【作者】 于士军；

【导师】 樊美珍；

【作者基本信息】 安徽农业大学 ， 微生物学， 2012， 博士

【摘要】 本研究针对蝙蝠蛾拟青霉（Paecilomyces hepiali Chen et Dai）发酵液多糖的活性和活性较高多糖组分的结构开展了一系列的实验研究，主要内容包括蝙蝠蛾拟青霉发酵液多糖的分级醇沉、不同分级醇沉粗多糖组分对果蝇寿命和果蝇体内SOD活力及MDA含量的影响，并从中选择活性最高的粗多糖进行进一步的研究。对活性最高的多糖组分进行了免疫调节活性、对佐剂性关节炎大鼠和失血性贫血小鼠的作用等方面的研究；然后用DE-52纤维素离子交换层析柱和Saphedax G-150交联葡聚糖凝胶层析柱对该粗多糖组分进行了分离纯化；之后，对分离纯化的多糖组分的抗肿瘤活性及结构分别进行了研究和分析。将蝙蝠蛾拟青霉发酵得到的发酵醪经16000rpm离心10min，除去菌丝；发酵液在45℃条件下真空旋转浓缩至原体积的1/4，分别依次加入乙醇使乙醇的终浓度为20%、30%、40%、50%、60%和70%分级沉淀得到粗多糖。将乙醇终浓度为20%、30%、40%、50%、60%和70%时，醇沉得到的蝙蝠蛾拟青霉胞外粗多糖组分分别标记为P20、P30、P40、P50、P60、P70，其得率分别为：0.13、0.08、0.06、0.78、0.45和0.18mg/mL。将不同乙醇浓度沉淀得到的粗多糖按1%（W/V）剂量分别添加到果蝇的饲料中，以不添加多糖组作为对照组（CK），进行饲养。实验结果表明粗多糖P60延缓衰老作用最好，对雌雄果蝇的半数死亡时间、平均寿命和平均最高寿命的延长率分别是6.47%、15.83%，17.04%、18.36%，28.81%和17.87%。P50和P60，可显著提高果蝇体内的SOD活性，并降低MDA含量，且P60作用效果较好。P60对雌雄果蝇体内SOD活力分别提高28.02%和26.85%，MDA含量分别降低50.67%和54.30%。表明蝙蝠蛾拟青霉胞外粗多糖P60具有良好的抗氧化和延缓衰老作用，将其选择作为进一步研究的对象。粗多糖P60的免疫调节实验结果表明：300mg/kg的蝙蝠蛾拟青霉胞外粗多糖P60能明显促进正常小鼠胸腺指数和脾脏指数的增长，增强小鼠的免疫功能。300mg/kg和450mg/kg均能提高免疫抑制的小鼠的免疫功能，也能抑制小鼠迟发型超敏反应；300mg/kg蝙蝠蛾拟青霉粗多糖P60能促进小鼠巨噬细胞增生，激活吞噬活性，增强小鼠吞噬异物能力。佐剂性关节炎大鼠模型实验结果表明：蝙蝠蛾拟青霉胞外粗多糖P60能抑制佐剂性关节炎大鼠左足趾继发性肿胀的程度，并对佐剂性关节炎大鼠血清内TGF-β1、TNF-α产生有一定的作用：促进佐剂性关节炎大鼠血清内TGF-β1的浓度升高，对佐剂性关节炎大鼠血清中促炎性细胞因子TNF-α的产生有一定的抑制作用；而对于IL-1β的产生无明显的影响作用。表明蝙蝠蛾拟青霉胞外粗多糖P60对佐剂性关节炎有一定的治疗作用。失血性贫血小鼠实验结果显示：用蝙蝠蛾拟青霉胞外粗多糖P60对失血性贫血小鼠进行灌胃28天后，低剂量组和高剂量组失血性贫血小鼠的外周血血红蛋白含量恢复正常，同时，外周血红细胞数也恢复至正常小鼠水平；并且有利于单核细胞和中性粒细胞恢复至正常小鼠水平；但是对小鼠血小板数和白细胞总数无明显的影响。粗多糖P60经Saveg试剂除蛋白，然后用DE-52纤维素离子交换层析柱进行初步纯化得到4个组分：PS1、PS2、PS3和PS4；将4个组分分别再经Sephadex G-150交联葡聚糖凝胶层析柱进行进一步的分离纯化，PS1和PS3仍是单一峰，PS2和PS4分别分离出2个组分：PS2-1、PS2-2和PS4-1、PS4-2。6个样品经Sephadex G-150交联葡聚糖凝胶层析检测和紫外200-400nm扫描初步判定均为均一性多糖。将粗多糖P60和分离纯化得到的纯化多糖组分分别进行对肺癌细胞A549、黑色素瘤细胞B-16、鼻咽癌细胞CNE和肝癌细胞HepG2增殖的抑制实验。实验结果表明：处理24h后，0.05μg/mL的粗多糖P60对A549细胞增殖的抑制作用较好，抑制率达到37.37%；浓度为5μg/mL多糖PS4-1对B-16细胞增殖的抑制作用达到32.54%；浓度为500μg/mL多糖PS3对CNE细胞增殖抑制作用较好，抑制率达到28.93%；浓度为50μg/mL多糖PS4-1对HepG2细胞增殖的抑制作用最好达到29.51%。处理48h后，0.05μg/mL粗多糖P60对A549细胞增殖的抑制作用较好，抑制率达到42.00%；浓度为5μg/mL多糖PS3对B-16细胞增殖的抑制作用最佳，抑制率达到47.15%；浓度为500μg/mL多糖PS3对CNE细胞增殖的抑制作用较好达到37.06%；浓度为500μg/mL多糖PS4-1对HepG2细胞增殖的抑制作用最高，抑制率达到42.51%。处理72h后，浓度为0.05μg/mL的粗多糖P60对A549抑制作用最佳，抑制率达到49.64%；浓度为5μg/mL多糖PS3对B-16细胞增殖的抑制作用达到52.81%；浓度为500μg/mL多糖PS4-1对CNE的抑制作用较好，抑制率达到59.84%；浓度为50μg/mL多糖PS3对HepG2细胞增殖的抑制作用达到56.17%。通过对多糖样品的FTIR、GC-MS、1H NMR、13C NMR、1H-1H COSY、HSQC和HMBC等分析，分别初步推测6个多糖样品的可能结构如下：PS1的分子量为361kDa，由葡萄糖、半乳糖和甘露糖组成，三者的摩尔比为5.15:0.30:0.10，其可能结构是以-1,6-D-Glcp连接形成主链，在-1,6-D-Glcp的3位处产生支链，与-1,3-D-Galp和β-1,3-D-Manp相连，端基由Glcp、Galp和Manp组成。其主链结构如下：→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→PS2-1的分子量为412kDa，由半乳糖、阿拉伯糖、葡萄糖、木糖和甘露糖组成，单糖的摩尔比为：3.98:1.00:0.28:0.33:0.18；其可能结构为以β-1,4-D-Galp连接形成主链，在β-1,4-D-Galp的3位处产生支链，与-1,3-D-Alap相连形成重复单位，含有少量的Xyl、Man和Glc残基。根据上述结果，PS2-1可能存在如下重复单元：PS2-2的分子量为48kDa，由甘露糖、葡萄糖和半乳糖组成，三者的摩尔比为：0.14:5.10:0.32；其可能结构为以-1,6-D-Glcp连接形成主链，在-1,6-D-Glcp的3位处产生支链，与-1,3-D-Galp和β-1,3-D-Manp相连，端基由Glcp、Galp和Manp组成。其主链结构如下：→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→PS3的分子量为536kDa，由阿拉伯糖、木糖、葡萄糖和半乳糖组成，四者的摩尔比为：0.62:0.34:0.13:4.62；其可能结构是以半乳糖残基通过β-1,4-糖苷键连接形成主链结构，葡萄糖残基通过β-1,3-糖苷键连接到半乳糖的C3上，部分阿拉伯糖残基或木糖残基通过α-1,3-糖苷键连接至葡萄糖残基的C3；其余阿拉伯糖残基或木糖残基通过β-1,3-糖苷键连接到半乳糖主链上。其主链结构式如下：→4)-β-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Galp-(1→PS4-1的分子量为729kDa，由阿拉伯糖、葡萄糖和半乳糖组成，三者的摩尔比为1.13:0.21:4.40；其可能结构是半乳糖残基通过β-1,4-糖苷键连接形成主链结构，阿拉伯糖残基和葡萄糖残基通过β-1,3-糖苷键连接到半乳糖主链上，每4个半乳糖残基连接1个阿拉伯糖残基。每5个上述半乳糖残基和阿拉伯糖残基的重复单元通过β-1,3-糖苷键连接1个葡萄糖残基。即每5个通过β-1-3糖苷键连接1个D-Glcp残基。PS4-2的分子量为668kDa，由阿拉伯糖、葡萄糖和半乳糖组成，三者的摩尔比为：0.62:0.57:4.46；其可能的结构是半乳糖残基通过β-1,4-糖苷键连接形成主链结构，阿拉伯和葡萄糖残基糖残基通过β-1,3-糖苷键连接到半乳糖主链上，约每15个半乳糖残基连接2个阿拉伯糖残基和2个葡萄糖残基。其可能的主要结构式如下：更多还原

【Abstract】 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:更多还原