The Functionalization Of Triple Helical Lentinan Based On Hydrogen Bonding Association

Fungal polysaccharides widely exist in the cell walls or cell culture media of fungi. Many kinds of fungal polysaccharides show anti-tumor, anti-virus or anti-oxidation bioactivities. They are ideal candidates as natural health products, drugs and food additives and have wide application prospect in the fields of medicine, food, daily necessities, and biological materials. Polysaccharides extracted from different resources vary in chemical structures, molecular size, and chain conformations, which show obvious influence on their bioactivities. Therefore, it’s a challenge and opportunity for us to develop effective extraction methods of polysaccharides and to resolve their chemical structures and chain conformations so that their practical values would be realized. Some progress has already been achieved in polysaccharides functionalization, but more work regarding the chemical and biological features of polysaccharides need to be done. In this thesis, we mainly focus on the chemical structures, conformations and functionalization of fungal polysaccharides. Our purpose is to resolve the chemical structures and conformations with the aid of different analytical methods and polymer solution theory. At the same time, we investigated the functionalization of polysaccharides by different means from nano to micro scale.The major innovations of this thesis include the followings:(1) We efficiently extracted lentinan from Lenlinus edodes with a green method, and established a kind of facile method for clarifing its chemical structures and triple helical conformation in water;(2) We successfully prepared Ag nanoparticles dispersible in water from the aqueous solution of triple helical lentinan, and studied the aggregation behavior of triple helical chain in base solution with Ag nanoparticles as labeling;(3) We observed the self-assembly of lentinan into a nanofilm for the first time and thus discovered a simple rapid method to prepare lentinan micro vesicles;(4) For the first time we fabricated nano vesicle and nanosphere from amphipathic lentinan and revealed the effect of chain stiffness on structures of self-assembly products;(5) We clarified the chemical structure and conformation of polyporus rhinoceros polysaccharides and found the formation of pine needle-like aggregation.The main contents and conclusions in this thesis are divided into the following parts. A kind of water-soluble polysaccharides was isolated from Lentinus edodes via a green method developed by the lab. From IR, NMR and GC-MS results, the chemical structure of the polysaccharide with high purity was analyzed to be a kind of β-1,3-D-glucans with two β-1,6-D-residues in interval of every five main-chain residues. The polysaccharide conformation was analyzed by using SEC-MALLS-RI-Visco combination method. The <s2>z1/2～Mw, equation and Mark-Houwink function were obtained by data fitting. The result revealed that the polysaccharide majorly exists as triple helical conformation in aqueous solution with high chain stiffness and few aggregations (12.6%) with coil conformation are also found in the solution. TEM and AFM observations further proved the results directly. Hydrophobic phthalocyanine could be incorporated into the one-dimensional hollow constructed by the polysaccharide triple helical chains to be water-soluble. The phenomena proved the existence of hydrophobic hollow in lentinan. This work provided a new kind of simple and reliable method, instead of trantional complex and time-consuming method, for characterizing the chemical structure and triple helical conformation of lentinan, which are import for deciding the structure of lentinan.Water-dispersible silver nanoparticles with mean radius of6nm were created in the triple helical polysaccharide aqueous solution. The results of TEM and DLS revealed that the silver nanoparticles were attached to the polysaccharide chains by the strong electrostatic interactions between the polysaccharides and the silver nanoparticles, leading to the good dispersion of the nanoparticles in the solution. Lentinan with abundant active hydroxyl groups not only supplied a matrix for the constructing of the silver nanoparticles, but also acted as a stabilizer for good dispersion of the nanoparticles in water. The silver nanoparticles could also exist stably in lentinan solution containing NaOH, and the aggregation of denatured polysaccharides did not affect the shape and size of the Ag nanoparticles. Moreover, the aggregation was related to the conformation transition of the polysaccharide from the triple helix to random coil in the solution. A new method to detect the aggregates and aggregation rate was established based on the intensity of the maximum absorption peaks of the polysaccharides labeled by Ag nanoparticles in the UV spectrum. This work provided important information for the creation of the silver nanoparticles via a simple and "green" pathway, and it will have wide applications in the biological field.Long-chain alkane was introduced to the reducing end of lentinan chains through the reaction between the aldehyde groups and the amine groups of laurylamine (C12H25NH2). The obtained sample (LNT-C12) was proved to form aggregation easily in water. TEM results showed the existing of single layer and multiple layer polysaccharides nano-sheets which were formed by the self-assembly of LNT-C12chains. Taking advantage of the LNT-C12self-assembly process, a simple and quick method was developed to obtain polysaccharides microcapsules. By changing the volume ratio (R) of aqueous phase (LNT-C12) versus organic phase (CH2G2), the type (O/W or W/O/W) and size of the microcapsules can be tuned. As the layer formed by LNT-C12was rather thin (about90nm at R=2) in the microcapsules, collapse happened during the evaporation of CH2Cl2. The stabilization of the microcapsules could be improved obviously after cross-linking by epoxy chloropropan (ECH). The drug-loading ability of the microcapsules was also studied by using polycaprolactone (PCL) as the target drug. The result revealed that PCL could be loaded into the microcapsules, and microspheres with PCL as core and LNT-C12as shell were formed. The size of the microspheres can also be tuned by changing the R value. This work provided a kind of facile method for constructing polysaccharide microcapsules, which can promote the application of letinan in the fields of drug delivery.Amiphiphilic polysaccharides (lentinan) were obtained with two different chain conformations (triple helical chains and random coil chains). The DLS, SLS and TEM results revealed that the amiphiphilic polysaccharides can self assembly into vehicles (triple helical lentinan) and spheres (random coil lentinan). The difference between the two assembly morphologies (vesicle and sphere) was original from the difference of chain stiffness. The high chain stiffness can prevent further collapse of the nanosphere structure, inducing the existence of vesicles. It proved the importance of chain stiffness on the self-assembly of amiphiphilic polysaccharides. By controlling the chain stiffness, we can tune the morphology and size of the obtained nanostructures. Moreover, the resulted polysaccharides vesicles and spheres can be used as drug carriers.A kind of water-soluble polysaccharide (PR-CA) from Polyporus rhinoceros was analyzed by FT-IR, NMR and GC-MS. The results indicated that PR-CA was a kind of β-(1,3)-D-glucans with β-1,6-D-residues branches. The degree of branch is calculated to be0.4. The conformation of PR-CA was proved to be random coil in DMSO and to be compact sphere conformation with smaller size in water. What’s more, PR-CA with low Mw could form pine needle-like nanostructures consisting of nano-needle structures. This work revealed the chemical structure and conformation, which is import for the further studying on the bioaetivity of PR-CA. The above results identified the chemical structures and conformations of lentinan and PR-CA. Meanwhile, we successfully prepared functional nano-materials based on lentinan with different sizes and morphologies, after which we studied the preliminary application prospect of these materials. This work provides profound data and academic supporting for studies involving polysaccharides’chemical structure, conformation, properties and their relationship. At the same time, we contributed to the industrialization of lentinan for expanded application.