Antinociceptive, anti-inflammatory, and antiophidic potential of Punica granatum leaves extract and fraction: an in vitro and in vivo evaluation

Punica granatum (Punicaceae) is a shrub with a worldwide distribution that has been used in folk medicine for the treatment of inflammatory disorders. The study investigated the therapeutic potential of spray-dried hydroethanolic extract (SDE) and spray-dried ethyl acetate fraction (EAF) from Punica granatum leaves in models of pain, inflammation, and snakebite envenomation. Phytochemical analysis highlighted the presence of polyphenols, particularly luteolin and hydrolysable tannins in SDE, and a significant concentration of flavonoids in EAF. Both samples demonstrated safety in murine fibroblast cultures, exhibiting no toxicity at concentrations up to 250 µg/mL. In vivo, EAF (25 mg/kg) showed remarkable peripheral antinociceptive activity, reducing abdominal writhings by 88.0%, comparable to morphine (87.0%), and superior to indomethacin (72.0%). In the formalin test, EAF reduced neurogenic and inflammatory pain by 80.9% and 87.3%, respectively, while SDE (200 mg/kg) reduced writhings by 76.7% and inflammatory pain by 57.1%. In the tail immersion test, both extracts increased latency time, confirming central antinociceptive effects. SDE and EAF also reduced carrageenan-induced edema and leukocyte migration by over 70%. In vitro, both samples completely inhibited the proteolytic and hyaluronidase activities of Bothrops brazili and Bothrops leucurus venoms and strongly inhibited PLA₂ activity. Notably, in vivo administration of SDE and EAF significantly reduced B. leucurus-induced paw edema and myeloperoxidase activity by up to 85.5%. These findings reinforce the traditional use of P. granatum leaves and provide the first report of their in vivo analgesic and antiophidic potential, supporting the role of their rich phenolic composition in modulating inflammatory and venom-induced responses.