Stage-dependent activity of curcumin-rosmarinic acid against Leishmania donovani: Synergy in amastigotes

BACKGROUND: Leishmania donovani causes visceral leishmaniasis, a severe and fatal parasitic disease that affects the world's most impoverished populations. Iron metabolism is fundamental in parasite survival and host immunity. In this study, we investigated the potential use of iron-chelating compounds to inhibit intracellular L. donovani through host regulation of iron metabolism and macrophage activation. METHODS: Four phenolic compounds (curcumin, rosmarinic acid, apigenin, and hesperidin) were screened individually and in fixed-ratio combinations against L. donovani promastigotes and intracellular amastigotes cultured in RAW 264.7 murine macrophages. Drug interactions were assessed using the overall mean fractional inhibitory concentration index ([Formula: see text]ƩFICI) averaged across four fixed ratios. Mechanistic approaches involved measuring intracellular iron, reactive oxygen species (ROS) and nitrite production, macrophage cytokine profiles, and iron-regulatory gene expression. RESULTS: The Curcumin-rosmarinic acid (Cur-Ros) combination exhibited stage-dependent activity: additive effects against promastigotes ([Formula: see text]ƩFICI = 0.93) but synergistic effects, though close to the threshold, against intracellular amastigotes ([Formula: see text]ƩFICI = 0.49), with selectivity indexes (SI) ≥10. Treatments enhanced pro-inflammatory macrophage activation characterized by increased secretion of Tumor Necrosis Factor-alpha (TNF-α) and Monocyte chemoattractant protein-1 (MCP-1). There were decreased interleukin-10 (IL-10) levels, enhanced ROS and nitrite production, and reduced intracellular iron availability. Gene expression analysis indicated upregulation of NRAMP1 (SLC11A1, a phagosomal divalent metal transporter) and downregulation of ferritin, consistent with altered iron homeostasis. CONCLUSIONS: In this study, curcumin-rosmarinic acid combinations inhibited growth of intracellular L. donovani amastigotes synergistically, correlating with macrophage activation and altered iron homeostasis. These findings identify curcumin-rosmarinic acid as a synergistic combination warranting further investigation with appropriate formulation and in vivo validation.