Plant Compound Andrograpanin Outperforms Insecticide Temephos in Mosquito Enzyme Docking Study

A compound extracted from the leaves of a medicinal herb long used in traditional Asian medicine may point the way toward a new generation of environmentally friendlier mosquito controls. In a study published in Discover Chemistry, researchers report the first molecular docking analysis of andrograpanin, a labdane diterpenoid purified from Andrographis paniculata, against acetylcholinesterase 1 (AChE1) from two of the world’s most medically important mosquito vectors: Aedes aegypti, the yellow fever mosquito, and Culex quinquefasciatus, the southern house mosquito. The computational results suggest that this plant molecule binds the mosquito nerve enzyme more favorably than temephos, the organophosphate insecticide that has anchored larval control programs for decades, though the authors are careful to stress that the mechanism remains a hypothesis awaiting laboratory confirmation.

The urgency behind the work stems from a familiar and worsening problem. Vector-borne diseases such as dengue, chikungunya, filariasis and West Nile fever continue to burden public health systems worldwide, while mosquito populations have grown steadily resistant to the synthetic chemicals deployed against them. Resistance typically arises through enhanced detoxification and reduced sensitivity of the target protein, eroding the effectiveness of organophosphates and carbamates that act on the cholinergic nervous system. Plant-derived phytochemicals, which are biodegradable, less persistent in the environment and often less toxic to non-target organisms, have attracted growing interest as alternatives. Previous studies have shown larvicidal effects of plant extracts ranging from Cassia fistula flowers to Calotropis procera leaves, but crude extracts blur the identity of the active molecule. What distinguishes the new study is its focus on a single, purified, structurally characterized compound.

Andrograpanin itself has an interesting pedigree. It is a deoxygenated analog of andrographolide, the principal bitter diterpenoid of A. paniculata, a plant widely known as the ‘king of bitters.’ For the study, fresh leaves were collected from Palayamkottai in Tamil Nadu, India, authenticated by the Siddha Central Research Institute in Chennai, and processed from 1.5 kilograms of shade-dried material. Successive Soxhlet extraction with hexane, chloroform and methanol yielded a methanol extract that showed the highest bioactivity. Silica gel column chromatography, eluted with a chloroform-ethyl acetate mixture, then delivered andrograpanin as a colorless crystalline solid melting at 103 to 104 degrees Celsius. Infrared, proton and carbon NMR, and GC-MS analyses confirmed a labdane diterpenoid with the formula C20H30O3 and a molecular mass of 318, featuring the gamma-lactone carbonyl and exomethylene groups characteristic of the compound.

Before any docking could begin, the team needed biological evidence that the molecule actually kills mosquitoes. Using standardized World Health Organization guidelines with five replicates per assay, and probit analysis to derive lethal concentrations, the researchers tested ovicidal, larvicidal and pupicidal activity against both species. The results were striking for a natural product. At just 2 parts per million, andrograpanin killed 72 percent of Ae. aegypti eggs and 78 percent of Cx. quinquefasciatus eggs. Larval LC50 values came in at 1.18 ppm for Ae. aegypti and 1.10 ppm for Cx. quinquefasciatus, with LC90 values of 2.74 and 2.55 ppm respectively. Pupae proved slightly hardier, with LC50 values of 1.33 and 1.25 ppm. These potencies, achieved by a single purified phytochemical at parts-per-million concentrations, provided the biological anchor for the computational question that followed: what molecular target might explain this activity?

The obvious candidate was acetylcholinesterase, the enzyme that sits at the heart of insect neurochemistry. Acetylcholinesterase hydrolyzes the neurotransmitter acetylcholine in the synaptic cleft, terminating each nerve impulse and resetting the synapse for the next signal. Block the enzyme, and acetylcholine accumulates, firing postsynaptic receptors continuously until the insect convulses, becomes paralyzed and dies. This is precisely the mechanism exploited by organophosphate and carbamate insecticides, which makes AChE1 a validated and well-characterized target. If andrograpanin could be shown to occupy the same binding pocket, it would generate a plausible mechanistic explanation for the observed mosquitocidal activity.

Because no experimental crystal structures existed for AChE1 from either mosquito species, the team built homology models using SWISS-MODEL, retrieving protein sequences from UniProtKB and selecting the crystal structure of an insecticide-resistant acetylcholinesterase mutant from Anopheles gambiae (PDB ID 6ARY) as the template. The resulting models captured the conserved catalytic domain, including the deep substrate-binding gorge where ligand recognition occurs. Validation was rigorous: Ramachandran plot analysis via PROCHECK placed 94.8 percent of Ae. aegypti residues in the most favored region with no disallowed positions, while the Cx. quinquefasciatus model showed 88.7 percent in the highly preferred region and also no disallowed residues. QMEAN scoring assessed global model quality, and CASTp analysis identified the principal binding pocket and the active-site residues involved in the enzyme’s hydrolase function, defining the docking arena.

The docking itself was performed with AutoDock Tools using a Lamarckian Genetic Algorithm, with each experiment comprising ten runs, 150 individuals in the population and 500,000 energy evaluations on a grid of 60 by 60 by 60 points at 0.375 angstrom spacing. The receptor was held rigid while the ligands were flexible. Andrograpanin emerged with a predicted binding energy of minus 6.36 kcal/mol against Ae. aegypti AChE1 and minus 6.45 kcal/mol against Cx. quinquefasciatus AChE1, with a ligand efficiency of 0.28 in both cases. In the Ae. aegypti model, the compound formed two conventional hydrogen bonds, one with the polar residue HIS47 at 3.02 angstroms and one involving the backbone of ILE91 at 2.11 angstroms, along with hydrophobic contact with TYR43 and carbon-hydrogen bonds with TYR45. In Cx. quinquefasciatus, it hydrogen-bonded with ASN65 and the GLY96 backbone, and made carbon-hydrogen contacts with SER44 and PRO64, all clustered around the catalytic gorge.

Temephos, docked under identical conditions as the reference standard, fared measurably worse. Against Ae. aegypti AChE1 it scored minus 4.36 kcal/mol with a ligand efficiency of 0.16, and against Cx. quinquefasciatus AChE1 it scored minus 4.06 kcal/mol with an efficiency of 0.20. Converted to inhibition constants through the thermodynamic relationship ΔG = RT ln(Ki) at 298.15 kelvin, the numbers diverge dramatically: andrograpanin’s Ki values ranged from 18.69 to 21.76 micromolar, while temephos ranged from 636.46 to 1056.07 micromolar, roughly a fifty-fold difference in predicted binding favorability. The comparison gains context from earlier docking studies of plant compounds against mosquito AChE1, which have reported scores from about minus 5.2 to minus 8.5 kcal/mol for molecules such as β-isocostic acid, longifolene and α-solanine. Andrograpanin’s values sit squarely within that range, though the authors caution that cross-study comparisons are complicated by differences in receptor structures, algorithms and scoring functions.

The novelty of the work lies in its integration. Rather than docking a hypothetical ligand or a crude extract, the team combined a purified, spectroscopically confirmed compound with experimentally validated ovicidal, larvicidal and pupicidal bioassays and a head-to-head computational comparison with a commercial insecticide under identical docking conditions. That convergence of biological and computational evidence strengthens the case for andrograpanin as a lead molecule for sustainable mosquito control, and the interaction profile, with hydrogen bonds and hydrophobic contacts surrounding the substrate-binding pocket, suggests the compound could interfere with substrate access or stabilization of the enzyme-substrate complex.

Yet the authors are unusually candid about the limits of their evidence. Molecular docking predicts a static binding pose and cannot fully capture protein flexibility, solvent effects or conformational dynamics, and the study lacks molecular dynamics simulations that would test whether the docked complexes remain stable over time. The absence of archived SWISS-MODEL project files also prevented reporting of detailed template alignment statistics. Most importantly, no direct acetylcholinesterase inhibition assay has yet been performed, so the proposed mechanism remains a computational hypothesis rather than demonstrated biochemistry. The docking energies, while more favorable than temephos’s, represent only moderate predicted affinities and should not be read as proof of potent enzyme inhibition. The path forward, the researchers argue, runs through enzyme inhibition assays, molecular dynamics simulations with MM/PBSA or MM/GBSA binding free-energy calculations, toxicity testing against non-target organisms and field trials. If those steps confirm what the docking suggests, a bitter leaf from southern India may yet yield a weapon against two of humanity’s most persistent disease vectors.

Subject of Research: Molecular docking of the plant diterpenoid andrograpanin with acetylcholinesterase 1 from Aedes aegypti and Culex quinquefasciatus mosquitoes

Article Title: Molecular docking analysis of andrograpanin interactions with acetylcholinesterase from Aedes aegypti and Culex quinquefasciatus

Article References: Vilvest, J., Milton, M. C. J., Yagoo, A., & Stalin, A. (2026). Molecular docking analysis of andrograpanin interactions with acetylcholinesterase from Aedes aegypti and Culex quinquefasciatus. Discover Chemistry, 3(1), Article 477. https://doi.org/10.1007/s44371-026-00933-w

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00933-w

Keywords: andrograpanin, acetylcholinesterase, molecular docking, Aedes aegypti, Culex quinquefasciatus, mosquito control, Andrographis paniculata, temephos, botanical insecticides, homology modeling, insecticide resistance, vector-borne disease