Oregano Extract’s Non-Polar Fraction Shows Genotoxic Effects on Algae

For centuries, oregano has graced kitchens and medicine cabinets alike, prized for its flavor and its remarkable array of biological activities. But a new study reveals a darker side to this beloved herb, one that could reshape how we think about its use as a natural pesticide and food additive. Researchers from the Institute of Biodiversity and Ecosystem Research at the Bulgarian Academy of Sciences have discovered that the non-polar fraction of a methanolic extract from Origanum vulgare spp. hirtum — commonly known as Greek oregano — possesses potent genotoxic, mutagenic, and DNA-damaging properties against the single-celled green alga Chlamydomonas reinhardtii. The findings, published in the journal BioRisk, mark the first time such effects have been documented for a carvacrol-rich fraction of oregano extract on a low eukaryote.

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