沃特曼内酯类化合物及其抗蠕虫活性研究
发布时间：2018-10-23 18:05
【摘要】：蠕虫病的危害历史悠久,迄今仍威胁着人类的健康,在世卫组织(WHO)确定的人类十大热带疾病中,排名第二位和第三位的都是蠕虫病。同时蠕虫病也给畜牧业带来巨大的损失,是威胁牲畜的头号疾病。目前可供选择治疗蠕虫感染的药物十分有限,且耐药性严重。NADH-延胡索酸还原酶(NFRD)主要存在于微氧环境中的成年蠕虫体内,这类酶系在蠕虫厌氧代谢途径中发挥着重要的作用。在正常的宿主细胞内此类酶系几乎不表达,因此该酶系可以作为抗蠕虫的特异性靶点。前期研究表明,来源于邬氏黄丝曲霉的次级代谢产物沃特曼内酯E、F具有较强的NFRD抑制活性。然而,已发现的沃特曼内酯类化合物偏少;其与NFRD的构效关系以及体内抗蠕虫活性的机理仍然不清楚;同时该类化合物在培养基中含量低,导致样品制备过程复杂且溶剂使用量大。针对以上问题,本论文主要从以下几个方面展开了研究。(1)采用化学表观遗传调控邬氏黄丝曲霉次级代谢产物的种类。考察了伏立诺他(SAHA,异羟肟酸类)、丁酸钠(脂肪酸类)和 cyclo(1-Am7(SAc)-Aib-l-Phe(pCl)-d-Pro)(CTSO,环肽类)三种组蛋白去乙酰化酶抑制剂对邬氏黄丝曲霉次级代谢产物的影响。结果表明,在邬氏黄丝曲霉培养基中添加100μM SAHA能够增加沃特曼内酯化合物的种类,并从该培养基中分离得到四个新的沃特曼内酯类化合物,分别命名为沃特曼内酯I、J、K、L。(2)采用单菌株多产物(one strain of many compounds,OSMAC)的方法增加邬氏黄丝曲霉次级代谢产物的种类。选择大米、玉米、麸皮和土豆四种不同的培养成分以及液体、固体和平板三种不同的培养形式进行组合,考察不同培养条件对邬氏黄丝曲霉次级代谢产物的影响。结果表明,不同培养条件下邬氏黄丝曲霉次级代谢产物的种类和产量具有明显的差异。从玉米浸提液平板培养的培养物中分离得到四个新的沃特曼内酯类化合物,分别命名为沃特曼内酯I1、12、13、14。(3)考察了海洋真菌DL1103(Aspergillussp.nov.F1)对沃特曼内酯F的生物转化作用。从转化产物中分离得到一种新的沃特曼内酯类化合物,并命名为沃特曼内酯Q。根据F与Q的结构差异,推测真菌DL1103能够产生还原沃特曼内酯F中杂环上羰基的羰基还原酶。(4)建立了三液相盐析萃取分离沃特曼内酯类化合物的方法,并考察了沃特曼内酯类化合物在该体系中的分配规律。考察了成相物质、温度、pH对沃特曼内酯类化合物分配系数的影响,确定了最优盐析萃取体系为20.0%(w/w)乙醇/12.9%(w/w)硫酸铵/25.9%(w/w)石油醚,操作条件为20 ℃C、pH=5.6。同时发现沃特曼内酯类化合物在乙醇/硫酸铵/石油醚体系中分配系数的自然对数值与化合物的ClogP具有线性关系,且其在上相的分配系数随系线长度的增加而减少。三液相盐析萃取体系提取沃特曼内酯F的收率为91.23%,HPLC纯度为5.53%,操作时间为0.5 h。利用三液相盐析萃取偶联柱层析方法制备得到了纯度大于95%的沃特曼内酯F。(5)考察了沃特曼内酯类化合物的体外和体内抗蠕虫活性,并分析了沃特曼内酯化合物与NFRD的构效关系。体外活性研究结果表明,沃特曼内酯化合物都具有较强的选择抑制蠕虫NFRD的活性。由构效关系分析得知,沃特曼内酯化合物中主要的活性基团是二氢吡喃环;此外,氧杂二环的4位酮羰基被还原为羟基后活性减弱,共轭四烯的长度对活性影响较小。体内活性评价显示,沃向感染旋毛虫小鼠灌胃200mg/kg沃特曼内酯F,其体内的减虫率与灌胃25 mg/kg阳性对照药物阿苯达唑相当;并且沃特曼内酯F对旋毛虫的组织发育产生显著影响。
[Abstract]:Worm disease has a long history, which has so far threatened human health, among the top ten tropical diseases identified by the World Health Organization (WHO), the second and third are helminths. At the same time, the worm disease also brings enormous losses to the animal husbandry, which is the number one disease that threatens livestock. currently available drugs for the treatment of worm infections are very limited and resistant to severe drug resistance. NADH-tetrahydrofolate reductase (NFRD) is mainly in adult worm in micro-oxygen environment, which plays an important role in worm anaerobic metabolism pathway. Such enzymes are hardly expressed in normal host cells, and therefore the enzyme system can act as a specific target for the anti-worm. Previous studies have shown that the secondary metabolite of Aspergillus flavus, Waltmann lactone E, F has stronger NFRD inhibitory activity. However, it has been found that there is less ester compound in Wutman, its relationship with the structure of NFRD and the mechanism of anti-worm activity in vivo is still unclear; meanwhile, the content of such compounds is low in the culture medium, which leads to the complicated preparation process of samples and the large amount of solvent used. In view of the above problems, the thesis focuses on the following aspects. (1) adopting the chemical apparent genetic regulation to control the species of the secondary metabolites of Aspergillus flavus. The effects of three histones of Vinochet (SAHA, hydroxamic acid), sodium butyrate (fatty acid) and cyclo (1-Am7 (SAc)-Aib-l-Phe (pCl)-d-Pro) (CTSO, cyclic peptide) on the secondary metabolites of Aspergillus flavus were investigated. The results show that the addition of 100.mu. M SAHA to Aspergillus flavus culture medium can increase the kind of Wutman lactone compound and separate the four new Wutman lactone compounds from the culture medium, and they are named Waltmann lactone I, J, K, respectively. L. (2) A single strain of polyproduct (OSMAC) was used to increase the species of secondary metabolites of Aspergillus flavus. Four different culture compositions of rice, maize, bran and potato were selected to study the effects of different culture conditions on the secondary metabolites of Aspergillus flavus. The results showed that the species and yield of the secondary metabolites of Aspergillus flavus were significantly different under different culture conditions. Four new Wutman lactone compounds were isolated from the culture of the corn extract plate culture, respectively named Wortmann lactone I1, 12, 13, 14. (3) The bioconversion of the marine fungus DL103 (Aspergillussp.nov.F1) to the Woltmann lactone F was investigated. According to the structural difference between F and Q, it is estimated that the fungus DL1 103 can produce a reducing enzyme in the heterocyclic ring in the reduction Woltmann lactone F. According to the structural difference of F and Q, the novel Wutman lactone Q is obtained. (4) Three liquid-phase salting-out extraction method was established, and the distribution of ester compound in the system was investigated. The influence of phase matter, temperature and pH on the distribution coefficient of ester compound in Wman was investigated. The optimum salting-out extraction system was found to be 20. 0% (w/ w) ethanol/ 12.9% (w/ w)/ 25.9% (w/ w) of petroleum ether, and the operating conditions were 20 鈩，
本文编号：2290060
[Abstract]:Worm disease has a long history, which has so far threatened human health, among the top ten tropical diseases identified by the World Health Organization (WHO), the second and third are helminths. At the same time, the worm disease also brings enormous losses to the animal husbandry, which is the number one disease that threatens livestock. currently available drugs for the treatment of worm infections are very limited and resistant to severe drug resistance. NADH-tetrahydrofolate reductase (NFRD) is mainly in adult worm in micro-oxygen environment, which plays an important role in worm anaerobic metabolism pathway. Such enzymes are hardly expressed in normal host cells, and therefore the enzyme system can act as a specific target for the anti-worm. Previous studies have shown that the secondary metabolite of Aspergillus flavus, Waltmann lactone E, F has stronger NFRD inhibitory activity. However, it has been found that there is less ester compound in Wutman, its relationship with the structure of NFRD and the mechanism of anti-worm activity in vivo is still unclear; meanwhile, the content of such compounds is low in the culture medium, which leads to the complicated preparation process of samples and the large amount of solvent used. In view of the above problems, the thesis focuses on the following aspects. (1) adopting the chemical apparent genetic regulation to control the species of the secondary metabolites of Aspergillus flavus. The effects of three histones of Vinochet (SAHA, hydroxamic acid), sodium butyrate (fatty acid) and cyclo (1-Am7 (SAc)-Aib-l-Phe (pCl)-d-Pro) (CTSO, cyclic peptide) on the secondary metabolites of Aspergillus flavus were investigated. The results show that the addition of 100.mu. M SAHA to Aspergillus flavus culture medium can increase the kind of Wutman lactone compound and separate the four new Wutman lactone compounds from the culture medium, and they are named Waltmann lactone I, J, K, respectively. L. (2) A single strain of polyproduct (OSMAC) was used to increase the species of secondary metabolites of Aspergillus flavus. Four different culture compositions of rice, maize, bran and potato were selected to study the effects of different culture conditions on the secondary metabolites of Aspergillus flavus. The results showed that the species and yield of the secondary metabolites of Aspergillus flavus were significantly different under different culture conditions. Four new Wutman lactone compounds were isolated from the culture of the corn extract plate culture, respectively named Wortmann lactone I1, 12, 13, 14. (3) The bioconversion of the marine fungus DL103 (Aspergillussp.nov.F1) to the Woltmann lactone F was investigated. According to the structural difference between F and Q, it is estimated that the fungus DL1 103 can produce a reducing enzyme in the heterocyclic ring in the reduction Woltmann lactone F. According to the structural difference of F and Q, the novel Wutman lactone Q is obtained. (4) Three liquid-phase salting-out extraction method was established, and the distribution of ester compound in the system was investigated. The influence of phase matter, temperature and pH on the distribution coefficient of ester compound in Wman was investigated. The optimum salting-out extraction system was found to be 20. 0% (w/ w) ethanol/ 12.9% (w/ w)/ 25.9% (w/ w) of petroleum ether, and the operating conditions were 20 鈩，
本文编号：2290060
本文链接：https://www.wllwen.com/shoufeilunwen/yxlbs/2290060.html
教材专著
