Synergistic anti-leishmanial effects of Rosmarinic acid and Meglumine Antimoniate against Leishmania major: Integrative in silico, in vitro, and immunomodulatory investigations.

Cutaneous leishmaniasis (CL), mainly caused by Leishmania major, remains a major public health concern, particularly in endemic regions. The clinical use of pentavalent antimonials such as meglumine antimoniate (MA) is limited by toxicity, resistance, and treatment failure. Rosmarinic acid (RMA), a natural polyphenolic compound, exhibits antimicrobial and immunomodulatory properties that may enhance antileishmanial efficacy. The antileishmanial activity of RMA alone and in combination with MA was evaluated in vitro against promastigote and intracellular amastigote forms of L. major using MTT and microscopic assays. Cytotoxicity was assessed in murine macrophages (J774-A1). Drug interactions were analyzed using the Chou-Talalay combination index (CI) method. Molecular docking was performed to explore the potential interaction of RMA with immune-related proteins (IL-12p40, IFN-γ, and iNOS). Immunomodulatory effects were evaluated by quantitative real-time PCR analysis of Th1 and Th2 cytokine gene expression in treated intra-macrophages. RMA exhibited dose-dependent antileishmanial activity with an appropriate safety index (SI > 10). The RMA-MA combination demonstrated synergistic effects (CI < 1) at several concentrations against both promastigote and amastigote forms. Docking analysis suggested favorable binding of RMA to immune-related targets. Gene expression analysis revealed significant upregulation of Th1-associated markers (IL-12p40, IFN-γ, iNOS) and downregulation of Th2-associated cytokines (IL-4 and IL-10). RMA enhances the antileishmanial efficacy of MA through synergistic antiparasitic activity and modulation of host immune responses. These findings support the potential of RMA as a natural adjunct to conventional antimonial therapy, warranting further in vivo investigation.