【作者】 吴艳；

【导师】 Steve W. Cui； 汤坚；

【作者基本信息】 江南大学 ， 食品科学， 2007， 博士

【摘要】 胖大海(Semen Sterculiae Lychnophera)为梧桐科苹婆属植物(Sterculia lychnophora Hance)的干燥成熟种子,是一种传统的清咽润喉的中药材,也是卫生部公布第三批药食两宜的资源。由于受生长地域环境和气候的限制,胖大海主要产于东南亚的热带地区,我国不产,于20世纪70年代从柬埔寨和泰国分别引进长粒和圆粒胖大海,只有在海南和西双版纳两地生长良好,但仅有长粒胖大海在西双版纳已结果多年,成片种植,故国内胖大海资源主要依靠从东南亚国家进口。目前国内外对胖大海的研究较少,仅限于做茶饮、甜点和含片等清咽润喉的保健品,开发利用程度较低,缺乏对其活性成分的系统研究和利用。胖大海多糖是胖大海的主要生物活性成分,本课题以越南产胖大海为原料,对胖大海多糖的提取、分离纯化,活性多糖的结构表征和溶液的性质进行研究,主要研究内容如下:对胖大海化学成分的初步分析:粗蛋白12.36%,粗脂肪5.89%,碳水化合物53.23%,还原糖29.45%,结果发现胖大海多糖含量丰富。采用去离子水和0.05 mol/L NaOH溶液提取胖大海粗多糖,对其得率、纯度和固有粘度进行比较,发现水溶性多糖(W-SP)的得率(13.8%)、纯度(69.7%)和固有粘度(1.71 dL/g)都明显高于NaOH提取的多糖(A-SP),分析原因可能在碱性溶液提取过程中部分多糖发生降解。对W-SP、A-SP和碱不溶性多糖(IMP)的化学成分、单糖组成以及抗炎活性进行研究。分别测得总糖含量为69.74%、44.14%、80.18%,糖醛酸含量为14.78%、7.35%、5.35%,蛋白质含量为9.12%、13.54%、3.45%;发现三种多糖均由Rha、Ara、Gal、Glc和Xyl单糖组成,只是含量有所不同,在W-SP和A-SP中Glc的含量均高于IMP多糖。以二甲苯致小鼠耳廓肿胀试验为模型对W-SP、A-SP和IMP的急性炎症抗炎活性进行筛选,结果发现,W-SP和A-SP均有抑制小鼠耳廓肿胀作用,但W-SP抑制率(17.84%)高于A-SP(4.15%),IMP基本无抗炎活性。运用响应面分析法以提取得率、纯度和相对粘度为指标,优化了水溶性粗多糖(W-SP)的提取工艺参数:提取温度60-65oC,提取时间2.3-3.1h,料水比75:1。水溶性粗多糖经过DEAE纤维素柱层析分离得到两个组分:W-SPN为中性多糖,W-SPA为酸性多糖。W-SPN的化学成分:总糖90.18%,蛋白质2.51%,灰分1.17%;主要是由Glc单糖组成。W-SPA的化学成分:总糖85.86%,蛋白质3.02%,灰分4.05%,糖醛酸40.13%;主要由Rha、Ara、Gal、Xyl和Glc单糖组成的;经酶法和HPAEC-PAD分析发现其糖醛酸主要由GalA(96.89%)组成,还有少量GlcA(3.11%)。经Sepharose CL-6B凝胶柱和HPSEC分析发现,W-SPN和W-SPA都是相对分子质量的均一组分,测得其相对分子质量分别为586,800 Da和1,125,000 Da;经过单糖组成和FT-IR分析确定W-SPA是一种果胶类多糖,并用FT-IR测得W-SPA的酯化度为68%。采用急性炎症抗炎模型对W-SPN和W-SPA进行活性筛选,结果表明,W-SPN对小鼠耳廓肿胀的抑制率(3.21%)远低于W-SPA(26.29%)。采用植入棉球致大鼠肉芽组织增生试验为模型,考察了W-SPA的慢性炎症抗炎活性,结果发现W-SPA能抑制肉芽肿28.38%,达到阿司匹林抑制率(34.38%)的82%,并且发现W-SPA对急、慢性炎症的抗炎活性都具有剂量依赖性。采用多糖部分酸水解和甲基化分析的化学方法,结合HPAEC、GC-MS和1D NMR、2D NMR技术对胖大海酸性多糖W-SPA进行结构分析,结果表明,W-SPA主要是由1,4-α-D-GalpA连接形成的半乳糖醛酸聚糖(HG)构成的“光滑区”和由1,4-α-D-GalpA通过O-4位与1,2-、1,2,4-α-L-Rhap的O-2交替连接形成的鼠李半乳糖醛酸聚糖-I(RG-I)构成的“毛发区”组成。其中,HG多糖链上有部分糖醛酸的羧基被甲酯化,部分的C-2和C-3位被乙酰基取代;RG-I链上有62.21%的鼠李糖在O-4位上发生取代。由T-, 1,3-, 1,5-L-Araf和T-, 1,4-, 1,6-, 1,3,6-D-Galp连接形成的阿拉伯聚糖、半乳聚糖和阿拉伯半乳聚糖构成了多糖W-SPA的支链。55.76%的L-Araf以非还原末端的形式连接在1,3-L-Araf的O-3、1,5-L-Araf的O-5以及1,6-D-Galp的O-3和主链鼠李糖的O-4位上;38.45%的L-Araf构成了阿拉伯聚糖。57.17%的D-Galp以非还原末端的形式连接,22.95%的1,6-D-Galp在O-3位上被取代。少量的蛋白质可能以O-糖苷的形式连接在W-SPA的多糖链上,但这还需要进一步确证。采用动态和静态光散射技术研究了酸性多糖大分子在溶液中的性质。经研究发现,W-SPA分子在水、5 mol/L尿素和100 mmol/L NaNO3溶液中均存在聚集,影响了多糖大分子的光散射分析,但在25 mmol/L NaOH溶液中能够消除分子的聚集现象。采用静态光散射技术测得W-SPA分子的重均分子量Mw为913,200 Da,旋转半径Rg为51.85 nm,第二维里系数A2为2.70×10-4 cm3 mol/g2。用动态光散射测得流体动力学半径Rh为39.3 nm,系数ρ=Rg/Rh为1.32表明W-SPA分子在25 mmol/L NaOH溶液中是一种多分支紧凑的无规线团构型。经过HPSEC分析测得W-SPA的Mark–Houwink-Sakurada方程指数α为0.586,表明W-SPA为紧凑的无规线团的分子链,ρ和α所反映的分子构型是基本一致的。对W-SPA的流变学性质进行了研究。通过稳态剪切流变实验发现,W-SPA在高浓度下表现的是剪切变稀的非牛顿流体,在低浓度下是典型的牛顿流体。与高甲氧基果胶类似,W-SPA本身不能形成胶体,在酸性和共溶质如糖的存在条件下才能形成胶体。在W-SPA的线性粘弹区采用动态流变分析了多糖的机械谱图,结果发现W-SPA的弹性模量(固态行为)和损失模量(流体行为)都依赖于多糖的浓度和切变频率。更多还原

【Abstract】 Boat-fruited sterculia seed (Semen Sterculiae Lychnophorae) is a traditional Chinese herb, specified as the ripe seeds of Sterculia lychnophora Hance in the Chinese pharmacopoeia, and is one of the third batch as both edible and medicinal resources published by Ministry of Health, China. Due to the limitation of region and climate, boat-fruited sterculia seed is mainly produced in the tropical zone of Southeast Asia. China is not the original production country of boat-fruited stercilia seed; it was not until the 1970s that Chinese scientists introduced and cultivated long grain seeds from Cambodia and round ones from Thailand. As a result, only in Hainan and Xishuangbanna the trees grew well and blossomed but the seeds were harvested only in Xishuangbanna. Therefore the seed resource in China depends on the import from Southeast Asia. So far boat-fruited sterculia seeds are primarily used for making tea, desserts and healthy buccal tablets etc, and only a little information has been published. Therefore, there is lack of systematic study of its bioactive components. Since polysaccharide is one of the main bioactive components in boat-fruited sterculia seeds, the objectives of the current project were to extract, isolate and purify the polysaccharides with anti-inflammatory bioactivity from boat-fruited sterculia seeds, and to study the structure and functional properties of the bioactive polysaccharide.Chemical composition analysis revealed that boat-fruited sterculia seeds imported from Vietnam contained 12.36% crude protein, 5.89% crude fat, 53.23% carbohydrate and 29.45% reducing sugar. The yield (13.8%), purity (69.7%) and intrinsic viscosity (1.71 dL/g) of crude polysaccharides (W-SP) from water extraction were higher than the polysaccharides (A-SP) extracted from water-insoluble residues with 0.05 mol/L NaOH at 40 oC, indicating that polysaccharides might be partially degraded in NaOH solution possibly due to beta-elimination.Chemical and monosaccharide compositions of W-SP, A-SP and alkali-insoluble mucilage polysaccharides (IMP) were analyzed and the results were as following: the total carbohydrate were 69.74%、44.14% and 80.18%, uronic acid were 14.78%, 7.35% and 5.35%, and protein were 9.12%、13.54% and 3.45%, respectively. They were complex heteropolymers containing a range of neutral sugars, including rhamnose, arabinose, galactose, glucose and small amounts of xylose.Acute anti-inflammatory activity tested in a model of ear edema formation in mice induced by dimethylbenzene was used to monitor the bioactivity of the three fractions. It was found that W-SP exhibited a significant inhibitory effect (inhibition rate 17.84%) compared to that of A-SP (4.15%) and IMP (0.67%). Hence, water extraction was used to obtain the crude polysaccharides and the extraction procedure was optimized using Surface Response Methodology (RSM). The obtained optimal parameters of the extraction procedure of W-SP by RSM were: temperature 60-65°C, time 2.3-3.1 h, and water to solid ratio 75:1. A neutral and an acidic fraction (W-SPN and W-SPA) were obtained from the crude polysaccharides (W-SP) following an ion exchange chromatography on a DEAE-Cellulose column. The neutral polysaccharide W-SPN was composed mainly of glucose (90.18%) and small amounts of protein and ash. The acidic fraction (W-SPA) consisted of galacturonic acids (39.30%), glucuronic acids (1.26%), rhamnose (11.36%), arabinose (17.46%), galactose (15.70%), xylose (0.63%) and glucose (0.35%), with a total carbohydrate of 85.86% and small amounts of protein and ash.Both polysaccharides were identified by gel filtration chromatography on Sepharose CL-6B column and HPSEC as homogeneous in molecular weight: their molecular weights were 586,800 Da and 1,125,000 Da, respectively. W-SPA was identified as a pectic polysaccharide confirmed by monosaccharide composition and FT-IR analysis. The degree of esterification (DE) value of W-SPA was 68% based on a FT-IR method.Acute anti-inflammatory activity of W-SPN and W-SPA was tested in a model of ear edema formation in mice. W-SPA exhibited a much higher anti-inflammatory activity (inhibition rate 26.29%) compared with W-SPN (3.21%). Further chronic anti-inflammatory activity of W-SPA was estimated by a well-accepted model of hyperplasia of granuloma tissue in rats: the inhibition rate of W-SPA was 28.38% which is 82% of the effect of aspirin (inhibition rate 34.38%). The current study revealed that W-SPA presented a significant dose-dependent inhibitory effect at low concentration range on both acute and chronic anti-inflammatory experiments. Therefore, W-SPA was chosen for further studies aimed at elucidation of its detailed structure, which was essential for the understanding of its mechanism of action.Structure characterization of W-SPA was carried out using partial acid hydrolysis and methylation analysis, combined with HPAEC-PAD, GC-MS and 1D & 2D NMR spectroscopy techniques. The backbone of W-SPA was found to contain two characteristic features: the first is homogalacturonan (HG) zones referred to as the‘smooth’regions characterized by a linear linkage of 1,4-α-D-GalpA; the second is rhamnogalacturonan-I (RG-I) zones, also called‘hairy’regions consisting of a←4)-α-D-GalpA-(1→2)-α-L-Rhap-α-(1→backbone partially substituted at O-4 of the rhamnose units. 20.8% of rhamnosyl residues were only 1,2- linked while 62.2% of residues were 1,2,4- linked (with the O-4 position substitution). On the HG chains carboxyl groups were partially methyl esterified and partially O-acetylated on C-2 and/or C-3. Side chains of W-SPA consisted of araban, galactan and arabinogalactan by the linkages of T-, 1,3-, 1,5-L-Araf and T-, 1,4-, 1,6-, 1,3,6-D-Galp, attached to the backbone via the O-4 position of the rhamnose residues. 55.8% of L-Araf were situated as the nonreducing terminals, which were linked to the O-3 position of 1,3-L-Araf , O-5 position of 1,5-L-Araf and O-3 position of the 1,6-D-Galp units except the O-4 position of the backbone rhamnosyl residues. Araban were composed mainly of 38.5% of 1,3-L-Araf and small amounts of 1,5-L-Araf. 57.2% D-Galp were situated as the nonreducing terminals, and in addition 23.0% of D-Galp units were the linkage of 1,3,6-D-Galp and 3.7% units were 1,6-D-Galp linkage. A small amount of proteins were possibly bonded with the W-SPA molecular chain by O-glycopeptide bonds, however, this speculation needed further confirmation.Solution properties of polysaccharide molecules were investigated by dynamic and static light scattering techniques. W-SPA molecules formed aggregates in pure water, 5 mol/L urea and 100 mmol/L NaNO3 solutions. These aggregates could be only partially eliminated by filtration of the solutions with 0.1μm membrane filter. However, when W-SPA was dissolved in 25 mmol/L NaOH solution at room temperature overnight, there was no observable aggregations: there was only one population of particles with a mean diameter of 51.8 nm in the dynamic light scattering measurement. The Zimm plot from static light scattering measurement for W-SPA in 25 mmol/L NaOH solution was obtained, and weight average molecular weight (Mw), radius of gyration (Rg) and the second virial coefficient (A2) were derived as 9.132×105 Da, 51.85 nm, 2.70×10-4 cm3 mol/g2, respectively. The hydrodynamic radius (Rh) was 39.3 nm by dynamic light scattering measurement. W-SPA in NaOH solution was found to be a branching and exhibited a relatively compact random coiled conformation as evidenced by the characteristic ratioρ=Rg/Rh=1.32. In Mark-Houwink-Sakurada equation,αvalue obtained by HPSEC was 0.586, indicating W-SPA molecule was a little compact random coiled chain, likely a consequence of molecular branching. This observation confirmed the compact random coiled conformation obtained from the characteristic ratioρ.Steady-shear rheological measurements of W-SPA showed a non-Newtonian shear-thinning flow behavior at high concentrations and a Newtonian flow behavior at low concentrations. Similar to other HM pectins, W-SPA could not form a gel on by itself; however, gelation occurred when a co-solute was added under acidic conditions. The mechanical spectra within the linear viscoelastic region were a function of W-SPA concentration and temperature. Both fluid-like (G") and solid-like (G′) behaviors were observed depending on concentration and frequency.更多还原