Pioneering Investigation on the Larvicidal Mechanism and Chemical Profile of Piper humillimum C.DC. (Piperaceae) Essential Oil: Integrating In Vivo, In Vitro, and In Silico Models Against Aedes aegypti (Linnaeus, 1762) and Anopheles darlingi Root, 1926 (Culicidae)

Aedes aegypti and Anopheles darlingi represent health challenges due to synthetic insecticide resistance. Hence, the essential oil from Piper humillimum is an alternative for vector control. In this study, the essential oil (3.5 ± 0.4% yield) alongside germacrene D (61.51%) and δ-cadinene (17.46%) showed larvicidal activity (LC50 of 34.75 to 46.04 µg/mL), accompanied by an increase of hydrogen peroxide (H2O2) production (36.67 ± 1.52 to 81.33 ± 1.52 µmol H2O2 min−1 mg−1 protein), causing lipid (43.3 ± 6.02 to 81.67 ± 3.05 nmol malondialdehyde mg−1 protein) and protein damages (61.67 ± 6.80 to 83.00 ± 2.64 nmol carbonyls mg−1 protein). Further triggering an increase in superoxide dismutase (83.31 ± 6.80 to 95.00 ± 3.60 U mg−1 protein) and catalase (74.31 ± 7.02 to 82.09 ± 1.00 µmol H2O2 min−1 mg−1 protein) activities. In addition, mixed-function oxidases (61.17 ± 11.37 to 73.52 ± 6.42 nmol cyt c min−1 mg−1 protein), α- and β-esterase (38.41 ± 4.04 to 61.31 ± 9.29 µmol min−1 mg−1 protein) levels increased. Conversely, glutathione S-transferase (GST) (11.01 ± 2.00 to 9.67 ± 3.05 µmol min−1 mg−1 protein) and acetylcholinesterase (AChE) (14.33 ± 3.78 to 17.00 ± 1.00 μmol min−1 mg−1 protein) were inhibited, corroborated by molecular docking, with germacrene D and δ-cadinene showing binding energies of −7.9 and −7.9 kcal/mol, 1.63 and 1.94 Ki for AChE, while for GST were −6.4 and 6.6 kcal/mol, and 20.5 and 15.50 Ki, respectively. These results demonstrate that the essential oil from P. humillimum is a promising multi-target alternative for the control of the investigated vectors.