Phytochemical Profile, Toxicity Evaluation and Antinociceptive Effect of the n-Butanolic Fraction from the Leaves of Calotropis procera (Aiton) W.T Aiton (Apocynaceae)

<i>Calotropis procera</i>, known as "Silk cotton", stands out for the presence of various classes of bioactive compounds responsible for its ethnopharmacological properties. The study aimed to conduct a phytochemical investigation, evaluating the in vitro and in vivo toxicity together with the antinociceptive potential of an n-butanolic fraction (FB) from the leaves. The crude ethanolic extract (CEE) was obtained by maceration in ethanol for 72 h. It was then partitioned using a gradual solvent sequence. The FB was analyzed by HPLC-ESI-MS/MS in negative mode and ¹H and ¹³C NMR. Toxicity was assessed by the erythrocyte hemolytic assay and acute oral toxicity test at a single dose of 300 mg·kg^-1. The antinociceptive effect was assessed by the acetic acid-induced abdominal writhing test and the formalin test in mice at doses of 3.75, 7.5 and 15 mg·kg^-1<i>per os</i>. HPLC-ESI-MS/MS analysis identified flavonoids, phenolic acids, and the megastigmane roseoside, isolated for the first time in <i>C. procera</i>. The FB did not cause hemolytic effects or behavioral or physiological changes in mice. It showed an antinociceptive effect at all doses, reducing abdominal writhing by up to 91.46% and the licking time in phases 1 and 2 of the formalin test by up to 63.83% and 91.73%, respectively. In this study, it was possible to determine that an FB of a crude extract of <i>C. procera</i> leaves has antinociceptive activity, possibly associated with the phenolic compounds and roseoside found, with a lack of toxicity <i>in vitro</i> and <i>in vivo</i>, validating its ethnopharmacological use.