Structures And Bioactivities Of Polysaccharides From Poria Cocos Mycelia With Cultivated Strain

Poria cocos has been long used in traditional Chinese medicine, and has pharmacological action of diuretic, antibacterial, immune, antitumor and complement activities etc. Polysaccharide is a kind of immunomodulator that can inhibit the tumor/cancer cell growth in vivo without any damage to the normal cell, which make it possible to produce healthy foods and new drugs in the future. Recently the antitumor activities of polysaccharides have been revealed to correlate with their chain conformation in solution. The polymer conformation is mainly characterized by polymer solution theory and modern methods in polymer science, so the research of the polysaccharides is performed by biotechnics combining with polymer physics. In this work, the mycelia of Poria cocos were cultivated through submerged fermentation. The water-soluble and alkali-soluble polysaccharides were extracted from the mycelia by biotechnics. Their chemical structure, chain conformation in solution and antitumor activity were studied. Therefore the study is in a cross field of polymer physics and biochemistry.The creative points of this work are as follows. (1) The chemical structures and weight-average molecular masses (M_w) of polysaccharides from the mycelium of Poria cocos with cultivated strain cultured in two different media were clarified, and the effects of the media were elucidated. (2) On the basis of polymer solution theory, (1→3)-α-D-glucan ab-PCM3-â…  and its sulfated derivative S-ab-PCM3-â…  were demonstrated to exist as extended random coil chain and semi-stiff chain in 0.25 M LiCl/dimethylsulfoxide (DMSO) and aqueous solution, respectively. (3) The chemical structure and M_w of exo-polysaccharide of Poria cocos were elucidated and its antitumor activity was evaluated.The main contents and conclusions in this thesis are divided into the following parts. Mycelium of a cultivated strain of Poria cocos was grown by submerged fermentation in a liquid medium containing corn steep liquor with orbital shaking. Six polysaccharides coded as ac-PCM1, ac-PCM2, ac-PCM3-â…  and â…¡, ac-PCM4-â…  and â…¡ were isolated from the mycelium by extracting with 0.9% NaCl aqueous solution, hot water, 0.5 M NaOH aqueous solution and 88% formic acid, respectively. Exo-polysaccharide was obtained from the culture medium and coded as ac-PCMO. The chemical structure and monosaccharide composition were characterized by using infrared spectroscopy (IR), gas chromatography (GC), ¹³C-NMR and elemental analysis (EA). These results indicated that ac-PCMO, ac-PCMl and ac-PCM2 are heteropolysaccharides containing glucose, galactose, mannose and fucose, while ac-PCM3-â…  and ac-PCM3-â…¡ mainly consist of D-glucose. The content of the glucose in the polysaccharides increased with the isolationprogress. Remarkably, a-glucan and /?-glucan coexist in the extract by NaOH aqueous solution (ac-PCM3), and could be separated into two fractions (ac-PCM3-I and ac-PCM3-II) after neutralizing with acetic acid. The protein in the ac-PCM polysaccharides cultured from the medium containing corn steep liquor was higher than that in the ab-PCM from the medium with bran extract. Mw and intrinsic viscosities ([n]) of these polysaccharides were characterized by using laser light scattering (LLS), size exclusion chromatography combined with laser light scattering (SEC-LLS), and viscometry. Therefore, the polysaccharides from Poria cocos mycelia cultured in different media have different chemical composition, Mw and conformations.The water-insoluble (1—>3)-a-D-glucan (ab-PCM3-I) was fractionated into ten fractions by non-solvent addition method in 0.25 M LiCl/DMSO. Seven fractions were treated with chlorosulfonic acid-pyridine complex to synthesis water-soluble sulfated derivatives (S-ab-PCM3-I). Their degrees of substitution ranged from 0.35 to 0.81. The solution characteristics were studied by SEC-LLS and viscometry at 25 °C. The Mark-Houwink equations for the native glucan and the sulfated derivatives were found to be [tj] = 9.92 x 10'3Mw077 (mL g"1) in the range of Mw from 1.08 to 23.1 x 104 and [rj] = 2.92 x lO^Mv109 (mL g"1) in the range of Mwfrom 0.74 to 5.80 x 104. Based on current theories for wormlike chain model, the conformation parameters of the glucan ab-PCM3-I and S-ab-PCM3-I were calculated to be 584 nm"1 and 834 nm'1 for molar mass per unit contour length (Mi), 3.7 nm and 6.8 nm for the persistence (q), 11.6 and 18.1 for the characteristic ratio (CK), respectively. The results indicated that the glucan ab-PCM3-I existed as an extended random coil chain in 0.25 M LiCl/DMSO, and S-ab-PCM3-I had good water-solubility and behaved as semi-stiff chain in 0.9% NaCl aqueous solution, which attributed to the steric hindrance and electrostatic repulsion of the sulfate groups.The water-soluble heteropolysaccharide ac-PCMO was fractionated into 11 fractions with narrow-distribution by preparative SEC column with double distilled water as eluent. It indicated that the preparative SEC column has good fractionation efficiency. The mannose content of the fractions increased, and the Mw values decreased with the process of the fractionation. The antitumor activities against Sarcoma 180 solid tumor both in vivo and in vitro, and cervical carcinoma Hela cells in vitro were tested using the fractions ac-PCM0-I^ac-PCM0-IV with the molecular mass of 5.45 x 104, 4.41 x 104, 2.95 x 104 and 0.93 x 104, respectively. The results from the antitumor assay suggested that the relatively higher Mw (5.45 x 104) seemed to increase the in vivo antitumor activities of the polysaccharides, and on the contrary the relatively lower Mw and higher mannose content were beneficial to the in vitro antitumor activities. The heteropolysaccharides ac-PCMO exhibited no antitumor activities in vitro against Hela cell strain, which indicated that ac-PCMO had the selective antitumor activities in vitro. The mice body-weightenhancement ratios of polysaccharides were much higher than that of 5-Fu, implying that the polysaccharides are not so toxic as 5-Fu, and the lethal dose 50% (LD50) of ac-PCMO was above 1250 mg/kg. The results mentioned above elucidated that ac-PCMO almost has no any cytotoxic effect to normal ells. This study suggested that the heteropolysaccharide had a potential application as natural antitumor drugs.The results of this foundation research mentioned above revealed effects of chain conformation in solution and the chemical structure of the polysaccharides from Poria cocos mecylia with cultivated strain on antitumor activity. It not only had academic significance and prospect of application, but also provided important scientific data for the exploration and application of Poria cocos.