Larvicidal mechanism of the rare sesquiterpene ishwarol B from Piper alatipetiolatum (Piperaceae) against Aedes aegypti, Anopheles darlingi, and Culex quinquefasciatus (Culicidae): oxidative damage, defense enzyme modulation, and acetylcholinesterase inhibition

Dengue, Oropouche, lymphatic filariasis, and malaria remain serious public health problems in Brazil, transmitted by Aedes aegypti, Culex quinquefasciatus, and Anopheles darlingi resistant to pyrethroid insecticides. As alternatives, plant-derived natural insecticides have gained attention for their potential. In this study, we evaluated ishwarol B for its larvicidal activity and explored its mechanism of action against these mosquitoes. Ishwarol B exhibited larvicidal activity against all larvae (LC₅₀ from 19.57 to 26.23 μg/mL) and a residual effect of approximately 50 % observed, accompanied by increased production of H₂O₂ (24.3 ± 5 to 41.0 ± 5 μmol of H₂O₂ per gram⁻¹), lipid peroxidation (11.00 ± 2 to 22.67 ± 2 ηmol of MDA per gram⁻¹), and protein oxidation (10.00 ± 1 to 17.00 ± nM of reactive carbonyls/mg protein). Additionally, there was an increase in the activity of SOD (14.33 ± 2 to 14.67 ± 8 mU/mg protein), CAT (10.47 ± 9 to 11.23 ± 1 mmol of H₂O₂ consumed/min/mg of protein), and GPx (11.91 ± 1 to 16.46 ± 2 mmol NADPH/min/mL). In contrast, AChE activity decreased (8.33 ± 1 to 6.67 ± 2 μmol/min/mg of protein), compared to negative control DMSO (105.30 ± 2 to 111.30 ± 3 μmol μmol/min/mg of protein), indicating enzymatic inhibition, further confirmed by an inhibition from 100 ± 0 to 4.41 ± 2 % (IC₅₀ of 3.46 μg/mL). Molecular docking revealed a binding energy of −8.4 kcal/mol and Ki = 0.695 μM, indicating a favorable interaction between ishwarol B and the AChE active site including Trp441, Tyr493, Tyr494, Ser280, Phe449, Ile446, Leu444, Gly445, Pro450, and Cys447. These findings confirm the broad mechanism of action of ishwarol B, highlighting its potential as a natural larvicide against the mosquito vectors investigated.