Bioactivities And Structure Elucidation Of Exopolysaccharides From Fermented Broth Of Paecilomyces Hepiali

In the present dissertation, a series of experiments were performed to study thebioactivities and structures of polysaccharide fractions from fermented broth ofPaecilomyces hepiali Chen et Dai. The crude polysaccharide fractions were precipitated bydifferent concentrations of ethanol from fermented broth continuously. The effects of crudepolysaccharide fractions on the lifespan of fruit flies and the activities of SOD and thelevels of MDA in fruit flies were studied and screened the polysaccharide fraction withbest bioactivities. And then the immunological modulation and effect on adjuvant arthritisrats and hemorrhage anaemia mice of the screened crude polysaccharides wereinvestigated, respectively. What’s more, the polysaccharide fraction was purified by DE-52cellulose anion exchange chromatography and Saphedax G-150gel chromatography. Theantitumor effects of purified polysaccharide fractions were also studied and their structureswere elucidated by the motheds of GC-MS, FTIR, NMR and so on.The fermented broth of P. hepiali was centrifuged at a speed of16000rpm for10minutes to remove the mycelia of P. hepiali and residues of media. The gained supernatantwas concentrated to one fourth of its original volume at45oC in vacuum conditions. Andthen ethanol was added to the concentrated supernatant and made the final concentration ofethanol to20%. The mixed solution was stored at4oC for12hours and was centrifuged ata speed of5000rpm for10minutes to get the precipitates and designate as crudepolysaccharides P20. And the supernatant was added more ethanol to30%and stored at4oC for12hours and centrifuged at a speed of5000rpm for10minutes to get theprecipitates and designate as crude polysaccharides P30. The above steps were repeatedseveral times to get crude polysaccharides P40, P50, P60and P70, respectively. And theiryield rates were0.13、0.08、0.06、0.78、0.45、0.18mg/mL.The crude polysaccharide fractions were added to the feed of fruit flies with a dose of1%(w/v) and the group without crude polysaccharides was used as control group. Theresults indicated that the crude polysaccharide P60could elongate the lethal date of halfindividual of female and male fruit flies by6.47%and15.83%, average lifespan17.04%and18.36%, highest average life28.81%and17.87%in comparison with the control group.Crude polysaccharide P50and P60both could not only enhance the activities of SOD, but also decrease the levels of MDA in fruit flies. And the activity of P60is better than that ofP50. The crude polysaccharide P60enhanced activities of SOD28.02%and26.85%anddecreases the levels MDA50.67%and54.30%of female and male fruit flies, respectively.All these results implied that crude polysaccharide P60had the effect of antioxidant anddelay aging and was chosen as the subject for further study.The results of immunological modulation experiment of the crude polysaccharideindicated that P60could increase the indexes of thymus and spleen significantly andpromote the immune function of mice at the dosage of300mg/kg. What’s more, P60couldpromote the immune function of immunosuppressed mice induced by cyclophosphamideand inhibit delayed type hypersensitivity induced by DNFB at the dosages of300mg/kgand450mg/kg. At the dosage of300mg/kg, P60could enhance macrophage function ofmice.The result of adjuvant arthritis model of rats indicated that crude polysaccharide P60could inhibit effectively the secondary edema in the left hind paw induced by Freund’scomplete adjuvant. Besides, it could promote the level of TGF-β1and decrease the level ofTNF-α in serum of model rats significantly. But it didn’t show significant impact on thelevel of IL-1β in serum of rats. It suggested that the crude polysaccharide fraction P60havesome therapeutic effect on adjuvant arthritis.The results of hemorrhage anaemia mice model indicated that haemoglobin anderythrocytes of hemorrhage anaemia mice recovered to normal level after the treatment ofcrude polysaccharide for28days. Besides, it helps mononuclear cells and neutrophilicgranulocytes of hemorrhage anaemia mice to recover to normal levels. But it didn’t showsignificant effect on platelet and total number of white blood cell in mice with hemorrhageanaemia.Firstly, protein of crude polysaccharide P60was removed by Sevag reagent. Then thepartly purified polysaccharide was purified by DE-52cellulose anion exchangechromatography. Four polysaccharide fractions were achieved and designated as PS1, PS2,PS3and PS4. These four polysaccharide fractions were further purified by SephadexG-150gel chromatography and PS1and PS3were still single peaks in the elution profiles.Two polysaccharide fractions were gained from PS2and PS4, respectively, and designatedas PS2-1, PS2-2, PS4-1and PS4-2. Furthermore, the purity of the six purifiedpolysaccharide fractions was tested by Saphedax G-150gel chromatography and UV scanprofile from200nm to400nm.The antitumor activities of crude polysaccharide P60and six purified polysaccharide fractions against A549, B-16, CNE and HepG2were determined. And the results indicatedthat, after treatment for24hours, inhibition rates of P60against the proliferation of A549reached to37.37%at a dose of0.05μg/mL. PS4-1inhibited the proliferation of B-16reached to32.54%at a dose of5μg/mL. The proliferation of CNE and HepG2wereinhibited28.93%and29.51%by PS3and PS4-1at doses of500μg/mL and50μg/mL,respectively. After treatment for48hours, the inhibition rate of P60against theproliferation of A549reached to42.00%at a dose of0.05μg/mL, PS3against B-16reached to47.15%at a dose of5μg/mL, PS3against CNE reached to37.06%at a dose of500μg/mL, PS4-1against HepG2reached to42.51%at a dose of500μg/mL. Theinhibition rate of P60against the proliferation of A549reached to49.64%at a dose of0.05μg/mL after treatment for72hours. The inhibition rate of polysaccharide fraction PS3against the proliferation of B-16and HepG2reached to52.81%and56.17%at doses of5μg/mL and50μg/mL, respectively. PS4-1inhibited the proliferation of CNE reached to59.84%at a dose of500μg/mL.Through the analysis of FTIR, GC-MS,1H NMR,13C NMR,1H-1H COSY, HSQC andHMBC of the purified polysaccharide fractions, we could deduce the possible structures ofthe six polysaccharide fractions as following:The polysaccharide fraction PS1, with a molecular weight361kDa, is composed ofglucose, galactose and mannose in the mole ratio of5.15:0.30:0.10. Its possible structure isthat-1,6-D-Glcp residues link together by-1,6-glucosic bond and form the backboneand-1,3-D-Galp and β-1,3-D-Manp residues are linked to the main chain at C3of glucoseresidue with-1,3-glucosic bond and β-1,3-glucosic bond, respectively. Its main chainstructure is as following:→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→PS2-1, with a molecular weight of412kDa, is composed of galactose, arabinose,glucose, xylose, and mannose in the mole ratio of3.98:1.00:0.28:0.33:0.18. NMR analysisshowed that the structure mainly consist of a backbone of (1,4)-β-D-Galp residues andsubstituted at C3of Galp by α-D-Arap residue. Small amount of xylose, mannose andglucose was linked to the backbone. Its possible repeat unit is as following structure:PS2-2, with a molecular weight48kDa, is composed of mannose, glucose and residues link together by-1,6-glucosidic bond, forming the backbone, and-1,3-D-Galpand β-1,3-D-Manp was linked to the backbone at C3of glucose residue with-1,3-glucosidic bond and β-1,3-glucosidic bond, respectively. Its main chain structure isas following:→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→PS3, with a molecular weight of536kDa, is composed of arabinose, xylose, glucoseand galactose in the mole ratio of0.62:0.34:0.13:4.62. NMR analysis showed that thestructure mainly consist of a backbone of (1,4)-β-D-Galp residues and substituted at C3ofGalp by α-D-Arap residue. Part of arabinose and xylose were linked to of glucose residuesby α-1,3-glucosidic bond. Other arabinose and xylose was linked to galactose residues ofbackbone by β-1,3-glucosidic bond. Its backbone structure is as following:→4)-β-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Galp-(1→PS4-1, with a molecular weight of729kDa, is composed of arabinose, glucose andgalactose in the mole ratio of1.13:0.21:4.40. NMR analysis indicates that the backbone ofPS4-1is D-Galp linked to each other by β-(1→4)-glucosidic bond and D-Arap and D-Glcpresidues were linked to backbone by β-(1→3)-glucosidic bond at C3of D-Galp residues.PS4-1consists of pentasaccharides repeating units with the following structure:PS4-2, with a molecular weight of668kDa, is consisted of rabinose, glucose andgalactose in the mole ratio of0.62:0.57:4.46. NMR analysis indicated that the structureconsist mainly of backbone of β-(1→4)-D-Galp residues and substituted at C3by β-D-Arapresidue or β-D-Glcp. Its possible structure is as following:...