The motivation behind the work is rooted in a growing set of problems with conventional sanitizers and disinfectants. Commercial germicidal products frequently rely on ethanol, isopropyl alcohol, and chemical preservatives, ingredients that the study’s authors note can cause skin dryness, irritation, burning, discoloration, dermatitis, and protein crosslinking with repeated use. Beyond the toll on human skin, there is an ecological dimension: disinfectants washed off floors and surfaces are entering sewage systems and polluting water bodies, placing aquatic ecosystems at risk. Perhaps most concerning, recent reports indicate that microorganisms are actively developing resistance to the chemicals used in sanitizers, echoing the well-known crisis of antibiotic resistance that has made pathogenic bacterial strains one of the defining public health challenges of the era.

Plant bioactive compounds have long been proposed as greener alternatives, and the researchers point out that they are less likely to induce resistance than many synthetic disinfectants. Rosemary is already known to contain rosmarinic acid, camphor, caffeic acid, ursolic acid, betulinic acid, carnosic acid, and carnosol, while hibiscus supplies saponins, flavonoids, and tannins, substances renowned for their membrane-disrupting and antimicrobial activities. Yet the team identified a gap in the literature: although antibacterial activity of plant extracts has been studied extensively, in silico analysis of plant bioactive compounds targeting specific bacterial proteins remains comparatively rare. Their study was designed to close that gap by pairing empirical assays with mechanistic computational predictions.

Methodologically, the team sourced rosemary leaves and hibiscus petals from Mumbai, Maharashtra, India, depositing voucher specimens for reproducibility. The plant material was ground into a fine powder and subjected to Soxhlet extraction with 70 percent ethanol, after which the extracts were concentrated by rotary evaporation under vacuum at a constant 40 degrees Celsius. Antibacterial activity was then measured using the agar well diffusion method against a formidable panel of eight pathogenic strains: Escherichia coli MTCC 2412, Staphylococcus aureus MTCC 2408, Salmonella species MTCC 4415, Shigella species MTCC 1457, Streptococcus pyogenes MTCC 442, Proteus vulgaris MTCC 426, Klebsiella pneumoniae MTCC 2716, and Bacillus subtilis MTCC 2010. Hydrogen peroxide served as the positive control and sterile saline as the negative control, providing benchmarks for comparison.

The results of the diffusion assays were striking, particularly for rosemary. Rosemary extract produced zones of inhibition ranging from 17.66 to 26.33 millimeters across the tested strains, while hibiscus extract produced zones ranging from 11.33 to 18.33 millimeters. Statistical analysis using one-way ANOVA followed by Tukey’s post hoc test in GraphPad Prism, with significance set at P less than 0.05, confirmed that the differences were meaningful. For rosemary, the analysis showed highly significant variation in activity across organisms (P less than 0.0001), with S. aureus emerging as the most sensitive strain, followed by moderate activity against S. pyogenes and Bacillus, while E. coli, Salmonella, Shigella, Proteus, and K. pneumoniae showed minimal or no inhibition. Hibiscus, by contrast, showed its strongest and most consistent effect against Salmonella, Shigella, S. pyogenes, Proteus, and Bacillus, with significantly lower inhibition of E. coli, S. aureus, and K. pneumoniae.

Minimum inhibitory concentrations told a complementary story about potency. Rosemary extract was effective at concentrations between 1.5 and 12.5 milligrams per milliliter, whereas hibiscus extract required 25 to 50 milligrams per milliliter to suppress bacterial growth. These values were determined using a resazurin dye reduction assay, in which the dye changes color as metabolically active bacteria reduce it, allowing growth inhibition to be tracked visually. The team also conducted preliminary phytochemical screening: rosemary revealed the presence of alkaloids, flavonoids, steroids, tannins, and saponins, while hibiscus contained alkaloids, flavonoids, glycosides, steroids, terpenoids, tannins, and saponins, a chemical arsenal consistent with the observed antibacterial effects.

To identify the specific molecules responsible, the researchers turned to gas chromatography-mass spectrometry using a Shimadzu GCMS-QP2010 Ultra instrument. The rosemary fingerprint profile revealed compounds including carbamic acid monoammonium salt, 1,2-dimethyl-3-nitrobenzene, n-hexadecanoic acid, 3-pentadecylphenol, 1,5,9,13-tetradecatetraene, tricyclo[20.8.0.0(7,16)]triacontane,1(22),7(16)-diepoxy, and 7-hexadecenal. Hibiscus yielded n-hexadecanoic acid, cis-vaccenic acid, 3-pentadecylphenol, 1,5,9,13-tetradecatetraene, bi-1-cycloocten-1-yl, alpha-santalol, and 1,3-diphenyl-1,2-butanediol. These molecular inventories then became the input for the computational phase of the study, in which AutoDock Vina, MGL Tools, and the PyMOL visualization tool were used to dock each compound against bacterial proteins selected because they are essential for cell survival or are established antibiotic targets.

The docking results provided the study’s headline finding. Tricyclo[20.8.0.0(7,16)]triacontane,1(22),7(16)-diepoxy from rosemary achieved a binding energy of minus 11.0 kilocalories per mole against the S. aureus protein (PDB: 3VSL), forming hydrogen bonds with Thr619, Thr621, Ser448, Glu623, Ser392, and Gln524 in Chain A, and with Thr621, Thr603, Ser392, and Glu623 in Chain B. The authors note that similar interactions have been observed with cefotaxime, a third-generation cephalosporin antibiotic. Meanwhile, 1,3-diphenyl-1,2-butanediol from hibiscus scored minus 7.5 kilocalories per mole against the K. pneumoniae protein (PDB: 2OV5), interacting with residues across three separate protein chains. Remarkably, these phytochemicals matched or exceeded the binding energies of the standard antibiotic meropenem, which ranged from minus 5.3 to minus 7.6 kilocalories per mole in comparative docking. Even more intriguingly, both lead compounds were relatively minor constituents of their extracts by GC-MS abundance, a phenomenon the authors note has been observed in earlier studies of essential oils, where trace compounds can exert outsized antimicrobial influence.

Before any compound can be considered a viable ingredient, however, it must pass pharmacological scrutiny. The team ran ADMET analysis, assessing absorption, distribution, metabolism, excretion, and toxicity, using the SwissADME platform. Both tricyclo[20.8.0.0(7,16)]triacontane,1(22),7(16)-diepoxy and bi-1-cycloocten-1-yl violated one of Lipinski’s rules of drug-likeness, but were deemed acceptable candidates on the strength of their bioavailability, with a bioavailability score of 0.55 indicating good absorption and distribution. The remaining bioactive compounds passed the ADMET filters cleanly, suggesting that the chemical repertoire of these two plants is not only antibacterial in principle but plausibly compatible with formulation into real products.

The broader implications reach into green chemistry and sustainable healthcare. Because plant-based bioactive compounds kill or inhibit microbes by destabilizing microbial membranes, disrupting proteins, and interfering with metabolic activities, they can serve as natural alternatives to alcohol in sanitizer formulations, which are typically built on gel or spray bases. Unlike alcohol, which dries and irritates the skin with regular use, plant-based formulations are described as gentler on both skin and environment, and their wide-spectrum activity makes them attractive for everyday hand hygiene. With recurring pandemics and epidemics underscoring the urgency of new antimicrobials, the identification of two lead candidates with antibiotic-comparable binding affinities gives the humble rosemary sprig and hibiscus bloom a new scientific pedigree, and offers formulators a concrete, evidence-backed starting point for alcohol-free germicidal products.

Subject of Research: Antimicrobial activity and molecular docking analysis of rosemary and hibiscus bioactive compounds for germicidal product development

Article Title: Exploring antimicrobial potency and in silico analysis of rosemary and hibiscus bioactive compounds to be used in germicidal product

Article References: Shedge, V., & Sambrani, S. (2025). Exploring antimicrobial potency and in silico analysis of rosemary and hibiscus bioactive compounds to be used in germicidal product. Discover Biotechnology, 2(1), Article 20. https://doi.org/10.1007/s44340-025-00028-9

Image Credits: AI Generated

DOI: 10.1007/s44340-025-00028-9

Keywords: rosemary, hibiscus, antimicrobial activity, molecular docking, bioactive compounds, plant extracts, hand sanitizer, antibiotic resistance, ADMET analysis, GC-MS, green chemistry, Staphylococcus aureus

The research, led by Hafs Essaadi and colleagues at Mohammed V University in Rabat, Morocco, together with collaborators at the Mohammed VI Center for Research and Innovation and UM6SS, focused on the viral 3C protease, an enzyme designated 3Cpro that is indispensable to the rhinovirus life cycle. After the virus infects a cell, its genome is translated into a single long polyprotein that must be cleaved into functional viral proteins, and the 3C protease performs most of these cuts. Because the catalytic architecture of 3Cpro is highly conserved across human rhinovirus species, a molecule that disables it could in principle suppress a wide range of rhinovirus strains, making the enzyme an attractive therapeutic target.

To find candidate inhibitors, the team screened a library of 49,437 compounds against the 3C protease of human rhinovirus species C, the rhinovirus group most frequently associated with severe asthma exacerbations. The docking calculations were carried out with AutoDock Vina, a widely used molecular docking engine that predicts how small molecules orient themselves within a protein binding pocket and estimates the binding affinity of each pose. The researchers then applied ADMET-based prioritization, filtering the top-scoring hits for acceptable absorption, distribution, metabolism, excretion, and toxicity profiles, a step designed to weed out compounds that might bind well in silico but fail as drug candidates.

The docking analysis converged on two lead compounds that occupied the catalytic pocket of the protease in orientations predicted to be highly favorable. Both molecules formed stabilizing interactions with key residues of the active site, including His40, Glu71, and Cys147, the latter being the catalytic cysteine that sits at the heart of the enzyme’s cleavage chemistry. When compared with rupintrivir, the best-known experimental rhinovirus 3C protease inhibitor, which functions as an irreversible covalent inhibitor, the two new compounds achieved comparable or better engagement of the catalytic site without forming covalent bonds, a property that could translate into improved safety profiles.

Docking scores alone are a crude measure of binding, so the team subjected the protein-ligand complexes to molecular dynamics simulations lasting 200 nanoseconds each, allowing the atoms to move under realistic physical forces and revealing whether the predicted binding poses remain stable over time. Both compounds stabilized the protease relative to the unbound, or apo, form of the enzyme. Compound 1 produced the lowest protein root-mean-square deviation, holding the overall structure of the protease at 1.21 plus or minus 0.22 angstroms from its starting conformation, while compound 2 yielded the lowest root-mean-square fluctuation for the critical Cys147 residue, at just 0.48 angstroms, indicating that the catalytic nucleophile itself was held unusually rigid in the presence of this ligand.

Ligand mobility within the binding pocket provided further evidence of durable binding. Over the course of the simulations, compound 1 displayed a ligand RMSD of 1.49 plus or minus 0.78 angstroms and compound 2 a value of 2.01 plus or minus 0.39 angstroms, whereas rupintrivir wandered considerably more, with a ligand RMSD of 4.83 plus or minus 0.89 angstroms. Lower ligand RMSD values indicate that a molecule stays anchored in its original binding pose rather than drifting or partially exiting the pocket, suggesting that the two new candidates maintain more persistent contact with the active site than the reference inhibitor under dynamic conditions.

To quantify binding strength more rigorously, the researchers applied the MM/PBSA method, which combines molecular mechanics energies with solvation models to estimate the free energy of binding from simulated trajectories. Both leads outperformed rupintrivir on this metric: compound 1 achieved an effective binding energy of minus 23.59 plus or minus 6.87 kilocalories per mole, and compound 2 reached minus 25.25 plus or minus 5.75 kilocalories per mole, compared with minus 19.43 plus or minus 4.46 kilocalories per mole for rupintrivir. The team complemented these calculations with free energy landscape analysis, a technique that maps the conformational states sampled during simulation and identifies the most thermodynamically stable configurations of each complex, providing an additional layer of confidence that the observed binding modes represent genuine energetic minima rather than transient artifacts.

The significance of a non-covalent mechanism deserves emphasis. Rupintrivir, which reached phase II clinical trials as a nasal spray, irreversibly modifies the catalytic cysteine, and while this reactivity underlies its potency, covalent inhibitors can raise concerns about off-target modification of human enzymes that rely on similar cysteine chemistry. Compounds that achieve strong binding through reversible, non-covalent interactions, as the two leads reported here appear to do, may offer a wider therapeutic window. The ADMET filtering applied during the study further suggests that the candidates were selected not only for potency but also for drug-like behavior, although the authors stress that computational predictions of this kind require experimental confirmation.

Indeed, the study stops short of laboratory validation, and the authors are explicit that compounds 1 and 2 should be regarded as promising candidates for further experimental testing rather than proven antivirals. Enzyme inhibition assays, antiviral activity measurements in infected cell cultures, and eventually pharmacokinetic and toxicity studies in vivo will be needed to determine whether the computational promise translates into real therapeutic value. The data generated during the study are available from the corresponding author upon request, and the work was supported in part by computational resources from the Pediatric Translational Clinical Research Unit.

Nevertheless, the study adds to a growing body of evidence that structure-based computational screening can accelerate antiviral discovery against rhinoviruses, a pathogen family that has historically frustrated drug developers because of its antigenic diversity. By anchoring the search on a conserved, essential enzyme and validating hits through a multi-layered pipeline of docking, long-timescale dynamics, free energy landscape mapping, MM/PBSA energetics, and ADMET profiling, the Moroccan team has delivered a shortlist of chemically tractable starting points. If subsequent experiments bear out the predicted potency of these molecules, the work could represent an early but meaningful step toward the first effective antiviral treatment for the infections that trigger many of the world’s asthma attacks and common colds.

Subject of Research: Computational discovery of non-covalent inhibitors targeting the human rhinovirus 3C protease

Article Title: Computational discovery of novel human rhinovirus 3 C protease inhibitors: molecular docking, dynamic simulations, free energy landscape, MMPBSA and ADMET analysis

Article References: Essaadi, H., Chourir, A., Kourou, J., Makhloufi, F., Hachlaf, O., Abidou, A., Boutayeb, S., Eljaoudi, R., Belyamani, L., Ibrahimi, A., Hakmi, M., & Hafidi, N. E. (2026). Computational discovery of novel human rhinovirus 3 C protease inhibitors: molecular docking, dynamic simulations, free energy landscape, MMPBSA and ADMET analysis. Molecular Diversity. https://doi.org/10.1007/s11030-026-11704-1

Image Credits: AI Generated

DOI: 10.1007/s11030-026-11704-1

Keywords: human rhinovirus, 3C protease, antiviral drug discovery, molecular docking, molecular dynamics simulations, MM/PBSA, free energy landscape, ADMET analysis, virtual screening, rupintrivir, common cold, asthma exacerbation