Isolation, Purification, Structure Elucidation And Biological Activities Of Polysaccharides From The Fruiting Bodies Of Pholiota Adiposa

Pholiota adiposa is a kind of edible and medicinal fungi. When cooked, it has tender flesh with a delicate flavor and rich nutrition. Polysaccharides extracted from Pholiota adiposa were reported to have immune-stimulatory and antitumor properties, as well as hypolipidemic effect, anti-oxidant and anti-aging activities. At present, researches on Pholiota adiposa were focused on extract technique for polysaccharides and biological activities of crude polysaccharides, however, up to now, no detailed investigation has been conducted on composition characterization, structure and activities of different purified polysaccharides isolated from the fruiting bodies of Pholiota adiposa. Therefore, the aim of present study is to characterize the compositions of polysaccharides extracted from the fruiting bodies of Pholiota adiposa and evaluate the antioxidant activities of these polysaccharides in vitro. It will be of scientific significance in researching and developing rare medicial fungi and be useful for searching new natural antioxidant. The main results are shown as following:1. Development of extract technique for polysaccharides from the fruiting bodies of Pholiota adiposaThrough single-factor experiments and orthogonal experiments, it was found that the best yield could be obtained by using water:raw material ratio 20:1 at 100℃for incubation for 4h, one time of extraction times. The yield of water-soluble polysaccharides was 8.19% in the technology conditions.2. Isolation and purification of polysaccharides from the fruiting bodies of Pholiota adiposaThe polysaccharide fractions from water extract of fruiting bodies of Pholiota adiposa were isolated and purified using DEAE Sepharose Fast Flow ion-exchange column chromatography and superdex^TM 200 column chromatography repeatedly. Three water-soluble homogeneous polysaccharides (PAP1-1, PAP1-2 and PAP2-1) were obtained. Lack of absorption at 280nm and 260nm by UV scanning indicated that they contained no protein and nucleic acid. HPSEC producing a single symmetrical peak, indicated each of them was homogeneous and their purity were 97.23%, 98.85% and 98.91%, respectively.3. Establishment of method for the analysis of monosaccharide compositions in polysaccharides(1) Acetylation derivatization and capillary gas chromatographyThe operation was performed using the following conditions: DB-23 capillary column (30m×0.25mm×0.25μm), carrier gas (high-purity nitrogen): 1.0mL/min, injection temperature: 250°C, detector temperature: 250°C, column temperature: 220°C, injection volume: 0.5μl, split ratio: 20:1. This method has realized the separation of 8 kinds of monosaccharides in 20 minutes.(2) HPLC-ELSD methodThe HPLC separation was performed on a Prevail Carbohydrate ES Column (4.6mm×250mm, 5μm) using acetonitrile:water (85:15) as mobile phase at a flow rate of 0.8mL/min. The drift tube temperature of ELSD was set at 80℃with the nitrogen pressure of 3.5bar. This method has realized the separation of glucuronic acid and 6 kinds of monosaccharides in 35 minutes.4. Studied on structure of the polysaccharides (PAP1-1, PAP1-2 and PAP2-1) from the fruiting bodies of Pholiota adiposaThe primary structures of the three homogeneous polysaccharides were analyzed using IR, HPSEC, GC, HPLC-ELSD and NMR (1H NMR and 13C NMR).(1) PAP1-1, with an average molecular weight of 2.3×10⁶Da, containedα-D-Glucose at a pyranose form and no uronic acid. NMR analysis further showed that the structure consist mainly of a backbone of (l, 4) and (l, 6)-linked glycosidic bond.(2) PAP1-2, with an average molecular weight of 8.8×10³Da, contained D-Glucose and L-rhamnose in the ratio 3.55:1 at a pyranose form. PAP1-1 contained no uronic acid and bothα- andβ-configuration glycosidic bond. NMR analysis further showed that the structure consist mainly of a backbone of (l, 6)-linked glycosidic bond.(3) PAP2-1, with an average molecular weight of 2.1×10⁶Da, containedα-D-Glucose at a pyranose form and no uronic acid. NMR analysis further showed that the structure consist mainly of a backbone of (l, 4) and (l, 6)-linked glycosidic bond. 5. Studied on biological activities of the polysaccharides from the fruiting bodies of Pholiota adiposa(1) To investigate the effects of polysaccharides of Pholiota adiposa (PAP) on the function of mouse peritoneal macrophages (PEMΦ), the results showed that PAP could promote the phagocytosis capability, enhance the production of NO and IL-1βand the activity of PEMΦkilling L1210 tumor cells. The peritoneal macrophages could be activated by PAP in mice.(2) The growth of Sl80 tumor in mice was inhibited when mice was fed by PAP. The inhibition rate was positively correlated to the amount of the PAP fed to mice. When the amount was 1.5mg/kg·d, the inhibition rate was 38.46%.(3) The ability of hypoxia tolerance of PAP was researched by animal experiment, different dosage groups could prolong survival time of mice; When the dosage was 1.5mg/kg·d, survival time of mice is longer than that of comparison group by 38.9%.(4) In vitro activities of anti-oxidation of refined PAP, PAP1-1, PAP1-2 and PAP2-1 were determined according to their scavenging capacities on the free-radicals of DPPH, superoxide anion and hydroxyl, using Vc as positive control. The results showed that the purified polysaccharide fractions exhibited less scavenging activity for DPPH radical than the crude polysaccharides (PAP). Antioxidant test in vitro also showed that PAP1-1 possessed stronger scavenging activity of superoxide radical and hydroxyl radical, especially scavenging activity of hydroxyl radical may be comparable to ascorbic acid, and should be explored as a novel potential antioxidant.