In Vitro Study of the Effect of Artimisinin on Promastigotes and Amastigotes of Leishmania major

Authors
1 Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2 Associated Professor, Department of Parasitilogy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Abstract
Objective: Cutaneous leishmaniasis is an endemic infectious disease considered to be a crucial health problem in many countries, including Iran. As such, there is a need for new medications with few side effects. In the present research we have studied the effect of artimisinin on Leishmania major (L. major) and cell death in vitro. Methods: A specific number of promastigotes of L. major were grown in the presence of different concentration of artimisinin to achieve IC50 of the drug. The MTT method was applied to evaluate the cytotoxic effect of the artiminisinin on L. major. Various densities of this drug were applied to study the induction of apoptosis by flow cytometry on L. major promastigotes. Results: We calculated the IC50 of artimisinin to be 25 μg/mlby promastigote assay. Promastigotes were incubated at 72 hours incubation with various doses of artimisinin (10, 25, 50 and 100 μg/ml). The dose 100 μg/ml showed the most apoptosis (68.16%) by Annexin-V FITC. Whereas the 10 μg/ml dose had the least apoptosis (12.78%). There was no change in the control group. According to MTT, the toxic effect of artiminisinin on L. major promastigotes increased with increasing drug concentration. Conclusions: This study revealed that artimisinin has a little toxic effect on macrophages. According to the flow cytometry and MTT results, artimisinin can be suggested as an appropriate drug for in vivo antileishmanial study.

Keywords


[1]     World Health Organization. Report on global surveillance of epidemic-prone infectious diseases- leishmaniasis.2006. http://www.who. int/csr/resources/publications/CSR_ISR_2000_1leish/en/index.html
[2]     World Health Organization. Control of leishmaniasis. Report by the secretariat. 2007. http://apps.who.int/gb/ebwha/pdf_files/WHA60/A60_10-en.pdf
[3]     Stratetegic Direction for Research leishmaniasis. Special Programme for Research and Training in Tropical Diseases Feb 2002. http://www. who.int/tdr
[4]     Desjeux P. The increase in risk factors for leishmaniasis worldwide. Trans R Soc Trop Med Hyg 2001; 95(3): 239-43.
[5]     Saghafipour A, Akbari A, Rasi Y, Mostafavi R. Epidemiology of Cutaneous Leishmaniasis in Qom Province, Iran, during 2003-2009.QomUniv Med Sci J 2012; 6(1): 83-8. (Persian)
[6]     Oliveira LF, Schubach AO, Martins MM, Passos SL, Oliveira RV, Marzochi MC, Andrade CA. Systematic review of the adverse effects of cutaneous leishmaniasis treatment in theNew World. Acta Trop 2011; 118(2): 87-96.
[7]     Sen R, Bandyopadhyay S, Dutta A, Mandal G, Ganguly S, Saha P, Chatterjee M. Artemisinin triggers induction of cell-cycle arrest and apoptosis in Leishmania donovani promastigotes. J med Microbiol 2007; 56(9): 1213-8.
[8]     Yang DM, Liew FY. Effects of qinghaosu (artemisinin) and its derivatives on experi-mental cutaneous leishmaniasis. Parasitology 1993; 106 (Pt 1): 7-11.
[9]     Marinho FA, Goncalves KCS, Oliveria SS, Oliviera ASC, Bellio M, d'Avila-Levy CM, Santos ALS, Branquinha MH. Miltefosine induces programmed cell death in Leishmania amazonensis promastigotes. Mem Inst Oswaldo Cruz 2011; 106(4): 507-9.
[10]  Shaha C. Apoptosis in Leishmania species & its relevance to disease Pathogenesis.India J Med Res 2006; 123(3): 233-44.
[11]  Meshnick SR. Artemisinin: mechanisms of action, resistance and toxicity. Int J Parasitol 2002; 32(13): 1655-60.
[12]  Ganguly S, Bandyopadhyay S, Sarkar A, Chatterjee M. Development of a semi-automated colorimetric assay for screening anti-leishmanial agents. J Microbiol Methods 2006; 66(1): 79-86.
[13]  Hepburn NC. Cutaneous leishmaniasis: an overview. J Postgrad Med 2003; 49(1): 50-4.
[14]  Rocha LG, Almeida JR, Macêdo RO, Barbosa-Filho JM. A review of natural products with antileishmanial activity. Phytomedicine 2005; 12(6-7): 514-35.

[15]  Räth K, Taxis K, Walz G, Gleiter CH, Li SM, Heide L. Pharmacokinetic study of artemisinin after oral intake of a traditional preparation of Artemisia annua L. (annual wormwood). Am J Trop Med Hyg 2004; 70(2): 128-32.


[16]  Gordi T, Lepist EI. Artemisinin derivatives: toxic for laboratory animals, safe for humans? Toxicol Lett 2004; 147(2): 99-107.


[17]  Li Y, Yu PL, Chen YX, Li LQ, Gai YZ, Wang DS, Zheng YP. [Studies on analogs of artemisinine. I. The synthesis of ethers, carboxylic esters and carbonates of dihydroartemisinine (author's transl)].Yao Xue Xue Bao 1981; 16(6):429-39.
[18]  Cooke DW, Lallinger GJ, Durack DT. In vitro sensitivity of Naegleria fowleri to qinghaosu and dihydroqinghaosu. J Parasitol 1987; 73(2): 411-3.
[19]  Klayman DL, Lin AJ, Acton N, Scovill JP, Hoch JM, Milhous WK, Theoharides AD, Dobek AS. Isolation of artemisinin (qinghaosu) from Artemisia annua growing in theUnited States. J Nat Prod 1984; 47(4): 715-7.
[20]  Krungkrai SR, Yuthavong Y. The antimalarial action on Plasmodium falciparum of qinghaosu and artesunate in combination with agents which modulate oxidant stress. Trans R Soc Trop Med Hyg 1987; 81(5): 710-4.
[21]  Avery MA, Gao F, Chong WK, Mehrotra S, Milhous WK. Structure-activity relationships of the antimalarial agent artemisinin. 1. Synthesis and comparative molecular field analysis of C-9 analogs of artemisinin and 10-deoxoartemisinin. J Med Chem 1993; 36(26): 4264-75.
[22]  Meshnick SR, Thomas A, Ranz A, Xu CM, Pan HZ. Artemisinin (qinghaosu): the role of intracellular hemin in its mechanism of antimalarial action. Mol Biochem Parasitol 1991; 49(2): 181-9.
[23]  Efferth T, Herrmann F, Tahrani A, Wink M. Cytotoxic activity of secondary metabolites derived from Artemisia annua L. towards cancer cells in comparison to its designated active constituent artemisinin. Phytomedicine 2011; 18(11): 959-69.