Evaluation of the Effects of Pomegranate Peel Aqueous Extract Against <i>Leishmania major</i>: A Randomized and Controlled Trial

Background: Leishmaniasis remains a major global health issue, with current treatments often limited by side effects, drug resistance, prolonged treatment durations, and high costs. These challenges have driven interest in alternative therapies. Objectives: The present study aimed to assess the in vitro and in vivo effects of pomegranate peel aqueous extract against Leishmania major. Methods: The study was a randomized, controlled, parallel-group trial with a 1:1:1:1:1 allocation ratio. Forty healthy female BALB/c mice (6 - 8 weeks old, 40 - 50 g) were subcutaneously infected with 2 × 106 L. major promastigotes. Once lesions developed, mice were randomly assigned to five groups (n = 8 per group). Randomization and blinding of outcome assessors were employed to minimize bias. Four groups received topical applications of pomegranate peel aqueous extract at concentrations of 2.5%, 5%, 20%, or 40%, while the control group received physiological serum. Treatments were applied directly to the lesion using a sterile cotton swab, twice daily for 30 days. The primary outcome was the change in lesion size, measured with a digital caliper at baseline and day 30. Secondary outcomes included the in vitro viability of L. major promastigotes after 72-hour exposure to extract concentrations (0.1 - 0.9 mg/mL), evaluated by trypan blue exclusion assay using a Neubauer chamber. Paired t-tests compared pre- and post-treatment lesion sizes within groups, and one-way ANOVA with post-hoc analysis compared between groups. A P ≤ 0.05 was considered statistically significant. Results: The extract inhibited L. major promastigote growth at concentrations between 0.3 and 0.9 mg/mL in vitro. In vivo, lesion size reductions were dose-dependent. Treatment with 20% extract led to a 27% reduction in lesion size (from 0.37 ± 0.03 cm to 0.27 ± 0.03 cm; mean difference = 0.10 cm, 95% CI: 0.07 - 0.13 cm, P < 0.05). The most significant effect was observed with 40% extract, resulting in an 84.7% reduction in lesion size (from 4.90 ± 0.75 cm to 0.75 ± 0.11 cm; mean difference = 4.15 cm, 95% CI: 3.85 - 4.45 cm, P < 0.05). Lower concentrations (2.5% and 5%) showed no statistically significant difference compared to the control group (P > 0.05). Conclusions: Pomegranate peel aqueous extract appears to be a promising treatment for cutaneous leishmaniasis (CL). However, the study has several limitations, including the absence of a standard anti-leishmanial drug as a positive control, potential imprecision in dosing due to percentage-based concentrations, and the use of a crude extract without quantification of active compounds. Further studies should compare extract types and identify specific therapeutic components.