The strongest activity was measured in essential oils of Monarda fistulosa, Satureja pilosa, Origanum vulgare subsp. hirtum and Thymus longidentatus, together with a methanolic extract of Leucojum aestivum. The study examined methanolic extracts and essential oils from 17 plant species in the Amaryllidaceae, Asteraceae, Brassicaceae and Lamiaceae families.

Plant material was collected from natural localities or obtained from the Institute of Biodiversity and Ecosystem Research and the Institute of Roses and Aromatic Plants. For methanolic extraction, 1 g of air-dried, powdered plant material was extracted with methanol for 24 hours at room temperature. After filtration, the solution was evaporated and the dry extract was stored at 4 °C until analysis. Essential oils were extracted by water distillation on a Clevenger apparatus using 50 g of dry plant material and 500 ml of water over two hours.

Acetylcholinesterase inhibition was measured using a modified version of Ellman’s colorimetric method in 96-well microplates. The assay used acetylcholinesterase from the electric eel Electrophorus electricus, acetylthiocholine iodide as the substrate, and 5,5′-dithiobis(2-nitrobenzoic acid). Samples were tested at concentrations between 0.001 and 1,000 µg/ml and incubated for 30 minutes at room temperature before substrate was added. Absorbance was measured at 405 nanometres after three minutes, and inhibition was calculated relative to a buffer control. Galanthamine served as the positive control.

Among the essential oils, M. fistulosa had the lowest reported half-maximal inhibitory concentration, or IC50, at 0.0042 mg/ml. The oil of S. pilosa followed at 0.0069 mg/ml. The O. vulgare subsp. hirtum and T. longidentatus oils had IC50 values of 0.30 and 0.72 mg/ml, respectively, while the L. aestivum methanolic extract had a value of 0.20 mg/ml. The galanthamine positive control had an IC50 of 0.0011 mg/ml, equivalent to 4.03 µM. IC50 values were measured in triplicate and reported as means.

A second group of seven methanolic extracts showed moderate activity, with IC50 values from 0.73 to 1.23 mg/ml. These extracts came from O. vulgare subsp. hirtum, Artemisia lerchiana, Artemisia santhonicum, Eupatorium cannabinum, Nepeta caria, Micromeria dalmatica and Salvia aethiopis. Several other samples had IC50 values above 1 mg/ml. For O. vulgare subsp. hirtum, the measured inhibition was stronger for the essential oil than for its methanolic extract.

The researchers used gas chromatography coupled with mass spectrometry to characterize compounds in the most active samples. Components were identified by comparing mass spectra and retention indices with authentic standards and a National Institute of Standards and Technology library. Carvacrol and thymol were major constituents of the essential oils of S. pilosa, O. vulgare subsp. hirtum and M. fistulosa. Carvacrol accounted for 51.18% of the oregano oil and 50.54% of the S. pilosa oil. Thymol represented 30.58% of the S. pilosa oil and 19.75% of the M. fistulosa oil. The M. fistulosa oil also contained thymoquinone at 25.41% and p-cymene at 21.82%.

In the T. longidentatus essential oil, the citral isomers neral and geranial accounted for 24.9% and 27.96% of the identified area. Galanthamine was the main alkaloid detected in the L. aestivum extract. The oregano methanolic extract contained carvacrol at 15.67%, compared with 51.18% in its essential oil, along with rosmarinic acid, flavonoid glycosides, malic acid and catechin. The authors noted that the difference in carvacrol content may be related to the difference in acetylcholinesterase inhibition, but the assay did not establish that individual compounds caused the measured effects.

The evidence in this study comprises in vitro enzyme measurements and GC/MS chemical characterization of selected samples. It does not demonstrate that the plant products kill or repel insects, protect crops under field conditions, or avoid effects on non-target organisms. The authors described the strong acetylcholinesterase activity as a prerequisite for potential insecticidal activity and recommended testing the plant products for insecticidal effects.

Subject of Research: In vitro acetylcholinesterase inhibition and chemical characterization of plant extracts and essential oils

Article Title: Plant products with acetylcholinesterase inhibitory activity for insect control

Article References: Georgiev, B., Nikolova, M., Aneva, I., Dzhurmanski, A., Sidjimova, B., & Berkov, S. (2022). Plant products with acetylcholinesterase inhibitory activity for insect control. BioRisk, 17, 309-315. https://doi.org/10.3897/biorisk.17.77052

Image Credits: AI Generated

DOI: 10.3897/biorisk.17.77052

Keywords: acetylcholinesterase inhibition, plant extracts, essential oils, in vitro assay, gas chromatography-mass spectrometry, insect control, carvacrol, thymol, galanthamine, Plant, products, acetylcholinesterase

The research team, working with plant material from the ex-situ collection of the Institute of Biodiversity and Ecosystem Research at the Bulgarian Academy of Sciences, focused on Origanum vulgare subsp. hirtum, a Greek oregano famous for its pungent, carvacrol-rich chemistry. Their earlier work had shown that both a methanolic extract and an essential oil from this plant could kill peach aphids, Myzus persicae, depending on the concentration applied. The critical question they now asked was whether doses causing even low levels of aphid mortality would also harm plant cells—either by poisoning them, breaking their DNA, or triggering mutations. Any herbicide candidate, after all, must damage weeds without leaving a legacy of genetic damage in the wider environment.

To probe these effects, the researchers turned to Chlamydomonas reinhardtii, a single-celled green alga that has served as a robust workhorse of environmental mutagenesis testing for years. Its cells form colonies predictably under standard conditions, which makes it possible to measure survival with precision, and its genome responds to DNA-damaging agents in ways that are well characterized. The team prepared the essential oil by water distillation in a Clevenger apparatus and the methanolic extract by classical maceration, then analyzed both with gas chromatography–mass spectrometry, identifying components against the NIST spectral database.

The chemical profiles of the two preparations turned out to be strikingly different, and those differences foreshadowed everything that followed. The essential oil was dominated by the monoterpenoid phenol carvacrol at 74.34 percent, flanked by γ-terpinene at 10.66 percent, p-cymene at 9.46 percent, and smaller amounts of pinenes and carvacrol methyl ether. The methanolic extract told another story entirely: it was rich in primary metabolites such as glucose, fructose, and sucrose, along with rosmarinic acid at 6.06 percent and carvacrol at just 15.67 percent, supplemented by organic acids, phenolic acids, and trace flavonoids. In essence, the extract offered a diluted, chemically complex mixture, while the oil delivered a concentrated carvacrol payload.

Toxicity testing used a clonal assay based on colony-forming ability, with cells exposed to concentrations of 250, 500, 750, and 1000 parts per million. The controls behaved as hoped: neither the DMSO solvent nor the commercial insecticide Nurelle D significantly reduced algal survival compared with the negative control medium. The treatments, however, separated cleanly. The essential oil significantly reduced survival starting at 250 parts per million, with the survival fraction falling from 0.51 at the lowest dose to 0.09 at 750 parts per million. The methanolic extract only showed statistically significant killing at 750 and 1000 parts per million, its dose–response curve forming a plateau in the lower range. Calculated lethal doses made the contrast quantitative: roughly two-fold lower concentrations of essential oil were needed to achieve 20, 50, and 80 percent lethality compared with the extract.

The study’s most technically revealing experiment measured DNA double-strand breaks using constant field gel electrophoresis, a method that separates fragmented DNA from intact genomic DNA trapped in agarose wells and quantifies the fraction released. Here the two preparations diverged dramatically. The methanolic extract showed no DNA-damaging capacity at any tested concentration, with double-strand break levels indistinguishable from spontaneously arising breaks in untreated controls. The essential oil, by contrast, produced around two-fold higher levels of double-strand breaks than the negative control across the entire concentration range, with every increase statistically significant. The authors report, for the first time, this DNA-damaging capacity of oregano essential oil, and they point to the strong correlation between rising break levels and falling survival, suggesting that carvacrol, the oil’s dominant constituent, is the likely culprit.

Mutagenicity told a different story. Using a test of visible mutations, which scores surviving colonies for changes in size, morphology, and pigmentation, the team calculated a mutagenicity index for each treatment. With a spontaneous mutation level of 0.136 percent, both the extract and the essential oil yielded indices below 2, the threshold beneath which no mutagenic effect is identified. This absence of mutagenic activity aligns with earlier findings in other systems, including the Ames test and the SMART assay in fruit flies, and it matters enormously for practical applications: compounds that kill cells without corrupting their genetic instructions are far more attractive for deployment in agriculture than those that risk seeding heritable changes.

The authors propose that the divergent behavior of the two preparations flows directly from their chemical composition. The essential oil’s concentrated carvacrol content plausibly drives both its superior toxicity—roughly 1.4 to 2 times more genotoxic than the extract—and its capacity to shatter DNA, while the extract’s abundance of benign primary metabolites dilutes its active principles. They caution, however, that further experiments are needed to disentangle the contributions of major and minor constituents, since essential oils act as complex mixtures whose components may interact synergistically or antagonistically.

The findings arrive at a moment when the search for bioherbicides has acquired real urgency. Oregano essential oil already has a documented portfolio of effects—phytotoxic, antimicrobial, antifungal, insecticidal, and active against plant pathogens—and this study extends that spectrum to include concentration-dependent DNA damage in a plant model system. Paradoxically, the very aggressiveness that makes the oil a candidate green herbicide is bounded by its lack of mutagenic potential, which suggests it could suppress weeds without generating resistant or otherwise altered plant lineages in treated fields.

For now, the Bulgarian team’s work stands as a template for how biopesticide candidates should be vetted: not just for killing power against target organisms, but for the precise cellular mechanisms by which they act. An oil that breaks DNA cleanly without inducing mutations occupies a sweet spot between efficacy and safety, and as agriculture retreats from synthetic chemistry, that combination of properties may prove exactly what green technologies have been looking for. Oregano, long valued for flavoring pizzas and salads, may soon earn a second career protecting the crops it seasons.

Subject of Research: Genotoxic and mutagenic effects of oregano extract and essential oil on a model green alga

Article Title: On the mode of action of Origanum vulgare spp. hirtum methanolic extract and essential oil on Chlamydomonas reinhardtii

Article References: Todorova, M. D., Parvanova, P. N., Todorova, T. I., Dronchev, G. D., Nikolova, M. T., Berkov, S. H., & Chankova, S. G. (2022). On the mode of action of Origanum vulgare spp. hirtum methanolic extract and essential oil on Chlamydomonas reinhardtii. BioRisk, 17, 179-190. https://doi.org/10.3897/biorisk.17.77313

Image Credits: AI Generated

DOI: 10.3897/biorisk.17.77313

Keywords: oregano, essential oil, carvacrol, genotoxicity, Chlamydomonas reinhardtii, biopesticide, green chemistry, DNA damage, herbicide resistance, Origanum vulgare, mutation assay, plant protection

The research team, led by Stephanka Chankova and colleagues, set out to test a straightforward hypothesis: if carvacrol, the phenolic monoterpene that gives oregano much of its antimicrobial punch, is responsible for the herb’s toxicity, then extract fractions rich in carvacrol should be more genotoxic than those without it. Previous work by the group had already shown that a methanolic extract containing 15.67% carvacrol was less toxic than the essential oil, which packs a staggering 74.34% carvacrol. To probe this question, the scientists collected aerial parts of Greek oregano during the flowering stage from an ex-situ collection at IBER-BAS, dried them, and extracted them with methanol over 24 hours. They then used liquid-liquid partitioning with water and chloroform to split the crude extract into two chemically distinct fractions — one polar, one non-polar — each carrying a different cargo of plant metabolites.

Gas chromatography-mass spectrometry (GC/MS) analysis, performed after derivatization with N,O-bis-(trimethylsilyl)trifluoroacetamide and deconvolution using the Automated Mass Spectral Deconvolution and Identification System (AMDIS), identified 34 compounds across the two fractions. The polar fraction was dominated by sugars, organic acids, and flavonoid glycosides — sucrose alone accounted for 26.28% of the chromatogram area, with a 6-hydroxyflavone glycoside contributing another 10.07%. Glucose, fructose, myo-inositol, and caffeic acid rounded out a profile rich in hydrophilic molecules. The non-polar fraction told a completely different story. Carvacrol reigned supreme at 37.08%, accompanied by fatty acids, sterols like β-sitosterol, triterpene acids, and flavonoid aglycones such as naringenin and taxifolin. The chemical contrast between the two fractions could not have been more pronounced — and, as the bioassays would soon reveal, neither could the biological one.

To evaluate toxicity and genotoxicity, the researchers turned to Chlamydomonas reinhardtii strain 137C+, a wild-type green alga widely used as a sensitive test system in radiobiology and toxicology. Cell suspensions at the end of the exponential phase were treated for just 10 seconds with six concentrations of each fraction, ranging from 250 to 5000 ppm. The team employed a battery of complementary assays: a spot test based on colony-forming ability, a clonal survival fraction (SF) calculation, a test for visible mutations in surviving colonies, and Constant Field Gel Electrophoresis (CFGE) to measure induced DNA double-strand breaks (DSBs). Negative controls included Sager-Granick medium and DMSO, while the insecticide Nurelle D served as a positive control.

The results were striking. The polar fraction showed no concentration-dependent toxicity whatsoever — spot intensity was identical across all samples, including controls, and none of the tested concentrations reduced cell survival below that of untreated cells. Mutagenicity and DNA damage were equally absent. The non-polar fraction, however, devastated the algal population. Even at the lowest concentration of 250 ppm, cell survival plummeted by 60%, yielding a survival fraction of just 0.41 ± 0.08. At concentrations of 500 ppm and above, lethality approached 100%. No colonies survived to form spots, and the spot test revealed single colonies instead of the dense growth seen with the polar fraction — a visual testament to the non-polar fraction’s lethal potency.

The mutagenicity assay added another layer of concern. Treatment with 250 ppm of the non-polar fraction induced a mild but measurable mutagenic effect, with a mutagenic index of 4.83 ± 0.004 — dramatically higher than the near-zero values recorded for the polar fraction at all concentrations. Two types of visible mutations appeared in surviving colonies: low-size mutants, attributed to delayed cell division, and pigmental mutants, linked to point mutations in nuclear or chloroplast DNA. The absence of visible mutations at higher concentrations of the non-polar fraction was not good news — it simply reflected the fact that no cells survived to express them.

The DNA-damaging capacity of the non-polar fraction was perhaps the most alarming finding. CFGE analysis revealed well-expressed, concentration-dependent induction of double-strand breaks, escalating to strong DNA fragmentation at higher doses. The polar fraction, by contrast, showed no statistically significant elevation of DSBs at any concentration. The concentration-effect curves for survival and DSB induction were not perfectly aligned, suggesting that mechanisms beyond double-strand break formation — perhaps membrane disruption or interference with cell cycle machinery — contribute to cell death. This mirrors the team’s earlier findings with the radiomimetic drug Zeocin, where survival did not correlate perfectly with DSB levels.

What explains the stark divergence between the two fractions? The researchers point to carvacrol as the prime suspect. As a lipophilic monoterpene, carvacrol can alter the physico-chemical properties of cell membranes, facilitating its own entry into cells and potentially disrupting membrane integrity. Previous studies have shown that essential oils and non-polar extracts are better absorbed into algal cells than their polar counterparts, and carvacrol has been classified with chronic aquatic toxicity under the European Chemicals Agency’s registration dossier. Evidence for carvacrol’s genotoxic properties has also been reported in plants like lettuce and sorghum, where it affects seed germination, root length, and the plant cell cycle. The fact that carvacrol is entirely absent from the polar fraction — which showed zero genotoxicity — strengthens the case that this single compound drives the non-polar fraction’s damaging effects.

The implications ripple outward. Oregano and its essential oils are increasingly promoted as natural biopesticides, food preservatives, and antimicrobial agents, with the global market for plant-derived bioactive compounds expanding rapidly. But this study suggests that carvacrol-rich preparations may carry genotoxic risks to non-target organisms, particularly aquatic and soil-dwelling eukaryotes like algae. The findings do not diminish oregano’s value as a culinary herb — the concentrations tested here far exceed anything encountered in food — but they do warrant caution in agricultural and environmental applications where carvacrol-rich extracts could contact non-target species. As the authors note, further experiments with purified carvacrol are needed to clarify the exact mechanism of action, whether it involves direct DNA interaction, membrane-mediated damage, or disruption of DNA repair pathways. For now, the message is clear: not all fractions of a natural extract are created equal, and the chemistry of a plant’s metabolites matters as much as the plant itself.

Subject of Research: Genotoxicity of oregano extract fractions on Chlamydomonas reinhardtii

Article Title: Polar and non-polar fraction from Origanum vulgare spp. hirtum methanolic extract – differences in their bioactivity on Chlamydomonas reinhardtii test system

Article References: Todorova, M. D., Parvanova, P. N., Todorova, T. I., Nikolova, M. T., Berkov, S. H., & Chankova, S. G. (2022). Polar and non-polar fraction from Origanum vulgare spp. hirtum methanolic extract – differences in their bioactivity on Chlamydomonas reinhardtii test system. BioRisk, 17, 191-200. https://doi.org/10.3897/biorisk.17.78169

Image Credits: AI Generated

DOI: 10.3897/biorisk.17.78169

Keywords: oregano, carvacrol, genotoxicity, Chlamydomonas reinhardtii, biopesticide, DNA damage, mutagenicity, BioRisk, Polar, non-polar, fraction, Origanum

The motivation behind the work lies in a well-known weakness of current influenza therapy. Drugs such as oseltamivir, zanamivir and peramivir all target neuraminidase, one of the two surface glycoproteins of the influenza virus, and single amino acid substitutions in that protein can render them ineffective. Influenza type A, the subtype responsible for pandemics since 1918, remains the most clinically significant of the influenza types, and the fragility of neuraminidase-targeted treatment has pushed researchers toward alternative targets and alternative chemical scaffolds. Hemagglutinin, the second surface glycoprotein, offers such a target: it carries the receptor-binding site that recognizes sialic acid on host cells and the fusion machinery that delivers the viral genome inside. Natural products, long valued as a reservoir of bioactive molecules including saponins, flavonoids, coumarins and stilbenoids, have repeatedly yielded compounds that interfere with viral proteins, and the authors set out to test whether thyme’s principal essential-oil constituents could do the same to hemagglutinin.

Thymus vulgaris, a woody member of the mint family native to southern Europe and the western Mediterranean, has a long history of medicinal use as an antiseptic, antimicrobial and tonic. Gas chromatography–mass spectrometry analyses of its essential oil identify five dominant constituents whose proportions vary widely between chemotypes: thymol, ranging from 6.7 to 66.10 percent; linalool, from 1.36 to 22.25 percent; carvacrol, from 2.33 to 20.03 percent; α-terpineol, from 0.15 to 11.61 percent; and geraniol, from 0.32 to 11.31 percent. Thymol and carvacrol are monoterpene phenols, linalool is a monoterpene alcohol, and all three have documented anti-inflammatory, antimicrobial and antioxidant activity. Curcumin, the polyphenolic yellow pigment of Curcuma longa, was included as a comparator because of its extensive literature on antiviral and anti-inflammatory effects, even though its clinical translation has been hampered by poor bioavailability and chemical instability.

The computational strategy rested on an atomistic model of the H1N1 hemagglutinin trimer, taken from the Protein Data Bank under entry 3LZG. The trimeric form is the biologically relevant prefusion assembly: each protomer consists of an HA1 chain forming the globular receptor-binding head and an HA2 chain containing the membrane-fusion stalk. The receptor-binding site sits at the distal tip of HA1 and is built from three structural elements—the 130-loop, the 190-helix and the 220-loop. Simulating the intact trimer matters because models that omit the trimeric interface cannot capture conformational stability, allosteric coupling between protomers, or the large-scale rearrangements that hemagglutinin undergoes when triggered by low pH to fuse viral and cellular membranes. The team therefore ran all-atom simulations in GROMACS 5.1.4 using the GROMOS54A7 force field, the SPC water model, a Nosé-Hoover thermostat at physiological temperature and a Berendsen barostat at 1 bar, with particle-mesh Ewald summation for electrostatics and a 1.2-nanometer cutoff for Lennard-Jones interactions.

Each simulation placed the hemagglutinin trimer in a cubic box seven nanometers on a side, solvated it, neutralized it and introduced three copies of one ligand at a concentration of 14 millimolar. Using multiple ligand molecules rather than a single one allowed the researchers to observe whether adsorption occurs competitively and which sites on the protein surface are preferred. After energy minimization, each system was equilibrated for 20 nanoseconds in the canonical ensemble and a further 20 nanoseconds under constant pressure, before a production run of 100 nanoseconds whose final 10 nanoseconds were analyzed in detail. Binding energies were decomposed with the MM-PBSA method, and the trajectories were visualized with VMD. Complementing the dynamics, density functional theory calculations at the B3LYP/6-31G(d,p) level in the GAMESS program yielded optimized geometries, HOMO and LUMO energies, dipole moments and polarizabilities, with partial atomic charges fitted to the electrostatic potential in an aqueous environment to mimic the biological milieu.

The docking-like outcome of the dynamics was striking in its site selectivity. Starting from random positions in the solvent, carvacrol and linalool both migrated to and adsorbed onto the 190-helix of HA1, a structural element that contributes directly to receptor binding, and onto the fusion-related stalk region of HA2. Thymol bound to the fusion-related stalk of HA2. Curcumin, by contrast, ended up at a site involving no functionally important residues. The energetic decomposition explained the difference. Thymol achieved the strongest Lennard-Jones interaction with the protein at −231.42 kJ/mol and the highest overall binding energy at −360.56 kJ/mol, followed by linalool at −351.21 kJ/mol and carvacrol at −229.71 kJ/mol. Curcumin’s binding energy of −167.28 kJ/mol was the weakest of the four, with both its dispersive and electrostatic contributions substantially smaller. Thymol also formed more hydrogen bonds with hemagglutinin than any other ligand tested.

Conformational analysis reinforced the picture. Relative to hemagglutinin in pure water, the root mean square deviation of the protein increased after adsorption of every thyme constituent, with thymol producing the largest structural drift, whereas curcumin’s trajectory closely resembled the ligand-free reference. Root mean square fluctuation profiles showed that the thyme compounds perturbed residues in both the receptor-binding HA1 head and the fusion-related HA2 stalk, while curcumin left the fluctuation pattern essentially unchanged. The radius of gyration, nearly constant between water and the curcumin-bound system at roughly 7.9 to 8.1 nanometers, decreased significantly when the thyme compounds adsorbed, indicating a more compact protein. Secondary-structure analysis added a further signature: thyme constituents reduced beta-sheet content and increased coil structures, whereas curcumin left the secondary structure largely intact. The thyme compounds also raised the protein’s solvent-accessible surface area and its number of hydrogen bonds with water—thymol lifted the hydrogen-bond count from 5623 to 5817—evidence that they remodel both the fold and the solvation shell of hemagglutinin.

Quantum chemical descriptors and pharmacokinetic screening filled in the electronic and drug-likeness context. Curcumin showed the smallest HOMO-LUMO band gap, 0.12263 Hartree, together with the largest dipole moment and polarizability, consistent with easier electron transfer and higher intrinsic reactivity; the three thyme compounds clustered at higher band gaps between 0.21645 and 0.23092 Hartree with lower polarizabilities. Yet the simulations showed that this electronic reactivity did not translate into stronger protein binding. On the pharmacokinetic side, linalool, thymol and carvacrol fully satisfied Lipinski’s Rule of Five, with molecular weights near 150 g/mol, single hydrogen-bond donors and acceptors, and log P values below 3.3, while curcumin violated one criterion with six hydrogen-bond acceptors, echoing its known bioavailability problems. None of the four compounds was predicted to be mutagenic or tumorigenic by the SwissADME toxicity models.

The authors are careful to frame the findings as computational hypotheses rather than therapeutic claims. Molecular dynamics and quantum mechanics offer atomic-resolution insight into binding geometry, energetics and conformational change, but phytochemical effects are notoriously context-dependent across in vitro, in vivo and clinical settings, and the study’s own limitations—a small number of ligand molecules per box and a 100-nanosecond timescale without an explicit viral membrane—leave room for refinement. Even so, the convergence of strong binding energies, occupation of the receptor-binding helix and the fusion stalk, drug-like physicochemical profiles and demonstrable structural destabilization makes the thyme constituents, and thymol above all, credible leads for experimental follow-up. Biochemical binding assays, antiviral testing in cell culture and ultimately in vivo studies will determine whether the simulated disruption of hemagglutinin can be reproduced where it matters: in a infected host, at the first step of the influenza life cycle.

Subject of Research: Computational evaluation of Thymus vulgaris constituents and curcumin as inhibitors of influenza H1N1 hemagglutinin

Article Title: Evaluating the antiviral potential of Thymus vulgaris constituents and curcumin against influenza hemagglutinin: A molecular dynamics study

Article References: Kordzadeh, A., Hosseinzade, Z., Hadi, A., & Ghaderi, S. (2026). Evaluating the antiviral potential of Thymus vulgaris constituents and curcumin against influenza hemagglutinin: A molecular dynamics study. Results in Chemistry, 31, Article 103943. https://doi.org/10.1016/j.rechem.2026.103943

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103943

Keywords: influenza, hemagglutinin, Thymus vulgaris, thymol, carvacrol, linalool, curcumin, molecular dynamics, quantum chemistry, antiviral, drug discovery, natural products

The research team began with a straightforward question: if edible mushrooms are increasingly recognized as reservoirs of bioactive anticancer compounds, what might P. eous hold? The species is known to be rich in phenolic and terpenoid constituents, two broad chemical families with well-documented biological activity, yet its potential against cancer cells had never been systematically evaluated. To answer the question, the investigators prepared a methanolic extract of the mushroom fruiting bodies and subjected it to gas chromatography-mass spectrometry, or GC-MS, a technique that vaporizes chemical compounds and separates them by mass, allowing researchers to identify individual molecules within a complex mixture. The spectral data, matched against reference libraries maintained by the National Institute of Standards and Technology, revealed a phytochemical profile that immediately caught the authors’ attention.

Among the compounds identified were carvacrol, quinic acid, and a derivative of cinnamic acid. Each of these molecules carries an established scientific pedigree. Carvacrol, the monoterpene phenol that gives oregano and thyme their characteristic aroma, has been repeatedly studied for antimicrobial, antioxidant, and pro-apoptotic effects in cancer models. Quinic acid, a cyclitol found in coffee and many plants, is a building block of chlorogenic acid and has been linked to anti-inflammatory and chemopreventive activity. Cinnamic acid derivatives, meanwhile, appear throughout the plant and fungal kingdoms and have been investigated for their ability to interfere with tumor cell proliferation. The presence of all three in a single mushroom extract suggested that P. eous might be more than an ordinary culinary fungus, prompting the team to move from chemical identification to biological testing.

The first biological assay targeted oxidative chemistry. The researchers used the DPPH assay, a widely employed colorimetric test in which the purple-colored free radical 2,2-diphenyl-1-picrylhydrazyl loses its absorbance when neutralized by antioxidants. The results were dramatic: at a concentration of 700 micrograms per milliliter, the P. eous extract scavenged 97.39 percent of the DPPH radicals, an efficiency approaching that of synthetic reference antioxidants. Complementing this, the team measured total phenolic content using the Folin-Ciocalteu assay, which quantifies phenolic compounds by their ability to reduce a phosphotungstic-phosphomolybdic acid reagent, expressed here in gallic acid equivalents. The extract yielded a total phenolic content of 2.80 plus or minus 0.04 milligrams of gallic acid equivalent per gram of dry weight. The strong radical-scavenging performance is consistent with the idea that phenolic compounds donate hydrogen atoms or electrons to stabilize reactive radicals, a mechanism thought to contribute to chemoprevention by protecting cellular DNA from oxidative damage.

The centerpiece of the study, however, was the cytotoxicity testing against MCF-7 cells, one of the most extensively characterized human breast cancer cell lines in existence. Originally isolated in 1970 from a patient with invasive breast carcinoma, MCF-7 cells are estrogen-receptor positive and serve as a standard in vitro model for hormone-responsive breast cancer, the most common malignancy in women worldwide. The researchers cultured the cells in Dulbecco’s Modified Eagle Medium supplemented with fetal bovine serum and exposed them to escalating concentrations of the mushroom extract. Cell viability was quantified with the MTT assay, a metabolic test in which living cells reduce a yellow tetrazolium salt into purple formazan crystals; the amount of formazan produced serves as a proxy for the number of metabolically active, surviving cells. Doxorubicin, a mainstay anthracycline chemotherapy drug, served as the positive control against which the extract’s performance could be benchmarked.

The outcome was unambiguous. The extract killed MCF-7 cells in a dose-dependent fashion, achieving a half-maximal inhibitory concentration, or IC50, of 82.69 micrograms per milliliter at the 24-hour time point. In pharmacological terms, the IC50 represents the concentration of a compound required to reduce cell viability by half, and values in the low tens to hundreds of micrograms per milliliter are considered meaningful starting points for natural product screening. Microscopic examination of the treated cultures revealed morphological changes characteristic of apoptosis, the programmed cell death pathway that cancer cells often evade. Cells exposed to higher concentrations of the extract displayed the shrinkage, rounding, and detachment that pathologists associate with cells undergoing self-destruction rather than simple toxic necrosis. Statistical analysis by analysis of variance followed by Tukey’s post-hoc test confirmed that the differences between treated and untreated groups were highly significant, with a p-value below 0.0001, meaning the probability that the observed effect arose by chance is vanishingly small.

The authors attribute the extract’s dual antioxidant and cytotoxic activity to its phenolic and terpenoid content, and the mechanistic logic is plausible. Phenolic compounds can act as pro-oxidants at high intracellular concentrations, tipping the redox balance of cancer cells, which already live under elevated oxidative stress, past the point of no return and triggering apoptotic signaling. Terpenoids such as carvacrol have been shown in other systems to disrupt mitochondrial membrane potential, activate caspase enzymes, and interfere with cell cycle progression. Quinic acid derivatives may contribute through modulation of inflammatory pathways that sustain tumor growth. It is important to stress, however, that the present study identifies associations rather than definitive mechanisms. The extract is a mixture of many compounds, and the specific molecule or combination of molecules responsible for killing MCF-7 cells has not yet been isolated or proven.

That caveat leads directly to the study’s stated limitations and next steps. The authors are explicit that further in vivo studies and compound purification are needed to confirm the observed effects and to clarify the underlying mechanisms. In vitro cell culture, while indispensable for screening, cannot capture the pharmacokinetics of a real organism: how a compound is absorbed, distributed, metabolized, and excreted, whether it reaches tumor tissue at active concentrations, and whether it harms healthy cells along the way. A crude methanolic extract that kills cancer cells in a dish may contain molecules that are too toxic, too unstable, or too poorly absorbed to ever become medicine. The purification pipeline, typically involving high-performance liquid chromatography fractionation followed by bioassay-guided testing, will be essential to isolate the active principles and determine whether a single compound or a synergistic ensemble is responsible.

Nevertheless, the study lands at a moment of surging interest in fungi as pharmaceutical factories. Mushrooms occupy a unique ecological niche as decomposers, and their evolutionary pressure to produce defensive secondary metabolites has equipped them with an extraordinary chemical arsenal. Established drugs derived from fungi include the statins, the immunosuppressant cyclosporine, and antibiotics such as penicillin, while mushroom-derived polysaccharides like lentinan from shiitake have been approved as adjuvant cancer therapies in some countries. Edible oyster mushrooms of the Pleurotus genus are particularly attractive candidates because they are cheap to cultivate on agricultural waste, fast-growing, and already consumed safely by millions of people, which simplifies the safety profile questions that plague many natural product candidates.

For now, the message for the public is one of measured enthusiasm rather than self-medication. Eating oyster mushrooms will not deliver the concentrated, chemically characterized extract tested in this study, and no clinical evidence yet supports any therapeutic use of P. eous in cancer patients. What the research does provide is a rigorous, statistically robust foundation for the next phase of investigation: isolating the active molecules, testing them against normal cells to establish selectivity, validating the findings in animal models, and ultimately, if the results hold, advancing toward clinical evaluation. Breast cancer remains one of the most prevalent malignancies worldwide, and the search for new agents, particularly those derived from accessible natural sources, remains urgent. A mushroom that most people walk past in the grocery aisle may, with enough scientific scrutiny, turn out to harbor chemistry worth far more than its weight in the produce bin. The journey from laboratory dish to pharmacy shelf is long and uncertain, but every credible candidate discovered along the way improves the odds.

Subject of Research: Anticancer and antioxidant potential of Pleurotus eous mushroom extract against human breast cancer MCF-7 cells in vitro

Article Title: Evaluation of the anticancer potential of Pleurotus eous against human breast cancer MCF-7 cell line

Article References: Al Qutaibi, M., & Kagne, S. R. (2026). Evaluation of the anticancer potential of Pleurotus eous against human breast cancer MCF-7 cell line. BMC Complementary Medicine and Therapies. https://doi.org/10.1186/s12906-026-05582-1

Image Credits: AI Generated

DOI: 10.1186/s12906-026-05582-1

Keywords: Pleurotus eous, oyster mushroom, breast cancer, MCF-7, anticancer, antioxidant, GC-MS, MTT assay, carvacrol, phenolic compounds, apoptosis, natural products