Larvicidal and Toxicological Properties of Insect-Repellent Essential Oils and their Main Constituents

Abstract

The female Anopheles mosquito, the principal vector of malaria, is increasingly developing resistance to conventional insecticides. As malaria cases continue to rise globally, there is a critical need to explore safer and more sustainable alternatives. In this context, natural products have emerged as a valuable source of bioactive molecules with potential insecticidal activity. Essential oils derived from plants possess diverse biological properties, including insecticidal activity, which is attributed to their chemical constituents. The aim of this study was to determine the larvicidal properties of insect-repellent essential oils and their key constituents, whilst determining their preliminary toxicity profile (haemolysis, Artemia franciscana nauplii lethality, free radical scavenging activity), possible mechanism of action (metal chelating properties) and in silico pharmacokinetic and toxicity studies. The essential oils selected for this study include black pepper (Piper nigrum), clove-leaf (Syzygium aromaticum), sweet basil (Ocimum basilicum), thyme (Thymus vulgaris) and oregano (Origanum vulgare). The main constituents of these essential oils were identified using Gas Chromatography-Mass Spectrometry (GC-MS). Additionally, eugenol, a common essential oil constituent, along with its derivatives, was investigated. Out of 17 tested compounds - including essential oils, essential oil constituents, eugenol and its derivatives, 16 exhibited potential larvicidal activity with LC50 values ranging from 8.66 μg/mL to 66.11 μg/mL at 72 hours of exposure, compared to standard insecticide, propoxur (LC50 value: 0.04 ± 0.01 μg/mL). Eugenol and its derivatives displayed low to moderate larvicidal activity, with isoeugenol exhibiting potential time-dependent mortality. Among the essential oils oregano (LC50 value: 10.42 ± 1.56 μg/mL) and thyme (LC50 value: 17.00 ± 4.58 μg/mL) were the most effective against the An. arabiensis larvae at 72 hours of exposure; however, they exhibited greater toxicity towards the Artemia franciscana with LC50 value of 4.21 ± 0.55 μg/mL and 2.42 ± 0.00μg/mL, respectively, compared to propoxur. The essential oils constituents, γ-terpinene (LC50 value: 8.66 ± 2.35 μg/mL at 72 hours) and p-cymene (LC50 value: 12.68 ± 1.28 μg/mL at 72 hours) emerged as the most potent larvicidal compounds while demonstrating lower toxicity to A. franciscana (% mortality range: 22.27% to 41.66%; from 24 to 72 hours) and minimal haemolytic (<22%) effects and low antioxidant activity (<0.1% to 0.20%) at high concentrations. When combined with propoxur at various concentration ratios, they demonstrated enhanced larvicidal activity. However, their mode of action against Anopheles vectors remains unclear, as they showed no significant metal- chelating activity (iron chelation: <0.1% to 0.60%; copper(II) chelation: <0.1 ± 0.01%). In silico studies found that the essential oil constituents are low class I toxic hazards with ECOSAR toxicity predicting higher toxicity than Artemia nauplii lethality results. Further research is necessary to optimise the efficacy of these essential oil constituents and elucidate their mechanism of action.