Structure And Bioactivities Of Polysaccharides From Ganoderma Lucidum

Ganoderma lucidum (Leyss, ex Fr.) Karst (G1), a basidiomycete funguns belongs to the Polyporaceae. The earliest record of Ganoderma lucidum was in the'Shen Nong's Materia Medica'about two thousands years ago in Han Dynasty of China. Even now, it is still used to treat gastric ulcers, chronic hepatitis, nephritis, hypertension, hyperlipemia, arthritis, insomnia, bronchitis, asthma and anorexia. It has been reported that Ganoderma Luciderma exhibits a high specific immunopotentiation, activation of the macrophage, inhibition of the proliferation of tumor cell, also promotes immuno-function of economically depressed people affected by cancer. Further, the derivatives of funguns polysaccharides could not only improve the water-solubility, but also enhance the anti-tumor and antioxidant activities. In this work, several polysaccharides were extracted from the fruit body of Ganoderma lucidum and the water-soluble derivatives were prepared. The relationship between chemical structure, molecular mass, chain conformation and the antitumor, antioxidant, and transfect efficiency were investigated. Therefore, this work is interdisciplinary involving polymer physics, biology and medicine, and also one of the frontiers of polymer science.The salient features of this work are as follows. (1) Sulfated, carboxymethylated, hydroxyethylated, hydroxypropylated and methylated polysaccharides were prepared successfully by using the water-insoluble Ganoderma Luciderma polysaccharides, and the anti-tumor activities and the mechanism of S-GL and CM-GL were investigated in vitro and in vivo. (2) The in vitro and in vivo antioxidant activities of S-GL and CM-GL were studied. (3) For the first time to the best of our knowledge, the cationic water-insoluble Ganoderma Luciderma polysaccharides were prepared successfully by using quaternization, and the cytotoxicity and transfect efficiency were investigated.The main contents and conclusions in this thesis are divided into the following parts. Five water-soluble heteropolysaccharides coded as GL-I to GL-V, were isolated from the cultured fruit body of Ganoderm Lucidum. The polysaccharides were analyzed using Fourier transform infrared spectroscopy, elementary analysis, gas chromatography, gas chromatography/mass spectrometry and 13C NMR techniques. The molecular mass, intrinsic viscosity and chain stiffness were determined with laser light scattering, size exclusion chromatography combined with laser light scattering and viscometry. GL-Ⅰ, GL-Ⅱand GL-Ⅲwere heteropolysaccharide-protein complexes with protein contents of 14.6%,5.7%, and 3.6%, respectively. Moreover, GL-Ⅰmainly contained galactose with the relatively high content of 52.0%, as well as less glucan, mannose, xylose and arabinose. With the progress of the isolation (1→3)-β-D--glucan increased, whereas the degree of branch of the heteropolysaccharides decreased. The relatively high branching of the heteropolysaccharide led to the improvement of water solubility. The samples of GL-Ⅰto GL-Ⅲexisted in the aqueous solution as compact coil conformation as a result of the relatively high branching. Chain conformation parameters detected by LLS supported the results obtained from AFM studies.A linear water-insoluble (1→3)-β-D-glucan, coded as GL-Ⅳ-Ⅰ, was isolated from the fruit body of Ganoderma lucidum by extracting with sodium hydroxide solution. Its derivatives were prepared by using sulfation, carboxymethylation, hydroxyethlation, hydroxypropylation and methylation, named them as S-GL, CM-GL, HE-GL, HP-GL and M-GL. The degrees of substitution of the derivatives were 0.94,1.18,0.53,0.44 and 0.74, separately. Their structure and chain conformation were investigated with infrared spectroscopy, elemental analysis, one and two-dimensional NMR, laser light scattering (LLS) and size-exclusion chromatography combined with laser light scattering (SEC-LLS). The reactivity of hydroxyl group of GL-Ⅳ-Ⅰwas in the order of C-6> C-4>C-2 in the five derivations. The weight-average molecular mass (Mw) of GL-IV-I, S-GL, CM-GL, HE-GL, HP-GL and M-GL were 13.3×104, 10.1×104,6.3×104,7.2×104,5.1×104, and 14.1×104, respectively. From data of Mw and the Z 1/2, the power low describing the relationship between Mw and z 1/2 ( 1/2=KMwa) can be created. Usually, the chain conformation of polymer in solution can be estimated by the value of exponentα. The values ofαS-GL, CM-GL, HE-GL, HP-GL and M-GL were found to be 0.58, 0.61,0.56,0.54 and 0.51 respectively. These results indicated that the derivatives existed as relatively expanded flexible chains in 0.9% NaCl aqueous solution, resulting from steric hindrance of the substituted groups introduced on the backbone.The anti-tumor activities, cell cycle arrest, inducement of apoptosis and inhibition effect against S-180 tumor cells of S-GL and CM-GL were investigated. The introduction of charge groups not only improved the water-solubility, but also enhanced the bioactivities evidently. The median inhibitory concentration (IC50) values of S-GL and CM-GL tested against S-180 cancer cells were 26 and 38μg/mL, respectively. The inhibition ratios against S-180 cancer cells of S-GL and CM-GL were 55.6±2.1% and 51.3±1.3%, respectively, which are only slightly lower than that of 5-Fu.The growth of the S-180 solid tumors was markedly inhibited by S-GL and CM-GL, the induce apoptosis in xenograft S-180 tumor cell by up-regulating Bax and down-regulating Bcl-2. The derivatives also could inhibit the growth of S-180 cells through cell-cycle arrest in G2/M phase and induced the apoptosis. Treatment with S-GL and CM-GL resulted in a significant increase apoptosis of S-180 cells. The percent of apoptosis in control S-180 cells (2.57±0.39) was significantly lower than that of S-GL (17.9±1.98) and CM-GL (14.1±2.67).A series of in vitro experiments were carried out to test the antioxidant activity of S-GL and CM-GL. Meanwhile, in order to further prove that the antioxidant activityof the sample, the in vivo antioxidant effect of the derivatives have been compared with those in vitro. S-GL and CM-GL showed good free radical scavenging effect in vitro. The derivatives also could course elimination of immunosuppression resulted from the treatment of 5-fluorouracil, the traditional anticancer medicine. Meanwhile, S-GL and CM-GL can enhanced immune function in mice; increase the content of SOD and GSH content in mice.Cationic Ganoderma lucidum polysaccharides were synthesized successfully by using 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) in NaOH aqueous solution, coded as QG, and their chemical structures were investigated. The QGs carry a strong positive charge and can form stable complexes with naked DNA (pGL-3 DNA). The transfect efficiency of the QGs was similar to that of PET with low side effect and can be explored as new generation transfect materials.In summary, this work dealt with the structure, molecular sise and the chain conformation of Ganoderm luciderma polysaccharides and its derivatives, their anti-tumor activities, immunomodulating, antioxidant and transfect efficiency. This work provided the scientific data for the research of life science and chemistry. It not only has significant academic value, but also has potential application prospect in the exploration of Ganoderm Luciderma as Chinese Traditional Medicine. Meanwhile, it has a positive role in promoting traditional and time-tested Chinese herbal medicine culture.