The Solution Properties Of Triple Helical Lentinan From Diluted To Concentrated Polymer Region

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.