Mechanistic evaluation of niosomal Rhoifolin in antileishmanial therapy and its additive potential with meglumine...

Leishmaniasis is a neglected tropical disease that urgently requires safer and more effective therapeutic strategies. Rhoifolin (RF), a natural flavonoid with anti-inflammatory and antiparasitic potential, has shown promising biological activities against several infectious diseases. In the present study, the antileishmanial effects of free RF and its niosomal formulation (NRF), alone and in combination with meglumine antimoniate (MA), were investigated against Leishmania major. Niosomes were prepared using Span 40, Tween 40, and cholesterol, resulting in high encapsulation efficiency (90.9%) and a sustained drug-release profile. The antileishmanial activity of RF, NRF, MA, and their combinations against promastigotes was evaluated using both MTT and trypan blue exclusion assays, while anti-amastigote activity was assessed in infected macrophages. NRF exhibited greater efficacy than free RF against both promastigote and amastigote forms of L. major. Combination treatments demonstrated additive interactions (combination index ≈ 1.0) and higher selectivity indices, indicating enhanced efficacy and safety. In addition, treatment with RF, NRF, and their combinations significantly increased LDH release and ROS production in infected macrophages compared with untreated controls. Molecular docking analysis revealed a strong binding affinity of RF toward caspase-3. Furthermore, treatment with RF and NRF significantly modulated apoptosis-related mediators, including increased expression of CASP3, CASP8, and Bax, along with reduced Bcl-2 expression. Immunological analyses also demonstrated macrophage polarization toward the M1 phenotype through upregulation of TNF-α, IFN-γ, IL-12, and iNOS/NO, accompanied by downregulation of IL-10, TGF-β, and ARG1. These immunomodulatory and apoptosis-associated effects were more pronounced in the NRF and NRF + MA groups. Overall, our findings suggest that niosomal RF represents a promising lipid-based delivery system for enhancing the antileishmanial efficacy of RF. The NRF + MA combination may provide a potential therapeutic strategy for improving treatment outcomes and reducing the limitations associated with current therapies for cutaneous leishmaniasis.