Limonene and carvacrol combined with drugs that inhibit ergosterol synthesis are potent against Leishmania major

Leishmaniases are neglected diseases widely distributed in tropical and subtropical countries, caused by parasites of the Leishmania genus. Current therapy includes pentavalent antimonial and amphotericin B, but adverse effects persist. The development of a new, more selective, and less toxic combination therapy is therefore a rational and promising approach. In this study, we report the effects of combinations of the monoterpenes limonene (Lim) and carvacrol (Car) with drugs targeting ergosterol biosynthesis, namely nystatin (Nys), tioconazole (Tio), and rosuvastatin (Ros), on Leishmania major, their cytotoxicity on macrophages, and we evaluate their synergism and mechanisms of action. The combinations inhibited the growth of L. major promastigotes, with Lim-Car combined with nystatin (3:2) exhibiting the highest activity, showing an IC₅₀ of 2.02 µg/mL. Furthermore, this combination demonstrated greater action against amastigotes (IC₅₀ of 0.53 µg/mL) and a high selectivity index (33.60). Low cytotoxicity was observed in murine macrophages (CC₅₀ 17.81 µg/mL), as well as a low hemolytic potential (CH₅₀ of > 100 µg/mL). The Lim-Car and nystatin (3:2) combination presented a synergistic effect, with a fractional inhibitory concentration index of 0.4. Changes in parasite membrane integrity were also observed, which may be linked to the interaction of nystatin with the enzyme 14-alpha demethylase, along with an increase in TNF-α expression and a reduction in IL-10 and IL-6 levels. These results suggest that the combination of Lim-Car with Nys (3:2) may exert a summative effect through multiple biochemical pathways and warrants further investigation as a potential combined therapy with antileishmanial activity.