First Evidence of Anti-Plasmodium vivax (Plasmodiidae): Activity of the Essential Oil and 6-Ishwarone Isolated from Piper alatipetiolatum Yunck. (Piperaceae)

<b>Background/Objectives</b>: 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 <i>Plasmodium vivax</i>, 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. <b>Methods</b>: In this context, we evaluated the anti-<i>Plasmodium</i> potential of <i>Piper alatipetiolatum</i> essential oil and its major constituent 6-ishwarone against <i>P. vivax</i>, including cytotoxicity in Vero and PBMCs, molecular docking on dihydrofolate reductase (DHFR) and lactate dehydrogenase (LDH), and in silico pharmacokinetic profiling. <b>Results</b>: Both the oil and 6-ishwarone inhibited <i>P. vivax</i> 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). <b>Conclusions</b>: Together, these findings demonstrate that essential oil and 6-ishwarone of <i>P. alatipetiolatum</i> are selective, bioavailable, and promising natural leads for antimalarial drug development.