本文選題：鐵皮石斛原球莖培養(yǎng)物 + 高速逆流色譜��； 參考：《山東師范大學(xué)》2017年碩士論文

【摘要】：鐵皮石斛是我國一種傳統(tǒng)的名貴中草藥,它主要的藥用部分是其新鮮或者干燥的莖。鐵皮石斛具有滋陰補(bǔ)陽,健胃補(bǔ)脾,潤肺止咳的功效,還可以抗癌防癌,延年益壽。面對(duì)巨大的社會(huì)需求量,導(dǎo)致了鐵皮石斛資源短缺而且價(jià)格昂貴,但是,鐵皮石斛原球莖培養(yǎng)物的出現(xiàn),可以讓目前市場(chǎng)上鐵皮石斛缺乏以及供不應(yīng)求的現(xiàn)狀得到一些緩解,通過研究鐵皮石斛原球莖培養(yǎng)物的化學(xué)成分,我們可以更好地了解人工栽培的鐵皮石斛原球莖培養(yǎng)物與純野外生長(zhǎng)的鐵皮石斛之間的區(qū)別,從而使鐵皮石斛原球莖培養(yǎng)物更好地更加充分地得以利用。本論文的主要研究?jī)?nèi)容有如下四點(diǎn):1.研究了通過高速逆流色譜技術(shù)從鐵皮石斛原球莖培養(yǎng)物中分離制備出芥子酸,此舉為石斛屬內(nèi)首次分離。分離采用的溶劑體系為石油醚-乙酸乙酯-甲醇-水(4:6:3:7,v/v),固定相流速為12 mL/min,流動(dòng)相流速為2 mL/min,單次進(jìn)樣200 mg,轉(zhuǎn)速800 rpm,檢測(cè)波長(zhǎng)280 nm,固定相保留率46.67%,一步分離得到純度為93.11%的芥子酸。體外抗氧化實(shí)驗(yàn)結(jié)果顯示,芥子酸具有較強(qiáng)的DPPH·自由基清除能力,計(jì)算可得其IC50值為0.046mg/m L。2.利用高速逆流色譜法進(jìn)行分離,優(yōu)化兩相溶劑系統(tǒng)后采用叔丁基甲醚-正丁醇-乙腈-水(5:1:2:6,v/v)的兩相溶劑體系,一步分離得到三種化合物,固定相流速為15 mL/min,流動(dòng)相流速為1 mL/min,單次進(jìn)樣150 mg,轉(zhuǎn)速800 rpm,檢測(cè)波長(zhǎng)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,純度為97.8%;化合物II是β-D-glucopyranose 1-[(E)-3-(3,4-dihydroxyphenyl)-2-propenoat,純度為98.4%;化合物III是1-O-sinapoyl glucopyranoside,純度為98.6%。通過HPLC測(cè)定三種化合物的純度,運(yùn)用ESI-MS,1 H NMR和13 C NMR鑒定這三種化合物的結(jié)構(gòu)。實(shí)驗(yàn)結(jié)果表明,三種化合物對(duì)DPPH·具有清除作用,在濃度為0.01mg/m L-0.2 mg/mL的時(shí)候,清除率隨著濃度的增加而增加,其中化合物I的效果較差,化合物II的效果最好,經(jīng)計(jì)算得化合物I,II和III清除DPPH·自由基的IC50值分別為0.6219 mg/m L,0.0497 mg/mL和0.1111mg/m L。3.優(yōu)化了鐵皮石斛原球莖培養(yǎng)物中多糖提取工藝及不同脫蛋白方法的比較。以提取溫度,提取時(shí)間,料液比三因素進(jìn)行正交試驗(yàn)得到多糖最佳提取工藝為提取溫度90℃、提取時(shí)間60 min、料液比1:40 g/mL,多糖提取率最高為78.51%。通過一系列單因素實(shí)驗(yàn)和正交優(yōu)化實(shí)驗(yàn),分析比較Sevage法、鹽酸法、三氯乙酸法和木瓜蛋白酶法四種脫蛋白工藝。結(jié)果顯示,木瓜蛋白酶法反應(yīng)時(shí)間90 min,溫度70℃、酶含量30 U時(shí),多糖含量85.01%,蛋白含量為5.89%,效果最為理想。Sevage溶劑脫蛋白中隨著洗脫次數(shù)的增加,多糖含量一直在減少,損失嚴(yán)重;鹽酸法中HCl濃度為25%時(shí)效果最好,多糖含量52.17%,蛋白含量為4.82%;三氯乙酸法中TCA濃度為0.03 g/m L時(shí),多糖含量最高35.50%,蛋白含量7.34%。4.鐵皮石斛原球莖培養(yǎng)物提取多糖脫蛋白后經(jīng)過DEAE-52陰離子交換柱層析純化,得到DOKM-A、DOKM-B、DOKM-C、DOKM-D四個(gè)洗脫峰。DOKM-A洗脫峰經(jīng)多次重復(fù)實(shí)驗(yàn)進(jìn)行收集,通過Sephadex G-75柱層析純化,得到DOKM-A-1,DOKM-A-2兩個(gè)洗脫峰,經(jīng)過島津高速液相檢測(cè)顯示DOKM-A-1相比DOKM-A-2純度較高,它的分子量是12 KDa。單糖組成分別為甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖,各單糖摩爾比為1:1.821:0.367:1.269:0.355:0.323:1.024。將得到的各部分多糖分別進(jìn)行DPPH·的清除作用實(shí)驗(yàn),鐵皮石斛原球莖培養(yǎng)物總多糖對(duì)DPPH·的清除作用較好,經(jīng)過DEAE-52陰離子交換柱層析純化所得的DOKM-A、DOKM-B、DOKM-C、DOKM-D這四種多糖對(duì)DPPH·的清除作用相對(duì)較差,DOKM-ADOKM-DDOKM-BDOKM-C,而多糖DOKM-A再次經(jīng)Sephadex G-75柱層析純化所得的DOKM-A-1對(duì)DPPH·的清除作用相對(duì)DOKM-A較差。
[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.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R284;O652

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 徐德林;儲(chǔ)士潤;肖世基;祝飛;錢剛;李林;;不同產(chǎn)地的鐵皮石斛和金釵石斛石斛多糖、石斛堿、氨基酸含量的比較[J];時(shí)珍國醫(yī)國藥;2016年11期

2 黎晶晶;李琳;徐柏頤;;一測(cè)多評(píng)法測(cè)定鐵皮石斛黃酮類共有成分[J];浙江農(nóng)業(yè)科學(xué);2016年09期

3 梁青青;李錫明;黃月圓;梁凌玲;黃鎖義;;野生鐵皮石斛色素超聲提取工藝的優(yōu)選[J];微量元素與健康研究;2017年01期

4 井鳳;劉峰;劉偉;;超高壓技術(shù)在中藥有效成分提取中的應(yīng)用[J];科學(xué)與管理;2016年04期

5 葉子;盧葉;薛亞甫;徐紅;王崢濤;;鐵皮石斛專屬性成分的分離制備及質(zhì)量標(biāo)準(zhǔn)研究[J];中國中藥雜志;2016年13期

6 韋菲;岑忠用;黃映恒;;鐵皮石斛人工種植技術(shù)研究[J];現(xiàn)代農(nóng)業(yè)科技;2016年11期

7 蘇丹;高玉橋;梁耀光;張?zhí)煊?梅全喜;;高速逆流色譜法分離制備山芝麻中的三萜類成分[J];中藥材;2016年05期

8 馬叢叢;許繼取;韓領(lǐng);田光晶;黃鳳洪;;芥子酸及其生物活性研究進(jìn)展[J];中國油脂;2016年05期

9 王雪竹;吳順紅;趙光遠(yuǎn);;超高壓提取女貞子總?cè)乒に嚄l件的優(yōu)化[J];安徽醫(yī)藥;2016年04期

10 胡奇杰;朱佳茜;王新財(cái);顧寬宇;;鐵皮石斛飲片中有機(jī)氯及擬除蟲菊酯農(nóng)藥多殘留檢測(cè)方法的研究[J];安徽農(nóng)業(yè)科學(xué);2016年07期

相關(guān)博士學(xué)位論文 前1條

1 包英華;鐵皮石斛種質(zhì)資源的鑒定與評(píng)價(jià)研究[D];廣州中醫(yī)藥大學(xué);2014年

，

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