铁皮石斛多糖结构及其硫酸化衍生物活性研究

发布时间：2018-11-03 21:55

【摘要】：目的:研究铁皮石斛中多糖大分子及其衍生物的结构特征与生物活性,为研究铁皮石斛中基于大分子多糖的生物活性物质基础及其相关质量控制以及基于石斛多糖创新药物研究奠定理论与物质基础。方法:本课题采用沸水提-乙醇沉、氢氧化钠提-乙醇沉的方法获得铁皮石斛水提粗多糖以及铁皮石斛碱提粗多糖。采用酶解法,阴离子交换法以及凝胶过滤色谱法分离纯化出均一多糖。采用糖组成分析,部分酸水解、甲基化、红外、核磁等化学和物理的方法对多糖性质及结构进行深入解析。采用氯磺酸-吡啶法(1:3)进行多糖的硫酸化衍生。人微血管内皮细胞(HMEC-1)的管腔形成实验以及划痕迁移实验用来考察铁皮石斛多糖及其硫酸化衍生物的抗血管生成能力。MTT法来检测铁皮石斛多糖及其衍生物对不同肿瘤细胞株增殖的抑制作用以及对人正常肝细胞LO2和HMEC-1细胞的毒性。结果:(1)从1.5 Kg铁皮石斛干燥茎中得到水提粗多糖DOW 304.0 g(得率为20.3%)和碱提粗多糖DOA 14.0 g(得率为0.9%)。(2)DOW经分离纯化后得到两种均一多糖5A以及5B,分子量分别为1.6×104 Da和8.2×103 Da。糖组成分析表明:5A由葡萄糖(glucose,Glc)、甘露糖(mannose,Man)、阿拉伯糖(arabinose,Ara)、半乳糖(galactose,Gal)、木糖(xylose,Xyl)组成,其摩尔比例为55.7:39.7:1.4:0.6:2.6,结构分析表明5A是以1,4-α-D-Glcp、1,4-β-D-Manp为主链,支链主要包括α和β构型的末端(terminal,T)-D-Glcp、T-Xyl以及T-Ara。支链连接于主链糖残基1,4-α-D-Glcp的C-6位;5B由Glc、Man和少量的Xyl组成,其摩尔比例为91.9:6.5:1.6,结构分析表明5B是以1,4-α-D-Glcp和极少量的1,4-β-D-Manp为主链,支链主要包含T-α-D-Glcp,木糖可能存在于支链上。支链连接于主链糖残基1,4-α-D-Glcp的C-6位。(3)DOA经分离纯化后得到两个均一酸性异木聚糖S32和S33S1,分子量分别为3.7×104Da和6.9×103 Da。糖组成分析结果表明:S32由Ara,Xyl,Glc,Gal,鼠李糖(rhamnose,Rha)和4-甲氧基-葡萄糖醛酸(4-methoxyl-glucuronic acid,4-MGA)组成,各单糖的比例为8.9:62.7:8.5:3.7:3.9:12.3;S33S1糖组成为Xyl,Ara,Glc和4-MGA,比例为6.1:79.8:1.2:12.9。结构分析表明S32和S33S1均是以1,4-β-D-Xylp为主链,分支连接于主链糖残基的C-2位。S32的分枝主要由T-4-Me O-α-D-Glc Ap、1,4-α-D-Glcp、1,3-α-L-Rhap、T-α-L-Araf、T-β-D-Galp以及T-β-D-Xylp组成。S33S1的支链较简单,主要包含T-4-Me O-α-D-Glc Ap、T-β-D-Xylp以及T-α-L-Araf。(4)5B和S32经硫酸化修饰分别得到S5B和S32S,分子量分别为1.8×104 Da和5.4×104 Da。氯化钡-明胶法测得二者硫酸基取代度分别为0.92和0.90。结构分析表明前者的硫酸化取代位点主要为1,4-α-D-Glcp和T-α-D-Glcp的C-6位。而后者硫酸化位点主要包括1,4-β-D-Xylp的C-2或者C-3位、少部分的1,2,4-β-D-Xylp的C-3位、T-4-MGA的C-3位以及1,4-α-D-Glcp的C-6位。(5)生物活性测试结果表明,未硫酸化的多糖5B和S32对HMEC-1细胞的管腔形成几乎没有影响,而硫酸化的多糖S5B和S32S均可以在低浓度(5.95μM,0.29μM)时呈现抑制HMEC-1细胞管腔形成和细胞迁移的活性,在较高浓度时1 mg/m L(47.62μM,18.52μM)对人正常的肝细胞LO2以及HMEC-1细胞几乎没有毒性。结论:(1)本课题获得4种均一多糖。从水提粗多糖中纯化出5A和5B;从碱提粗多糖中纯化出S32和S33S1。(2)5A和5B主要是以1,4-α-D-Glcp和1,4-β-D-Manp为主链的甘葡聚糖;S32和S33S1是以1,4-β-D-Xylp为主链的酸性异木聚糖。(3)铁皮石斛多糖硫酸化衍生物S5B以及S32S具有抗血管生成能力,硫酸基团对生物活性的发挥有着重要的作用。
[Abstract]:Objective: To study the structural characteristics and biological activities of polysaccharide macromolecules and their derivatives in Dendrobium candidum. METHODS: The crude polysaccharides of Dendrobium candidum and crude polysaccharides of Dendrobium candidum were obtained by boiling water extraction-ethanol precipitation, sodium hydroxide extraction-ethanol precipitation. The polysaccharide was isolated and purified by enzymatic hydrolysis, anion exchange and gel filtration chromatography. The properties and structure of polysaccharides were analyzed by chemical and physical methods such as sugar composition analysis, partial acid hydrolysis, methylation, infrared, nuclear magnetic and other chemical and physical methods. Sulfation of polysaccharides was carried out by chlorosulfonic acid-Fenton method (1: 3). The tube cavity formation experiment and scratch migration experiment of human microvascular endothelial cells (HMEC-1) were used to investigate the anti-angiogenic ability of Dendrobium candidum polysaccharide and sulfated derivatives. MTT assay was used to detect the inhibitory effect of Dendrobium candidum polysaccharide and its derivatives on the proliferation of different tumor cell lines, as well as the toxicity to human normal hepatocytes LO2 and HMEC-1 cells. Results: (1) The DOW 304. 0g of the crude polysaccharide was obtained from the dried stem of Dendrobium candidum 1. 5Kg (the yield was 20.3%) and the DOA of the crude polysaccharide was 14.0g (the yield was 0. 9%). (2) DOW was isolated and purified to obtain two homogeneous polysaccharides (5A and A21), and their molecular weights were 1. 6, 104 Da and 8. 2, 103 Da, respectively. The composition analysis indicated that 5A was composed of glucose (Glc), mannose (Mannose, Man), arabinose, Ara, galactose, xylose, xylose and Xyl. Its molar ratio was 55. 7: 39. 7: 1. 4: 0. 6: 2.6. The structure analysis showed that 5A was the main chain of 1,4-dioxane-D-Glcp, 1,4-dioxane-D-Manp. The branched chain mainly includes terminal (T)-D-Glcp, T-Xyl, and T-Ara. the branched chain is connected to the C-6 position of the main chain sugar residue 1,4-dioxane-D-Glcp; the branched chain is composed of Glc, Man and a small amount of Xyl, the molar ratio of which is 91.9: 6.5: 1.6, and the structure analysis shows that the branched chain is a main chain of 1,4-dioxane-D-Glcp and a very small amount of 1,4-dioxane-D-Manp, and the branched chain mainly comprises T-MAA-D-Glcp, xylose may be present on the branched chain. The branched chain is connected to the C-6 position of the main chain sugar residue 1,4-dioxane-D-Glcp. (3) The DOA was separated and purified to obtain two homogeneous acidic xylanase S32 and S33S1 with a molecular weight of 3. 7-104Da and 6. 9-0103 Da, respectively. The results of the composition analysis showed that: S32 was composed of Ara, Xyl, Glc, dioxane, nose (rhamnose, Rha) and 4-methoxy-glucononic acid (4-MGA). The ratio of each monosaccharide was 8. 9: 62. 7: 8. 5: 3. 7: 3. 9: 12. 3; S33S1 sugar was composed of Xyl, Ara, Glc and 4-MGA, and the ratio was 6. 1: 79. 8: 1. 2: 12. 9. The structural analysis shows that S32 and S33S1 are the main chains of 1,4-dioxane-D-Xylene, and the branches are connected to the C-2 position of the main chain sugar residue. The branches of S32 are mainly composed of T-4-Me O-A-D-Glc Ap, 1,4-dioxane-D-Glcp, 1,3-dioxane-L-Rhap, T-Et-L-Araf, T-GaP-D-Galp, and T-GaP-D-Xylene. The branched chain of S33S1 is relatively simple, mainly containing T-4-Me O-A-D-Glc Ap, T-Et-D-Xylene and T-Et-L-Araf. (4) S300 and S32S, respectively, were obtained by sulfation modification of quercetin and S32, and the molecular weights were 1. 8 to 104 Da and 5. 4 to 104 Da, respectively. The substitution degree of sulfuric acid was 0.992 and 0.990, respectively. The structural analysis indicated that the former was the C-6 position of 1,4-dioxane-D-Glcp and T-GaP-D-Glcp. The latter mainly consists of the C-2 or C-3 bits of 1,4-dioxane-D-Xylene, the C-3 position of the small part of 1, 2, 4-GaP-D-Xylp, the C-3 position of T-4-MGA, and the C-6 position of 1,4-dioxane-D-Glcp. (5) The results of biological activity test showed that the non-sulfated polysaccharides S300 and S32 had little effect on the tube cavity formation of HMEC-1 cells, but the sulfated polysaccharides S32S and S32S could inhibit the formation of HMEC-1 cell and the activity of cell migration at low concentration (5.95. mu.M, 0. 29. mu.M). At higher concentrations, 1 mg/ m L (47. 62. mu.M, 18. 52. mu.M) had little toxicity to human normal liver cells LO2 and HMEC-1 cells. Conclusion: (1) Four kinds of polysaccharides were obtained in this study. 5A and S33S1 were purified from the crude polysaccharide of water; S32 and S33S1 were purified from the crude polysaccharide. (2) 5A and S33S1 mainly consist of 1,4-dioxane-D-Glcp and 1,4-dioxane-D-Manp as the main chain; S32 and S33S1 are acidic isoxylans with 1,4-dioxane-D-Xylene as the main chain. (3) The sulfated derivatives SS32S and S32S of Dendrobium candidum have anti-angiogenic ability, and sulfuric acid groups play an important role in the biological activity.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.12

【相似文献】