哈薩克藥材別克參多糖分離純化、抗炎免疫活性及藥材質(zhì)量標(biāo)準(zhǔn)研究
發(fā)布時(shí)間：2018-08-31 15:42
【摘要】：目的:通過(guò)提取、分離、純化獲得別克參均一多糖,對(duì)均一多糖理化性質(zhì)及一級(jí)結(jié)構(gòu)進(jìn)行初步表征,為別克參藥材物質(zhì)基礎(chǔ)研究奠定基礎(chǔ);考察別克參多糖體內(nèi)、體外免疫調(diào)節(jié)及抗炎活性,為該藥材生物活性研究提供理論支持;建立別克參藥材質(zhì)量控制方法,保證該藥材臨床用藥的安全、有效、可控、穩(wěn)定。方法:(1)運(yùn)用響應(yīng)曲面法及四因素三水平的Box-Behnken中心組合實(shí)驗(yàn)設(shè)計(jì)方法優(yōu)化別克參多糖成分的熱水提取工藝,通過(guò)DEAE Sepharose CL-6B和Sephadex G-100對(duì)提取后的粗多糖進(jìn)行分離純化,采用高效凝膠色譜法分析純化后均一多糖的純度及相對(duì)分子質(zhì)量;(2)運(yùn)用苯酚-硫酸法、間羥聯(lián)苯法、碘顯色法、柱前衍生-HPLC法測(cè)定別克參均一多糖中總糖含量、糖醛酸、淀粉含量及單糖組成;結(jié)合紅外、Smith降解、甲基化實(shí)驗(yàn)、部分酸水解及NMR分析初步鑒別均一多糖的結(jié)構(gòu);(3)通過(guò)小鼠體內(nèi)碳粒廓清實(shí)驗(yàn)考察別克參粗多糖對(duì)免疫低下小鼠非特異性免疫調(diào)節(jié)功能;通過(guò)二甲苯致小鼠耳腫脹、小鼠棉球肉芽腫及冰醋酸致小鼠扭體實(shí)驗(yàn)考察別克參粗多糖抗炎鎮(zhèn)痛功效;通過(guò)測(cè)定別克參多糖組分對(duì)RAW264.7細(xì)胞活性、吞噬能力、分泌細(xì)胞因子及介質(zhì)能力的影響,考察體外免疫調(diào)節(jié)能力;通過(guò)測(cè)定別克參多糖組分對(duì)LPS誘導(dǎo)的RAW264.7炎癥細(xì)胞分泌炎癥因子及介質(zhì)的影響,考察體外抗炎能力;(4)藥材質(zhì)量標(biāo)準(zhǔn)研究:分析10批別克參藥材中水分、灰分;建立微波消解ICP-MS法測(cè)定別克參中微量元素、重金屬含量的方法;建立乙腈超聲萃取、GC-MS法分析別克參藥材中農(nóng)藥殘留的方法;建立別克參藥材中氨基酸成分、多糖成分的薄層鑒別方法;建立柱前衍生-HPLC法測(cè)定藥材中多糖含量的方法。結(jié)果:(1)響應(yīng)曲面法優(yōu)化后的別克參粗多糖提取工藝為:提取時(shí)間4.28 h,提取溫度90℃,料液比37 ml/g,提取次數(shù)3次。驗(yàn)證試驗(yàn)所得產(chǎn)率(37.25%±0.17%)與回歸模型預(yù)測(cè)值37.465%吻合。別克參粗多糖經(jīng)DEAE Sepharose CL-6B和Sephadex G-100分離純化后,得4個(gè)均一多糖(ESBP1-1、ESBP1-2、ESBP2-1、ESBP3-1),高效凝膠色譜法表明分子量分別為1.3×104、1.7×104、9.4×105、4.1×105 Da。(2)苯酚-硫酸法結(jié)果表明ESBP1-1、ESBP1-2、ESBP2-1、ESBP3-1總糖含量分別為97.8%、98.7%、99.3%、97.0%;間羥聯(lián)苯法結(jié)果表明ESBP2-1、ESBP3-1糖醛酸含量分別為0.02%、1.9%,ESBP1-1、ESBP1-2中不含糖醛酸;碘顯色法結(jié)果表明ESBP1-1、ESBP1-2含淀粉分別為0.5%、0.3%,ESBP2-1、ESBP3-1中未檢出淀粉;柱前衍生-HPLC法結(jié)果表明ESBP1-1、ESBP1-2均由葡萄糖組成,ESBP2-1主要由葡萄糖、半乳糖、阿拉伯糖組成(摩爾比24.3∶1.1∶1),ESBP3-1主要由葡萄糖和半乳糖組成(摩爾比14.8∶1);紅外、Smith降解、甲基化實(shí)驗(yàn)、部分酸水解及NMR分析結(jié)果表明別克參4種均一多糖的結(jié)構(gòu)具有共性,4種均一多糖主鏈均由(1→4)-α-D-Glcp構(gòu)成,均從6-O位上分支出側(cè)鏈,4種均一多糖側(cè)鏈結(jié)構(gòu)存在不同。(3)小鼠體內(nèi)碳粒廓清實(shí)驗(yàn)表明別克參粗多糖具有提高免疫抑制小鼠臟器指數(shù)及單核巨噬細(xì)胞廓清指數(shù)的能力,提示別克參多糖具有一定非特異性免疫調(diào)節(jié)功能;二甲苯致小鼠耳腫脹、小鼠棉球肉芽腫及冰醋酸致小鼠扭體實(shí)驗(yàn)表明別克參粗多糖具有明顯抑制耳腫脹、降低肉芽質(zhì)量,減少扭體次數(shù)的能力,提示別克參多糖具有明顯抗炎鎮(zhèn)痛功效;體外細(xì)胞實(shí)驗(yàn)表明,別克參粗多糖ESBP及均一多糖ESBP1-1、ESBP1-2、ESBP2-1及ESBP3-1能促進(jìn)RAW264.7細(xì)胞增殖,提高其吞噬活力,上調(diào)NO、IL-1β及TNF-α的釋放,顯現(xiàn)免疫增強(qiáng)作用;LPS誘導(dǎo)的RAW264.7細(xì)胞炎癥反應(yīng)實(shí)驗(yàn)表明別克參多糖可抑制由LPS誘導(dǎo)的過(guò)度免疫而產(chǎn)生的NO、IL-1β、TNF-α的分泌,顯現(xiàn)抗炎效果。(4)質(zhì)量控制方法研究:建立了別克參藥材中微量金屬元素、農(nóng)藥殘留的檢查方法,建立了別克參藥材中氨基酸成分、多糖成分的薄層鑒別方法,建立了藥材中多糖成分的含量測(cè)定方法,完成藥材質(zhì)量標(biāo)準(zhǔn)草案及起草說(shuō)明的撰寫(xiě)。結(jié)論:(1)運(yùn)用響應(yīng)曲面法提取別克參多糖,建立的模型準(zhǔn)確可靠,可以用于別克參多糖的提取工藝;(2)運(yùn)用DEAE Sepharose CL-6B和Sephadex G-100分離純化別克參粗多糖,方法可行;純化出4種均一多糖,4種均一多糖主鏈結(jié)構(gòu)具有共性;(3)別克參多糖組分具有增強(qiáng)免疫力的功效,可能與別克參多糖可促進(jìn)細(xì)胞增殖、增強(qiáng)吞噬能力及上調(diào)細(xì)胞因子及介質(zhì)有關(guān);同時(shí),別克參多糖具有抗炎活性,可能與別克參多糖可抑制由過(guò)度免疫引起的炎性因子及介質(zhì)的分泌有關(guān)。綜合分析別克參多糖的這兩種表現(xiàn),說(shuō)明別克參多糖具有免疫調(diào)節(jié)的雙向性,其抗炎活性與免疫調(diào)節(jié)有關(guān);(4)建立了別克參藥材的質(zhì)量標(biāo)準(zhǔn),通過(guò)方法學(xué)考察及系統(tǒng)驗(yàn)證,表明方法操作簡(jiǎn)單、穩(wěn)定、可行,填補(bǔ)了該藥材質(zhì)量控制方面的空白。
[Abstract]:OBJECTIVE: To obtain homogeneous polysaccharides from Buick Ginseng by extraction, isolation and purification, and to characterize the physicochemical properties and primary structure of homogeneous polysaccharides so as to lay a foundation for the study on the material basis of Buick Ginseng. Methods: (1) Response surface methodology and four factors and three levels of Box-Behnken central composite experimental design method were used to optimize the hot water extraction process of Polysaccharides from Buick Ginseng. DEAE Sepharose CL-6B and Sephadex G-100 were used to extract crude polysaccharides from Buick Ginseng. The purity and molecular weight of the purified polysaccharides were analyzed by high performance gel chromatography. (2) The contents of total sugar, glucuronic acid, starch and monosaccharide in the homogeneous polysaccharides were determined by phenol-sulfuric acid method, m-hydroxybenzene method, iodine coloration method, pre-column derivatization-HPLC method. Partial acid hydrolysis and NMR analysis were used to identify the structure of the homogeneous polysaccharides. (3) Carbon clearance test in mice was used to investigate the non-specific immune regulation function of Buick Ginseng crude polysaccharides on immunosuppressed mice. The effects of Polysaccharides from Buick Ginseng on the activity, phagocytosis, cytokine and mediator secretion of RAW264.7 cells were studied to investigate the immunomodulatory ability in vitro. Standard study: analysis of water and ash in 10 batches of Buickshen medicinal materials; establishment of microwave digestion ICP-MS method for determination of trace elements and heavy metals in Buickshen; establishment of acetonitrile ultrasonic extraction, GC-MS method for analysis of pesticide residues in Buickshen medicinal materials; establishment of Buickshen medicinal amino acid components, polysaccharide components TLC identification method; Results: (1) Response surface methodology was used to optimize the extraction process of crude polysaccharides from Buick Ginseng. The extraction time was 4.28 h, the extraction temperature was 90 ~C, the ratio of material to liquid was 37 ml/g, and the extraction times were 3 times. The yield (37.25%+0.17%) was in agreement with the predicted value of regression model (37.465%). After separation and purification of AE Sepharose CL-6B and Sephadex G-100, four homogeneous polysaccharides (ESBP1-1, ESBP1-2, ESBP2-1, ESBP3-1) were obtained. The molecular weights of these polysaccharides were 1.3 x 104, 1.7 x 104, 9.4 x 105, 4.1 x 105Da. (2) The results of phenol-sulfuric acid method showed that the total sugar contents of ESBP1-1, ESBP1-2-1, ESBP2-1 and ESBP3-1 were 97.8%, 98.7%, 99.3% and 97.0% respectively. The content of ESBP2-1, ESBP3-1 glucuronic acid was 0.02%, 1.9%, ESBP1-1 and ESBP1-2 were free of glucuronic acid, while the content of ESBP1-1, ESBP1-2 were 0.5%, 0.3%, 0.3%, ESBP2-1 and ESBP3-1 were not detected by hydroxybenzene method, respectively. ESBP3-1 was composed of glucose, galactose and arabinose (molar ratio 24.3:1.1:1), and ESBP3-1 was mainly composed of glucose and galactose (molar ratio 14.8:1); IR, Smith degradation, methylation, partial acid hydrolysis and NMR analysis showed that the structures of four homogeneous polysaccharides of Buicksan were similar, and the main chains of the four homogeneous polysaccharides were composed of (1_4) - alpha-D-Glcp. All the four homogeneous polysaccharides branched from the 6-O position and had different side chains. (3) Carbon clearance test in mice showed that the crude polysaccharides of Buick Ginseng could improve the organ index and monocyte-macrophage clearance index of immunosuppressed mice, suggesting that Buick Ginseng polysaccharides had some non-specific immunomodulatory function. Swelling, cotton ball granuloma and writhing test in mice induced by glacial acetic acid showed that the crude polysaccharide of Buick Ginseng had the ability to inhibit ear swelling, reduce the quality of granulation and writhing times, suggesting that Buick Ginseng polysaccharide had obvious anti-inflammatory and analgesic effects; in vitro cell experiments showed that Buick Ginseng crude polysaccharide ESBP and homogeneous polysaccharide ESBP1-1, ESBP1-2, ESBP2-1 And ESBP3-1 can promote the proliferation of RAW264.7 cells, increase their phagocytic activity, up-regulate the release of NO, IL-1 beta and TNF-alpha, showing immunopotentiatory effect; LPS-induced inflammatory response of RAW264.7 cells showed that Buickshen polysaccharide can inhibit the secretion of NO, IL-1 beta and TNF-alpha produced by LPS-induced excessive immunity, showing anti-inflammatory effect. METHODS: A TLC method for the determination of trace metal elements and pesticide residues in Buickshen was established. A TLC method for the identification of amino acids and polysaccharides in Buickshen was established. A method for the determination of polysaccharides in Buickshen was established. The draft quality standard of Buickshen and the drafting instructions were completed. Buick ginseng polysaccharides were extracted by DEAE Sepharose CL-6B and Sephadex G-100, and the established model was accurate and reliable, which could be used for the extraction process of Buick ginseng polysaccharides. (2) The crude polysaccharides were isolated and purified by DEAE Sepharose CL-6B and Sephadex G-100, and the method was feasible. The effect may be related to Buick Ginseng polysaccharide can promote cell proliferation, enhance phagocytosis and up-regulate cytokines and mediators; at the same time, Buick Ginseng polysaccharide has anti-inflammatory activity, which may be related to Buick Ginseng polysaccharide can inhibit the secretion of inflammatory factors and mediators caused by overimmunity. Keshen polysaccharide has two-way immunoregulation, and its anti-inflammatory activity is related to immunoregulation. (4) The quality standard of Bukeshen was established. The method was proved to be simple, stable and feasible by methodological investigation and systematic verification, which filled the blank in quality control of Bukeshen.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R29
本文編號(hào)：2215438
[Abstract]:OBJECTIVE: To obtain homogeneous polysaccharides from Buick Ginseng by extraction, isolation and purification, and to characterize the physicochemical properties and primary structure of homogeneous polysaccharides so as to lay a foundation for the study on the material basis of Buick Ginseng. Methods: (1) Response surface methodology and four factors and three levels of Box-Behnken central composite experimental design method were used to optimize the hot water extraction process of Polysaccharides from Buick Ginseng. DEAE Sepharose CL-6B and Sephadex G-100 were used to extract crude polysaccharides from Buick Ginseng. The purity and molecular weight of the purified polysaccharides were analyzed by high performance gel chromatography. (2) The contents of total sugar, glucuronic acid, starch and monosaccharide in the homogeneous polysaccharides were determined by phenol-sulfuric acid method, m-hydroxybenzene method, iodine coloration method, pre-column derivatization-HPLC method. Partial acid hydrolysis and NMR analysis were used to identify the structure of the homogeneous polysaccharides. (3) Carbon clearance test in mice was used to investigate the non-specific immune regulation function of Buick Ginseng crude polysaccharides on immunosuppressed mice. The effects of Polysaccharides from Buick Ginseng on the activity, phagocytosis, cytokine and mediator secretion of RAW264.7 cells were studied to investigate the immunomodulatory ability in vitro. Standard study: analysis of water and ash in 10 batches of Buickshen medicinal materials; establishment of microwave digestion ICP-MS method for determination of trace elements and heavy metals in Buickshen; establishment of acetonitrile ultrasonic extraction, GC-MS method for analysis of pesticide residues in Buickshen medicinal materials; establishment of Buickshen medicinal amino acid components, polysaccharide components TLC identification method; Results: (1) Response surface methodology was used to optimize the extraction process of crude polysaccharides from Buick Ginseng. The extraction time was 4.28 h, the extraction temperature was 90 ~C, the ratio of material to liquid was 37 ml/g, and the extraction times were 3 times. The yield (37.25%+0.17%) was in agreement with the predicted value of regression model (37.465%). After separation and purification of AE Sepharose CL-6B and Sephadex G-100, four homogeneous polysaccharides (ESBP1-1, ESBP1-2, ESBP2-1, ESBP3-1) were obtained. The molecular weights of these polysaccharides were 1.3 x 104, 1.7 x 104, 9.4 x 105, 4.1 x 105Da. (2) The results of phenol-sulfuric acid method showed that the total sugar contents of ESBP1-1, ESBP1-2-1, ESBP2-1 and ESBP3-1 were 97.8%, 98.7%, 99.3% and 97.0% respectively. The content of ESBP2-1, ESBP3-1 glucuronic acid was 0.02%, 1.9%, ESBP1-1 and ESBP1-2 were free of glucuronic acid, while the content of ESBP1-1, ESBP1-2 were 0.5%, 0.3%, 0.3%, ESBP2-1 and ESBP3-1 were not detected by hydroxybenzene method, respectively. ESBP3-1 was composed of glucose, galactose and arabinose (molar ratio 24.3:1.1:1), and ESBP3-1 was mainly composed of glucose and galactose (molar ratio 14.8:1); IR, Smith degradation, methylation, partial acid hydrolysis and NMR analysis showed that the structures of four homogeneous polysaccharides of Buicksan were similar, and the main chains of the four homogeneous polysaccharides were composed of (1_4) - alpha-D-Glcp. All the four homogeneous polysaccharides branched from the 6-O position and had different side chains. (3) Carbon clearance test in mice showed that the crude polysaccharides of Buick Ginseng could improve the organ index and monocyte-macrophage clearance index of immunosuppressed mice, suggesting that Buick Ginseng polysaccharides had some non-specific immunomodulatory function. Swelling, cotton ball granuloma and writhing test in mice induced by glacial acetic acid showed that the crude polysaccharide of Buick Ginseng had the ability to inhibit ear swelling, reduce the quality of granulation and writhing times, suggesting that Buick Ginseng polysaccharide had obvious anti-inflammatory and analgesic effects; in vitro cell experiments showed that Buick Ginseng crude polysaccharide ESBP and homogeneous polysaccharide ESBP1-1, ESBP1-2, ESBP2-1 And ESBP3-1 can promote the proliferation of RAW264.7 cells, increase their phagocytic activity, up-regulate the release of NO, IL-1 beta and TNF-alpha, showing immunopotentiatory effect; LPS-induced inflammatory response of RAW264.7 cells showed that Buickshen polysaccharide can inhibit the secretion of NO, IL-1 beta and TNF-alpha produced by LPS-induced excessive immunity, showing anti-inflammatory effect. METHODS: A TLC method for the determination of trace metal elements and pesticide residues in Buickshen was established. A TLC method for the identification of amino acids and polysaccharides in Buickshen was established. A method for the determination of polysaccharides in Buickshen was established. The draft quality standard of Buickshen and the drafting instructions were completed. Buick ginseng polysaccharides were extracted by DEAE Sepharose CL-6B and Sephadex G-100, and the established model was accurate and reliable, which could be used for the extraction process of Buick ginseng polysaccharides. (2) The crude polysaccharides were isolated and purified by DEAE Sepharose CL-6B and Sephadex G-100, and the method was feasible. The effect may be related to Buick Ginseng polysaccharide can promote cell proliferation, enhance phagocytosis and up-regulate cytokines and mediators; at the same time, Buick Ginseng polysaccharide has anti-inflammatory activity, which may be related to Buick Ginseng polysaccharide can inhibit the secretion of inflammatory factors and mediators caused by overimmunity. Keshen polysaccharide has two-way immunoregulation, and its anti-inflammatory activity is related to immunoregulation. (4) The quality standard of Bukeshen was established. The method was proved to be simple, stable and feasible by methodological investigation and systematic verification, which filled the blank in quality control of Bukeshen.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R29
【相似文獻(xiàn)】
相關(guān)期刊論文 前2條
1 陳春麗;羅曉湄;候弈;尚德軍;熱娜·卡斯木;;微波消解法測(cè)定別克參中微量元素的含量[J];中國(guó)實(shí)驗(yàn)方劑學(xué)雜志;2014年16期
2 ;[J];;年期
相關(guān)會(huì)議論文 前2條
1 ;富山俱樂(lè)部啟動(dòng)儀式及“選富山衣車(chē),開(kāi)別克轎車(chē)”開(kāi)獎(jiǎng)儀式在福建晉江拉開(kāi)帷幕[A];中國(guó)縫制械行業(yè)“十一五”發(fā)展戰(zhàn)略高層論壇論文集[C];2005年
2 ;路易德·布瑞禮·別克,93歲 不落人后的硬漢[A];首屆中國(guó)老年人才論壇論文集[C];2005年
相關(guān)重要報(bào)紙文章 前10條
1 記者黃慧;哈依爾別克致富不忘扶貧幫困[N];亞洲中心時(shí)報(bào)(漢);2009年
2 趙正;別克：用平面創(chuàng)造品牌形象[N];中國(guó)工商報(bào);2001年
3 袁曉明（作者為埃森哲美國(guó)公司高級(jí)信息管理咨詢顧問(wèn)）;通用汽車(chē)會(huì)對(duì)別克“忍痛割?lèi)?ài)”嗎[N];第一財(cái)經(jīng)日?qǐng)?bào);2005年
4 本報(bào)記者 王保純;別克汽車(chē)品牌是如何植根中國(guó)的[N];光明日?qǐng)?bào);2004年
5 王政;上海通用全線調(diào)整別克系列產(chǎn)品價(jià)格[N];人民日?qǐng)?bào);2004年
6 記者 趙永平;上海通用推出新款別克[N];市場(chǎng)報(bào);2000年
7 上海通用汽車(chē)公關(guān)部提供;好車(chē)好服務(wù) 別克對(duì)未來(lái)的承諾[N];新華每日電訊;2002年
8 朗明;上海通用首創(chuàng)“別克品牌日”[N];中國(guó)信息報(bào);2004年
9 蔣婷婷;通用加速別克中美子公司產(chǎn)品共享[N];第一財(cái)經(jīng)日?qǐng)?bào);2007年
10 崔學(xué)雪;從牧民到商人[N];經(jīng)理日?qǐng)?bào);2007年
相關(guān)博士學(xué)位論文 前1條
1 陳春麗;哈薩克藥材別克參多糖分離純化、抗炎免疫活性及藥材質(zhì)量標(biāo)準(zhǔn)研究[D];新疆醫(yī)科大學(xué);2017年
相關(guān)碩士學(xué)位論文 前3條
1 卡爾漢（qia er han）;哈薩克英雄敘事詩(shī)《賈尼別克》的文本研究[D];西北民族大學(xué);2015年
2 趙景陽(yáng);秦皇島別克汽車(chē)市場(chǎng)發(fā)展研究[D];河北科技師范學(xué)院;2014年
3 余志勇;上海通用“別克”汽車(chē)營(yíng)銷(xiāo)案例[D];鄭州大學(xué);2004年
，本文編號(hào)：2215438
本文鏈接：http://sikaile.net/shoufeilunwen/yxlbs/2215438.html
最近更新
教材專(zhuān)著
