哈萨克药材别克参多糖分离纯化、抗炎免疫活性及药材质量标准研究

发布时间：2018-08-31 15:42

【摘要】：目的:通过提取、分离、纯化获得别克参均一多糖,对均一多糖理化性质及一级结构进行初步表征,为别克参药材物质基础研究奠定基础;考察别克参多糖体内、体外免疫调节及抗炎活性,为该药材生物活性研究提供理论支持;建立别克参药材质量控制方法,保证该药材临床用药的安全、有效、可控、稳定。方法:(1)运用响应曲面法及四因素三水平的Box-Behnken中心组合实验设计方法优化别克参多糖成分的热水提取工艺,通过DEAE Sepharose CL-6B和Sephadex G-100对提取后的粗多糖进行分离纯化,采用高效凝胶色谱法分析纯化后均一多糖的纯度及相对分子质量;(2)运用苯酚-硫酸法、间羟联苯法、碘显色法、柱前衍生-HPLC法测定别克参均一多糖中总糖含量、糖醛酸、淀粉含量及单糖组成;结合红外、Smith降解、甲基化实验、部分酸水解及NMR分析初步鉴别均一多糖的结构;(3)通过小鼠体内碳粒廓清实验考察别克参粗多糖对免疫低下小鼠非特异性免疫调节功能;通过二甲苯致小鼠耳肿胀、小鼠棉球肉芽肿及冰醋酸致小鼠扭体实验考察别克参粗多糖抗炎镇痛功效;通过测定别克参多糖组分对RAW264.7细胞活性、吞噬能力、分泌细胞因子及介质能力的影响,考察体外免疫调节能力;通过测定别克参多糖组分对LPS诱导的RAW264.7炎症细胞分泌炎症因子及介质的影响,考察体外抗炎能力;(4)药材质量标准研究:分析10批别克参药材中水分、灰分;建立微波消解ICP-MS法测定别克参中微量元素、重金属含量的方法;建立乙腈超声萃取、GC-MS法分析别克参药材中农药残留的方法;建立别克参药材中氨基酸成分、多糖成分的薄层鉴别方法;建立柱前衍生-HPLC法测定药材中多糖含量的方法。结果:(1)响应曲面法优化后的别克参粗多糖提取工艺为:提取时间4.28 h,提取温度90℃,料液比37 ml/g,提取次数3次。验证试验所得产率(37.25%±0.17%)与回归模型预测值37.465%吻合。别克参粗多糖经DEAE Sepharose CL-6B和Sephadex G-100分离纯化后,得4个均一多糖(ESBP1-1、ESBP1-2、ESBP2-1、ESBP3-1),高效凝胶色谱法表明分子量分别为1.3×104、1.7×104、9.4×105、4.1×105 Da。(2)苯酚-硫酸法结果表明ESBP1-1、ESBP1-2、ESBP2-1、ESBP3-1总糖含量分别为97.8%、98.7%、99.3%、97.0%;间羟联苯法结果表明ESBP2-1、ESBP3-1糖醛酸含量分别为0.02%、1.9%,ESBP1-1、ESBP1-2中不含糖醛酸;碘显色法结果表明ESBP1-1、ESBP1-2含淀粉分别为0.5%、0.3%,ESBP2-1、ESBP3-1中未检出淀粉;柱前衍生-HPLC法结果表明ESBP1-1、ESBP1-2均由葡萄糖组成,ESBP2-1主要由葡萄糖、半乳糖、阿拉伯糖组成(摩尔比24.3∶1.1∶1),ESBP3-1主要由葡萄糖和半乳糖组成(摩尔比14.8∶1);红外、Smith降解、甲基化实验、部分酸水解及NMR分析结果表明别克参4种均一多糖的结构具有共性,4种均一多糖主链均由(1→4)-α-D-Glcp构成,均从6-O位上分支出侧链,4种均一多糖侧链结构存在不同。(3)小鼠体内碳粒廓清实验表明别克参粗多糖具有提高免疫抑制小鼠脏器指数及单核巨噬细胞廓清指数的能力,提示别克参多糖具有一定非特异性免疫调节功能;二甲苯致小鼠耳肿胀、小鼠棉球肉芽肿及冰醋酸致小鼠扭体实验表明别克参粗多糖具有明显抑制耳肿胀、降低肉芽质量,减少扭体次数的能力,提示别克参多糖具有明显抗炎镇痛功效;体外细胞实验表明,别克参粗多糖ESBP及均一多糖ESBP1-1、ESBP1-2、ESBP2-1及ESBP3-1能促进RAW264.7细胞增殖,提高其吞噬活力,上调NO、IL-1β及TNF-α的释放,显现免疫增强作用;LPS诱导的RAW264.7细胞炎症反应实验表明别克参多糖可抑制由LPS诱导的过度免疫而产生的NO、IL-1β、TNF-α的分泌,显现抗炎效果。(4)质量控制方法研究:建立了别克参药材中微量金属元素、农药残留的检查方法,建立了别克参药材中氨基酸成分、多糖成分的薄层鉴别方法,建立了药材中多糖成分的含量测定方法,完成药材质量标准草案及起草说明的撰写。结论:(1)运用响应曲面法提取别克参多糖,建立的模型准确可靠,可以用于别克参多糖的提取工艺;(2)运用DEAE Sepharose CL-6B和Sephadex G-100分离纯化别克参粗多糖,方法可行;纯化出4种均一多糖,4种均一多糖主链结构具有共性;(3)别克参多糖组分具有增强免疫力的功效,可能与别克参多糖可促进细胞增殖、增强吞噬能力及上调细胞因子及介质有关;同时,别克参多糖具有抗炎活性,可能与别克参多糖可抑制由过度免疫引起的炎性因子及介质的分泌有关。综合分析别克参多糖的这两种表现,说明别克参多糖具有免疫调节的双向性,其抗炎活性与免疫调节有关;(4)建立了别克参药材的质量标准,通过方法学考察及系统验证,表明方法操作简单、稳定、可行,填补了该药材质量控制方面的空白。
[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.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R29

【相似文献】