| Mushroom has been used as food and drugs for human in the world about several thousand years already, and many nutrients and compounds with medicinal use, including carbohydrates, protein, vitamins, thiamine, riboflavin, ascorbic acid, vitamin D, and minerals, etc, were found in the mushroom. Modern medical research shows that the mushroom polysaccharides possess the bioactivities of immunomodulatory, anti-tumor, antioxidant, cholesterol-lowering, hypoglycemic, anti-bacterial, anti-virus, anti-allergic and anti-inflammatory, hepatoprotectivity and intestinal prebiotics. Pholiota dinghuensis Bi is a new species in Pholiota family, and is only found in Guangdong Province of China. As a specific kind of mushroom, its bioactive polysaccharides should be paid more attention. However, little information is available compared with those of other Pholiota mushrooms. Therefore, studies about polysaccharide of from the mycelium of Pholiota dinghuensis Bi, on the optimization of fermentation conditions, separation and purification, physicochemical properties and in vivo hepatoprotective effects on CC14-induced acute liver injury, in vitro anticancer activities, prevention of cell proliferation and induction of apoptosis in human breast cancer MCF-7cells were studied. The main findings are as following:1. Optimization of submerged fermentation conditions for crude PDPFirstly, optical medium composition and fermentation conditions were screened by single factor experiments for the submerged fermentation of crude polysaccharide (PDP) from mycelia of Pholiota dingensis Bi. The primary conditions for crude PDP production were set as of SCS40g/L, SNS4g/L, inoculum volume15%(v/v), culture temperature25℃and shaking speed140rpm.Based on the results of single factor experiments, a fractional factorial design (FFD) was initially employed to screen the most significant factors based on our previous study, and the glucose, corn flour and yeast extract were selected as the most significant factors. And then, a path of steepest ascent experiment was carried out for the suitable concentrations for the selected factors, and the central points of the concentrations of glucose, corn flour and yeast extract were set35,10and5g/L, respectively.After that, a central composite design (CCD) of RSM was applied to study the optimal medium compositions for maximum PDP production. As results, the optimal conditions for crude PDP production were determined as (g/L):glucose36.0, corn flour11.8, peptone3.0, yeast extract5.4, KH2PO41.0and MgSO41.5. Using these optimal conditions, the crude PDP yield of757±38mg/L was obtained, which is in a good correlation with the prediction of this model. The statistical analysis of this model showed an effective prediction of the crude PDP.Mycelia of Pholiota dingensis Bi were dried, powdered, and extracted by hot water. The supernatant were precipitated by ethanol, and then centrifuged. The precipitate were collected and dried for crude PDP.2. Separation and purification, and physicochemical properties of PDPCrude PDP was sequentially purified by DEAE-52cellulose ion-exchange chromatography and Sephadex G-100size-exclusion chromatography, then three fractions (PDP-1, PDP-2and PDP-3) were obtained, with a recovery rate of60.25%.23.69%and4.61%, respectively. PDP-1, PDP-2and PDP-3were further confirmed as homogeneous polysaccharides using HPGPC, and its relative molecular weight was1.59x105,7.20x105and3.53×105Da, respectively.The contents of carbohydrate, uronic acid, protein and sulfate in polysaccharide samples were determined according to the reported methods of sulfuric acid-phenol coloration, the m-hydroxybiphenyl colourimetric procedure, coomassie brilliant blue coloration and barium chloride-gelatin, respectively. The contents of carbohydrate of crude PDP, PDP-1, PDP-2and PDP-3were86.95%,97.97%,94.46%and85.58%, respectively. In the purified fractions, PDP-3represents higher uronic acid, protein and sulfate content than PDP-1and PDP-3, indicating PDP-3was a complex of polysaccharide and protein or peptide.The monosaccharide compositions of crude PDP and its purified fractions (PDP-1, PDP-2and PDP-3) were carried out by GC according to the methods of aldonitrile acetate derivatives. Notably, glucose was found to be the most abundant monosaccharide, in a ratio of92.54%,92.24%,88.65%and67.83%, respectively, for crude PDP, PDP-1, PDP-2and PDP-3. For crude PDP, it was composed of arabinose, fucose, xylose, mannose, glucose and galactose in relative percent of2.18,1.06,1.48,0.92,92.54and1.82, respectively. However, the monosaccharide composition of PDP-3was different from that of PDP-1or PDP-2. Interestingly, xylose and rhamnose were found only to be present in PDP-3. In addition, the contents of fucose, mannose and galactose in PDP-3were relatively higher than those in PDP-1or PDP-2.The FT-IR spectra of crude PDP, PDP-1, PDP-2, and PDP-3were by the potassium bromide (KBr) pellet method. All the absorption peaks of tested samples indicated the characteristic absorptions of polysaccharides. Furthermore, specific absorbance of COO-and S=O indicated PDP-2and PDP-3were acidic polysaccharides and sulfated polysaccharides. What’s more, the relative stronger absorption peak of N-H bending vibration might be related to the higher content of protein in PDP-3. UV spectrum results indicated that there was some protein absorption in all samples, but higher protein absorption in PDP-3was found.3. In vivo hepatoprotective effects of crude PDP against carbon tetrachloride-induced acute liver injury in miceThe hepatoprotective effects of PDP against carbon tetrachloride (CC14)-induced acute liver damage in Kunming female mice were investigated. As a result, PDP-3significantly prevented the increase of serum ALT and AST activities, and enhanced the SOD and GSH-Px activities in CC14-induced mice. The formation and accumulation of MDA in the liver of CC14-induced mice was significantly prevented by the pretreatment of PDP-3. The histopathological results showed, pretreatment with crude PDP at200mg/kg BW per day restored from the CC14-induced liver jury, which depicted in the model control with marked and massive inflammation, infiltration and color changes of tissue, indicating a similar effect of pretreatment with silymarin in the positive control group. All the results showed that crude PDP represents a relative hepatoprotective effect against carbon tetrachloride (CC14)-induced acute liver damage in mice.4. In vitro antiproliferative activities and its mechanism of PDPs to the human cancer cells MTT assay for human gastric cancer BGC-823cells, and methylene blue staining for human liver cancer HepG2cells, were used to screen in vitro antiproliferative activities toward human tumor cells of crude PDP and its purified fractions (PDP-1, PDP-2and PDP-3). The results showed the PDP-3represented the highest antiproliferative activity than PDP-1and PDP-2. And then, human breast MCF-10A cells, human breast cancer MDA-MB-231and MCF-7cells were selected to screen the in vitro antiproliferative activity of PDP-3toward human breast cells. As a result, a little prevention of proliferation of MCF-10A cells was found when pretreated with PDP-3, while PDP-3showed significant antiproliferative activities toward human breast cancer MDA-MB-231and MCF-7cells. However, higher antiproliferative activity of PDP-3in estrogen-dependent human breast cancer MCF-7cell, when compared to the estrogen-independent human breast cancer MDA-MB-231cell. To further investigation showed PDP-3up-regulated the protein expression of p21and down-regulated the protein expression of PCNA and Cyclin D1and CDK4in human breast cancer MCF-7cells.5. In vitro apoptosis of human breast cancer MCF-7cells and its mechanism induced by PDP-3Western-blot analysis were used to determine the effect of protein expression of Bcl-2, Bax, Caspase-9, Caspase-3, p-p53, p-p38, ASK1, TRAF2, nuclear and plasmic ER-α in the human breast cancer MCF-7cells pretreated by PDP-3. The results showed the PDP-3activated p38/MAPK pathway in human breast cancer MCF-7cells. For the cell proliferation, PDP-3increased the p21protein expression and reduced human breast cancer MCF-7cells and cell proliferation (PCNA) and cell cycle-related protein (Cyclin D1and CDK4) expression; For the cell apoptosis, PDP-3increased Bax and lowered Bcl-2expression, and also activated the expression of Caspase family proteins (caspase-9and Caspase-3). Results of upstream protein expression showed PDP-3increased p-p53, p-p38and ASK1protein expression, and reduced the TRAF2protein expression. What’s more, PDP-3down-regulated the protein expression of nuclear and plasmic ER-α in human breast cancer MCF-7cells. The results of TUNEL assay and DNA fragmentation confirmed the apoptosis of human breast cancer MCF-7cells pretreated by the PDP-3. The signaling pathway of PDP-3induced antiproliferation and apoptosis in human breast cancer MCF-7cells was summed up, based on all of the relative results in the fifth and sixth chapter of this thesis. |
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