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伊贝母多糖的分离分析及其生物活性研究
阿衣吐逊·阿布都外力
Thesis Advisor阿布力米提·伊力
2018-06-03
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Name硕士
Degree Discipline有机化学
Keyword伊贝母 多糖 分离纯化 结构分析 生物活性
Abstract

贝母是百合科(Liliaceae)贝母属(Fritillaria)植物的干燥鳞茎。作为常用的药用植物,具有清热、化痰、止咳、润肺的作用,在我国具有悠久的药用历史,已收载于《中国药典》2015年版和《维吾尔药志》等。目前为止,对平贝母、川贝母及浙贝母等的药效物质基础及作用机制研究已较为深入,从贝母属植物中分离并鉴定的化合物包括生物碱、黄酮、甾体、皂苷和多糖等。新疆贝母属资源极为丰富,也开始了人工种植栽培。但有关伊贝母化学成分的提取分离和结构鉴定及其相关的药理学研究尚处于初始阶段,尤其是对其多糖的研究报道鲜见。本文以伊贝母为主要原料,对伊贝母多糖进行了较系统的研究,包括提取条件的优化、分离纯化、结构鉴定及生物活性筛选等方面的内容。1. 用传统热水提取法对伊贝母多糖(FPSP)进行提取工艺优化,以FPSP得率为指标,进行单因素实验及正交实验,得到最佳提取工艺。在最佳提取条件基础上分别用不同温度的水及柠檬酸对FPSP进行连续提取,得到水提多糖(FPSP-C)、热水提多糖(FPSP-H)及酸提多糖(FPSP-A)。结果表明:最佳提取条件为料液比1︰30(w/v),提取温度 90℃,提取时间 2 h,醇沉比1︰5(v/v)。连续提取后FPSP-C、FPSP-H及FPSP-A的提取率分别为4.82%,4.46%,21.40%。2. 对粗多糖进行了分离纯化及结构初探。醇沉所得的粗多糖经脱蛋白、透析除杂后,用阴离子交换树脂和葡聚糖凝胶柱层析法进行了分离纯化,得到了四种多糖组分,命名为FPSP-C2-1、FPSP-H1-1、FPSP-H2-1及FPSP-A1-1-1。初步研究发现FPSP-C2-1是糖蛋白。采用UV、FT-IR、HPLC、GC-MS、SEM-EDX和NMR等表征手段对FPSP-H1-1、FPSP-H2-1及FPSP-A1-1-1进行结构初探。结果表明,三种多糖组分相对分子量分别为19.01、12.74、39.90 kDa。FPSP-H1-1由摩尔比为0.42:1.36:1:0.155:10.26:1.38的鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖及半乳糖组成;FPSP-H2-1由摩尔比为0.75:1.35:1:1.02:2.34的鼠李糖、阿拉伯糖、木糖、葡萄糖及半乳糖组成;FPSP-A1-1-1由葡萄糖组成。除了FPSP-H1-1属于α-构型和β-构型的吡喃多糖外,其余均是α-构型的吡喃多糖。SEM-EDX 分析表明所有的组分均有特定结构。利用1H-NMR,13C-NMR,1H-1H-COSY, HMBC 及HSQC进一步分析了FPSP-H1-1的结构。3. 对粗多糖FPSP-C、FPSP-H及组分FPSP-H1-1 和FPSP-H2-1进行了抗氧化活性和抑菌活性实验。结果表明上述多糖均对DPPH及羟自由基有一定的体外抗氧化活性,但抑菌活性不明显。

Other Abstract

Fritillaria pallidiflora schrenk, belonging to the Fritillaria genus of Liliaceae family, is among one of the most well-known traditional medicinal plants. F. pallidiflora (FPS) is widely used as an important source for anti-asthmatic, anti-tussive and expectorant agents in traditional Chinese medicine. It was recorded in Chinese Pharmacopoeia (2015) and Uyghur Medicine. Since ancient times, bulbs of FPS have been used for the treatment of several diseases. Although many biologically active compounds including alkaloids, proteins and peptides were isolated, few reports have been focused on the purification and structural analysis of polysaccharide constituents from FPS. This paper contains three aspects:isolation, structural analysis and biological activity of polysaccharides from FPS.1.The extraction of Fritillaria pallidiflora schrenk polysaccharide (FPSP) was optimized using traditional hot water extraction method through single factor and orthogonal tests depending on extraction rate. Successive extractions were conducted using water and citric acid on the basis of optimum extraction conditions, and yielded cold-water soluble polysaccharide (FPSP-C), hot-water soluble polysaccharide (FPSP-H) and acid extracted polysaccharide (FPSP-A). The results showed that optimum conditions were, solid-liquid ratio 1:30 (w/v), extraction tempereture 90℃, extraction time 2 h, extract-ethanol ratio 1:5 (v/v). The extraction rate for FPSP-C, FPSP-H and FPSP-A were about 4.82%,4.46%,21.40% , respectively. 2. Purification and structural analysis of obtained polysaccharides were investigated. The crude polysaccharides were deproteinized and dialyzed, then purified by anion-exchange chromatography and gel-permeation chromatography, and obtained four polysaccharide fractions, namely FPSP-C2-1, FPSP-H1-1, FPSP-H2-1 and FPSP-A1-1-1. Structures of FPSP-H1-1、FPSP-H2-1 and FPSP-A1-1-1 were characterized by UV、FT-IR、HPLC、GC-MS、SEM-EDX and NMR. The results showed that FPSP-C2-1 was a glycoprotein, the other three fraction were heteropolysaccharides with an average molecular weight of 19.01, 12.74 and 39.90 kDa, respectively. FPSP-H1-1 contained Rha, Ara, Xyl, Man, Glc and Gal with a molar ratio of 0.42:1.36:1:0.155:10.26:1.38. FPSP-H2-1 contained Rha, Ara, Xyl, Glc and Gal with a molar ratio of 0.75:1.35:1:1.02:2.34. FPSP-A1-1-1 was mainly composed of Glc. The polysaccharide fractions were further characterized by the combination of FT-IR and SEM-EDX. FT-IR spectra showed that all of the fractions were in a pyranose rings. SEM micrographs revealed that the isolated fractions have different morphology characteristics. The structural characteristics of the FPSP-H1-1 was further investigated by 1H, 13C-NMR, HSQC, HMBC and H-H COSY spectrum. 3. Antioxidant and antimicrobial activity of FPSP-C, FPSP-H, FPSP-H1-1 and FPSP-H2-1 were evaluated in vitro. The results showed all of the fractions exhibited certain antioxidant activities to DPPH and hydroxyl free radicals, with no obvious antimicrobial activity.

Document Type学位论文
Identifierhttp://ir.xjipc.cas.cn/handle/365002/5447
Collection资源化学研究室
Recommended Citation
GB/T 7714
阿衣吐逊·阿布都外力. 伊贝母多糖的分离分析及其生物活性研究[D]. 北京. 中国科学院大学,2018.
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