Supercritical CO2 oregano extracts: antimicrobial and antibiofilm activity studied in the laboratory

In practical terms, the extracts hindered the growth of the microorganisms examined, though not all with the same intensity. Bacteria were generally found to be more sensitive than fungi. For bacteria, the minimum inhibitory concentration—the lowest amount capable of preventing visible growth—was between 0.25 and 1.0 mg/mL. For some Fusarium species, however, 7.5 or 15 mg/mL were required.

The extract produced at 40 °C showed the most thoroughly evaluated effect on biofilm. At 2×MIC, it completely prevented biofilm formation by L. monocytogenes and S. aureus, while against Pseudomonas aeruginosa, the inhibition was 34%. On mature biofilms, which are more difficult to remove, the effect was partial, ranging from 18% to 52%. The observed differences indicate that the microbial species, dosage, and extract preparation influence the result.

The research is primarily relevant to the microbiological safety of food and surfaces, not for the treatment of infections. It shows that a plant extract is not a fixed substance: temperature, solvent, and extraction process can change the profile of the recovered molecules and the measured activity. The same principle also emerged in a previous study on oregano fractions obtained with supercritical CO2 [1].

The result regarding biofilm deserves attention because these structures make microorganisms more difficult to control. Another study on the essential oil of O. vulgare subsp. hirtum observed activity against free-floating cells and biofilms of Salmonella and Listeria [2]. However, essential oils and supercritical extracts have different compositions and concentrations: they are not equivalent products.

The main limitation is that all the evidence was conducted in vitro. An effect in a petri dish does not prove that the extract prevents or cures infections in people, nor that it is safe to ingest, inhale, or apply to the skin. No individuals, animals, absorption, metabolism, clinical toxicity, or drug interactions were studied.

Furthermore, the effect was measured against specific strains and conditions. The presence of phenolic acids, flavonoids, and triterpenes documents a complex composition, but it does not identify with certainty which molecule is responsible for the activity. It is also not possible to equate these extracts to the common food spice: the experimental concentrations and the form of preparation are different.

The chain of confirmation begins with preparation. Santoyo and colleagues compared oregano leaf fractions obtained via supercritical CO2 in an experiment on six microorganisms: all were active, but the most effective fraction was the one rich in volatile components obtained with specific parameters [1]. This directly supports the idea that extraction modifies activity, even while using different equipment and microbial species.

The next step concerns the biofilm. Kolypetri and colleagues studied an essential oil of O. vulgare subsp. hirtum in vitro against two foodborne pathogens. The minimum inhibitory concentrations were 0.03-0.06%, and those required to prevent biofilm were 0.06-0.13%; treatment at 6.25% for 15 minutes eliminated biofilm cells in their model [2]. The confirmation is moderate because it concerns an oil, not the substance extracted by Gwiazdowska.

A third study analyzed an ethanolic extract against twelve oral isolates and found biofilm inhibition that varied between strains and the surface model [3]. It therefore confirms that the effect can extend to adherent microbial communities, but also emphasizes that it is not uniform and remains confined to laboratory tests.

The data do not indicate equal activity for all preparations. Stamenic and colleagues compared supercritical extracts of Greek oregano with oil obtained via hydrodistillation. The supercritical extracts were more active against staphylococci, while the distilled oil was more effective against Escherichia coli, Salmonella, and Klebsiella pneumoniae [4]. This result is relevant because it demonstrates a dependency on the extraction method and the bacterial species.

Even more clearly, Sahin and colleagues detected essential oil activity against 10 bacteria and 15 fungi or yeasts, but no antimicrobial activity from the methanolic extract of the plant's aerial parts [5]. No directly conflicting evidence sufficiently relevant to each individual data point from Gwiazdowska and colleagues was identified; this does not prove the absence of data contradicting what was observed. Overall, the literature suggests that composition, dose, and microorganism strongly influence the outcome.