In vitro activity and mode of action of phenolic compounds on Leishmania donovani

Background Leishmaniasis is a disease caused by the protozoan parasite, Leishmania. The disease remains a global threat to public health requiring effective chemotherapy for control and treatment. In this study, the effect of some selected phenolic compounds on Leishmania donovani was investigated. The compounds were screened for their anti-leishmanial activities against promastigote and intracellular amastigote forms of Leishmania donovani. Methodology/Principal findings The dose dependent effect and cytotoxicity of the compounds were determined by the MTT assay. Flow cytometry was used to determine the effect of the compounds on the cell cycle. Parasite morphological analysis was done by microscopy and growth kinetic studies were conducted by culturing cells and counting at 24 hours intervals over 120 hours. The cellular levels of iron in promastigotes treated with compounds was determined by atomic absorption spectroscopy and the effect of compounds on the expression of iron dependent enzymes was investigated using RT-qPCR. The IC50 of the compounds ranged from 16.34 M to 124 M compared to amphotericin B and deferoxamine controls. Rosmarinic acid and apigenin were the most effective against the promastigote and the intracellular amastigote forms. Selectivity indexes (SI) of rosmarinic acid and apigenin were 15.03 and 10.45 respectively for promastigotes while the SI of 12.78 and 5.20 respectively was obtained for intracellular amastigotes. Morphologically, 70% of rosmarinic acid treated promastigotes showed rounded morphology similar to the deferoxamine control. About 30% of cells treated with apigenin showed distorted cell membrane. Rosmarinic acid and apigenin induced cell arrest in the G0/G1 phase in promastigotes. Elevated intracellular iron levels were observed in promastigotes when parasites were treated with rosmarinic acid and this correlated with the level of expression of iron dependent genes. Conclusions/Significance The data suggests that rosmarinic acid exerts its anti-leishmanial effect via iron chelation resulting in variable morphological changes and cell cycle arrest. Author summary The available drugs used in treatment of leishmaniasis, a disease caused by the Leishmania parasite, include pentavalent antimonials and amphotericin B. The cost and associated toxicity of the drugs, side effects and the emergence of drug resistance parasites, as well as the discomfort in drug administration suggests the need for new and better chemotherapeutic interventions. Iron is involved in several biological processes in the Leishmania parasite and it's important for its growth and survival. The effects of using iron chelators to deprive parasite of iron was investigated in this study. Treatment of L. donovani with rosmarinic acid and apigenin inhibited the growth of promastigotes and intracellular amastigotes in a dose dependent manner. Morphologically, the compounds induced alterations in the parasites upon treatments. Rosmarinic acid was observed to cause the dysfunction of the mitochondria and alter the expression of iron dependent enzymes. Rosmarinic acid ability to chelate iron could be responsible for the changes in cell morphology and cell cycle observed.