【摘要】：蠕蟲病的危害歷史悠久,迄今仍威脅著人類的健康,在世衛(wèi)組織(WHO)確定的人類十大熱帶疾病中,排名第二位和第三位的都是蠕蟲病。同時(shí)蠕蟲病也給畜牧業(yè)帶來(lái)巨大的損失,是威脅牲畜的頭號(hào)疾病。目前可供選擇治療蠕蟲感染的藥物十分有限,且耐藥性嚴(yán)重。NADH-延胡索酸還原酶(NFRD)主要存在于微氧環(huán)境中的成年蠕蟲體內(nèi),這類酶系在蠕蟲厭氧代謝途徑中發(fā)揮著重要的作用。在正常的宿主細(xì)胞內(nèi)此類酶系幾乎不表達(dá),因此該酶系可以作為抗蠕蟲的特異性靶點(diǎn)。前期研究表明,來(lái)源于鄔氏黃絲曲霉的次級(jí)代謝產(chǎn)物沃特曼內(nèi)酯E、F具有較強(qiáng)的NFRD抑制活性。然而,已發(fā)現(xiàn)的沃特曼內(nèi)酯類化合物偏少;其與NFRD的構(gòu)效關(guān)系以及體內(nèi)抗蠕蟲活性的機(jī)理仍然不清楚;同時(shí)該類化合物在培養(yǎng)基中含量低,導(dǎo)致樣品制備過(guò)程復(fù)雜且溶劑使用量大。針對(duì)以上問(wèn)題,本論文主要從以下幾個(gè)方面展開(kāi)了研究。(1)采用化學(xué)表觀遺傳調(diào)控鄔氏黃絲曲霉次級(jí)代謝產(chǎn)物的種類�？疾炝朔⒅Z他(SAHA,異羥肟酸類)、丁酸鈉(脂肪酸類)和 cyclo(1-Am7(SAc)-Aib-l-Phe(pCl)-d-Pro)(CTSO,環(huán)肽類)三種組蛋白去乙酰化酶抑制劑對(duì)鄔氏黃絲曲霉次級(jí)代謝產(chǎn)物的影響。結(jié)果表明,在鄔氏黃絲曲霉培養(yǎng)基中添加100μM SAHA能夠增加沃特曼內(nèi)酯化合物的種類,并從該培養(yǎng)基中分離得到四個(gè)新的沃特曼內(nèi)酯類化合物,分別命名為沃特曼內(nèi)酯I、J、K、L。(2)采用單菌株多產(chǎn)物(one strain of many compounds,OSMAC)的方法增加鄔氏黃絲曲霉次級(jí)代謝產(chǎn)物的種類。選擇大米、玉米、麩皮和土豆四種不同的培養(yǎng)成分以及液體、固體和平板三種不同的培養(yǎng)形式進(jìn)行組合,考察不同培養(yǎng)條件對(duì)鄔氏黃絲曲霉次級(jí)代謝產(chǎn)物的影響。結(jié)果表明,不同培養(yǎng)條件下鄔氏黃絲曲霉次級(jí)代謝產(chǎn)物的種類和產(chǎn)量具有明顯的差異。從玉米浸提液平板培養(yǎng)的培養(yǎng)物中分離得到四個(gè)新的沃特曼內(nèi)酯類化合物,分別命名為沃特曼內(nèi)酯I1、12、13、14。(3)考察了海洋真菌DL1103(Aspergillussp.nov.F1)對(duì)沃特曼內(nèi)酯F的生物轉(zhuǎn)化作用。從轉(zhuǎn)化產(chǎn)物中分離得到一種新的沃特曼內(nèi)酯類化合物,并命名為沃特曼內(nèi)酯Q。根據(jù)F與Q的結(jié)構(gòu)差異,推測(cè)真菌DL1103能夠產(chǎn)生還原沃特曼內(nèi)酯F中雜環(huán)上羰基的羰基還原酶。(4)建立了三液相鹽析萃取分離沃特曼內(nèi)酯類化合物的方法,并考察了沃特曼內(nèi)酯類化合物在該體系中的分配規(guī)律�？疾炝顺上辔镔|(zhì)、溫度、pH對(duì)沃特曼內(nèi)酯類化合物分配系數(shù)的影響,確定了最優(yōu)鹽析萃取體系為20.0%(w/w)乙醇/12.9%(w/w)硫酸銨/25.9%(w/w)石油醚,操作條件為20 ℃C、pH=5.6。同時(shí)發(fā)現(xiàn)沃特曼內(nèi)酯類化合物在乙醇/硫酸銨/石油醚體系中分配系數(shù)的自然對(duì)數(shù)值與化合物的ClogP具有線性關(guān)系,且其在上相的分配系數(shù)隨系線長(zhǎng)度的增加而減少。三液相鹽析萃取體系提取沃特曼內(nèi)酯F的收率為91.23%,HPLC純度為5.53%,操作時(shí)間為0.5 h。利用三液相鹽析萃取偶聯(lián)柱層析方法制備得到了純度大于95%的沃特曼內(nèi)酯F。(5)考察了沃特曼內(nèi)酯類化合物的體外和體內(nèi)抗蠕蟲活性,并分析了沃特曼內(nèi)酯化合物與NFRD的構(gòu)效關(guān)系。體外活性研究結(jié)果表明,沃特曼內(nèi)酯化合物都具有較強(qiáng)的選擇抑制蠕蟲NFRD的活性。由構(gòu)效關(guān)系分析得知,沃特曼內(nèi)酯化合物中主要的活性基團(tuán)是二氫吡喃環(huán);此外,氧雜二環(huán)的4位酮羰基被還原為羥基后活性減弱,共軛四烯的長(zhǎng)度對(duì)活性影響較小。體內(nèi)活性評(píng)價(jià)顯示,沃向感染旋毛蟲小鼠灌胃200mg/kg沃特曼內(nèi)酯F,其體內(nèi)的減蟲率與灌胃25 mg/kg陽(yáng)性對(duì)照藥物阿苯達(dá)唑相當(dāng);并且沃特曼內(nèi)酯F對(duì)旋毛蟲的組織發(fā)育產(chǎn)生顯著影響。
[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，

本文編號(hào)：2290060