In vitro and in vivo anti-inflammatory and antiophidic effects of the extract and fraction of Eugenia unihora

Ethnopharmacological relevance: Eugenia uniflora Linn, popularly known as 'pitanga', is a native plant endemic to Brazil that belongs to the Myrtaceae family. Its traditional use (leaves infusion) has been reported for the treatment of different diseases, including hypertension, inflammation, and as a diuretic agent. Considering the snakebite problem and the rich molecule repertoire of this herbal species, studies that evaluate its antiophidic potential are relevant for a broad social impact. Aim of the study: This approach aims to evaluate the anti-inflammatory and antiophidic potential in vitro and in vivo of the extract (aqueous) and a fraction (ethyl acetate) of E. unihora leaves against Bothrops leucurus and Bothrops brazili venoms. Materials and methods: Extract and fraction from E. unihora leaves were obtained by turbo-extraction and partitioning. The cytotoxicity was assayed on normal cell lines (Vero E6 and 3T3) using the 3-methyl-[4-5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method. The anti-inflammatory activity of the aqueous extract was analyzed in vivo in the zymosan-induced air pouch model, and the leukocytes migration and other molecular inflammatory mediators quantified (myeloperoxidase, total protein, pro-inflammatory cytokine, malondialdehyde, and glutathione). In vitro, the antiophidic effect was evaluated by the ability of the E. unihora extract and fraction to inhibit the enzymatic action (proteolytic, phospholipase A2, and hyaluronidase) of B. leucurus and B. brazili venoms. In addition, the antiophidic action in vivo was investigated after treatment with E. unihora extract and fraction (50, 100, and 200 mg/kg) in the B. leucurus venom-induced paw edema with an evaluation of the antiedematogenic effect and quantification of myeloperoxidase (MPO) and pro-inflammatory cytokine levels. Results: The E. unihora leaves extract (7.8-125 mg/mL) revealed no toxicity in cell culture, but reduced MTT by 47% at the highest concentration (250 mg/mL) in Vero E6 cells. In contrast, the E. unihora fraction (7.8-250 mg/ mL) showed no cytotoxicity for both cell lines. In the air pouch model, E. unihora leaves extract demonstrated anti-inflammatory activity, reducing cell migration, MPO activity, protein, malondialdehyde, and proinflammatory cytokines, and increased glutathione levels. Evaluating the antiophidic action in vitro, E. unihora extract and fraction inhibited the proteolytic, phospholipase, and hyaluronidase effects of B. leucurus and B. brazili venoms at low concentrations. In addition, the extract and fraction also demonstrated in vivo antiophidic activity by reducing edema in the first 0.5 h after treatment, besides reducing MPO and proinflammatory cytokines levels. Conclusion: E. unihora leaves extract showed cytotoxicity only at the highest concentration while the fraction revealed no toxic effect in vitro. This approach showed for the first time that the aqueous extract and ethyl acetate fraction of E. unihora leaves has similar antiophidic action in vitro and in vivo, with antiedematogenic and antiinflammatory effects and the ability to inhibit the enzymatic action of B. leucurus and B. brazili venoms. Therefore, this study points to the presence of bioactive components in the leaves of E. unihora useful for the