Toxicological and Sedative Effects of Chipilin (Crotalaria longirostrata) Leaf Extracts Obtained by Maceration and Supercritical Fluid Extraction

Chipilin (Crotalaria longirostrata) is consumed as a vegetable in the preparation of traditional dishes. As a folk medicine, Chipilin extracts are used as a hypnotic and sedative agent; however, there are few reports that support these uses. This study aimed to characterize the compounds present in Chipilin leaf extracts and to investigate their sedative effect using zebrafish as an in vivo model. Extracts were obtained by maceration with water (H₂O), ethanol (EtOH), and EtOH-H₂O, while oleoresin was obtained by supercritical fluid extraction (SFE). Total phenolic and flavonoid contents were quantified by colorimetric methods. Phytochemical constituents were identified by gas chromatography-mass spectrometry (GC-MS) analysis. The chronic and acute toxicities of Chipilin extracts were tested in zebrafish embryos and larvae, respectively. Chipilin sedative effect was tested by the larvae response to dark-light-dark transitions. EtOH-H₂O extracts had the highest value of total phenolics (5345 ± 5.1 μg GAE/g), followed by water and oleoresin (1815 ± 5.1 and 394 ± 5.1 μg GAE/g, respectively). In water extracts were identified the alkaloid trachelanthamidine, 1,2β-epoxy- and the alkyl ketone 7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, while oleamide, α-monostearin, and erucamide were detected in all samples except in water extracts. Oleoresin extract had the lowest embryotoxicity (LC₅₀ = 4.99 μg/mL) and the highest sedative effects. SFE is a green alternative to obtain Chipilin extracts rich in erucamide, an endocannabinoid analogue, which plays an important role in the development of the central nervous system and in modulating neurotransmitter release.