamnose, galacononic acid, glucose, galactose, xylose, Arabia sugar, and mole ratio of each monosaccharide to 1:1.821:0.367:1.269:0.355:0.323:1.024.. The scavenging effect of polysaccharides was carried out by DPPH. The scavenging effect of the total polysaccharide on the protocorm culture of Dendrobium officinale was better. The four kinds of polysaccharides obtained by DEAE-52 anion exchange column chromatography were relatively poor in the clearance of DPPH. DOKM-ADOKM-DDOKM-BDOKM-C, while the polysaccharides DOKM-A were again Sep. The scavenging effect of DOKM-A-1 from hadex G-75 column chromatography on DPPH was poorer than that of DOKM-A.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284;O652

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