【作者】 丛媛媛；

【导师】 热娜·卡斯木；

【作者基本信息】 新疆医科大学 ， 药理学， 2008， 博士

【摘要】 甘草(Glycyrrhiza spp.)是传统常用中草药,具有清热解毒、健脾补气、润肺止咳之功效,民间用于治疗乳腺炎、胃及十二指肠溃疡、慢性气管炎、咳嗽、气喘、慢性咽喉炎、食物中毒等症。甘草也是新疆具有区域特色的民族药资源和重要的大宗药材之一。甘草的现代化学及药理活性研究重点多集中在甘草酸、甘草次酸等三萜皂苷类化合物和甘草苷等黄酮类化合物上,而对甘草中另一类重要的生物活性物质甘草多糖(Glycyrrhiza Polysacchiade,GPS)的研究报道相对较少。本文以新疆甘草中资源最为丰富的主要原料草-胀果甘草(Glycyrrhiza inflata Bat.)为研究对象,首次系统研究胀果甘草多糖的提取、分离、纯化、理化性质、一级结构,对不同结构多糖的免疫活性、抗氧化活性以及抗肿瘤活性进行研究,并探讨活性多糖的构效关系。目的对新疆胀果甘草多糖进行系统的研究,阐明新疆胀果甘草多糖的化学结构和生物活性,探讨其构效关系,为研发多糖类新药和开发利用甘草多糖提供科学依据。方法1.胀果甘草多糖的提取分离和纯化:采用水提醇沉方法从胀果甘草中提取粗多糖,透析后经DEAE纤维素离子交换柱色谱分级,各级分采用Sephadex、Sepharose等凝胶色谱填料进行分离纯化,HPGPC法检测纯度并测定平均分子量。2.胀果甘草多糖的结构鉴定:采用GC、HPLC、GC-MS、IR、NMR、MS等现代色谱、光谱技术,结合酸水解、部分酸水解、甲基化、酶解等经典化学方法进行多糖的结构鉴定。3.胀果甘草多糖的生物活性研究:通过小鼠脾细胞体外增殖实验,对各多糖组分的免疫活性进行比较研究;通过4种肿瘤细胞株的体外抑制实验对胀果甘草粗多糖及其分离纯化得到的均一多糖进行抗肿瘤活性实验;运用化学发光体系产生自由基模型研究胀果甘草多糖清除自由基的能力,以评价其抗氧化活性。4.胀果甘草多糖的构效关系研究:采用部分酸水解、酶解及糖醛酸还原方法处理胀果甘草活性多糖,对各结构片断进行活性研究,确定活性片段,阐明构效关系。结果1.胀果甘草通过水提醇沉,再以Sevag法和反复醇洗除去蛋白质和色素,得到粗多糖。经DEAE-52离子交换柱色谱及Sepharose CL-6B和Sephadex G-50凝胶柱色谱反复分离纯化,首次从该植物中得到13个均一多糖组分,并研究分析了胀果甘草粗多糖和各均一多糖的性质和化学结构。分离纯化得到的13个均一多糖中,Gi-A1和Gi-A2为中性阿拉伯半乳葡聚糖;Gi-A3和Gi-A4为中性阿拉伯半乳聚糖; Gi-B1、Gi-C1、Gi-D1具有典型的RGⅠ型果胶多糖的结构特征:其主链包括1→4连接的α-D-GalpA构成的无分支的光滑区和由1,2-连接的α-L-Rhap和1,4-连接的的α-GalAp交替连接构成的带分支的毛发区,在Rha的的4位有分支;其它的包括2个中性杂多糖和4个酸性杂多糖。2.对胀果甘草粗多糖及各个分离部分进行免疫调节、抗肿瘤及抗氧化活性研究。小鼠脾细胞增殖实验结果表明,粗多糖中的透析袋内级分具有更好的免疫活性。经DEAE柱色谱分离,从水和低浓度盐洗脱的多糖级分以及从这些级分中分离纯化的多糖具有较高的体外免疫活性。体外肿瘤细胞抑制实验结果表明透析带外粗多糖以及通过较高浓度盐洗脱级分中分离纯化的大分子量多糖具有较好的抗肿瘤活性。清除自由基实验的结果表明,胀果甘草粗多糖及各均一多糖对DPPH自由基、羟自由基(·OH)和超氧自由基(O2·)具有一定的清除作用,提示胀果甘草多糖具有一定的抗氧化活性。3.探讨胀果甘草多糖的构效关系。对果胶多糖Gi-B1、Gi-C1、Gi-D1及其还原产物、部分酸水解产物、酶解产物的免疫活性及抗肿瘤活性等进行比较研究,探讨果胶多糖结构中中性糖支链、鼠李糖-半乳糖醛酸核心区及糖醛酸基团对其活性的影响,结果表明,去掉部分阿拉伯糖或半乳糖支链后的产物的免疫活性显著下降;酸性果胶多糖在糖醛酸被还原后,对小鼠脾细胞增殖能力显著下降;果胶多糖中Rha-GalA核心损失,其整体活性降低。抗肿瘤活性实验结果也证实了果胶多糖结构中中性糖支链、鼠李糖-半乳糖醛酸核心区及糖醛酸基团对其活性具有相似的影响作用。结论1.首次从新疆胀果甘草中分离纯化得到13个均一多糖组分,并对其一级结构进行了解析,发现胀果甘草多糖均为多分支的杂多糖,结构类型包括阿拉伯半乳葡聚糖、阿拉伯半乳聚糖和以多聚半乳糖醛酸夹杂有鼠李半乳糖醛酸聚糖为主链的果胶多糖等。2.新疆胀果甘草多糖具有多种生物活性,对小鼠脾细胞体外增殖反应具有显著的促进作用,体外抗肿瘤实验表明其对人胃癌细胞株BGC823等四种癌细胞株的生长具有一定的抑制作用,清除自由基实验表明其具有一定的抗氧化活性。3.胀果甘草中活性果胶多糖的构效关系实验证实了其结构中的部分活性片段:中性糖支链、鼠李糖-半乳糖醛酸核心及糖醛酸基团,这些活性片段的存在与胀果甘草果胶多糖体外免疫活性和抗肿瘤活性的表达有关。以上研究丰富了胀果甘草化学成分研究的内容,填补了我国对该种植物多糖研究的空白,明确了胀果甘草多糖的结构、生物活性及其构效关系,为阐明甘草药理、药效作用的物质基础研究提供了新资料,也为今后甘草的合理用药、综合利用提供了科学依据。更多还原

【Abstract】 As an important traditional Chinese medicine, the roots and rhizomes of Glycyrrhiza spp. have been used for centuries with the activities of heat-clearing and detoxifying, invigorating the spleen and replenishing qi, moistenning lung, relieving cough and alleviate the drug property, etc. Glycyrrhiza spp. are also the ethnic medicine resource with the reginal characteristics and the famous medicinal materials in Xinjiang. Morden researches of chemistry and pharmacology of Glycyrrhiza spp. have focused on the chemical constituents, such as triterpenoids and flavonoids. Meanwhile, there are less researches of another active constituent-Glygyrrhiza polysaccharide(GPS). We have studied for the first time the structural elucidation, biological activities and structure-activity relationships of polysaccharides from Glycyrrhiza. inflata Bat.-the main resource of Xinjiang licorice.ObjectiveTo systematically study the polysaccharides from Glycyrrhiza inflata Bat. in Xinjiangand clarify the chemical structure, biological activities and structure-activity relationship of the polysaccharides, which will provide scientific basis for new polysaccharide drug research and comprehensive utilization of Glycyrrhiza polysaccharide.Methods1 . Extraction, isolation and purification of GPS. Water extracting-alcohol precipitating method was applied to extract crude polysaccharide and dialysis and DEAE cellulose ion-exchange chromatography were applied to fractionalize. Every fraction was purified by Sepharose and Sephadex gel chromatography.Purity and molecular weight of every homogeneous polysaccharide were determined by HPGPC method.2 . Structural elucidation of homogeneous polysaccharide.Based on chemical methods (including sugar compositin analysis, methylation analysis, uronic acid reduction, partial acid hydrolysis and enzymatic digestion) and spectral analysis (IR, NMR,GC-MS), the structural characteristics of homogeneous polysaccharide was investigated.3.Study on the biological activities of GPS.Mouse splenocyte proliferation assay in vitro, free-radicals scavenging assay and antiblastic assay of cancer cell in vitro.were applied respectively to investigate the immunological activity, antioxidation activity and antitumor activity.4.Study on the activity-structure relationship of active polysaccharides. Partial acid hydrolysis, uronic acid reduction and enzymatic digestion were used to treat the active polysaccharides from Glycyrrhiza Inflata Bat.To compare the immunostimulating and antitumor activities between the structural fragments and the original polysaccharides and clarify the active fragments and structure-activity relationship.Results1.The roots and rhizomes of Glycyrrhiza Inflata Bat.were extracted with distilled water and precipitated with ethnol. The deproteinized and decolored residues were separated and purified by DEAE ion-exchange chromatography and Sepharose CL-6B, Sephadex G-50 gel-filtration chromatography repeatedly. Thirteen homogeneous polysaccharides were obtained for the first time from Glycyrrhiza inflata Bat. Their properties and structures were identified on the basis of chemical methods and spectral methods. Among the 13 homogeneous polysaccharides, Gi-A1 and Gi-A2 were neutral arabinogalactoglucan; Gi-A3 and Gi-A4 were neutral arabinogalactan; Gi-B1,Gi-C1,Gi-D1 were typical RGⅠtype pectic polysaccharides,which contained a backbone of the repeating disaccharide[→4)-α-D-GalAp-(1→2)-α-L-Rhap-(1→],with substitution at O-4 of the rhamnopyranosyl residues; the rest included 2 neutral complicated heteropolysaccharides and 4 acidic complicated heteropolysaccharides.2.The results of the biological activities indicated that the crude GiP and its purified fractions had remarkable immunostimulating activities, especially the H2O and lower concentration NaCl eluate fractions from DEAE ion-exchange chromatography and their purified fractions. The results of antitumor tests on GiP and its purified fractions showed only the higer concentration NaCl eluate fractions had certain antitumor activities. Additionally, GiP and some purified fractions showed certain activities on scavenging free-radicals, such as DPPH·,·OH, and O2·, which indicated they would have antioxidation activity.3.Compared with their reduced products, partial hydrolysis products and enzymatic digestion products, three typical pectic polysaccharides Gi-B1, Gi-C1 and Gi-D1 have showed more potent biological activities. The results of the studies on the relationships between the structures and the activities of the active polysaccharides from Glycyrrhiza inflata Bat.indicated that the neutral glycosyl side chains, the uronic acid unit and the rhamnogalacturonan core of the active pectic polysaccharide were each involved in the expression of biological activities.Conclusion1.13 homogeneous polysaccharides were obtained from Glycyrrhiza inflata Bat.and their structure were elucidated for the first time.All of them were complicated heteropolysaccharides with multi-branch,including arabinogalactoglucan, arabinogalactan and pectin possessed anα-(1→4)-galacturonan backbone with some insertions ofα-(1→2)-rhamnopyranosyl residues.2.Crude and homogeneous polysaccharides from Glycyrrhiza inflata Bat.had various biological activities.They could stimulate mouse slenocyte proliferation in vitro, inhibitate the growth of some cancer cell in vitro and scanvenge free-radicals, such as DPPH,·OH, and O2·.3.Some active fragments of pectic polysaccharides from Glycyrrhiza inflata Bat. were revealed. The neutral glycosyl side chains, the uronic acid unit and the rhamnogalacturonan core of the active pectic polysaccharide were each involved in the expression of biological activities.For the first time, the structural characterizations and activities of polysaccharides of Glycyrrhiza inflata Bat. were reported. These researches enriched the studies on chemical constituents of Glycyrrhiza inflata Bat., filled the gaps in the studies on the polysaccharide of Glycyrrhiza inflata Bat., identifed the chemical structure, biological activities and the structure-activity relationship of the polysaccharides from Glycyrrhiza inflata Bat., which would provide research data for clarifying the efficient substance and for the comprehensive utilization of Glycyrrhiza.更多还原