Isolation, Purification, Structural Elucidation And Biological Activities Of Mycelial Polysaccharides From Phellinus Baumii Pilat

Phellinus baumii Pilat, a basidiomycete fungus belonging to the genus Phellinus in the family of Hymenochaetaceae, is a precious and highly acclaimed medicinal fungus in traditional Chinese medicine. Its fruiting body is called "Sanghuang" in Chinese, Recently, it has been reported that the extracts from the fruiting body of P. baumii Pilat possess potential antioxidant, immunostimulating, anti-tumor anti-inflammatory, anti-mutation and anti-diabetic effect. At present, researches on P. baumii Pilat have been developed, and most of them were focused on the crude polysaccharides from P. baumii Pilat. However, there is still lack of knowledge concerning the isolation, purification, structural elucidation, and bioactivity of polysaccharides from P. baumii Pilat mycelia.As we know, the cultivation of the fruiting body of P. baumii Pilat requires 5-6 months and its product quality is difficult to control when P. baumii Pilat is traditionally cultivated in solid culture. In contrast, submerged culture has potential advantages for higher mycelial and polysaccharide production in a more-compact space over a shorter incubation time with less chances of contamination, and availability of convenient control. Therefore, submerged culture has become a promising alternative for efficient production of its valuable metabolites, especially polysaccharide. In order to investigate systemically the chemical structure and bioactivity of polysaccharide from P. baumii Pilat, the isolation, purification, structural elucidation, and bioactivity of polysaccharides from P. baumii Pilat mycelia were carried.In this dissertation, there are four parts including optimization of culture conditions, isolation and purification, physicochemical characterization, structure, free radical scavenging activities, effect on oxidative stress in D-Galactose-induced aging mice, immunomodulation and anticancer activity of polysaccharides from P. baumii Pilat mycelia. The main results are shown as follows: 1. Optimization of culture conditions for PBMP production in submerged cultureThe significant influence of glucose, yeast extract, peptone and MgSO4 on PBMP production was evaluated by a 2(7-3) fractional factorial design. Then the optimum levels of these significant components were determined by employing a central composite design in response surface methodology, resulting in an optimal medium as following (g/1):glucose 35.36, yeast extract 2.88, peptone 2.73, MgSO41.47, KH2PO41, VB1 0.0075 and diammonium oxalate monohydrate 0.3.Under the optimal medium composition, the optimum levels of temperature and pH were determined by using a central composite design as following:pH 6.0, temperature 28℃. To investigate the effects of all optimal cultivation conditions (e.g. optimal culture medium, initial pH and temperature) on simultaneous production of mycelia and polysaccharide by P. baumii Pilat, the fermentation time courses, including residual sugar concentration, pH, mycelial dry weight and mycelial polysaccharide, were recorded and analyzed at 1-day interval during the whole fermentation process. The results showed the maximum PBMP production indicated 0.936 g/1 at 144 h of the cultivation while the mycelial growth reached 13.69 g/1. Therefore, the present work has also proved that statistical optimization techniques could be used as a valuable and dependable tool for the optimization of PBMP production from P. baumii Pilat in general.2. Isolation, purification, physicochemical characterization and structural elucidationThe dry mycelia were extracted with distilled water, precipitated by using 75% ethanol, washed with acetone and ether respectively, and dried to afford crude PBMP. The extraction yield of PBMP was about 6.72%.The crude PBMP was separated firstly through DEAE-cellulose 52 column, and preliminary isolated fractions (F1, F2 and F3) were collected. F1, F2 and F3 were further purified through Sephadex G-100 column respectively, and four purified fractions, named as PBMP-1, PBMP-2a, PBMP-2b and PBMP-3, were obtained respectively. The recovery rates of PBMP-1, PBMP-2a, PBMP-2b and PBMP-3 based on the amount of crude PBMP used were 32.57%,4.13%,5.08% and 0.79%, respectively.The contents of carbohydrate, protein, and uronic acid in PBMP-1, PBMP-2a, PBMP-2b and PBMP-3 were determined according to the reported methods of sulfuric acid-phenol coloration, coomassie brilliant blue coloration, and the m-hydroxybiphenyl colourimetric procedure, respectively. The contents of carbohydrate were 99.49%,77.02%, 92.03% and 71.19%, respectively. Protein contents were not detected,4.99%,1.54% and 4.45%, respectively. Uronic acid contents were 2.01%,3.74%,4.29% and 5.27%, respectively.Purity of PBMP-1, PBMP-2a, PBMP-2b and PBMP-3 was further confirmed by using HPGPC, and results showed that PBMP-1, PBMP-2a, PBMP-2b and PBMP-3 were homogeneous polysaccharides, respectively. The relative molecular weight of PBMP-1, PBMP-2a, PBMP-2b and PBMP-3, determined by HPGPC, was 2.95×103kDa,1.17×103 kDa,16.29 kDa and 1.54×103kDa, respectively.Monosaccharide composition analysis by GC was carried out according to the methods of aldonitrile acetate derivatives. PBMP-1 was composed of fucose, glucose and galactose with a molar ratio of 1.00:24.54:0.29. PBMP-2a was composed of fucose, mannose, glucose and galactose with a molar ratio of 0.56:0.76:3.23:1.00. PBMP-2b was composed of fucose, mannose, glucose and galactose in the molar rate of 0.23:1.29:1.00: 1.01. PBMP-3 was composed of rhamnose, fucose, mannose, glucose and galactose in the molar rate of 0.52:0.58:2.51:1.44:1.00.The infrared spectra of PBMP were obtained by grinding a mixture of sample with dry potassium bromide powder and pressing in a mold. Characteristic absorptions of polysaccharides, carboxyl group and pyranose ring were observed in PBMP-1, PBMP-2a and PBMP-2b. PBMP-1 showed feature of a-glycosidic bond, PBMP-2a and PBMP-2b exhibited character of both a-andβ-glycosidic bond. UV spectrum showed that there was no protein absorption in PBMP-1, but weaker protein absorption in PBMP-2a and PBMP-2b.The major structural features of PBMP-1, PBMP-2a and PBMP-2b were elucidated by employing periodate oxidation, Smith degradation, methylation analysis, GC/MS,1H and 13C NMR. According to results obtained above, it was concluded that PBMP-1 contained a backbone of (1,4)-linkedα-D-glucosyl and (1,6)-galactosyl residues with small amounts of the nonreducing end L-fucose, also contained a minor (1,4,6)-and (1,3,4)-D-glucose residue.PBMP-2a contained a backbone of (1,4)-linkedα-D-glucosyl and (1,6)-galactosyl residues with small amounts of the nonreducing end L-fucose, D-mannose and D-glucose residue, also contained a minor (1,2,6)-β-D-mannose residue, (1,2)-β-mannose residue, (1,6)-, (1,4,6)-and (1,3,4)-D-glucose residue.PBMP-2b has a backbone of (1,3,4)-linked a-D-glucopyranosyl and (1,2,6)- mannose residues, with Fuc (1â†'6)-Gal-(1â†'), Mannose (1â†'6)-Gal-(1â†') or Glc (1â†'6)-Gal-(1â†') side chain substituted at O-3 of a-D-glucopyranosyl and branch substituted at O-6 ofβ-D-mannose. 3. Free radical scavenging propertis of polysaccharides from Phellinus baumii Pilatmycelia and its effect on oxidative stress in D-Galactose-induced aging miceScavenging free radical activities in vitro of crude PBMP and PBMP-1 were measured by using the chemical methods. Results showed that crude PBMP and PBMP-1 could scavenge free radicals of 02,OH·and RCOO·in dose-dependent manner.The effect in vivo of crude PBMP and PBMP-1 on oxidative stress in D-Galactose-induced aging mice was carried out. The results showed that the administration of PBMP could attenuate oxidative stress of D-Galactose-induced aging mice, increase the activities of antioxidant enzymes (SOD, CAT and GSH-Px), decrease the levels of malondialodehyde (MDA), and enhance total antioxidant capabilities (TOAC) in livers and serums of D-galactose-induced mice. In addition, crude PBMP and PBMP-1 could increase thymus and spleen index in D-Galactose-induced aging mice.4. Immunomodulatory and anticancer in vitro effect of polysaccharides isolated from Phellinus baumii Pilat myceliaThe assay of splenocyte proliferation in vitro of PBMP-1, PBMP-2a, PBMP-2b and PBMP-3 was manipulated as described by Mosmann using MTT-based colorimetric method. Results showed that PBMP-1, PBMP-2a and PBMP-2b could enhance both the LPS-induced B cells and Con A-induced T cells proliferation. On the contrary, PBMP-3 could inhibit both the LPS-induced B cells and Con A-induced T cells proliferation.The ability of PBMP-1, PBMP-2a, PBMP-2b and PBMP-3 from P. baumii Pilat to stimulate acid phosphatase activity and devour neutral red in peritoneal macrophages was investigated. Results showed that PBMP-1, PBMP-2a, PBMP-2b and PBMP-3 could enhance activity of acid phosphatase in peritoneal macrophages and increase ability of devouring neutral red of peritoneal macrophages in vitro in dose-dependent manner. Therefore, it was evident that the mycelial polysaccharide from P. baumii Pilat strongly induced the activation of macrophage and possessed higher immunomodulatory potential.MTT assay was used to investigate the anti-proliferation activities in vitro of PBMP-1, PBMP-2a, PBMP-2b and PBMP-3. The results showed that PBMP-1, PBMP-2a, PBMP-2b and PBMP-3 significantly inhibited proliferation of human gastric carcinoma cell BGC-823 in dose-dependent and time-dependent manner after 24,48,72 h treatment, respectively. The percentages of inhibition were 81.98%,90.68%,90.27% and 90.05%(50μg/ml), respectively, after 72 hours treatment. The inhibitory effect of PBMP-2a, PBMP-2b and PBMP-3 have been reached or closed to that of cisplatin (5μg/ml). PBMP-1, PBMP-2a, PBMP-2b and PBMP-3 could exhibit stronger anticancer in vitro activity in some extent.