1. Introduction
Origanum vulgare L. is among plants with a broad spectrum of uses. This plant is native to Europe and Central Asia but has escaped gardens and naturalized in parts of the eastern and far western U.S. and Canada. From a botanical point of view, oregano is a perennial plant belonging to the
Lamiaceae, with a woody base and herbaceous stems that grow up to 20–80 cm. The species is highly variable in its morphological traits and chemical composition. According to the latest taxonomy, six subspecies have been distinguished based on morphological features, i.e.,
O. vulgare L. subsp. glandulosum (Desfontaines) Ietswaart,
O. vulgare L. subsp. hirtum (Link) Ietswaart,
O. vulgare L. subsp. gracile (Koch) Ietswaart,
O. vulgare L. subsp. virens (Hoffmannsegg et Link) Ietswaart,
O. vulgare L. subsp. vulgare L., and
O. vulgare L. subsp. viride (Boissier) Hayek [
1]. Oregano is a source of substances that give it its characteristic aroma and are responsible for its valuable antioxidant, antibacterial, and anti-inflammatory properties [
2,
3,
4,
5,
6,
7]. The phytochemical composition of the raw material depends on many factors. Both genetic and environmental conditions affect the qualitative and quantitative characteristics of phytoactive substances contained in the plant [
8,
9] and agronomic factors such as water and nitrogen supply, harvest time, and plant maturity stage [
10]. The phytochemical composition of extracts may vary depending on the plant subspecies and habitat type [
11].
Chemical substances such as carvacrol, thymol, c-terpinene, and p-cymene contained in the aerial part of oregano are believed to determine its high quality and commercial suitability. Other components in oregano, such as acyclic or bornane-type compounds, significantly influence the raw material features desirable in the pharmaceutical and cosmetic industries [
12,
13,
14]. The properties of oregano raw material and its extracts are widely described in the scientific literature [
15,
16,
17]. Much scientific evidence indicates high antioxidant [
17,
18] and antimicrobial [
19,
20] properties of oregano extracts. Other components of oregano that affect the growth of cancer cells are also the subject of research analyses. Chemical compounds contained in oregano seem to affect many mechanisms of cancer cell development. Numerous studies confirm its anticancer effects. Di Liberto et al. [
21] have proven that the extract in the form of Sicilian
Origanum vulgare L. oil induces apoptosis in breast cancer cells (human breast cancer epithelial cell line estrogen (ERα)-positive receptor MCF-7 and triple-negative human breast cancer cell line MDA-MB-231 (ER-, PR-, HER-2-negative). In addition, the anticancer effect of oregano extract on gastric cancer (apoptotic effect on AGS cancer cell lines, reduced ability of cancer cell migration, and thus prevention of cancer cell proliferation) [
22], breast adenocarcinoma (reduced viability and proliferation of MCF-7 cancer cells) [
23], cervical adenocarcinoma [
24,
25], and colon adenocarcinoma [
26] have been demonstrated. In order to effectively inhibit the development and growth of cancer cells, plant extracts must be as selective as possible about these cells. Studies conducted by Savini et al. [
27] on human colon cancer (Caco 2 cell line) show that oregano oil has such properties. Moreover, the entire extract, not a specific component, may be responsible for the observed cytotoxic effects. Studies by Di Liberto et al. [
21] on breast cancer cells (MDA-MB-231 and MCF-7 cell lines) also indicate that the cytotoxicity of oregano oil towards cancer cells is mainly due to the combination of its various components. In the presented studies, the effect of oregano extract on the viability of human cancer cells (Mcf-7 breast cancer cell line, Caco-2 c colon cancer cell line, Dld-1, Ht-29, Ls180, U87mg glioblastoma multiforme cell line, U251mg astrocytoma cell line, Sk-mel-28 melanoma cell line, and AGS gastric cancer cell line) and its cytotoxicity towards healthy cells (CCD841CoN colon epithelial cell line) were assessed.
Plant breeders are successively introducing new cultivars and hybrids of known herbal plants to make the offer of ornamental and functional plants at home more attractive. The described cultivars of origanum are valued because of their new attractive appearance (origanum ‘Aureum’) or taste and smell, so they are used as ornamental or spice plants (origanum ‘Hot & spicy’ has a spicy flavor, origanum ‘Margerita’ has a mild taste). Phytochemical substances are responsible for the intensity of the aroma. The same substances also determine the biological properties of the raw material. The preliminary analysis of the composition and properties of
Origanum vulgare grown in Poland shows a large variability of the composition for individual cultivars [
28].
Oregano cultivation in Poland is less widespread than in other southern European countries, but the climatic and soil conditions allow for high yields. Therefore, it seems that oregano grown in Poland could be an alternative to
O. vulgare varieties grown in regions with a Mediterranean climate. The possibility of growing oregano in climatic conditions other than those in the Mediterranean region and its good antioxidant and antimicrobial properties are essential signals for plant breeders as the demand for this raw material is growing. There are few origanum plantations in Poland, and the raw material is imported from abroad. However, the Joint Research Centre of the European Commission (JRC) published a report in 2021 on the authenticity of herbs and spices that can be purchased in the European Union. After conducting nearly 10,000 analyses, its authors determined that the percentage of samples considered “at risk of adulteration” was 17% for pepper, 14% for cumin, 11% for turmeric, 11% for turmeric, and 11% for saffron. In the case of oregano, this percentage was as high as 48% [
29]. Therefore, this work aimed to characterize the polyphenolic composition, antioxidant, and antimicrobial properties of different
O. vulgare varieties grown in Poland. All these analyses provided a detailed description of the features of
O. vulgare extracts. They allowed for identifying a variety that may be important for the pharmaceutical and food industries producing functional foods.
3. Discussion
Extracts from the tested oregano cultivars show antioxidant properties at the level of 1007.62–1143.18 [µmol TE/g] in the reduction of ABTS
•+ and 1167.76–2156.16 [µmol TE/g] in the reduction in the DPPH
• radical. Relating the obtained results to the results of other authors [
41], it should be stated that suitable antioxidant parameters characterize oregano grown in Poland. The obtained oregano can be a successful alternative to
O. vulgare cultivated in regions with a Mediterranean climate. Antioxidant activity, verified in numerous tests, including using the DPPH radical, as well as in the FRAP, ChP, and CUPRAC methods, showed that four cultivars:
O. vulgare L. (sample O1), Greek oregano
O. vulgare spp. hirtum (Link) Ietsw. (sample O2),
O. vulgare L. ‘Margerita’ (sample O3), and
O. vulgare L. ‘Aureum’ (sample O5) showed a similar level of antioxidant properties, exceeding the antioxidant activity of the
O. vulgare L. ‘Hot & spicy’ (sample O4). Total phenolic content analysis indicated that Greek oregano (sample O2) and the ‘Margerita’ (sample O3) cultivars had the highest polyphenol content. Large amounts of polyphenols were identified in the raw materials of these cultivars, such as isosalvianolic acid B and sagerinic acid, which were also present in the remaining cultivars but in smaller amounts. Studies by other authors indicate that these substances have been identified in other oregano species, such as
O. majorana [
42,
43]. Among the identified polyphenols, the indicated two—sagerinic acid and yunnaneic acid E—have not been identified in
O. vulgare. At the same time, it should be added that their identification was based on identical compounds, the presence of which was confirmed in Sage species, which belongs to the same botanical family as oregano [
39]. There is a high probability that the newly identified polyphenols may also occur in the characterized oregano genus. The dominant polyphenolic compounds in all oregano cultivars were rosmarinic acid, 4′-O-glucopyranosyl-3′, 4′-dihyroxybenzyl-protocatechuate, and luteolin O-di-glucuronide-O-di-pentoside, which is also confirmed by the work of other researchers [
44,
45]. Sarrou et al. [
46] indicated that rosmarinic acid is the main phenolic acid found in the raw material of
O. vulgare species. Yfanti et al. [
47] found that oregano is only sometimes a raw material rich in rosmarinic acid. However, many factors, e.g., agronomic or genetic, influence the phytochemical composition of the raw material [
48].
Polyphenols containing at least one phenolic hydroxyl group in their structure may have antioxidant activity—one of the mechanisms of cell protection against oxidative stress. In the conducted research,
O. vulgare L. (sample O1) and Greek oregano
O. vulgare spp. hirtum (Link) Ietsw. were characterized by an exceptionally high ability to engage in radical scavenging activity (sample O2). Since these cultivars show significant differences in the quantitative composition of polyphenols, the ability of these cultivars to have such high radical scavenging activity may be related to the activity of other substances that were not determined in the tests. The antioxidant potential of oregano extracts should also be considered, taking into account the characteristics of the oil substances, which is confirmed by the work of other researchers [
49,
50].
Studies on cancer cell lines have shown that the best cytotoxicity was characteristic of
O. vulgare L. extracts ‘Hot & spicy’ (sample O4). This cultivar was characterized by the best results in free radical scavenging tests and the ChP test. At the same time, the amount of identified polyphenols in extracts from this oregano cultivar was the lowest compared to other extracts. Dawra et al. [
51] indicate that the antiproliferative effect of oregano extracts on cancer cell lines may be related to the presence of polyphenols in the extracts, especially 4-coumaric acid or naringenin. Small amounts of coumaric acid were determined in the tested ‘Hot & spicy’ cultivar, and naringenin was not detected.
The use of cell lines for the assessment of in vitro cytotoxicity allows obtaining basic but important information on the effect of phytochemicals on the sensitivity of cancer cells [
52,
53]. Nine cancer cell lines were used in the conducted research, with the Caco-2 cell line being the most sensitive to the tested extracts. At the same time, the most significant cytotoxic effect was obtained for oregano extract, the ‘Hot & spicy’ variety, on the gastric cancer cell line. Extracts from the ‘Hot & spicy’ oregano cultivar showed satisfactory results in studies on reducing the invasive potential of gastric cancer cells and in migration and colony formation tests. The 100 g/mL extract dose reduced cell invasiveness by up to 84.98% and decreased migration by up to 85.32% compared to control tests. In the colony formation assay, complete inhibition of single AGS cells from forming colonies was observed at the dose used.
Many studies indicate that
O. vulgare extracts inhibit the growth of pathogenic microorganisms [
54,
55,
56]. The research showed that extracts from various oregano cultivars strongly limit the cell viability of
Staphylococcus aureus and
Pseudomonas aeruginosa bacteria. The ‘Hot & spicy’ cultivar had excellent killing properties against these two species of bacteria. According to Fouromiti et al. [
57], oregano extracts can be used as an antimicrobial agent against Gram-positive and Gram-negative bacteria. The antibacterial effect is mainly attributed to oil ingredients such as carvacrol, thymol, and gamma-terpinene [
58]. These substances cause the loss of cytoplasmic material from cells by leaking ions and intracellular compounds [
59].
In contrast to the results of other researchers [
60], extracts from the tested oregano cultivars showed a weak inhibitory effect on the growth of
C. albicans fungal cells. Souza et al. [
61] found that oregano oils could reduce the size of the
C. albicans inoculum by 99.9%. Also, Walasek-Janusz et al. [
62] proved that small amounts of extracts from various oregano cultivars, at 0.06–0.125 mg/mL, are sufficient to inhibit the development of
C. albicans. The reason for the poor fungicidal properties of the analyzed oregano cultivars requires a deeper analysis. Still, it seems that the concentration of the extract and volatile substances in the extract may be the reason for lower fungicidal values compared to the results achieved by other researchers. Lower fungicidal activity may also depend on the extraction method of biologically active substances. Bhat et al. [
63] showed in their research on the influence of oregano extracts on the growth of
C. ablicans fungi that extracts obtained using the maceration technique were characterized by worse fungicidal activity compared to the essential oil.
4. Materials and Methods
4.1. Materials and Reagents
Gallic acid, neocuproine, crystal violet, paraformaldehyde, EDTA (ethylenediaminetetraacetic acid disodium salt dihydrate), ferrozine, 2-Deoxy-D-ribose, NBT (nitrotetrazolium blue chloride), NADH (β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate), PMS (phenazine methosulfate), phosphate-buffered saline (PBS), RPMI-1640 medium, Dulbecco’s Modified Eagle Medium (DMEM), McCoy’s 5A medium, antibiotics (100 U/mL penicillin and streptomycins), fetal bovine serum (FBS), 0.25% trypsin-EDTA, 0.4% trypan blue solution, and Matrigel were purchased from Sigma-Aldrich (Steinheim, Germany). CellTiter 96®® AQueous Cell Proliferation Assay was purchased from Promega (Madison, WI, USA). All other chemicals were purchased from Chempur (Piekary Śląskie, Poland).
4.2. Plant Material
The plant material was obtained from five origanum cultivars: common oregano O. vulgare L. (sample O1); greek oregano O. vulgare spp. hirtum (Link) Ietsw. (sample O2); O. vulgare L. ‘Margerita’ (sample O3); O. vulgare L. ‘Hot & spicy’ (sample O4); and O. vulgare L. ‘Aureum’ (sample O5).
Plants of all cultivars were obtained from the collection of the Garden of Cosmetic Plants and Raw Materials of the Research and Science Innovation Center in Wola Zadybska near Lublin (Poland) (51°44′49″ N 21°50′38″ E). Botanical identification was performed by the Curator of the Department of Aromatic Plants, Dr. Anna Kiełtyka-Dadasiewicz. The plants were grown in clay soil of loess origin. The upper, leafy, flowering shoots were collected from a 2-year-old plantation in June 2022, in the full flowering phase. The samples were dried for 3 h to achieve a constant humidity level of 12%. After drying (at 35 °C), the stems were manually separated and discarded as the least valuable parts of the raw material. The crushed herb was used as a raw material to obtain extracts. Voucher specimens were deposited in the Research and Science Innovation Center.
4.3. Preparation of the Extract
Dried oregano was ground, mixed with 50% methanol, and subjected to extraction assisted by ultrasonic waves (30 °C, 20 min, 50 Hz) (Sonic 10, Polsonic, Poland) [
64]. After this time, the samples were centrifugated (Centrifuge 5430, Eppendorf, Hamburg, Germany) at 10,000×
g for 10 min, and received supernatants were pre-evaporated on a rotary evaporator (R-215 Rotavapor System, Buchi, Switzerland) and lyophilized (ALPHA 1–2 LD plus, Osterode, Germany). The obtained powders were dissolved in water and analyzed.
4.4. Identification of Phenolic Compounds
Polyphenolic compounds were determined using ultra-performance liquid chromatography (UPLC-PDA-MS/MS) of the Waters ACQUITY system (Waters, Milford, MA, USA). The UPLC system (UPLC-PDA-MS/MS) is equipped with a binary pump manager, column manager, sample manager, a photodiode array detector (PDA), and a tandem quadrupole mass spectrometer (TQD) with an electrospray ionization source (ESI). The separation of polyphenols was performed using a 1.7 µm, 100 mm × 2.1 mm UPLC BEH RP C18 column (Waters, Milford, MA, USA). For separation, the mobile phase consisted of 0.1% formic acid in water, v/v (solvent A), and 0.1% formic acid in 40% acetonitrile, v/v (solvent B). The flow rate was kept constant at 0.35 mL/min for a total duration of 8 min. The system was run with the following gradient program: from 0 min 5% B, from 0 to 8 min linearly to 100% B, and from 8 to 9.5 min for washing and returning to the initial conditions. The sample injection volume was 5 μL, and the column was maintained at 50 °C. The following TQD parameters were used: cone voltage 30 V, capillary voltage 3500 V, source and desolvation temperatures 120 °C and 350 °C, respectively, and desolvation gas flow rate 800 L/h. Characterization of individual polyphenolic compounds was carried out on the basis of retention time, mass-to-charge ratio, fragment ions, and comparison with data obtained from commercial standards and literature results. The obtained data were processed in the Waters MassLynx v.4.1 (Waters, Milford, MA, USA). The method was validated for parameters such as linearity, accuracy (relative error, RE), limit of detection (LOD), limit of quantification (LOQ), and precision (relative standard deviation, RSD). Quantification was performed by injecting solutions with known concentrations from 0.05 to 5 mg mL−1 (R2 ≤ 0.999) of the following phenolic compounds as standards: protocatechuic acid, rosmarinic acid, caffeic acid, p-coumaric acid, salvianolic acid B, and Luteolin 7-O-glucoside (Extrasynthese, Genay Cedex, France). Stock-standard solutions of polyphenols were prepared using methanol. Six calibrators determined the peak area ratio of each polyphenol to its nominal concentration. The regression equation was obtained using weighted (1/c2) least squares linear regression. LOD was defined as a signal-to-noise (S/N) ratio of 3:1, and LOQ was defined as a signal-to-noise ratio >10. An allowable RE within ± 20% and intra- and inter-day variability were determined using the relative standard deviation values (RSD), which was determined using relative standard deviation (RSD) values that were <3.5% for all compounds analyzed.
4.5. Total Phenolic Content
The total phenolic content was assessed according to the method of Gao et al. [
65]. Briefly, 2.0 mL of water, 0.2 mL of Folin–Ciocalteu reagent, and 1.0 mL of 20% sodium carbonate solution were added to the tested extracts. After 60 min, the absorbance of 765 nm was measured (UV–VIS spectrometer, Type UV2900, Hitachi, Japan). The results were expressed in mg gallic acid (GAE)/g dm.
4.6. Evaluation of Antioxidant Activity
4.6.1. Superoxide (O2•−) Radical Scavenging Activity Assay
Superoxide radical scavenging activity was assessed according to the method of Robak and Gryglewski [
66]. Oregano extracts were mixed with 1.0 mL of 150 µM NBT, 1.0 mL of 468 µM NADH, and 1.0 mL of 60 µM PMS and left for 5 min. The absorbance was measured at 560 nm. The results were expressed as IC
50 (µg/mL).
4.6.2. Hydroxyl (OH•) Radical Scavenging Activity Assay
Hydroxyl radical scavenging activity was assessed according to the method of Halliwell et al. [
67]. Oregano extracts were mixed with 0.1 mL of 0.2 mM 2-deoxyribose, 0.1 mL of 1.0 mM iron ammonium sulfate, 0.1 mL of 1.04 mM EDTA, 0.01 mL of 1.0 mM ascorbic acid, 0.01 mL of 0.1 M perhydrol, 1.0 mL of 2.8% trichloroacetic acid, and 0.5 mL of 1% thiobarbituric acid and heated to 100 °C for 15 min. After this time, the samples were cooled to room temperature, and the absorbance was measured at 532 nm. The results were expressed as IC
50 (µg/mL).
4.6.3. Chelating Potential of Ferrous Ion
The chelating ability of ferrous ions was assessed according to the method of Żurek et al. [
68]. Oregano extracts were mixed with 0.4 mL of 0.25 mM ferrozine and 0.2 mL of 0.1 mM iron II sulfate and left for 10 min. The absorbance was measured at 562 nm. The results were expressed as IC
50 (µg/mL).
4.6.4. ABTS•+ Radical Scavenging Activity
The scavenging activity of extracts on ABTS
•+ radicals was assessed according to the method of Re et al. [
69]. Oregano extracts were mixed with 0.03 mL of ABTS
•+ solution and left for 6 min. The absorbance was measured at 734 nm. The results were expressed as Trolox Equivalent (mmol TE/g dm).
4.6.5. Determination of Copper Ion Reduction
The copper ion reduction was assessed according to the method of Apak et al. [
70]. Oregano extracts were mixed with 1.0 mL of 7.5 mM neocuproine, 1.0 mL of 10 mM copper chloride, and 1.0 mL of 1 M acetate buffer and left for 30 min. The absorbance was measured at 450 nm. The results were expressed as Trolox Equivalent (mmol TE/g dm).
4.6.6. DPPH Test
The DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity test was performed according to the modified method of Brand-Williams [
71]. Samples of oregano extracts were diluted to an appropriate concentration within the calibration curve. Measurements were made by placing 0.5 mL of the diluted extract into spectrophotometric cuvettes, adding 2 mL of DPPH solution to them, and mixing after 10 min of incubation in the dark. Spectrophotometric measurements were made at a wavelength of λ = 517 nm against 96% methanol. The results were converted to mmol TE/g dm and as IC
50 (µg/mL).
4.6.7. Determination of Ferric Reducing Power
The iron-reducing antioxidant power (FRAP) level was determined by reducing Fe
2+ to Fe
3+ in an acidic medium using a modified method of Benzie and Strain [
72]. To prepare the FRAP reagent, 300 mM acetate, pH 3.6 acetic acid buffer, ferric chloride (FeCl
3·6H
2O), and 10 mM 4,6-triridyl-triazine (TPTZ) were prepared in 40 mM HCl. All three solutions were freshly mixed together at 10:1:1 (
v/
v/
v). Performing the determination: 3 mL of FRAP reagent solution was added to the collected 0.5 mL samples and mixed thoroughly. After 10 min at 37 °C, the absorption was measured at a wavelength of 593 nm against distilled water. The results were converted into Trolox Equivalents (mmol TE/g dm).
4.7. Cell Culture
The human cell lines Mcf-7 (breast cancer), Caco-2, Dld-1, Ht-29, Ls180 (colorectal cancer), U87mg (glioblastoma), U251mg (astrocytoma), Sk-mel-28 (melanoma), AGS (gastric cancer), and CCD841CoN (colon epithelial cells) were used in the analysis. Selected cell lines were cultured in DMEM, RPMI 1640, or McCoy’s 5A medium, which were enriched with 10% FBS and 1% antibiotics. Cells were incubated at 37 °C in a humidified atmosphere under 5% CO2 (incubator CB170, Binder, Tuttlingen, Germany).
4.8. Cell Viability Assay
Cell viability was assessed using the MTS test, as described in another study by Żurek et al. [
73]. Briefly, the analyzed cell lines were seeded in a 96-well plate at a concentration of 8000 cells/well and incubated for 24 h for cell adhesion. The cells were exposed to five concentrations of oregano extracts (10, 100, 250, 500, and 750 µg/mL). After 48 h, the extracts were removed, the cells were washed with PBS, a fresh medium was added, and the MTS assay was performed according to the manufacturer’s instructions (Promega). Absorbance was measured at 490 nm using a microplate reader (SmartReader 96, Accuris Instruments, St. Louis, MO, USA). The results were expressed as the IC
50 (µg/mL). Cisplatin was used as a standard (
Table S3).
4.9. Wound Scratch Test
The wound healing test was performed according to the method of Liang et al. [
74]. AGS cells were seeded in 24-well plates at a concentration of 400,000 cells/well and cultured until they reached 80–90% confluence. Then, a wound was made using a sterile pipette tip, and the cells were washed with PBS and treated with extracts at a concentration below the IC
50 value. Cells were photographed using inverted light microscopy (Oxion Inverso, Euromex, Mataró, Spain) until the wound was closed entirely. Results were expressed as % wound closure.
4.10. Migration and Invasion Cell Assay
The ability of cells to migrate and invade was assessed using the transwell chamber assay. Cells (20,000 cells/well) were seeded on a 24-well plate with transwell chambers (pore size 8 μm) and treated with the tested extracts. For the invasion assay, the transwell chamber was coated with 25 µL of Matrigel two hours before seeding. After 48 h, the medium was collected, and the cells were washed with PBS, fixed in 4% paraformaldehyde, treated with ethanol, and stained with 0.5% crystal violet. The stained cells were photographed under a microscope at 20× magnification. The results were expressed as % of migrating and invasive cells relative to the control.
4.11. Colony Formation Assay
AGS cells were seeded in a 6-well plate at a concentration of 500 cells/well and placed in the incubator for 24 h. After this time, the medium was removed, and the cells were treated with oregano extract for 48 h. The extracts were then removed, fresh medium was added, and culture was continued for 14 days, changing the medium every 3/4 days. The formed colonies were fixed with methanol, stained with 0.5% crystal violet, and counted under an inverted microscope. Results are expressed as % colony formation relative to control.
4.12. Evaluation of Antimicrobial Properties
Antimicrobial properties were assessed using the PrestoBlue viability assay (Cat. No. A13261, Invitrogen, Waltham, MA, USA). The microorganism strains used in the study were Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Candida albicans (ATCC 90028). Microorganism cultures were diluted in sterile buffer saline to adjust the optical density to 0.5 on the McFarland scale. A total of 90 μL of each microorganism suspension was added into 96-well plates, and 10 μL of each extract was added to final concentrations [%] as follows: 2.5; 1.25; 0.625; 0.3125; 0.156; 0.078. Wells with microorganisms with the addition of 10 μL sterile buffer saline to replace extracts were control samples. Plates were incubated for 24 h at 37 °C. After that, 10 μL of PrestoBlue reagent was added into each well and incubated for 10 min at 37 °C protected from the light. After the incubation, fluorescence was measured (λex = 570 nm, λem = 600 nm) using a microplate reader (ELISA) (Infinite M200, Tecan, Durham, NC, USA). The results were repeated a minimum of three times for each group. Results were calculated as a percentage of the control samples for each microorganism strain. Results are presented as mean values ± standard deviation.
4.13. Statistical Analysis
For the PrestoBlue test, a one-way analysis of variance with Tukey’s posthoc test (HSD) was performed using GraphPad Prism 9 software (version 9.2.0, San Diego, CA, USA). The statistically significant differences were considered at p-value ≤ 0.05.
Statistical analysis of the results was performed in Statistica version 13 (TIBCO Software Inc., Palo Alto, CA, USA). Analysis of variance (ANOVA) was used to test (α = 0.05) significant differences between oregano cultivars. A comparison of means was performed using the Tukey test, where α = 0.05.
5. Conclusions
The tested oregano cultivars are an essential source of antioxidants. In particular, the cultivars O. vulgare L. (sample O1) and Greek oregano O. vulgare spp. hirtum (Link) Ietsw (sample O2) were characterized by high antioxidant potential, so they could be successfully used where protection against oxidative processes is required; it is worth including these varieties in the daily diet or as food additives. Oregano is also a raw material rich in antioxidant phytochemicals that maintain health properly.
The most important conclusions from the conducted studies are:
- -
the dominant polyphenol in all oregano extracts tested was rosmarinic acid. The largest amounts were identified in extracts from the O. vulgare spp. hirtum (Link) Ietsw. (sample O2), and the smallest amounts in O. vulgare L. ‘Hot & spicy’ extracts (sample O4);
- -
polyphenols such as sagerinic acid and yunnaneic acid E were identified in the extracts, which had not been identified in O. vulgare before;
- -
the prepared extracts show similar antioxidant activity determined in the ABTS•+, FRAP, DPPH, and CUPRAC tests;
- -
the most effective in O2•− and OH• radical scavenging activity was the extract the ‘Hot & spicy’ cultivar (sample O4);
- -
extracts from the ‘Hot & spicy’ cultivar (sample O4) were characterized by the best cytotoxic potential with respect to selected cancer cell lines;
- -
oregano extracts, especially from the ‘Hot & spicy’ cultivar, have an inhibitory effect on the viability of S. aureus and P. aeruginosa. At the same time, they show mild fungicidal activity against C. albicans.
Therefore, future work should focus on the precise characterization of this oregano variety and the extracts obtained from it. Further detailed studies evaluating the effect of extracts from the ‘Hot & spicy’ cultivar on the cytotoxicity of cancer cell lines should be continued.
At the same time, the characteristics of all oregano cultivars analyzed in this publication require further detailed studies. In particular, a complete quantitative and qualitative characterization of newly identified phenolic compounds is indicated, including their isolation and confirmation of chemical structure using NMR analysis.
It seems that oregano grown in Poland can be successfully used by various industries that use this raw material. At the same time, good antioxidant and antimicrobial properties are essential signals for plant breeders because the demand for this raw material is growing. All the tested cultivars can be grown on a large scale if a suitable application is found.