Analysis of the active fraction of Iranian Naja naja oxiana snake venom on the metabolite profiles of the malaria parasite by 1HNMR in vitro

Document Type : Original Article


1 Medical Parasitology Department, School of Medicine -International Campus, Iran University of Medical Sciences, Tehran, Iran

2 Parasitology and Mycology Department, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran

3 Biochemistry Department, Pasteur Institute of Iran, Pasteur Avenue, Tehran. Iran

4 Laboratory of Venom and Bio Therapeutics Molecules, Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

5 Department of Parasitology, Faculty of Medical Sciences, Tarbiat modares University Tehran, Iran


Objective(s): Malaria is an important parasitic disease with high morbidity and mortality in tropical areas. Resistance to most antimalarial drugs has encouraged the development of new drugs including natural products. Venom is a complex mixture of active pharmaceutical ingredients. The purpose of this study was to investigate the antimalarial activity of purified fractions of Naja naja oxiana.
Materials and Methods: Lyophilized venom was purified with a Sephacryl S-200 HR column and the fractions lyophilized and inhibitory concentration 50% against Plasmodium falciparum 3D7 in vitro obtained. The 4th fraction was run on a Mono Q column, and activity against P. falciparum was detected by lactate dehydrogenase assay and purity by SDS PAGE. Large scale culture of the parasite was carried out with and without the active fraction on the ring stage for 48 hr. The parasites were collected and lyophilized and analyzed by 1HNMR. Chemometrics studies were performed using MATLAB, differentiating metabolites were identified by Human Metabolic Database, and metabolic pathways by the Metaboanalyst online package.
Results: The active fraction from the ion exchange column had a 50% inhibitory concentration of 0.026 µg/ml on P. falciparum in vitro (P<0.001) with molecular weight of 63 kDa by SDS-PAGE and no hemolytic activity. Metabolomics studies on the two groups with and without the fraction identified 5 differentiating metabolites and a number of related pathways.
Conclusion: The metabolites were succinic acid, l-glutamic acid, pyruvic acid, cholesterol, and NAD. The changes in the Krebs cycle and metabolism pathways of nicotinamide and pyruvate were noticeable.


1. Organization WHO. World malaria report 2017.
2. Organization WHO. World malaria report 2015.
3. Norouzinejad F, Ghaffari F, Raeisi A. Epidemiological status of malaria in Iran, 2011–2014. Asian Pac J Trop Med 2016;9:1055-1061.
4. Elmi T, Hajialiani F, Asadi M R, Orujzadeh F, Kalantari Hesari A, Rahimi Esboei B et al . A Study on the Effect of Zingiber Officinale Hydroalcoholic Extract on Plasmodium berghei in Infected Mice: An Experimental Study. JRUMS 2019;18:353-364.
5. Thangam R, Gunasekaran P, Kaveri K, Sridevi G, Seundarraj S, Paulpandi M, et al. A novel disintegrin protein from Naja naja venom induces cytotoxicity and apoptosis in human cancer cell lines in vitro. Process Biochem 2012;47:1243-1249.
6. Castillo J, Vargas L, Segura C, Gutiérrez M, Pérez JC. In vitro antiplasmodial activity of phospholipases A2 and a phospholipase homologue isolated from the venom of the snake Bothrops asper. Toxins 2012;4:1500-1516.
7. Maluf S, Dal Mas C, Oliveira E, Melo P, Carmona A, Gazarini M, et al. Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom. Peptides 2016;78:11-16.
8. Zerez C, Roth E, Schulman S, Tanaka K. Increased nicotinamide adenine dinucleotide content and synthesis in Plasmodium falciparum-infected human erythrocytes. Blood 1990;75:1705-1710.
9. Parvazi S, Sadeghi S, Azadi M, Mohammadi M, Arjmand M, Vahabi F, et al. The effect of aqueous extract of cinnamon on the metabolome of Plasmodium falciparum using 1HNMR spectroscopy. J Trop Med 2016; 2016:1-5.
10. Griffin JL. Metabonomics: NMR spectroscopy and pattern recognition analysis of body fluids and tissues for characterisation of xenobiotic toxicity and disease diagnosis. Curr Opin Chem Biol 2003;7:648-654.
11. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680.
12. Memar B, Jamili S, Shahbazzadeh D, Bagheri K. The first report on coagulation and phospholipase A2 activities of Persian Gulf lionfish, Pterois russelli, an Iranian venomous fish. Toxicon 2016;113:25-31.
13. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science 1976;193:673-675.
14. Radfar A, Méndez D, Moneriz C, Linares M, MarínGarcía P, Puyet A, et al. Synchronous culture of Plasmodium falciparum at high parasitemia levels. Nat Protoc 2009;4:1899.
15. Tasanor O, Noedl H, Na-Bangchang K, Congpuong K, Sirichaisinthop J. An in vitro system for assessing the sensitivity of Plasmodium vivax to chloroquine. Acta Trop 2002;83:49-61.
16. D’alessandro S, Silvestrini F, Dechering K, Corbett Y, Parapini S, Timmerman M, et al. A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection. J Antimicrob Chemother 2013;68:2048-2058.
17. E.D B. High Resolution NMR. 2, editor. New York: academic press 2007.
18. Utkin YN. Animal venom studies: Current benefits and future developments. World J Biol Chem 2015;6:28.
19. Sherman D. Drug Derived From Snake Venom May Help Stroke Patients. Jama Jam Med Assoc 2016.
20. Liu C, Yang H, Zhang L, Zhang Q, Chen B, Wang Y. Biotoxins for cancer therapy. Asian Pac J Cancer Prev 2014;15:4753-4758.
21. Dhananjaya B, Sivashankari PJ. Snake venom derived molecules in tumor angiogenesis and its application in cancer therapy; an overview. Curr Top Med Chem 2015;15:649-657.
22. S Liberio M, A Joanitti G, Fontes W. Anticancer peptides and proteins: a panoramic view. Protein Pept Lett 2013;20:380-391.
23. ROY A. Structural and Functional Characterization of Haditoxin, a novel neurotoxin isolated from the venom of Ophiohagus Hannah (King Cobra). J Biol Chem 2011.;285: 8302–8315.
24. Samel M, Tõnismägi K, Rönnholm G, Vija H, Siigur J, Kalkkinen N, et al. L-Amino acid oxidase from Naja naja oxiana venom. Comp Biochem Physiol B Biochem Mol Biol 2008;149:572-580.
25. Zieler H, Keister D, Dvorak J, Ribeiro JM. A snake venom phospholipase A2 blocks malaria parasite development in the mosquito midgut by inhibiting ookinete association with the midgut surface. J Exp Biol 2001;204:4157-4167.
26. Tischfield J. A reassessment of the low molecular weight phospholipase A2 gene family in mammals. J Biol Chem 1997;272:17247-17250.
27. Ginsburg HJ. Metabolism: Malaria parasite stands out. Nature 2010;466:702.
28. Olszewski K, Mather M, Morrisey J, Garcia B, Vaidya AB, Rabinowitz J, et al. Branched tricarboxylic acid metabolism in Plasmodium falciparum. Nature 2010;466:774.
29. Olszewski K, Llinás M. Central carbon metabolism of Plasmodium parasites. Mol Biochem Parasitol 2011;175:95-103.
30. Roth J. Plasmodium falciparum carbohydrate metabolism: a connection between host cell and parasite. Blood Cells 1990;16:453-460.
31. Sampaio S, SousaeSilva M, Borelli P, Curi R, Cury Y. Crotalus durissus terrificus snake venom regulates macrophage metabolism and function. J Leukoc Biol 2001;70:551-558.
32. O’Hara J, Kerwin L, Cobbold S, Tai J, Bedell T, Reider P, et al. Targeting NAD+ metabolism in the human malaria parasite Plasmodium falciparum. PLoS One 2014;9: e94061.
33. Ghosh S, Sengupta A, Sharma S, Sonawat H. Metabolic fingerprints of serum, brain, and liver are distinct for mice with cerebral and noncerebral malaria: a 1H NMR spectroscopy-based metabonomic study. J Proteome Res 2012;11:4992-5004.
34. Malleswari M, Josthna P, Doss PJ. Orally administered venom of Naja naja alters protein metabolic profiles in the liver of albino rats. Int J Life Sci Biotechnol Pharma Res 2015;4:10.
35. Guggisberg A, Amthor R, Odom AR. Isoprenoid biosynthesis in Plasmodium falciparum. EC. Eukaryot Cell 2014;13:1348-59
36. Müller T, Johann L, Jannack B, Brückner M, Lanfranchi DA, Bauer H, et al. Glutathione reductase-catalyzed cascade of redox reactions to bioactivate potent antimalarial 1, 4-naphthoquinones–a new strategy to combat malarial parasites. J Am Chem Soc 2011;133:11557-11571.
37. Meissner PE, Mandi G, Witte S, Coulibaly B, Mansmann U, Rengelshausen J, et al. Safety of the methylene blue plus chloroquine combination in the treatment of uncomplicated falciparum malaria in young children of Burkina Faso. Malar J 2005;4:45.
38. Al-Quraishy S, Dkhil M, Moneim A. Hepatotoxicity and oxidative stress induced by Naja haje crude venom. J Venom Anim Toxins Incl Trop Dis 2014;20:42.
39. Hussain T, Yogavel M, Sharma AJ. Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases. Antimicrob Agents Chemother 2015;59:1856-67.
40. Pham J, Dawson K, Jackson K, Lim E, Pasaje C, Turner K, et al. Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites. Int J Parasitol Drugs Drug Resist 2014;4:1-13.