Extraction, Separation,structure Characterization And Bioactivities Of Polysaccharides From Ganoderma Lucidum

In this dissertation, a series of study of water-soluble polysaccharides by ultrasonic/microwave assisted (UCP) and alkali-soluble polysaccharides(ACP) by alkali were conducted on Ganoderma lucidum. In order to further study the sturecure of UCP, it was isolated , purified and determined with a series of instruments analysis technique. UCP, ACP and their derivates'immune activities were investigated in vitro and in vivo. The following results were achieved.1. The content of total sugar, reducing sugar,crude fiber, protein, fat ,ash and moisture from Ganoderma lucidum of different producing places were studied , results indicated that non-reducing suger content in Longquan,Zhejiang is the highest, reach 20.4%, which indicated the content of polysaccharides from Ganoderma lucidum of Zhejiang is the highest. Besides, in order to investigate the accumulating mechanism of active componets in Ganoderma lucidum, the levels of triterpenoids and protein content were analyzed, results indicated that with the growth of Ganoderma lucidum, the content of triterpenic acid and protein fell from 1.39% to 0.845% and 20.82% to 10.30, respectively, and polysaccharides raised from 0.25% to 0.61% then fall to 0.53%.The content of Ganoderma lucidum polysaccharides raised to the highest of 0.61% when it reached mature stage( about 50 to 80 days curtivated under land) ,then gradually reduced after it entered period of decline(after 80days curtivated under land).2.Water-soluble polysaccharide(sUCP)from Ganoderma lucidum of longquan,Zhejing were extracted by ultrasonic/microwave,the results indicated the optimal extraction conditions were microwave power of 284 W, ultrasonic power of 50 W, extraction time of 12 min and water/solid ratio of 11.5:1, respectively. Using a short application of ultrasound, the yield of water-soluble polysaccharide was 3.35%, which is 115.56% above that of classical hot water extraction. From the residue of Ganoderma lucidum by ultrasonic/microwave extraction, NaOH solution were used to extract alkali-soluble polysaccharides(ACP),the results indicated that optimum conditions were: extraction temperature of 60℃, extraction time of 80 minutes, sodium hydroxide (NaOH) concentration of 5.0% and material/ liquid ratio of 1:20, the yield of alkali-solublepolysaccharide was 8.04%.3. A water-soluble Ganoderma lucidum polysaccharide extracted by ultrasound (UCP) was isolated from the fruiting bodies of Ganoderma lucidum by polyamide, DEAE Sepharose Fast Flow and Sephacryl S-500 High Resolution Chromatography, five fragments were obtained from UCP-F1-1 to UCP-F1-5. The homogeneity of UCP-F1-1and UCP-F1-2 were evaluated by HPLC and capillary electrophoresis(CE).The results indicated that both UCP-F1-1and UCP-F1-2 were homogeneous components, their peak molecular weight (Mp) were approximately 2.52×106 Da and 2.34×106Da. The peak molecular weight of UCP-F1-3 and UCP-F1-4 were 2.27×106 Da and 2.19×106Da.4. 1H and 13C NMR spectroscopy in combination with GC–MS technique indicated that UCP-F1-1 had a backbone chain of 1, 4-disubstituted-β-glucoseopyranose and 1, 4, 6-trisubstituted-β-glucoseopyranosyl, every ten 1, 4-disubstituted-β-glucoseopyranose has a branch on the sixth site of the backbone chain, while the branched chains were mainly composed of 1, 6-disubstituted-β-glucopyranosyl and 1, 4-disubstituted-β- galactoseopyranosyl residues and their mol ratio is 6:1. UCP-F1-2 had a backbone chain of 1, 4-disubstituted-β-glucoseopyranose, every six 1, 4-disubstituted-β-glucoseopyranose has a branch on the sixth site of the backbone chain, while the branched chains were mainly composed of 1, 6-disubstituted-β-glucopyranosyl.5. The immunological assays results in mice demonstrated that there were significant differences between serum hemolysin of UCP and ACP in high,medium,low dosages treatment;murine delayed type hypersensitivity ability of ACP in high,medium,low dosages and UCP in high dosage ; murine monocyte-macrophage carbon clearance capacity of ACP in high dosage; NK cell activity of UCP in high dosage and ACP in high,medium dosages; thymus gland index of UCP in low dosage treatment were significantly higher than that of the contrasted groups (ρ<0.05).This result indicated the two Ganoderma lucidum polysaccharides may enhance immune function in mice , and the effects of ACP is better than that of UCP.It also indicated that the higher doses of Ganoderma lucidum polysaccharide not means the better effect of immune enhancement of mice, it should be a suitable dose. There were no significant differences among the prolfiration effect of lymphotocyte of UCP and its components (UCP-F1-1 to UCP-F1-5)with three dosages.6.In order to improve immunce enhancement activity of UCP and ACP, carboxymethylation and salicylic acid modification were used to obtain four polysaccharide derivates: carboxymethylated water-soluble Ganoderma lucidum polysaccharide(CUCP), water-soluble Ganoderma lucidum polysaccharide salicylic acid salicylate (AUCP), carboxymethylated alkai-soluble Ganoderma lucidum polysaccharide (CACP), alkali-soluble Ganoderma lucidum polysaccharide salicylic acid salicylate (AACP). Proliferation of lymphtocyte induced by ConA in rat of four polysaccharide derivates were studied, the results indicated that proliferation of lymphtocyte induced by ConA in rat of CUCP and CACP in medium dosage,AACP in high,medium dosages were significantly higher than that of the contrasted groups,which indicated that these polysaccharide derivates have immune enhancement activity in rat. The immune enhancement activity of CUCP was higher that UCP,and it means both carboxymethylation and salicylic acid salicylate modification can improve immune activity of Ganoderma lucidum polysaccharides .