Aedes aegypti and Anopheles darlingi are the primary vectors of dengue and malaria in Brazil. Natural products are currently regarded as promising alternatives for their control, offering environmentally friendly solutions for larval management due to their low toxicity to non-target organisms. Thus, Piplartine, isolated for the first time from Piper purusanum, exhibited larvicidal activity against Ae. aegypti and An. darlingi (LC₅₀ of 14.56 and 26.44 μg/mL), occasioned by the overproduction of reactive oxygen and nitrogen species (66.67 ± 7% and 86.33 ± 6%). Furthermore, piplartine enhanced the activity of key detoxifying enzymes, including catalase (87.00 ± 9 and 94.67 ± 9 μmol of H₂O₂ consumed per minute per mg of protein), glutathione S-transferase (76.00 ± 1 and 134.00 ± 1 μmol/min/mg), mixed-function oxidase (26.67 ± 5 and 55.00 ± 1 nmol cti mg⁻¹ protein), α-esterase, and β-esterase (27.67 ± 7 to 46.33 ± 1 nmol cti mg⁻¹ protein). In contrast, piplartine inhibited acetylcholinesterase activity (43.33 ± 7 and 48.00 ± 2 μmol/min/mg) compared to the negative control DMSO (87.33 ± 1 and 146.30 ± 3 μmol/min/mg). It is important to highlight that piplartine showed no lethal effects on non-target aquatic insects, with 100% survival observed at a concentration of 264.4 μg/mL. In contrast, α-cypermethrin demonstrated acute and rapid toxicity to non-target organisms, with only 9.1% survival. These findings highlight piplartine as a promising larvicide with selective toxicity and low environmental impact, suitable for integrated larval management strategies.

Background/Objectives: In the Brazilian Amazon, which accounts for over 99% of national malaria cases, 34,260 cases were reported as of August 2025, predominantly caused by Plasmodium vivax, responsible for 86.69% of the infections. The increasing resistance of the parasite to conventional therapies highlights the urgent need for novel control strategies, with essential oils and plant-derived substances emerging as promising alternatives. Methods: In this context, we evaluated the anti-Plasmodium potential of Piper alatipetiolatum essential oil and its major constituent 6-ishwarone against P. vivax, including cytotoxicity in Vero and PBMCs, molecular docking on dihydrofolate reductase (DHFR) and lactate dehydrogenase (LDH), and in silico pharmacokinetic profiling. Results: Both the oil and 6-ishwarone inhibited P. vivax dose-dependently (2.1 ± 1 to 100%), with IC₅₀ values of 9.25 µg/mL and 3.93 µg/mL, respectively. Importantly, no cytotoxic effects were observed at 24 h, with cell viability ranging from 94.7% to 98.3%, highlighting the selectivity of these compounds towards the parasite over mammalian cells. Docking studies indicated selective binding of 6-ishwarone to DHFR (−7.7 kcal/mol; Ki = 2.27 µM) with key interactions (Trp816, Lys820, Tyr819, Asn823, Thr865), whereas binding to LDH was weaker (−6.2 kcal/mol; Ki = 28.10 µM), suggesting DHFR as the primary molecular target. In silico ADMET predictions and experimental data indicated favorable drug-like properties: TPSA = 20.23 Ų, moderate lipophilicity (LogP = 3.37), soluble (ESOL Log S = −3.58; Ali Log S = −3.89; Silicos-IT Log S = −2.84), high gastrointestinal absorption, BBB permeability (0.985), not a P-glycoprotein substrate (0.11), and low likelihood of CYP inhibition. Toxicity predictions showed non-mutagenic and non-hepatotoxic effects, low cardiotoxicity (hERG inhibition risk 0.08–0.32), low reproductive toxicity (0.03), moderate neurotoxicity (0.28), low acute toxicity (oral LD₅₀ = 2.061 mol/kg), and low chronic toxicity (LOAEL = 1.995 log mg/kg/day). Conclusions: Together, these findings demonstrate that essential oil and 6-ishwarone of P. alatipetiolatum are selective, bioavailable, and promising natural leads for antimalarial drug development.