Pinus sp. leaf extracts exert antileishmanial effects against Leishmania donovani by targeting trypanothione reductase

The urgent need for new antileishmanial drugs with novel mechanisms of action is driven by the limited efficacy, high toxicity, and growing resistance to currently available treatments. This study investigates the antileishmanial potential of extracts from Pinus sp., a plant widely used in the Cameroonian pharmacopoeia to treat various infections, including cough and fever conditions. The antileishmanial activity of the ethanol, methanol, hydro-ethanol, and hydro-methanol extracts of Pinus sp. leaves were evaluated against Leishmania donovani promastigotes and amastigotes using a resazurin-based assay. The most active extract was then subjected to UHPLC-LC-MS/MS metabolomic profiling. Cytotoxicity, immunomodulatory, antioxidant, and anti-inflammatory properties of Pinus sp. extracts were also investigated. Moreover, the pharmacokinetic profile and molecular interactions of the identified compounds were predicted through in silico experiments using trypanothione reductase as the target enzyme. The acute toxicity study of the most promising extract (ethanol extract) was performed at 2000 and 5000 mg/kg using the organization for economic co-operation and development (OECD) guidelines, number 423. The plant extracts inhibited the growth of L. donovani with IC50 values ranging from 6.45 to 15.78 {micro}g/mL and 16.11 to 24.44 {micro}g/mL in promastigotes and amastigotes, respectively. These extracts also showed antioxidant, immunomodulatory, and anti-inflammatory effects. The extracts indicated no cytotoxicity and high selectivity (SI >10) toward Raw264.7 and Vero CRL1586 cells. The ethanolic extract showed a lethal dose (LD) value greater than 5000 mg/kg, thus highlighting its non toxicity upon the acute toxicity test. The metabolomic profiling of the ethanol extract identified several compound classes, including flavanone O-glycosides (e.g. epiafzelechin trimethyl ether), alkaloids (e.g. harmane), and diterpenes (e.g. abietic acid). Further molecular docking studies revealed strong binding affinities of these compounds to trypanothione reductase, thus suggesting the inhibitory potential of these compounds toward the target enzyme. Overall, extracts from Pinus sp. leaves demonstrate promising antileishmanial effects. However, isolation and characterization of the antileishmanial compounds from Pinus sp. leaf, detailed pharmacokinetics, and mechanisms of action are warranted for the successful utilization of this plant in antileishmanial drug discovery.