【作者】 李永裕；

【导师】 吴少华；

【作者基本信息】 福建农林大学 ， 果树学， 2010， 博士

【摘要】 余甘(Phyllanthus emblica L.)是大戟科叶下珠属植物,是世界卫生组织指定在世界范围内推广种植的3种保健植物之一,2002年被我国卫生部确定为药食两用植物,是我国的传统民族用药。余甘多糖是余甘的主要活性成分,具有抗肿瘤和抗氧化生物活性。目前对余甘多糖的研究主要侧重于精多糖药理活性探索,对其活性级分及结构研究较少,且不够深入。本文以余甘主栽品种“粉甘”果实为研究对象,对余甘多糖的提取技术、分离纯化方法、结构信息以及多糖抗氧化活性进行了系统研究,研究结果如下:从余甘中提取出粗多糖,其糖含量为31.13%;粗多糖经冻融分级、脱色素、除蛋白、透析和凝胶柱层析等初步分级纯化后,得白色丝状余甘精制多糖EPS,多糖收率10.33%;EPS的化学组成为:中性糖61.67%,糖醛酸26.04%,蛋白质1.98%,中性糖由Gal、Rha、Ara、Glc、Xyl和少量Man组成,糖醛酸为GalA。研究了余甘多糖的超声波强化提取和微波辅助萃取工艺。超声波提取最佳工艺参数:超声波提取温度80℃、超声波时间70 min、超声波功率475 W、料液比1:20;微波前处理-热水浸提法最佳工艺参数:微波前处理功率480W,微波处理时间60s,热水浸提温度90℃,浸提时间4h。对水提余甘精多糖的分级纯化方法和化学组成进行了研究。EPS经DEAE-Sepharose CL-6B柱层析分级后得到2个多糖级分EPS1和EPS2;EPS1为中性多糖,经HPGFC分级后得到2个多糖级分EPS1-1和EPS1-2;EPS2为分子量均一的酸性多糖,;EPS1-1、EPS1-2和EPS2分别占总洗脱量的3.39%、19.60%和77.01%,总回收率92.32%。EPS1-1是一种中性多糖,相对分子质量约为123 KDa,纯度98.53%,总糖95.09%,主要由Gal、Glc组成;EPS1-2总糖含量96.66%,纯度98.91%,分子量约为15 KDa,是一种中性多糖,主要由Gal、Glc和Xyl组成;IR光谱显示EPS1-1和EPS1-2是β构型的吡喃醛糖。EPS2是一种酸性多糖,含有79.01%的半乳糖醛酸,总糖含量97.53%,相对分子质量约为140 KDa,纯度98.96%,主要由GalA、Gal和Rha组成,还有少量的Ara;单糖组成结合IR图谱确定EPS2是一种果胶多糖,多糖分子以α-糖苷键为主,并含有一定量的β-糖苷键。应用NMR、GC-MS、FT-IR、GC、HPLC等仪器分析技术,配合部分酸水解、甲基化分析等经典化学方法研究了余甘多糖EPS1-2和EPS2的化学结构。EPS1-2的结构特征为:糖残基构型主要是1, 4-,1, 6-β-D-Galp、T-,1, 4-,1, 3, 6-β-D-Glcp以及1, 3, 6-,1, 2-β-D-Manp;主链由Galp和Glcp连接形成半乳葡聚糖,(1→)-β-D-Glcp为主链的非还原性末端残基,主链每五个糖残基中有一个分支;由1, 6-β-D-Glcp通过O-3位与1, 3, 6-,1, 2-β-D-Manp等中性糖侧链相连;Manp残基处于支链位置,1, 3, 6-Manp处于支链的核心区域。EPS1-2主要由β-构型构成,含有少量α-构型。部分糖残基被乙酰基取代。EPS1-2重复结构单位可能为:EPS2一种典型的果胶多糖,主链由1, 4-D-GalpA线性连接形成半乳糖醛酸聚糖,由1, 4-D-GalpA通过O-4位与1, 2-、1, 2, 4-L-Rhap的O-1位交替连接形成含有较多分支的鼠李糖半乳糖醛酸聚糖。半乳糖醛酸聚糖链部分羧基被甲酯化,部分O-2或O-3位被乙酰基取代。T-、1, 6-、1, 3, 6-α-Galp和T-、1, 5-α-Araf聚合形成的阿拉伯半乳聚糖以及半乳聚糖是EPS2侧链的主要组成,通过Rhap残基的O-4位与主链相连。T-α-GalpA、T-α-L-Araf和T-β-Galp位于非还原性末端。EPS2可能的一种分子结构为:通过检测EPS、EPS1、EPS1-2和EPS2对羟基自由基、DPPH自由基、超氧阴离子自由基的清除能力以及抗脂质过氧化和还原能力,对余甘多糖的体外抗氧化活性进行了研究,并与阳性对照BHT进行比较。结果表明,EPS1-2和EPS2均表现出很强的抗氧化活性,各级分多糖的抗氧化活性大小为EPS2 > EPS1-2 > EPS1 > EPS,均多糖的活性大于杂多糖;EPS2的还原能力强于BHT。酸性多聚糖EPS2的抗氧化活性高于中性多聚糖EPS1-2和EPS1,这与酸性多糖含有较多的带负电荷的糖醛酸有关;EPS1-2的抗氧化活性强于EPS1,表明多糖的抗氧化活性与分子量大小有关;多糖的糖单元类型、糖苷键构型、取代基、空间结构等结构因素也是影响多糖抗氧化活性的主要因素。更多还原

【Abstract】 The emblica, which is the fruit of the plant Phyllanthus emblica L., in the genus Phyllanthus of Euphorbiaceae, is the traditional ethnic medicine of China. Not only is the emblica designated one of the three health protection plants by WHO, but also is the appointed plant both of medicine and food by Chinese ministry of health in 2002. The major active component in emblica fruits is emblica polysaccharides (EPS), which has been discovered to have biological activities of anti-oxidation and anti-tumor. For now, the studies of EPS are focused mostly on pharmacology. The functional groups and structure of EPS, however, remain poorly understood. In this paper, the extraction, isolation and purification, structure and anti-oxidation activity of EPS were studied systematically. The results were summarized as follows:The crude EPS was firstly extracted containing a sugar content of 31.13%. After freezing and thawing fractionation, depigmentation, deproteinization, dialysis and gel column chromatography, the crude EPS was purified into refined EPS exhibiting white filamentous structure, with a yield efficiency of 10.33%. The chemical composition of EPS consisted of 61.67% of neutral sugar which contained Gal, Rha, Ara, Glc, Xyl as well as few Man, 26.04% of GalA-rich uronic acid and 1.98% of protein.Ultrasonic intensification and microwave-assisted methods for ESP extraction were compared. The optimal parameters of ultrasonic intensification extraction were 80℃for extraction temperature, 70 min for ultrasonic treatment, 475 W for ultrasonic power and 1:20 for solid– liquid ratio. The optimal parameters of microwave pretreatment– hot water extraction were 480 W for microwave pretreatment power, 60 s for microwave treatment and 90℃for 4 h for extraction temperature.The stepwise purification procedure and chemical composition of water-extracted refined ESP were studied. Two fractions of EPS1 and EPS2 were obtained after fractionating through DEAE-Sepharose CL-6B column. The neutral polysaccharide EPS1 could be divided into two fractions of EPS1-1 and EPS1-2 after fractionating through HPGFC, while EPS2 was an acid polysaccharide having narrow molecular weight distribution. The rates of EPS1-1, EPS1-2 and EPS2 in elution amount were 3.39%, 19.60% and 77.01%, respectively, and the total yield efficiency was 92.32%. The neutral polysaccharide EPS1-1, 98.53% in purity and 95.09% in total sugar content, consisted mostly of Gal and Glc, with a relative molecular weight of 123 KDa. The other neutral polysaccharide EPS1-2, 98.91% in purity and 96.66% in total sugar content, consisted mostly of Gal, Glc and Xyl, with a relative molecular weight of 15 KDa. The IR spectrum showed that both of EPS1-1 and EPS1-2 wereβ-pyran aldoses.The acid polysaccharide EPS2 containing 79.01% galacturonic acid, 98.96% in purity and 97.53% in total sugar content, consisted mostly of GalA, Gal and Rha as well as few Ara, with a relative molecular weight of 140 KDa. Combination of monosaccharide composition analysis and IR spectrum revealed that EPS2 was a pectin polysaccharide polymerized majorly byα-glycosidic bonds as well as a certain amount ofβ- bonds.By using technology of NMR, GC-MS, FT-IR, GC and HPLC and cooperating with classical chemical methods of partial acid hydrolysis and methylation analysis, etc. the chemical structure of EPS1-2 and EPS2 were studied.The major configuration of glycoside residues of EPS1-2 were 1, 4-, 1, 6-β-D-Galp, T-, 1, 4-, 1, 3, 6-β-D-Glcp, and 1, 3, 6- and 1, 2-β-D-Manp. The galacto-glucan of main chain, of which the non-reducing terminal residue was (1→)-β-D-Glcp, was formed through the connection between Galp and Glcp. The branch, which was formed through the connection between 1,6-β-D-Glcp, at the position of O-3, and neutral sugar, such as 1, 3, 6- and 1, 2-β-D-Manp, appeared in every five glycoside residues. The residue of Manp located at the side chain, with 1, 3, 6-Manp at the core region. EPS1-2 was majorly composed ofβ-configuration as well as fewα- structure, parts of glycosyl residues on which were replaced by acetyl. The possible repeat units of EPS1-2 were:The EPS2 was a typical pectic polysaccharide, with a galacturonoglycan main chain formed through linear connection of 1, 4-GalpA. The branched rhamnogalacturonan of main chain was formed through the alternative connection between 1, 4-GalpA at position of O-4 and 1, 2, 4-Rhap at O-2. Parts of carboxyls on the polysaccharide were methylated, while some were replaced by acetyl at the position of O-2 or O-3. The side chain of EPS2, linking to the main chain through Rhap residues at the position of O-4, consisted of arabinogalactan and galactan which were polymerized by T-, 1, 6- and 1, 3, 6-α-Galp and T-, 1, 5-α-Araf residues. T-α-GalpA, T-α-L-Araf and T-β-Galp were located at the non-reducing terminal. The possible molecular configuration of EPS2 was:In vitro activities of anti-oxidation of refined EPS, EPS1, EPS1-2 and EPS2 were determined according to their scavenging capacities on the free-radicals of hydroxyl, DPPH and superoxide anion, and their anti-lipid peroxidation and reduction efficacy, using BHT as positive control. The results showed that both of EPS1-2 and EPS2 exhibited strong anti-oxidation activity. The degrees of different fractions of EPS on the anti-oxidation activity were in order of EPS2, EPS1-2, EPS1 and refined EPS, indicating the bioactivity of homopolysaccharide was higher than that of heteropolysaccharide. In addition, the reduction activity of EPS2 was higher than that of BHT.Compared with neutral polysaccharide, EPS1-2 and EPS1, the higher anti-oxidation activity of EPS2 was likely related to the abundance of negative electricity-carrying uronic acid on this acid polysaccharide. The higher anti-oxidation activity of EPS1-2 over EPS1 suggested the molecular weight of polysaccharide influenced the corresponding bioactivity. Finally, the structural factors such as types of sugar units, configuration of glycosidic bond, substituent group and spatial structure of polysaccharide also play an important role in anti-oxidation activity.更多还原