| Helminthiasis has a long history and still threaten human health,dates from WHO indicated that helminthiasis rank second and third in the top ten human tropical diseases.At the same time,as a major livestock disease,it has brought huge losses to the livestock industry.At present,only few types of drugs can be used to treat helminthiasis,and many helminth are resistant to these drugs.NADH-fumarate reductase(NFRD)exists in most adult helminth and plays an important role in the anaerobic energy metableolism of helminth.However,this enzyme system is rarely founded in host normal cells,therefore,it could be regarded as specific target for antihelminth.Wortmannilactone E and F isolated from secondary metabolites of Talaromyces wortmannii by our group,showed strong inhibitory activity against NFRD.However,few of this class compounds had been obtained,the structure activity relationship between wortmannilactones and NFRD and activity in vivo are still unknown.On the other hand,the content of the wortmannilactone compounds in the broth is very low,the preparation process is complex with low recovery and consumes a large amount of organic solvent.In order to solve the above problems,the main work of this thesis is as follows.(1)Secondary metabolites of Talaromyces wortmannii were manipulated by small chemical molecules.Three histone deacetylase inhibitors,including SAHA(hydroxamic acids),sodium butyrate(aliphatic acid)and cyclo(1-Am7(SAc)-Aib-L-Phe(pCl)-D-Pro),were used to investigate the regulation effect on secondary metabolites of Talaromyces wortmannii.According to HPLC analysis,significant changes were only observed in the medium with 100 jaM SAHA.Four new wortmannilactones I,J,K,L are isolated from medium with 100 μM SAHA(2)Secondary metabolites of Talaromyces wortmannii were manipulated by one strain of many compounds(OSMAC).Four different culture medium composition,included rice,corm,wheat bran and potatoes,three different types of culture methods,included liquids,solids,and plate were selected for studying the effects of culturing condition on secondary metabolites of Talaromyces wortmannii.According to HPLC analysis,change of medium could influence the productivity and diversity of wortmannilactones.Four new wortmannilactones Ⅱ,12,13,14 are isolated and identified.(3)The structural modification of wortmannilactone F was investigated by fungi(DL1103),identified as Aspergillus sp.nov.FI.The transformation products of wortmannilactone F was isolated and identified as new wortmannilactone,named wortmannilactone Q.The structure analysis indicated that DL1103 might be capable of producing carbonyl reductase.(4)The method for separation and extraction of wortmannilactone compounds by three liquid phase salting out extraction was established,and the partition behavior of wortmannilactone compounds in the system was investigated.The effect of phase-forming substance,temperature and pH on partition behavior of wortmannilaetones were studied,optimal extraction conditions are determined as 20℃,pH=5.6,20.0%(w/w)ethanol/12.9%(w/w)ammonium sulfate/25.9%(w/w)petroleum ether.Meanwhile,the partition behavior of wortmannilactones indicated that partition coefficient of all the studied products are correlated with tie line length and their ClogP.The partition coefficient of wortmannilactones in the upper phase decreased with the increase of the length of the tie lines.Using this method,the wortmannilactone F could be separated and purified with the system,recovery rate of 91.2%and operation time of 0.5 hour.Using the method of combining three-liquid-phase salting-out extraction with ODS column chromatography,wortmannilactone F was obtained with purity of over 95%.(5)Anthelminthic activity of wortmannilactones in vivo and vitro and structure-activity relationship between wortmannilactones and NFRD were investigated.The wortmannilactones showed selective inhibition against NFRD in vitro.Study of structure-activity relationship indicated that dihydropyrane ring play main role in activity.Reduction of ketone carbonyl in oxabicyclo[2.2.1]heptane moiety could also reduce activity.The tetraen strain didn’t showed obvious effects on the activity.Wortmannilactone F showed anthelmintic activity for trichinella spiralis in mice in vivo,200 mg/kg wortmannilactone F(p.o.)showed similar cut worm mice with that of 25 mg/kg albendazole.The sections of different tissues indicated wortmannilactone F also can affect the histological development of Trichinella spiralis significantly. |
