超声对麦冬多糖结构、溶液行为及生物活性影响的研究

【作者】 王小梅；

【导师】 孙润广；

【作者基本信息】 陕西师范大学 ， 声学， 2013， 博士

【摘要】 本文用热水提取法与超声提取法提取麦冬多糖,并对热水提取的麦冬多糖进行超声处理。比较研究了超声提取与超声处理麦冬多糖对多糖一级结构的影响,并对热水提取与超声提取麦冬多糖的形貌特征、溶液构象及生物活性进行了比较研究,旨在揭示超声对麦冬多糖结构及活性的影响。主要研究内容结果如下：(1)研究超声提取功率对麦冬多糖结构及活性的影响,实验选用五种功率超声提取麦冬多糖,并利用季铵盐沉淀法对其进行分离,分别得到五种AP1和AP3多糖,然后利用高效液相色谱法和气相色谱法分别对分离得到多糖的分子量分布、单糖组成进行了分析研究。结果表明,随着超声功率的增大,不断有较大分子量的多糖被提取出来,但当超声功率进一步增大时,大分子量的多糖分子量减半,被降解成较小分子量的多糖。气相色谱研究表明,随着超声提取功率的不断增大,嵌入麦冬原材料中较稳定、用小功率很难被提取出来单糖组分,如鼠李糖、阿拉伯糖和木糖均都被提取出来。对得到的多糖的抗氧化活性研究结果表明,随着超声功率的增大,麦冬多糖的抗氧化活性呈现出先增大后减小的趋势,且超声功率为400W时其抗氧化活性最高。因此,后续实验中超声提取麦冬多糖的功率选用400W。(2)对热水提取的麦冬多糖WPOJ与超声提取的麦冬多糖UPOJ的结构及生物活性进行比较研究,结果发现：UPOJ的提取率明显高于WPOJ;利用红外光谱对两者的官能团进行检测发现,WPOJ中可以检测到β-葡萄吡喃糖,而UPOJ中未检测到,说明超声提取对麦冬多糖的结构产生了影响；两者的生物活性结果表明,UPOJ的体外抗氧化活性及抗糖尿病活性均高于WPOJ,而对K562细胞的抗肿瘤活性却相反。(3)两种多糖经DEAE-cellulose52色谱柱层析与Sephacryl S-300凝胶色谱柱层析纯化得到的WPOJ-DS与UPOJ-DS均为单一组分。利用化学分析法(包括高碘酸氧化、Smith降解、酸水解、甲基化分析等)及仪器分析法(包括气相、液相、紫外、红外、质谱、核磁共振等)对这两种组分的一级结构进行研究,结果显示,WPOJ-DS与UPOJ-DS的主链结构存在差异,WPOJ-DS的主链由1,6-α-D-Glcf,1,6-β-D-Glcp,1,3,6-α-D-Glcf和1,3-a-D-Araf组成,其摩尔比为5:1:3:2, UPOJ-DS的主链由1,6-α-D-Glcf,1,3,6-α-D-Glcf和1,3-α-D-Araf组成,其摩尔比为5：4：2,说明WPOJ-DS中1,6-β-D-Glcp在UPOJ-DS中变为1,3,6-α-D-Glcf; WPOJ-DS支链及链木端主要由1,6-α-D-Glcp,1,2-α-D-Fruf和1-a-D-Araf组成,其摩尔比为2：3：5,UPOJ-DS支链及链末端主要由1,6-α-D-Glcp,1,2-α-D-Fruf和1-α-D-Araf组成,其摩尔比为3：4：6,说明UPOJ-DS的侧链结构比WPOJ-DS丰富。因此,相对于热水提取法,利用超声提取法可以将麦冬中嵌入较深层次、支链较丰富的麦冬多糖提取出来。(4)对WPOJ-DS进行超声处理后记为WPOJ-DS-U,将WPOJ-DS-U与WPOJ-DS的一级结构进行比较研究,结果表明,超声处理后,麦冬多糖的分子量、单糖组成、摩尔比及连连接方式几乎没有变化,但是超声处理作用对热水提取的麦冬多糖的p构型产生了影响,使其转变为α构型,主要表现为→6)-β-D-Glcp(1→转变为→6)-a-D-Glcp(1→。通常β-D-Glc要比α-D-Glc稳定,但超声作用的巨大能量促使麦冬多糖结构处于亚稳定状态,提高了其生物活性。(5)对WPOJ-DS与UPOJ-DS的溶液行为与链构象进行比较研究。粒度分析结果表明,UPOJ-DS的有效直径相对于WPOJ-DS较小,且两者均在酸性条件下粒度较小,而在碱性条件下容易发生聚集。刚果红实验结果表明,UPOJ-DS具有单螺旋结构,而WPOJ-DS则不具有单螺旋结构。圆二色性结果表明,Ca2+与刚果红的加入,对WPOJ-DS的构象影响较小,而对UPOJ-DS影响较大；DMSO的加入对两种多糖的构象影响均较大,使两种多糖的不对称性明显增加；两者在酸碱性溶液中构象都会发生突变,但在碱性溶液中更加明显。X射线衍射结果显示,WPOJ-DS与UPOJ-DS均具有晶体结构,且UPOJ-DS的晶格常数略大于WPOJ-DS。通过测定TG-DTG与DSC曲线,对两种多糖的热稳定性进行研究,结果表明,两种多糖的热性质基本相似,但UPOJ-DS相对于WPOJ-DS热稳定性较强。对两种多糖的原子力显微镜及扫描电镜观察结果表明,超声提取的麦冬多糖UPOJ-DS相对于热水提取的麦冬多糖WPOJ-DS的分子聚集性明显减少,同时原子力显微镜下观察到了UPOJ-DS明显的螺旋结构,这与刚果红实验结果一致。云母片试样与粉末试样的扫描电镜结果均表明UPOJ-DS相对于WPOJ-DS分子聚集程度明显减少,说明超声的巨大能量使麦冬多糖的结构发生了变化,使得分子间作用力减小,分子交联程度减弱,进而导致麦冬多糖聚集体的形态发生变化。(6) WPOJ-DS与UPOJ-DS抗氧化活性结果表明,WPOJ-DS与UPOJ-DS对·OH、O2-·与DPPH·均有清除作用,且UPOJ-DS对这三者的清除效果都比WPOJ-DS要好,结合两种多糖的一级结构、形貌特征和溶液行为来分析,UPOJ-DS的分子的侧链结构比WPOJ-DS的侧链结构丰富；UPOJ-DS的原子力显微镜下可以观测到螺旋结构,且分子分布较为分散,因此推测UPOJ-DS的较强的体外抗氧化活性与其分子的结构、形貌、溶液行为是密切相关的。更多还原

【Abstract】 In this paper, polysaccharides were extracted from ophiopogon japonicus by hot water and ultrasound. The polysaccharides extracted by hot water were subjected to ultrasonic treatment. The effect on primary structure of the polysaccharides extracted by hot water and subjected to ultrasonic treatment was studied. Morphology, conformation and biological activity of the polysaccharides extracted by hot water and ultrasound were studied to reveal the effect of ultrasound on the structure and activity of ophiopogon japonicus polysaccharides. The main research contents and results are as follows:(1) The effect of ultrasonic power on the structure and activity of ophiopogon japonicus polysaccharides was studied. We selected five kinds of ultrasonic power to extract polysaccharides from ophiopogon japonicus root. The polysaccharides of API and AP3were obtained applying quaternary ammonium salt to isolate the five kinds of polysaccharides, respectively. Then molecular weight distribution and monosaccharide composition of API and AP3were detected by high performance liquid chromatography and gas chromatography, respectively. The results showed that larger molecular weight polysaccharides were obtained with the increasing of ultrasonic power, but degradation phenomenon that large molecular weight cut in half and degraded to small molecular weight emerged with the further increasing of ultrasonic power. Gas chromatography studies showed that, monosaccharide components, such as rhamnose, arabinose and xylose, which were steadily embedded in ophiopogon japonicus raw material and difficult to extract using small power obtained with the increase of the ultrasonic power. Study on antioxidant activity showed that antioxidant activity of the polysaccharides increased to the maximum and then decreased with the increasing ultrasonic power. The antioxidant activity was strongest when the ultrasonic power was400W. Therefore, the ultrasonic power of400W was selected in follow-up experiments.(2) Structure and biological activity of polysaccharides WPOJ extracted by hot water and UPOJ extracted by ultrasound were studied. The results showed that the extraction rate of UPOJ was significantly higher than that of WPOJ. The functional groups of WPOJ and UPOJ were detected with IR spectrum. The result showed (3-glucopyranose was detected in WPO but not in UPOJ, indicating that the structure of ophiopogon japonicus polysaccharides was affected with ultrasound. The result of bioactivity showed the antioxidant activity and antidiabetic activity in vitro of UPOJ were higher than that of WPOJ. But the antitumor activity on K562cells of UPOJ was lower than that of WPOJ.(3) The homogeneity of WPOJ-DS and UPOJ-DS were purified by a combination of DEAE-52anion-exchange column chromatography and repeated Sephacryl S-300HR gel chromatography from WPOJ and UPOJ. The primary structure of WPOJ-DS and UPOJ-DS were studied by chemical analysis (including periodate oxidation, Smith degradation, hydrolysis with acid and methylation analysis) and instrumental analysis (including GC, HPLC, UV, IR, MS and NMR). The results showed that the backbone of WPOJ-DS was different with UPOJ-DS. The backbone of WPOJ-DS was1,6-α-D-glucopyranose,1,6-β-D-glucopyranose,1,3,6-α-D-glucofuranose and1,3-α-D-arabinofuranose in the molar ratio of5:1:3:2, while the backbone of UPOJ-DS was1,6-α-D-glucopyranose,1,3,6-α-D-glucofuranose and1,3-a-D-arabinofuranose in the molar ratio of5:4:2, indicating1,6-β-D-glucopyranose in WPOJ-DS was instead of1,3,6-α-D-glucofuranose in UPOJ-DS. The branched chains and the ends of the backbone of WPOJ-DS were mainly composed of1,6-α-D-glucopyranose,1,2-α-D-fructofuranose and1-α-D-arabinofuranose, in the molar ratio of2:3:5, while the branched chains and the ends of the backbone were mainly composed of1,6-α-D-glucopyranose,1,2-α-D-fructofuranose and1-α-D-arabinofuranose, in the molar ratio of3:4:6, demonstrating the branched chains of UPOJ-DS were richer than that of WPOJ-DS. Therefore, the polysaccharides including richer branched chains, embedded in ophiopogon japonicus could be extracted by ultrasound but not by hot water.(4) WPOJ-DS was treated by ultrasound and named as WPOJ-DS-U. The primary structures of WPOJ-DS-U and WPOJ-DS were studied comparatively. Analysis showed that the molecular weight, monosaccharide composition, molar ratio, and chain connections of WPOJ-DS-U were not in the least different with WPOJ-DS. β-configuration in WPOJ-DS was affected with ultrasound. β-configuration in WPOJ-DS had changed to α-configuration in WPOJ-DS-U. The main effect was→6)-β-D-Glcp(1→in WPOJ-DS had changed to→6)-a-D-Glcp(1→in WPOJ-DS-U. Generally, β-D-Glc is more stable than α-D-Glc. Therefore, the tremendous energy of ultrasound causes the structure of ophiopogon japonicus polysaccharide in a metastable state and the increasing of the biological activity.(5) The solution behavior and chain conformation of WPOJ-DS and UPOJ-DS were studied comparatively. The analysis result of particle size showed the effective diameter of UPOJ-DS was smaller than that of WPOJ-DS. Both of them had smaller effective diameter under acidic conditions and aggregation under alkaline conditions. The result of Congo red experiment showed UPOJ-DS had single spiral structure, while the WPOJ-DS had not. The result of circular dichroism showed it had larger effects on the conformation of UPOJ-DS but smaller effects on the conformation of WPOJ-DS when Ca2+and Congo red were joined in. It had larger effects on the conformation and increasing the asymmetry of both WPOJ-DS and UPOJ-DS when DMSO was joined in. The conformation of WPOJ-DS and UPOJ-DS changeed in acid and alkaline solution and the change was more obvious in alkaline solution. The result of X ray diffraction showed WPOJ-DS and UPOJ-DS had crystal structure, and the crystal lattice constant of UPOJ-DS was slightly larger than that of WPOJ-DS. TG-DTG and DSC curve and the thermal stability of the two kinds of polysaccharides were determined. Results indicated that thermal properties of the two kinds of polysaccharides were basically similar, but the UPOJ-DS relative to WPOJ-DS had a good thermal stability.The results of atomic force microscopy and scanning electron microscopy showed the molecules aggregation of UPOJ-DS significantly reduced relative to WPOJ-DS. The spiral structure was observed in UPOJ-DS by atomic force microscopy, which was consistent with the result of Congo red experiment. The results of SEM (includes mica samples and powder sample s) showed molecular aggregation behavior of UPOJ-DS significantly weakened relative to WPOJ-DS, indicating the tremendous energy of ultrasound had changed the structure of ophiopogon japonicus polysaccharide. The intermolecular force and molecular cross-linking degree in UPOJ-DS reduced, causing the change on the aggregates of the polysaccharide.(6) Antioxidant activity results of WPOJ-DS and UPOJ-DS showed that both of them had scavenging effects on·OH, O2-· and DPPH·. The scavenging effect of UPOJ-DS was better than that of UPOJ-DS. Combine the results of primary structure, morphology and solution behavior of the two kinds of polysaccharides that the branched chains of UPOJ-DS were richer than that of WPOJ-DS, UPOJ-DS had the spiral structure observed by atomic force microscopy, and the molecular distribution was more dispersed. Therefore, it was speculated that the higher antioxidant activity is closely related to the structure, morphology and solution behavior.更多还原