The study, published in Frontiers in Parasitology, examined ethyl acetate extracts prepared from Terminalia catappa (Indian almond) leaves and from the seeds and stems of Eucalyptus citriodora (lemon-scented gum). Researchers led by Eze Elijah Ajaegbu of David Umahi Federal University of Health Sciences set out to fill a notable gap in the botanical pesticide literature, which has historically favored leaf assays while largely ignoring other plant organs. Their comparative evaluation across three medically important mosquito species—Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus—provides one of the more comprehensive simultaneous bioassays reported to date.

The team collected plant material from two Nigerian locations: T. catappa foliage from Trans-Ekulu in Enugu State and E. citriodora stems and seeds from Lakokari Farm in Gombe State. After taxonomic authentication and voucher deposition, the dried materials were milled into fine powder and subjected to cold maceration with ethyl acetate for 48 hours. Extraction yields varied by organ: the seed extract (LEEE) produced the highest recovery at 4.7 percent by weight, the leaf extract (TLEE) yielded 3.3 percent, and the stem extract (LSEE) gave the lowest at 2.8 percent. Interestingly, yield did not predict potency—a theme that would recur throughout the study.

Larvicidal bioassays were conducted on fourth-instar larvae using a concentration gradient spanning 125 to 1000 parts per million, with mortality assessed at 24 and 48 hours. Groups of 25 larvae were exposed in quadruplicate, and results were corrected using Abbott’s equation. The findings were striking. Against Ae. aegypti, the dengue and Zika vector, the eucalyptus stem extract proved the most aggressive toxicant, achieving complete 100 percent mortality at 1000 ppm within 24 hours and posting an LC50 of 172.851 ppm. By 48 hours, its LC50 had dropped further to 144.393 ppm, with total population elimination persisting down to 500 ppm.

The malaria vector An. gambiae showed even greater vulnerability over longer exposure. After 48 hours, the T. catappa leaf extract recorded an extraordinary LC50 of just 13.271 ppm, killing 96 percent of larvae at a mere 125 ppm and wiping out entire cohorts at 250 ppm and above. The eucalyptus stem extract was nearly as potent, with an LC50 of 20.370 ppm and complete mortality from 250 ppm upward. The seed extract, while weaker, still reached an LC50 of 84.841 ppm at the same time point. These figures suggest that even modest concentrations of these botanical preparations could deliver lethal blows to malaria-transmitting larvae given sufficient contact time.

Culex quinquefasciatus, the filariasis vector, presented a different vulnerability hierarchy. The T. catappa leaf extract dominated here, eradicating entire cohorts at 1000 ppm within 24 hours and maintaining an LC50 of 20.370 ppm at 48 hours with complete mortality from 250 ppm onward. The eucalyptus seed extract consolidated its performance over time, reaching an LC50 of 49.090 ppm by 48 hours, while the stem extract lagged behind with an LC50 of 302.323 ppm. The species-specific differences likely reflect variations in larval detoxification enzymes, cuticular permeability, and receptor sensitivities—factors that underscore the importance of testing botanical larvicides against multiple vector species before field deployment.

Phytochemical screening revealed the chemical underpinnings of these effects. Tannins appeared uniformly across all three extracts, but the eucalyptus derivatives carried a richer metabolic load, concentrating flavonoids, resins, and steroids. Alkaloids were detected exclusively in the stem tissue, while saponins were absent from all samples. The researchers explain that these compound classes act through complementary mechanisms: alkaloids inhibit acetylcholinesterase, causing continuous nerve firing, muscle spasms, and paralysis; flavonoids and tannins bind digestive enzymes in the larval midgut, disrupting nutrient assimilation and inducing oxidative stress; and steroids and resins can compromise membrane integrity and arrest developmental processes.

Gas chromatography–mass spectrometry (GC-MS) profiling of the T. catappa leaf extract resolved 16 discrete volatile compounds, dominated by fatty acids and their methyl esters—including n-hexadecanoic acid, 9,12-octadecadienoic acid, and hexadecanoic acid methyl ester—alongside nitrogenous heterocycles such as 2-methyl-7-phenylindole and 7-methyl-7H-purin-6-amine. Prior research links these lipophilic fatty acid configurations to membrane disruption and energy production failure in insects. The authors argue that the broad-spectrum toxicity likely arises from synergistic interactions among multiple metabolites rather than the action of any single molecule, a hallmark of botanical insecticides that also complicates the evolution of resistance in target populations.

The study’s implications extend beyond the laboratory. With malaria claiming approximately 608,000 lives and causing 249 million cases in 2022 according to the World Health Organization, and dengue now endemic in over 100 countries, the search for sustainable alternatives to synthetic neurotoxicants has become urgent. Botanical larvicides degrade rapidly in the environment, pose reduced risks to non-target organisms, and employ multiple modes of action that delay resistance development. The U.S. Environmental Protection Agency has already classified lemon eucalyptus oil as a biopesticide candidate for integrated vector management, lending regulatory precedent to this approach.

Nevertheless, the authors caution that laboratory results do not automatically translate to field success. Sunlight, water chemistry, microbial degradation, and temperature fluctuations may all alter larvicidal performance under natural breeding conditions. Moreover, because the extracts were evaluated as complex mixtures, the individual contribution of each identified compound remains unknown. Future work, they note, should focus on bioassay-guided fractionation, purification of active constituents, elucidation of specific mechanisms of action, toxicity assessment toward non-target aquatic organisms, formulation studies to improve stability, and ultimately field evaluation. For now, the findings position Terminalia catappa and Eucalyptus citriodora as promising candidates in the ongoing quest for eco-friendly mosquito control tools.

Subject of Research: Larvicidal activity and GC-MS chemical profiling of Terminalia catappa leaf and Eucalyptus citriodora seed and stem extracts against Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus mosquitoes

Article Title: GCMS profiling and larvicidal potentials of the extracts of Terminalia catappa leaf, Eucalyptus citriodora seed and stem against malarial, dengue and filarial vector mosquitoes (Diptera: Culicidae)

Article References: Ajaegbu, E. E., Nwigwe, J. O., Chikezie, C. C., & Enyi, N. C. (2026). GCMS profiling and larvicidal potentials of the extracts of Terminalia catappa leaf, Eucalyptus citriodora seed and stem against malarial, dengue and filarial vector mosquitoes (Diptera: Culicidae). Frontiers in Parasitology, 5, Article 1934443. https://doi.org/10.3389/fpara.2026.1934443

Image Credits: AI Generated

DOI: 10.3389/fpara.2026.1934443

Keywords: mosquito larvae, botanical insecticides, Terminalia catappa, Eucalyptus citriodora, larvicidal activity, GC-MS analysis, vector control, malaria, dengue, lymphatic filariasis, phytochemicals, insecticide resistance

Natural deep eutectic solvents, or NaDES, have emerged in recent years as one of the most promising tools in green chemistry. These liquids are formed when certain natural compounds, such as sugars, alcohols, and organic acids, are mixed together in specific ratios, causing them to melt into a stable liquid through an extensive network of hydrogen bonds. Unlike many conventional organic solvents, NaDES are typically non-toxic, biodegradable, inexpensive, and tunable in polarity, which makes them attractive replacements for the volatile organic solvents that have long dominated phytochemical extraction. The United Nations has explicitly called for a transition away from traditional organic solvents as part of its 2030 Agenda for Sustainable Development, and NaDES chemistry is one of the leading answers to that call.

The research team selected two contrasting NaDES for their investigation. The first was a hydrophilic mixture of glycerol and choline chloride, abbreviated GCC, prepared by heating the two components in a one-to-three molar ratio at 70 degrees Celsius until a clear viscous liquid formed. The second was a hydrophobic blend of camphor and thymol, abbreviated CT, which liquefied within ten minutes at 35 to 40 degrees Celsius in a one-to-one ratio. Because deep eutectic solvents are inherently viscous and struggle to penetrate plant tissue on their own, the researchers diluted the GCC system with water and the CT system with acetonitrile, lowering viscosity and improving the solvents’ ability to reach the phytochemicals locked inside the leaf matrix.

To squeeze the maximum performance out of each solvent system, the team turned to microwave-assisted extraction, a technique that heats the solvent and plant material from the inside out. Microwaves interact with polar molecules by inducing dipole rotation, generating heat directly within the extraction medium. This localized heating builds internal pressure inside plant cells, rupturing cell walls and releasing intracellular compounds far faster than conventional stirring or reflux methods. The approach conserves energy, slashes extraction times to mere seconds, and reduces the risk of degrading heat-sensitive molecules because the total exposure window is so short. In this study, powdered leaves were combined with 30 milliliters of the solvent-diluent mixture in a conical flask and irradiated in a domestic microwave oven at controlled power and duration settings.

Optimizing the process required a statistical framework capable of mapping how multiple variables interact simultaneously. The researchers employed response surface methodology using a Box-Behnken design, which generated 17 experimental runs per solvent system with five replicates at central points. Three factors were varied across three levels each: the concentration of the NaDES in its diluent, the microwave power ranging from 220 to 440 watts, and the irradiation time ranging from 20 to 40 seconds. Five responses were measured for every run: total phenolic content expressed as gallic acid equivalents, total flavonoid content expressed as rutin equivalents, DPPH radical scavenging activity expressed as ascorbic acid equivalents, ABTS radical cation scavenging activity expressed as Trolox equivalents, and metal iron chelating activity expressed as EDTA equivalents, all normalized to grams of dry leaf powder.

The results exposed a striking polarity-driven divide between the two solvent systems. The hydrophilic GCC system proved dramatically superior for phenolic recovery, achieving a predicted optimal total phenolic content of 137.49 milligrams of gallic acid equivalents per gram of dry weight, nearly three times the 48.35 milligrams obtained with the hydrophobic CT system. The GCC extracts also dominated in ABTS radical scavenging, reaching 11.57 milligrams of Trolox equivalents per gram, while the CT extracts managed only 0.17 milligrams. This makes chemical sense: the ABTS assay operates through single-electron transfer and preferentially detects highly polar antioxidant molecules, which dissolve readily in the hydrogen-bonding, water-compatible environment that glycerol and choline chloride provide.

The story reversed for flavonoids. The hydrophobic camphor-thymol system extracted 61.72 milligrams of rutin equivalents per gram, roughly five times the 12.17 milligrams achieved by GCC. Many flavonoids possess substantial hydrophobic aromatic ring systems, and the non-polar character of camphor and thymol enables favorable hydrophobic and pi-pi interactions with these structures. The CT system also edged out GCC slightly in DPPH radical scavenging, at 28.38 versus 27.08 milligrams of ascorbic acid equivalents per gram, and in metal chelating activity, at 23.39 versus 21.38 milligrams of EDTA equivalents per gram, suggesting that a meaningful fraction of the leaf’s lipophilic antioxidants and metal-binding ligands are better captured in the hydrophobic medium. Both systems produced comparable DPPH responses overall, indicating that radical-scavenging compounds were extracted efficiently across most processing conditions.

The statistical models underlying these findings proved highly reliable, with coefficients of determination exceeding 0.91 for every response and non-significant lack-of-fit tests confirming that the quadratic equations adequately described the experimental data. Solvent concentration emerged as the most influential variable, followed by microwave power and extraction time. For the GCC system, moderate DES concentration around 75 percent paired with 220 watts and 40 seconds produced optimal results with a desirability value of 1.0, and experimental validation showed relative standard deviations between 0.15 and 5.10 percent. For the CT system, the optimum landed at 60 percent DES concentration in acetonitrile, 220 watts, and 30 seconds, with validation deviations between 0.16 and 6.25 percent. The researchers explain that too much water in the GCC mixture dilutes its solubilizing power for moderately polar polyphenols, while too little water leaves the solvent too viscous to penetrate the biomass, so an intermediate composition balances fluidity and polarity. Similarly, acetonitrile lowers the viscosity of the hydrophobic CT solvent while adding just enough polarity to assist extraction.

Beyond simple solubility, the study highlights a deeper mechanism by which NaDES enhance recovery: direct attack on the plant cell wall. The chloride ions of the GCC mixture can form hydrogen bonds with the hydroxyl groups of cellulose, disrupting the wall’s architecture, while camphor and thymol interact with cell wall components in their own way. Combined with the internal pressure buildup from microwave dielectric heating, this dual assault on cellular structure accelerates the desorption and release of target compounds from the plant matrix. The authors conclude that microwave-assisted extraction coupled with carefully chosen NaDES offers a sustainable, rapid, and selective strategy for recovering antioxidant phytochemicals from Eucalyptus citriodora leaves, and that the choice between hydrophilic and hydrophobic solvents should be guided by the specific class of biomolecules a manufacturer or researcher wishes to target, whether for nutraceutical, pharmaceutical, or food applications.

Subject of Research: Microwave-assisted extraction of phenolic and antioxidant compounds from Eucalyptus citriodora leaves using hydrophilic and hydrophobic natural deep eutectic solvents

Article Title: Microwave-assisted extraction of phenolic and antioxidant compounds from Eucalyptus citriodora leaves using hydrophilic and hydrophobic natural deep eutectic solvents

Article References: Asif, M., & Ahmed, D. (2026). Microwave-assisted extraction of phenolic and antioxidant compounds from Eucalyptus citriodora leaves using hydrophilic and hydrophobic natural deep eutectic solvents. Discover Green Chemistry, 1(1), Article 21. https://doi.org/10.1007/s44509-026-00025-z

Image Credits: AI Generated

DOI: 10.1007/s44509-026-00025-z

Keywords: Eucalyptus citriodora, natural deep eutectic solvents, microwave-assisted extraction, green chemistry, polyphenols, flavonoids, antioxidant activity, response surface methodology, glycerol-choline chloride, camphor-thymol, DPPH, ABTS