In Vitro Mechanistic Study of Anti-Leishmanial Activity of Novel Tetracyclic Iridoid Compounds Isolated from Morinda Lucida

ABSTRACT

Leishmaniasis is widely considered among neglected tropical disease and threatens about 350 million people globally. In spite of advances in drug discovery, high toxicity, and drug resistance limits current drugs. With current emphasizes on use of medicinal plants worldwide, three novel tetracyclic iridoids, Molucidin, ML-2-3 and ML-F52 were identified from Morinda lucida, a medicinal plant, to have anti-trypanosomal activity. This study sought to determine the activity of iridoids against Leishmania spp. and to determine the mechanism of action of active compounds. The activity of tetracyclic iridoids against Leishmania donovani (D10) and L. major (NR48815) were studied using promastigotes and intracellular amastigotes. Infectivity and cytotoxicity assays were performed with RAW 264.7 macrophage cells using Amphotericin as reference drug. The mechanisms of action were analyzed by performing Nexin Assay, Immunohistochemistry (IHC), and Cell cycle analysis. A 50% inhibitory concentration of compounds was determined by Alamar blue assay. Molucidin and ML-F52 inhibited the growth of promastigote in L. donovani (Molucidin; IC50 = 2.94±0.60 μM, ML-F52; IC50 = 0.91±0.50 μM) and L. major (Molucidin; IC50 = 1.85± 0.20 μM, ML-F52; IC50 = 1.77± 0.20 μM). ML-F52 had a 10-fold cytotoxic effect on parasites relative to normal cell lines. Against intracellular forms, Molucidin and ML-F52 inhibited intracellular amastigote replication and infectivity. Amphotericin B, Molucidin and MLF52, induced a dose-dependent apoptotic effect on promastigotes. Molucidin and ML-F52 induced apoptotic mechanism of cell death in 38.9 % and 12.8% of promastigote culture, respectively. Both compounds inhibited cytokinesis and induced phenotypic changes in promastigote forms. Molucidin also induced ‘’nectomonad-like’’ forms and loss of kDNA, ML-F52 induced ‘cellrounding’ with loss of flagellum. Molucidin further induced cell growth arrest at G2-M phase (54.5%). A significant induction of apoptosis (P = 0.05) was shown by an enhanced peak in the sub-G1 confirming the apoptotic inducing properties of Molucidin and ML-F52. This study shows the antileishmania activity of tetracyclic iridoids which could be further investigated for the development of new chemotherapy against leishmaniasis.