香菇葡聚糖稀至浓溶液的行为研究
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摘要
三螺旋香菇β-(1→3)-D-葡聚糖(lentinan)表现高度特异性免疫增强作用,能活化巨噬细胞,增强细胞杀伤性,并能激活机体带癌状态下的低免疫功能。多糖的生物活性与它在生物体内特殊的空间伸展状态和独特的分子识别能力有关。然而,至今仍不清楚香菇葡聚糖在溶液中不同条件下的分子链构象转变和氢键作用。本论文主要从香菇中提取和纯化得到三螺旋链香菇葡聚糖,采用高分子物理理论和表征方法研究它在不同浓度区间的分子尺寸和形态,以及分子间相互作用及导致的链构象变化。本工作涉及高分子物理与生物化学交叉学科领域,也是国际前沿研究领域之一。
本论文的主要创新包括以下几点：(1)用高分子物理方法首次证实短的三螺旋链彼此紧密平行排列形成有序的“柴火棒状”团簇；(2)首次证明lentinan水溶液可发生溶液-凝胶转变行为,并提出其凝胶化机理为：高度缠结的Lentinan三螺旋链形成连续的网络,导致凝胶化,但不存在交联点和明显的聚集体,因此它是不具有流动性的浓溶液；(3)首次发现,在7-9℃区间内,lentinan水溶液粘度随温度出现反常变化,归因于三螺旋侧链的有序—无序变化；(4)证实lentinan与聚核苷酸形成由氢键力驱动的新的三螺旋复合物,而且复合物中聚核苷酸的热稳定性明显增强。
本论文主要研究内容和结论概括如下。采用改进的碱液浸提法从香菇子实体中成功提取出水溶性的β-(1→3)-D-葡聚糖,命名为lentinan。该法具有高纯度、高产率、低成本、零污染等诸多优点。利用动态激光光散射仪(DLS)成功地分析lentinan水溶液中单条三螺旋和聚集体对应的双模式峰,并将LLS和DLS结果相结合,弄清单条的三螺旋链和它所形成的物理聚集体的分子参数。运用“快”和“慢”模型成功地描述了稀溶液中lentinan三螺旋链的短链构象,指出其中占主要成分的三螺旋链与少量聚集体共存于水溶液体系中。DLS和原子力显微镜(AFM)结果揭示短的三螺旋链彼此紧密平行排列形成有序的“柴火棒状”团簇。同时证明,只有低分子量的棒状链才能在较强相互作用和最小的空间位阻下形成有序排列的聚集体。
运用流变仪证实lentinan水溶液在25℃时发生溶液—凝胶转变。依据Winter-Chambon提出的凝胶化转变附近频率与损耗模量tanδ无依赖关系的理论确定了该体系的凝胶点cgel。同时发现,Winter-Chambon确定凝胶点的方法可适用于本体系。Lentinan具有大量羟基而容易聚集,而且三螺旋的构象使其分子链具有很高的刚性。因此,lentinan不同于大多数合成高分子和其它柔顺链多糖,具有较低的cgel,且随着分子量的增加,cgel显著降低。流变测量结果指出该溶液的损耗模量(G”)和储能模量(G’)与频率的依赖关系服从标度定律(G’～ωn),且标度指数n显著依赖于多糖浓度及分子量。Lentinan水溶液在低频时即表现出剪切变稀行为,归因于其刚性的分子链在剪切应力作用下的取向。与非凝胶多糖xanthan和schizophyllan相比,Lentinan水溶液在更低浓度时就形成凝胶。
运用流变仪成功地确定了lentinan水溶液冷致弹性凝胶的形成,以及不同恒定温度下G’,G”和tanδ的变化规律。温度降低时lentinan试样均形成弱凝胶,升温即溶解,且lentinan水体系溶液/凝胶转变是热可逆的。依据tanδ与温度的依赖关系用Winter-Chambon方法准确确定体系的临界凝胶化转变温度Tgel,并证明该法有效。同时发现,Tgel随着分子量或聚合物浓度减小而降低。凝胶点时的标度指数n值随聚合物浓度增大而减小,但是与分子量没有依赖关系。Lentinan水体系凝胶机理可推测如下：高度缠结的Lentinan三螺旋链形成连续的网络,导致凝胶性质化,但不存在交联点和明显的聚集体,因此它是非流动性的浓溶液。
流变实验证明,25℃时lentinan在稀溶液中为典型的牛顿流体,而在半稀溶液区间呈现剪切变稀行为。零切粘度的浓度和分子量依赖关系证明lentinan表现出棒状高分子各相同性溶液的特征,但是不能用棒状高分子的管道模型描绘,这归因于lentinan具有较高的分子量。随着溶液浓度增加,弱凝胶形成,并且可以观察到凝胶结构中由很长的多糖链形成缠结网络结构。储能模量(G’)的反常变化以及复合黏数η*在7-9℃出现极大值,说明lentinan凝胶相中形成了更刚性的结构。Lentinan侧链的葡萄糖残基与主链周围的水分子通过由于氢键作用形成了更刚性结构,即发生了从无序到有序的分子内构象转变。首次发现,侧链的葡萄糖残基在高于9℃时相对自由,而当温度低于9℃时,氢键形成,导致链刚性和直径增加,并且更加固定。另外,这种有序—无序的分子内构象转变具有溶剂依赖性。
利用差示扫描微量热法成功地表征了溶剂种类和分子量对有序-无序转变的影响。Lentinan可以从有序的三螺旋Ⅰ型转变到相对无序的三螺旋Ⅱ型的构象。结果表明这种链构象的低温转变明显依赖于溶剂种类,同时转变温度的高低直接取决于溶剂本身的热力学性质。低温转变在未达到临界浓度之前无浓度依赖性,分子量对转变温度的影响较小。Lentinan级试样发生有序—无序构象转变的协同单元分子量约为13×104,该值与溶剂种类几乎无关。试样分子量越小,协同单元分子量越大,但是与其它氢键诱导的构象转变相比,低温转变协同性较差。激光光散射测定结果证明lentinan在侧链相对固定时,分子链形成更加伸展的刚性棒状链构象。基于实验结果,提出一个涉及不同分子尺寸的链构象变化模型来描述lentinan有序—无序构象转变。直观地解释了lentinan发生从有序的三螺旋Ⅰ型到相对无序的三螺旋Ⅱ型的构象转变时,协同单元分子量与溶剂种类和分子链尺寸无关。
利用圆二色谱和紫外光谱成功地表征了香菇三螺旋链多糖lentinan与聚核苷酸poly(A)及poly(C)在Vw=0.936的DMSO/水混合溶剂的相互作用。实验结果证明lentinan与聚核苷酸形成由氢键力驱动的复合物,而且复合物中聚核苷酸的热稳定性明显增强。同时,动态光散射测量结果示出lentinan与poly(C)形成复合物的动态过程,表明poly(C)能迅速参与lentinan的复性过程,并与lentinan形成较稳定的新的三螺旋复合物。由此本工作为弄清多糖参与生命活动的形式和功能提供了重要科学依据。
通过上述基础研究结果确定了lentinan在不同浓度区间的动力学及热力学性质以及分子间相互作用及诱导的链构象变化。本工作完善了用高分子物理理论确立生物大分子的尺寸、形状及其链构象转变的方法,并且对它们在生命过程中的作用提供了有价值的科学数据,具有重要学术意义和应用前景。同时,也为香菇多糖进一步开发为保健品和天然药物提供大量有价值的科学数据,具有应用前景。
Lentinan, existing as triple helical chains in water, mainly refers to theβ-(1→3)-D-glucan in the fruiting bodies of lentinus edodes. It has been reported that lentinan not only exhibits a high specificity immunopotentiation, activing of the macrophage, inhibiting of the proliferation of tumor cell, but also processes immune function of economy depressed by cancer. These significant bioactivities are related to the space extension state of polysaccharide in organism and its unique molecular recognition ability. However, the conformation transition of molecular chains and hydrogen bonding interactions of lentinan in different solutions, as well as the relationship of the chain structure to bioactivities are still unknown. In this thesis, triple helical lentinan was extracted and purified from Lentinus edodes. The molecular size and chain conformation in different concentration region were investigated by polymer physical theories and methods. Therefore, this work is in a cross field of polymer physics, biology and medicine, and also one of the frontiers of polymer science.
The innovative points of this work are as follows. (1) It was, for the first time, confirmed by using polymer physics method that the short triple helical chains parallel aligned to each other and close packed to form ordered "faggot-like" clusters. (2) Lentinan fractions formed weak gels and the gelation mechanism is proposed as follows:the extremely entangled lentinan chains make a continuous network, conferring to the system the gel-like properties, and there were no contain junction zones and pronounced aggregates in lentinan gel. The Lentinan aqueous gel is more like a very concentrated solution that is unable to flow within a timescale of usual observation. (3) It was found firstly the abnormal experimental phenomenon that the storage modulus G' and complex viscosityη* went through a maximum at 7-9℃was ascribed to the order-disorder structure transition of the side glucose residues. (4) Lentinan could form a higher-order structure with polynucleotide through hydrogen-bonding and hydrophobic interactions, and the thermostability of polynucleotide in the complex was dramatically increased.
The main contents and conclusions in this project are divided into the following parts. Lentinan, aβ-(1→3)-D-glucan, was isolated from Lentinus edodes by using an improved extraction and purification method to show good water solubility and high yield. Dynamic light scattering was successfully used to detect the bimodal peaks corresponding to individual triple helical chains and their aggregates in the lentinan aqueous system. A combination of static and dynamic LLS results created their molecular characteristic parameters of both the fast mode and the slow one, which are corresponding to the relaxation of individual triple helical lentinan and their physical associations, respectively. By using fast and slow modes we successfully described the chain conformation of triple helical lentinan in dilute aqueous solution, indicating co-existence of the predominant triple helical chain and few aggregates. The results from DLS and AFM revealed that the short triple helical chains parallel aligned to each other and close packed to form ordered "faggot-like" clusters. Only the rod-like chains of lentinan having low molecular weight could form ordered aggregates, as a results of the strongest interaction and the smallest steric hindrance between each chains.
The solution-gel transition of Lentinan in water at 25℃was observed rheologically. The gel point cgei was determined by using the Winter-Chambon method (frequency-independent of tanδin the vicinity of the solution-gel transition). It was found that the Winter-Chambon criterion worked well in determining the critical gelation point of the present system although the system behaved as a weak gel before gelation. The cgei was relatively lower than the general synthesized polymers and other flexible polysaccharides, resulting from the high stiffness of triple helix and strong aggregation as a result of abundant hydroxyl groups. The cgei decreased sharply with increasing molecular weight. G'and G " were found to follow a power law behavior as a function of frequency (G'-ωn), and the exponents n are strongly dependent on concentration and molecular weight. The Lentinan solution exhibited shear-thinning behavior at low frequency because of the orientation of the stiff chains under shear force. Compared to the non-gelling Xanthan gum and schizophyllan, Lentinan could form gels more easily at low concentration.
The cold-set formation of an elastic fractal gel for triple helical Lentinan in water was investigated rheologically from measurements of G',G" and tanδunder isothermal conditions at different constant temperatures. The results showed that Lentinan fractions formed weak gels with decreasing temperature, and the sol-gel transition was thermally reversible. The critical gelation temperature Tgel was accurately determined by the Winter-Chambon method from the temperature dependence of tanδ, indicating the validity of the Winter-Chambon criterion. It was found that Tgel decreased with decreasing molecular weight and polymer concentration, and that the exponent (n) values at the gel point decreased with increasing polymer concentration but showed an independence of molecular weight. The gelation mechanism for lentinan fractions in water is proposed as follows:the extremely entangled lentinan chains make a continuous network, conferring to the system the gel-like properties, and there were no contain junction zones and pronounced aggregates in lentinan gel. The Lentinan aqueous gel is more like a very concentrated solution that is unable to flow within a timescale of usual observation.
The experimental results and their analysis presented above have led us to conclude that the polysaccharide Lentinan exhibits Newtonian-flow behavior in dilute solution and shear-thinning behavior in semidilute concentration domain at 25℃. The concentration and molecular weight dependences of the zero-shear viscosity appear common to viscous properties of isotropic solutions of rodlike polymers, but they could not described by the tube model for rodlike polymers. This was attributed to the high molecular weight of Lentinan used here. With increasing concentration, weak gel formed and the gel structure was observed to be the entangled network. The abnormal experimental phenomenon that the storage modulus G' and complex viscosityη* went through a maximum at 7～9℃was ascribed to the formation of more rigid structure in the lentinan gel state.. Moreover, the order-disorder structure transition associating with the hydrogen-bond interaction between the side glucose residues and the water molecules surrounding the polysaccharide backbone was also observed in this temperature range. Namely, the side glucose residues were free at higher than～9℃, and the hydrogen-bonds were formed as temperature lower than～9℃, resulting in the increase in chain stiffness and diameter. Moreover, this order-disorder intramolecular conformation is solvent-dependent. This result further proved that lentinan adopts a triple-helical conformation in water.
Lentinan could transit from triple helix-I to triple helix-II, namely from a highly immobilization of the backbone to a relatively rotating one. The influence of solvent and molecular weight to the order-disorder conformation transition were investigated by US-DSC. The result revealed that the order-disorder transition depended on solvent kinds dramatically, and the transition temperature was directly laid on the thermodynamic property of solvent. Moreover, the transition had no polymer concentration dependence and the influence of molecular weight was tiny. The molecular weight of each cooperative unit for lentinan sample was about 13×104, and the value was slightly affected by solvent kinds. Moreover, the molecular weight of cooperative units elevated with a decrease of lentinan's Mw. However, compared to other hydrogen-bond induced conformation transition, the cooperativity of lentinan in low temperature was poor. The results of dynamic light scattering revealed that when the side chain was relatively immobilizated, lentinan polymer chains formed a more stiff and extend rod-like conformation. On the basis of the experiment results, a schematic diagram to describe the effect of molecular size to the order-disorder transition was proposed, and clearly revealed that the molecular weight of cooperative units has no dependence on solvent kinds and molecular weight.
The circular dichroism (CD) and ultraviolet absorbance (UV) were used to investigate the interaction between triple helical lentinan and polynucleotide (poly(A) and poly(C)) in ww=0.93 water/DMSO system. The results showed that lentinan could formed a higher-order structure with polynucleotide through hydrogen-bonding and hydrophobic interactions, and the thermostability of polynucleotide in the complex was dramatically increased. Moreover, dynamic light scattering was used to detect the dynamic procedure of the interaction between lentinan and polynucleotide. The results indicated that poly(C) took part in the renaturation of lentinan chains rapidly, and formed a new triple helical complex.
This foundation research mentioned above focus on the thermodynamics and dynamics properties of lentinan in different concentration region, along with the intermolecular interaction and the conformation transition led by. This work provided important scientific data determining the macromolecule size, shape and conformation transition by using polymer physics method as well as their function in life process. It not only has scientific significance, but also provides important scientific data for the exploration and application of lentinan.
本论文的主要创新包括以下几点：(1)用高分子物理方法首次证实短的三螺旋链彼此紧密平行排列形成有序的“柴火棒状”团簇；(2)首次证明lentinan水溶液可发生溶液-凝胶转变行为,并提出其凝胶化机理为：高度缠结的Lentinan三螺旋链形成连续的网络,导致凝胶化,但不存在交联点和明显的聚集体,因此它是不具有流动性的浓溶液；(3)首次发现,在7-9℃区间内,lentinan水溶液粘度随温度出现反常变化,归因于三螺旋侧链的有序—无序变化；(4)证实lentinan与聚核苷酸形成由氢键力驱动的新的三螺旋复合物,而且复合物中聚核苷酸的热稳定性明显增强。
本论文主要研究内容和结论概括如下。采用改进的碱液浸提法从香菇子实体中成功提取出水溶性的β-(1→3)-D-葡聚糖,命名为lentinan。该法具有高纯度、高产率、低成本、零污染等诸多优点。利用动态激光光散射仪(DLS)成功地分析lentinan水溶液中单条三螺旋和聚集体对应的双模式峰,并将LLS和DLS结果相结合,弄清单条的三螺旋链和它所形成的物理聚集体的分子参数。运用“快”和“慢”模型成功地描述了稀溶液中lentinan三螺旋链的短链构象,指出其中占主要成分的三螺旋链与少量聚集体共存于水溶液体系中。DLS和原子力显微镜(AFM)结果揭示短的三螺旋链彼此紧密平行排列形成有序的“柴火棒状”团簇。同时证明,只有低分子量的棒状链才能在较强相互作用和最小的空间位阻下形成有序排列的聚集体。
运用流变仪证实lentinan水溶液在25℃时发生溶液—凝胶转变。依据Winter-Chambon提出的凝胶化转变附近频率与损耗模量tanδ无依赖关系的理论确定了该体系的凝胶点cgel。同时发现,Winter-Chambon确定凝胶点的方法可适用于本体系。Lentinan具有大量羟基而容易聚集,而且三螺旋的构象使其分子链具有很高的刚性。因此,lentinan不同于大多数合成高分子和其它柔顺链多糖,具有较低的cgel,且随着分子量的增加,cgel显著降低。流变测量结果指出该溶液的损耗模量(G”)和储能模量(G’)与频率的依赖关系服从标度定律(G’～ωn),且标度指数n显著依赖于多糖浓度及分子量。Lentinan水溶液在低频时即表现出剪切变稀行为,归因于其刚性的分子链在剪切应力作用下的取向。与非凝胶多糖xanthan和schizophyllan相比,Lentinan水溶液在更低浓度时就形成凝胶。
运用流变仪成功地确定了lentinan水溶液冷致弹性凝胶的形成,以及不同恒定温度下G’,G”和tanδ的变化规律。温度降低时lentinan试样均形成弱凝胶,升温即溶解,且lentinan水体系溶液/凝胶转变是热可逆的。依据tanδ与温度的依赖关系用Winter-Chambon方法准确确定体系的临界凝胶化转变温度Tgel,并证明该法有效。同时发现,Tgel随着分子量或聚合物浓度减小而降低。凝胶点时的标度指数n值随聚合物浓度增大而减小,但是与分子量没有依赖关系。Lentinan水体系凝胶机理可推测如下：高度缠结的Lentinan三螺旋链形成连续的网络,导致凝胶性质化,但不存在交联点和明显的聚集体,因此它是非流动性的浓溶液。
流变实验证明,25℃时lentinan在稀溶液中为典型的牛顿流体,而在半稀溶液区间呈现剪切变稀行为。零切粘度的浓度和分子量依赖关系证明lentinan表现出棒状高分子各相同性溶液的特征,但是不能用棒状高分子的管道模型描绘,这归因于lentinan具有较高的分子量。随着溶液浓度增加,弱凝胶形成,并且可以观察到凝胶结构中由很长的多糖链形成缠结网络结构。储能模量(G’)的反常变化以及复合黏数η*在7-9℃出现极大值,说明lentinan凝胶相中形成了更刚性的结构。Lentinan侧链的葡萄糖残基与主链周围的水分子通过由于氢键作用形成了更刚性结构,即发生了从无序到有序的分子内构象转变。首次发现,侧链的葡萄糖残基在高于9℃时相对自由,而当温度低于9℃时,氢键形成,导致链刚性和直径增加,并且更加固定。另外,这种有序—无序的分子内构象转变具有溶剂依赖性。
利用差示扫描微量热法成功地表征了溶剂种类和分子量对有序-无序转变的影响。Lentinan可以从有序的三螺旋Ⅰ型转变到相对无序的三螺旋Ⅱ型的构象。结果表明这种链构象的低温转变明显依赖于溶剂种类,同时转变温度的高低直接取决于溶剂本身的热力学性质。低温转变在未达到临界浓度之前无浓度依赖性,分子量对转变温度的影响较小。Lentinan级试样发生有序—无序构象转变的协同单元分子量约为13×104,该值与溶剂种类几乎无关。试样分子量越小,协同单元分子量越大,但是与其它氢键诱导的构象转变相比,低温转变协同性较差。激光光散射测定结果证明lentinan在侧链相对固定时,分子链形成更加伸展的刚性棒状链构象。基于实验结果,提出一个涉及不同分子尺寸的链构象变化模型来描述lentinan有序—无序构象转变。直观地解释了lentinan发生从有序的三螺旋Ⅰ型到相对无序的三螺旋Ⅱ型的构象转变时,协同单元分子量与溶剂种类和分子链尺寸无关。
利用圆二色谱和紫外光谱成功地表征了香菇三螺旋链多糖lentinan与聚核苷酸poly(A)及poly(C)在Vw=0.936的DMSO/水混合溶剂的相互作用。实验结果证明lentinan与聚核苷酸形成由氢键力驱动的复合物,而且复合物中聚核苷酸的热稳定性明显增强。同时,动态光散射测量结果示出lentinan与poly(C)形成复合物的动态过程,表明poly(C)能迅速参与lentinan的复性过程,并与lentinan形成较稳定的新的三螺旋复合物。由此本工作为弄清多糖参与生命活动的形式和功能提供了重要科学依据。
通过上述基础研究结果确定了lentinan在不同浓度区间的动力学及热力学性质以及分子间相互作用及诱导的链构象变化。本工作完善了用高分子物理理论确立生物大分子的尺寸、形状及其链构象转变的方法,并且对它们在生命过程中的作用提供了有价值的科学数据,具有重要学术意义和应用前景。同时,也为香菇多糖进一步开发为保健品和天然药物提供大量有价值的科学数据,具有应用前景。
Lentinan, existing as triple helical chains in water, mainly refers to theβ-(1→3)-D-glucan in the fruiting bodies of lentinus edodes. It has been reported that lentinan not only exhibits a high specificity immunopotentiation, activing of the macrophage, inhibiting of the proliferation of tumor cell, but also processes immune function of economy depressed by cancer. These significant bioactivities are related to the space extension state of polysaccharide in organism and its unique molecular recognition ability. However, the conformation transition of molecular chains and hydrogen bonding interactions of lentinan in different solutions, as well as the relationship of the chain structure to bioactivities are still unknown. In this thesis, triple helical lentinan was extracted and purified from Lentinus edodes. The molecular size and chain conformation in different concentration region were investigated by polymer physical theories and methods. Therefore, this work is in a cross field of polymer physics, biology and medicine, and also one of the frontiers of polymer science.
The innovative points of this work are as follows. (1) It was, for the first time, confirmed by using polymer physics method that the short triple helical chains parallel aligned to each other and close packed to form ordered "faggot-like" clusters. (2) Lentinan fractions formed weak gels and the gelation mechanism is proposed as follows:the extremely entangled lentinan chains make a continuous network, conferring to the system the gel-like properties, and there were no contain junction zones and pronounced aggregates in lentinan gel. The Lentinan aqueous gel is more like a very concentrated solution that is unable to flow within a timescale of usual observation. (3) It was found firstly the abnormal experimental phenomenon that the storage modulus G' and complex viscosityη* went through a maximum at 7-9℃was ascribed to the order-disorder structure transition of the side glucose residues. (4) Lentinan could form a higher-order structure with polynucleotide through hydrogen-bonding and hydrophobic interactions, and the thermostability of polynucleotide in the complex was dramatically increased.
The main contents and conclusions in this project are divided into the following parts. Lentinan, aβ-(1→3)-D-glucan, was isolated from Lentinus edodes by using an improved extraction and purification method to show good water solubility and high yield. Dynamic light scattering was successfully used to detect the bimodal peaks corresponding to individual triple helical chains and their aggregates in the lentinan aqueous system. A combination of static and dynamic LLS results created their molecular characteristic parameters of both the fast mode and the slow one, which are corresponding to the relaxation of individual triple helical lentinan and their physical associations, respectively. By using fast and slow modes we successfully described the chain conformation of triple helical lentinan in dilute aqueous solution, indicating co-existence of the predominant triple helical chain and few aggregates. The results from DLS and AFM revealed that the short triple helical chains parallel aligned to each other and close packed to form ordered "faggot-like" clusters. Only the rod-like chains of lentinan having low molecular weight could form ordered aggregates, as a results of the strongest interaction and the smallest steric hindrance between each chains.
The solution-gel transition of Lentinan in water at 25℃was observed rheologically. The gel point cgei was determined by using the Winter-Chambon method (frequency-independent of tanδin the vicinity of the solution-gel transition). It was found that the Winter-Chambon criterion worked well in determining the critical gelation point of the present system although the system behaved as a weak gel before gelation. The cgei was relatively lower than the general synthesized polymers and other flexible polysaccharides, resulting from the high stiffness of triple helix and strong aggregation as a result of abundant hydroxyl groups. The cgei decreased sharply with increasing molecular weight. G'and G " were found to follow a power law behavior as a function of frequency (G'-ωn), and the exponents n are strongly dependent on concentration and molecular weight. The Lentinan solution exhibited shear-thinning behavior at low frequency because of the orientation of the stiff chains under shear force. Compared to the non-gelling Xanthan gum and schizophyllan, Lentinan could form gels more easily at low concentration.
The cold-set formation of an elastic fractal gel for triple helical Lentinan in water was investigated rheologically from measurements of G',G" and tanδunder isothermal conditions at different constant temperatures. The results showed that Lentinan fractions formed weak gels with decreasing temperature, and the sol-gel transition was thermally reversible. The critical gelation temperature Tgel was accurately determined by the Winter-Chambon method from the temperature dependence of tanδ, indicating the validity of the Winter-Chambon criterion. It was found that Tgel decreased with decreasing molecular weight and polymer concentration, and that the exponent (n) values at the gel point decreased with increasing polymer concentration but showed an independence of molecular weight. The gelation mechanism for lentinan fractions in water is proposed as follows:the extremely entangled lentinan chains make a continuous network, conferring to the system the gel-like properties, and there were no contain junction zones and pronounced aggregates in lentinan gel. The Lentinan aqueous gel is more like a very concentrated solution that is unable to flow within a timescale of usual observation.
The experimental results and their analysis presented above have led us to conclude that the polysaccharide Lentinan exhibits Newtonian-flow behavior in dilute solution and shear-thinning behavior in semidilute concentration domain at 25℃. The concentration and molecular weight dependences of the zero-shear viscosity appear common to viscous properties of isotropic solutions of rodlike polymers, but they could not described by the tube model for rodlike polymers. This was attributed to the high molecular weight of Lentinan used here. With increasing concentration, weak gel formed and the gel structure was observed to be the entangled network. The abnormal experimental phenomenon that the storage modulus G' and complex viscosityη* went through a maximum at 7～9℃was ascribed to the formation of more rigid structure in the lentinan gel state.. Moreover, the order-disorder structure transition associating with the hydrogen-bond interaction between the side glucose residues and the water molecules surrounding the polysaccharide backbone was also observed in this temperature range. Namely, the side glucose residues were free at higher than～9℃, and the hydrogen-bonds were formed as temperature lower than～9℃, resulting in the increase in chain stiffness and diameter. Moreover, this order-disorder intramolecular conformation is solvent-dependent. This result further proved that lentinan adopts a triple-helical conformation in water.
Lentinan could transit from triple helix-I to triple helix-II, namely from a highly immobilization of the backbone to a relatively rotating one. The influence of solvent and molecular weight to the order-disorder conformation transition were investigated by US-DSC. The result revealed that the order-disorder transition depended on solvent kinds dramatically, and the transition temperature was directly laid on the thermodynamic property of solvent. Moreover, the transition had no polymer concentration dependence and the influence of molecular weight was tiny. The molecular weight of each cooperative unit for lentinan sample was about 13×104, and the value was slightly affected by solvent kinds. Moreover, the molecular weight of cooperative units elevated with a decrease of lentinan's Mw. However, compared to other hydrogen-bond induced conformation transition, the cooperativity of lentinan in low temperature was poor. The results of dynamic light scattering revealed that when the side chain was relatively immobilizated, lentinan polymer chains formed a more stiff and extend rod-like conformation. On the basis of the experiment results, a schematic diagram to describe the effect of molecular size to the order-disorder transition was proposed, and clearly revealed that the molecular weight of cooperative units has no dependence on solvent kinds and molecular weight.
The circular dichroism (CD) and ultraviolet absorbance (UV) were used to investigate the interaction between triple helical lentinan and polynucleotide (poly(A) and poly(C)) in ww=0.93 water/DMSO system. The results showed that lentinan could formed a higher-order structure with polynucleotide through hydrogen-bonding and hydrophobic interactions, and the thermostability of polynucleotide in the complex was dramatically increased. Moreover, dynamic light scattering was used to detect the dynamic procedure of the interaction between lentinan and polynucleotide. The results indicated that poly(C) took part in the renaturation of lentinan chains rapidly, and formed a new triple helical complex.
This foundation research mentioned above focus on the thermodynamics and dynamics properties of lentinan in different concentration region, along with the intermolecular interaction and the conformation transition led by. This work provided important scientific data determining the macromolecule size, shape and conformation transition by using polymer physics method as well as their function in life process. It not only has scientific significance, but also provides important scientific data for the exploration and application of lentinan.
引文
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