Screening Inhibitors From Crude Herbal Extracts And Field Diagnosis For Lactate Dehydrogenase Of Plasmodium Falciparum

Malaria is a kind of insect-mediated infectious disease that severely threatens human health with obvious elevation in the case numbers of the disease besides AIDS,claiming 3 million victims' death worldwide annually.The potential of malarial parasites to generate multidrug resistance to current used antimalarial drugs is a major scourge for patients in endemic regions and the multiple targets of antimalarials are crucial to avoid or retard occurrence of such drug resistance.Facing to the challenges come from the prevalent multidrug resistant malaria,extensive search for novel anti-malarial drugs that act on the new targets is necessary.As an essential metabolic enzyme for energy generation in the parasite,lactate dehydrogenase of Plasmodium falciparum(PfLDH)is produced through the erythrocyte stage and represents an appreciable target for antimalarial action.PfLDH utilizes 3-acetylpyridine adenine dinucleotide(APAD),an analogue of oxidized nicotiamide adenine dinucleotide(NAD),as a coenzyme to involve anerobic glycolysis and generates adenosine triphosphorilate(ATP)to supply energy for parasite existence.For the anaerobic malarial parasite,PfLDH is essential for its growth and propagation.Therefore,compounds that inhibit PfLDH can also kill the parasite,thereby providing a feasiability to use PfLDH as an alternative target for screening novel antimalarial prodrugs.To establish a platform for high throughput screening and in vitro evaluating novel metabolic enzyme-targeted inhibitors toward novel anti-malarial drugs,PfLDH gene was amplified from the Hainan isolate FCC1/HN by polymerase chain reaction(PCR)and cloned into pTARGET vector for full-length gene sequencing,which has been accessed in GenBank with the accession number of DQ825436.By BLAST browsing,PfLDH gene of FCC/HN isolate was found to share homology of 99.79%with that of K1 isolate.To recover large quantities of soluble PfLDH,two fusion expression vectors,pGEX-2TK and pET-29a(+),were applied to introducing PfLDH gene into Escherichia coli strains,BL21 and BL21(DE3),for microbial overexpression.Consequently,PfLDH fusion protein generated by pGEX-PfLDH mainly presented in the state of inclusion bodies,while expression of PfLDH cloned in pET-29a(+) formed large quantities of the soluble form of PfLDH,demonstrating the latter vehicle might be more suitable for PfLDH gene expression.The affinity chromatography-purified recombinant PfLDH proteins were immobilized on the 96-well microplates and the crude extracts from four species of medicinal plants,Changshan(D.febrifuga),Gegeng(P.lobata), Sharen(A.villosum)and Huzhang(P.cuspidatum),were filled into the microwells for in vitro screening of the inhibitory component(s)in above crude extracts.The result has shown that except for Changshan,the crude extracts of other three species of medicinal plants demonstrate potent inhibition to PfLDH.Moreover,by supplementing all those medicinal plant crude extracts to the micro-scale antimalarial evaluation system,besides the undetectable Huzhang due to technical reason,Changshan and Gegeng but not Sharen were discovered to suppress malaria growth,thereby indicating that the crude extract from Gegeng is both an inhibitor of PfLDH activity and a suppressor of malaria growth. Furthermore,results from organic sovent extraction of Gegeng crude extract and PfLDH activity assay have revealed that the water phase,n-butanol partition and ice acetic acid partition exhibit more powerful inhibition to PfLDH,the ethanol partition has only weak inhibition,whereas acetoacetate partition does not have significant inhibitory role.On the other hand,the recombinant PfLDH protein available from this experiment has been exploited as an antigen to raise PfLDH-derived polyclonal antibody and a simplified malaria diagnostic kit preliminarily manufactured,by which PfLDH has been successfully detect in both blood-supplemented malaria culture suspention and malaria-infected patients' serum samples.In summary,the platform established in present study for antimalarial herbal drug screening and malaria infection diagnosis warrant further research and development due to its notable application values with merits of low cost,high efficiency and sensitivity.