【作者】 金洁；

【导师】 时连根；

【作者基本信息】 浙江大学 ， 特种经济动物科学， 2009， 博士

【摘要】 白僵蚕是蚕蛾科家蚕(Bombyx mori)幼虫感染白僵菌(Beauveria bassiana (Bals.) Vuill)后致死的干燥菌虫体,作为我国名贵的传统中药资源,其利用已有数千年历史。白僵蚕的药用目前仍十分广泛,513余种人用成药中含有白僵蚕的有49余种,其中64余种儿科成药中有29余种用白僵蚕。白僵蚕味咸,性微温,具有解毒、降胆固醇、抗惊厥、镇静、祛痰等作用,临床上用于治疗小儿惊痫、痉挛夜啼、中风、喉痹、野火丹毒、金疮、半身不遂等症,对腮腺炎、癫痫、脑炎后遗症、部分动脉硬化、癌症、糖尿病等也均有一定疗效。现代药理学研究发现,白僵蚕的药理作用与白僵蚕的活性成分密切相关。我国是世界上最大的养蚕国,蚕区地域广阔,地理环境差异很大,这会形成许多不同的白僵菌菌株。不同白僵菌菌株在蚕体内繁殖代谢产生的活性成分会存在差异,这进而影响白僵蚕的药效。查明白僵菌菌株间的系统发育差异以及白僵蚕的药效机理与活性成分,可为寻找优质白僵蚕中药资源,提高白僵蚕的药用范围与药用水平等,提供科学依据。本研究利用随机扩增多态性DNA (RAPD)技术和核糖体(rDNA)内转录间隔区(ITS)序列分析,研究了家蚕病原白僵菌的系统发育和菌株间亲缘关系,并以抗氧化性为中心,采用测定、分离、纯化等方法,研究了白僵蚕的抗氧化、抗癌、降血糖等药理作用,并对其中的活性物质进行了分析。主要研究结果如下：1、筛选出13个随机引物,对从我国不同蚕区分离的21株白僵菌菌株进行RAPD分析。结果得到的扩增图谱中,共有92个多态性位点,其中来自辽宁沈阳、山西夏县和吉林永吉蚕区的白僵菌菌株(Bb13、Bb15和Bb20)的带谱相近,但与其他18株白僵菌菌株间有明显差异。结果表明,不同地理来源的家蚕病原白僵菌,尽管大多数菌株间的遗传信息还基本一致,但每个菌株间表现出一定的多态性,出现了一定的遗传分化。基于白僵菌菌株间的相似性系数分析和应用非加权算术平均数聚类法(Unweighted Pair-Group Method using Arithmetic Average, UPGMA),构建了家蚕病原白僵菌的UPGMA聚类树状图。结果发现,21株家蚕病原白僵菌菌株间亲缘关系的远近,与其采集的地理分布呈现一定的相关性,不同地理来源的家蚕病原白僵菌虽然寄主相同、表型相似,但已呈现一定的DNA多态性,RAPD可以很好地检测出其遗传物质的变化。对21株家蚕病原白僵菌菌株的ITS区(ITS1.5.8S rDNA.ITS2)进行序列测定,所有序列经排列并用Mega 3.0软件进行分析,得到了最简约的系统发育树。结果采集自广西南宁、贵州遵义和云南蒙自的白僵菌菌株(Bb03、Bb06和Bb07)的ITS区序列,与其他白僵菌菌株间的差异显著,在NCBI GenBank中进行序列比对,发现其与球孢虫草(Cordyceps bassiana)同源性为100％(accession number:AB027382);采集自吉林永吉和山东烟台的白僵菌菌株(Bb20和Bb21)有较多位点的突变,而其他白僵菌菌株ITS区序列完全一致或仅有几个位点的碱基对缺失或变异。结果表明,家蚕病原白僵菌的ITS序列在种内水平相对保守,菌株间差异不显著。对家蚕病白僵菌菌株间的遗传变异及亲缘关系的分析,ITS序列不如RAPD敏感,但由于其种内的保守性,使其对相似种间的分类鉴定更加可靠。2、以氯仿、乙酸乙酯、丙酮、甲醇和蒸馏水为溶剂,依次抽提白僵蚕,测定各提取物中酚类物质的含量,结果白僵蚕的甲醇提取物中酚类物质含量最高,为10.75±0.35 mg GAE／g,其次为乙酸乙酯提取物和丙酮提取物,酚类物质含量分别为4.95±0.11 mg GAE／g和4.55±0.03 mg GAE／g,氯仿提取物中酚类物质含量最低,为2.78±0.39 mg GAE／g.测定白僵蚕各提取物的DPPH自由基清除活性、还原能力、铁离子络合能力以及亚油酸系统抑制过氧化反应能力,结果除氯仿提取物外,其余各提取物均表现出良好的抗氧化活性,并与浓度呈现良好的量效关系,其中甲醇提取物的抗氧化能力最强,其清除DPPH自由基、络合亚铁离子、抑制过氧化反应的半抑制浓度(IC50)分别为0.20±0.00 mg／mL、0.39±0.01 mg／mL、0.71±0.03 mg／mL。结果表明,白僵蚕中的活性物质可以有效地清除自由基,参与氧化还原反应,抑制脂质过氧化反应,显示出明显的抗氧化作用,其抗氧化活性可能与酚类物质含量相关。3、选取人肝癌细胞株Hep G2、人宫颈癌细胞株HeLa、人髓性白血病细胞株HL-60、人乳腺癌细胞MCF-7进行体外细胞培养,通过MTT实验,测定白僵蚕的氯仿、乙酸乙酯、丙酮、甲醇和蒸馏水提取物对这4株癌细胞生长的抑制作用。结果白僵蚕各提取物均能体外抑制HepG2、Hela、MCF-7和HL-60细胞的生长,其中氯仿提取物的抑制作用最强,其IC50值依次为0.34 mg/mL、0.49mg/mL、0.77 mg/mL和1.16 mg/mL,其次为乙酸乙酯提取物、甲醇提取物和蒸馏水提取物,丙酮提取物的抑制作用最弱,其IC50值均>2.5 mg/mL。结果表明,白僵蚕具有一定的抗肿瘤活性,作为天然抗肿瘤药物筛选的资源,具有一定的价值。4、采用正交实验法对白僵蚕多糖的测定条件进行优化,确立了白僵蚕粉过160目、超声波提取50 min、在100℃水浴中提取60 min、重复抽提2次,为白僵蚕多糖的最佳提取方案。建立了白僵蚕多糖含量测定的分光光度法,该方法的重现性、稳定性、加样回收率等均符合测定要求。白僵蚕富含多糖,其含量随家蚕感染病原白僵菌后天数的增加而显著提高,从感染第1天的2.07±0.24 mg／g提高到感染第7天的11.84±0.44 mg／g,提高了约6倍。5、以清除DPPH自由基、过氧化氢、羟自由基和超氧阴离子自由基的活性为指标,测定了白僵蚕多糖的体外抗氧化活性。结果白僵蚕多糖对DPPH自由基、过氧化氢、羟自由基和超氧阴离子自由基具有一定的清除作用,其IC50值分别为0.15 mg/mL、>1 mg/mL、0.98 mg/mL和0.85 mg/mL,并且其清除作用与其浓度呈一定的量效关系。结果表明,白僵蚕多糖具有一定的体外抗氧化活性。6、给小鼠灌胃白僵蚕多糖,通过小鼠血清和肝脏中超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量和谷胱甘肽(GSH)含量的测定,研究白僵蚕多糖的体内抗氧化活性。结果白僵蚕多糖能提高小鼠血清及肝脏中的SOD活性、GSH含量,并降低小鼠血清及肝脏中的MDA含量。结果表明,白僵蚕多糖可明显增强小鼠内源性氧自由基清除系统的功能,减少由于自由基作用于膜脂质化合物而生成脂质过氧化物MDA,调节机体内自由基水平及氧化还原信号的传递,表现出生物抗氧化活性。7、以腹腔注射链脲佐菌素(STZ)制备糖尿病小鼠模型,灌胃给药,通过小鼠体重、摄食量、饮水量、血糖值、总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL)、胰岛素含量等指标测定,研究白僵蚕多糖的降血糖功能。结果白僵蚕多糖能明显改善糖尿病模型小鼠的消瘦症状,调节糖尿病小鼠的饮食水平,降低血糖浓度,降低血清总胆固醇水平和提高高密度脂蛋白水平,但对血清甘油三酯水平和胰岛素含量无明显的影响。结果提示,白僵蚕多糖对糖尿病具有一定的治疗作用,其治疗机理可能是通过调节机体糖代谢、促进肝糖原合成、减少肝糖原分解来实现的,而不是通过刺激胰岛β细胞分泌胰岛素来作用的。8、对白僵蚕常规成分、微量元素、氨基酸及脂肪酸的含量进行定量分析,发现白僵蚕的水分、灰分、粗脂肪和粗蛋白的含量分别为8.62%、8.40%、4.9%和69.3%；白僵蚕富含微量元素,所测定的12种微量元素中,磷、钙、镁、锌、锰的含量较高,分别为4911.7mg/kg、4610.8 mg/kg、3346.1 mg/kg、121.4 mg/kg、30.4 mg/kg,硒的含量为0.24 mg/kg,比富硒抗癌中药黄芪(含硒0.08 mg/kg)的高3倍；白僵蚕中含有天门冬氨酸、丝氨酸、谷氨酸等17种氨基酸,总含量为32.87%,其中甘氨酸含量最高(4.89%),必需氨基酸占总氨基酸的比率为27,60%；白僵蚕脂肪酸中不饱和脂肪酸的含量高达78.86%,其中亚油酸、亚麻酸等必须脂肪酸的含量占77.63%,尤其是n-3系列的多不饱和脂肪酸亚麻酸的含量为最高,达到了69.98%。结果表明,白僵蚕含有丰富的营养物质,具有广阔的深度开发前景。更多还原

【Abstract】 Jiangcan. MTT assays were used to explore their inhibiting effect on the proliferation of Hep G2, HeLa, MCF-7 and HL-60 human cancer cell lines. The cytotoxicity effect was evaluated using growth inhibition ration and IC50 value. The chloroform extract exhibited the most effective antitumor activity, with the IC50 value of 0.34 mg/mL, 0.49 mg/mL,0.77 mg/mL and 1.16 mg/mL for Hep G2, HeLa, MCF-7 and HL-60 cells respectively. Acetone extract showed the weakest inhibitory effects in antitumor tests. The results proved that Jiangcan had antitumor activity.4. The present study optimized the condition of the determination of the polysaccharide in Jiangcan with the orthogonal methods. The content of polysaccharide could be exactly and stably measured with spectrophotometer after it was treated with the following process:it was sifted with 160 item, extracted with the ultrasonic for 50 min and the extraction for 60 min at 100℃in water for twice. Jiangcan was rich in polysaccharide. The content of polysaccharide significantly increased after inoculated with Beauveria bassiana, from 2.07±0.24 mg/g at the 1st day after inoculation up to 11.84±0.44 mg/g at the 7th day.5. The in vitro free radical scavenging tests indicated the polysaccharide from Jiangcan had scavenging abilities in DPPH radicals, hydrogen peroxide, hydroxyl radicals and superoxide radicals, with the IC50 value of 0.15 mg/mL,>1 mg/mL, 0.98 mg/mL and 0.85 mg/mL respectively, and exhibited does-dependant relationship.6. In vivo antioxidant analysis showed that the polysaccharide from Jiangcan enhanced SOD activity, increased GSH content and reduced the MDA level in liver and serum of mice. The results indicated that the polysaccharide from Jiangcan could enhance the function of endogenous oxygen radical scavenging system of mice, decrease the production of lipid peroxidation, regulate the free radical levels and redox-signaling transmission in organism, and perform bio-antioxidative activities.7. It was also discovered that the polysaccharide from Jiangcan could increase the weight of streptozotocin-induced diabetic mice, decrease the water and food intake of diabetes mice. By determination of blood glucose (BG), total cholesterol (TC), total triglyceride (TG) and high density lipoprotein cholesterol (HDL) levels, it revealed that polysaccharide from Jiangcan had beneficial effects on the improvement in the control of blood glucose and serum lipid level. The insulin level in streptozotocin-induced diabetic mice was not significantly affected by the polysaccharide from Jiangcan treatment. All the results suggested reduce hyperglycemia by regulating glycometabolism, accelerating hepatic glycogen synthesis and reducing hepatic glycogen decomposition, but not stimulating isletβcells to secrete insulin.8. The content of moisture, ash, crude fat and crude protein in Jiangcan powder was determined, which was 8.62%、8.40%、4.9%and 69.3%respectively. Within 12 kinds of trace elements determined, Jiangcan powder contained P, Ca, Mg, Zn and Mn at high level, for 4911.7 mg/kg、4610.8 mg/kg、3346.1 mg/kg、121.4 mg/kg%和30.4 mg/kg respectively, and Se for 0.24 mg/kg.17 species of amino acid were detected in Jiangcan powder, such as ASP, SER, GLU et al. The total content was 32.87% and the ratio of essential amino acid reached up to 27.60%. Up to 78.86% of unsaturated fatty acid was detected in Jiangcan powder, especially linolenic acid, whose content reached up to 69.98%.更多还原