1. Introduction
Throughout human history, medicinal plants have played a crucial role in the prevention and treatment of various diseases [
1]. The increasing prevalence of diseases in modern times has led to a renewed interest in the medicinal properties of these plants for therapeutic purposes [
2]. One such plant with notable antimicrobial and antioxidant properties is
Origanum onites, commonly known as Turkish oregano.
The use of medicinal plants dates back to ancient civilizations, where they were employed to treat a wide range of ailments [
3]. Traditional systems of medicine, such as Ayurveda, Traditional Chinese Medicine, and Unani, have relied heavily on the therapeutic properties of plants [
4]. Today, many modern pharmaceuticals are derived from plant sources, highlighting the continued importance of medicinal plants in healthcare [
1].
The rise in antibiotic-resistant pathogens has become a significant global health concern [
5]. As a result, researchers are increasingly exploring the antimicrobial properties of medicinal plants as potential alternatives to conventional antibiotics [
6]. Numerous studies have demonstrated the effectiveness of plant extracts and essential oils against a variety of pathogenic microorganisms, including bacteria, fungi, and viruses [
7].
The antioxidant properties of medicinal plants are also of great interest due to their potential role in preventing chronic diseases such as cancer, cardiovascular disease, and neurodegenerative disorders [
8]. Oxidative stress, caused by an imbalance between the production of reactive oxygen species (ROS) and the body’s antioxidant defenses, has been implicated in the pathogenesis of these diseases [
9]. Plant-derived antioxidants, such as flavonoids, phenolic acids, and terpenoids, can help neutralize ROS and protect cells from oxidative damage [
8].
There are about 23 species and 32 taxa related to the genus
Origanum (Lamiaceae) in Turkey [
10,
11]. In Anatolia, members of the genus
Origanum are often used as culinary herbs, spices, and herbal tea, and are called kekik [
10,
12,
13,
14].
O. onites is called by names such as ari kekik, bilya kekik, bilyali kekik, incir kekigi, izmir kekigi, kirkbas kekik, tokali kekik, and yemis kekigi in Turkish [
15,
16,
17].
O. onites is a steno-endemic taxon with a narrow distribution area covering only the Eastern Mediterranean region [
18]. The leaves of this plant are widely used in traditional medicine due to their antimicrobial and antioxidant properties [
19].
The antimicrobial properties of
O. onites are attributed to its bioactive compounds, such as phenolic acids and terpenoids, which demonstrate effectiveness against various pathogenic microorganisms [
20]. Numerous studies have shown that extracts of the plant inhibit the growth of bacteria and fungi and, in some cases, even kill them [
21]. In particular, the antimicrobial properties of
O. onites have enabled its use as a natural preservative in the food industry [
22].
The antioxidant properties of
O. onites are significant due to their ability to neutralize free radicals [
19]. Free radicals are molecules that can cause cellular damage and aging. The antioxidant properties of
O. onites, attributed to its high content of flavonoids, phenolic acids, and terpenoids, may play a potential role in preventing cancer, heart disease, and other chronic diseases [
23].
Among the medicinal uses of
O. onites is the treatment of various ailments, such as respiratory infections, digestive system issues, pain, and inflammation [
20]. Additionally, due to its antioxidant properties, the plant can also be used for skin health [
23].
According to previous research,
O. onites has been found to contain a variety of significant essential oils, including carvacrol,
p-cymene, and
γ-terpinene, all of which are present in amounts exceeding 1%. Additionally, notable hydrophilic compounds such as rosmarinic acid, 4-hydroxybenzoic acid, caffeic acid, gentisic acid, apigenin-7-glucoside, 4-hydroxybenzaldehyde, and vanillic acid have been identified [
24].
In conclusion, the antimicrobial, antioxidant, and medicinal uses of
O. onites have been investigated by numerous researchers. Extracts obtained from the leaves of the plant can be used as natural preservatives in the food industry due to their effectiveness against various pathogenic microorganisms [
22]. Furthermore, the antioxidant properties of the plant may play a potential role in preventing various diseases [
19]. Its medicinal uses include the treatment of respiratory infections, digestive system issues, and pain-related ailments [
20]. However, there is a need for further investigation of the antimicrobial activity of
O. onites ethanol extract (OOEt) against a wider range of microorganisms, including multidrug-resistant strains, to better understand its therapeutic potential. The primary aim of this study is to investigate the antimicrobial activity of
O. onites ethanol extract (OOEt) against a wide range of microorganisms, including multi-drug-resistant strains, as well as its antioxidant activity, which has not been adequately studied in previous literature.
3. Results
The effectiveness of OOEt in inhibiting various microorganisms can be observed through the inhibition zone diameters presented in
Table 1. With different concentrations (50 µL, 100 µL, and 200 µL) tested against a wide range of bacterial and fungal strains, the data suggest that the antimicrobial activity of OOEt is concentration-dependent and exhibits varying levels of inhibition against each tested microorganism. The negative controls showed no activity, and the ANOVA test showed that there were no significant differences between parallels (
p > 0.05) in the antimicrobial activity tests. The overall Pearson correlation coefficient for the concentration increase and the average inhibition zone diameters across about 73% of all microorganisms is higher than 0.8000, indicating a strong positive correlation (
Table 2).
In order to better understand the effectiveness of OOEt against MDR strains and the resistance levels of the MDR strains used in this study, the effects of various antibiotics on these strains were investigated. The findings of this analysis are presented in
Table 3, which illustrates the susceptibility of the MDR strains to a wide range of antibiotics.
The minimum inhibitory concentration (MIC) test results for a range of microorganisms, as presented in
Table 4, reveal varying susceptibilities to OOEt. These microorganisms include both Gram-positive and Gram-negative bacteria, as well as yeasts.
B. subtilis DSMZ 1971,
C. albicans DSMZ 1386, and
L. innocua (FI) all demonstrated MIC values of 4.28 mg/mL when exposed to OOEt. On the other hand, higher MIC values of 34.3 mg/mL were observed for
S. enteritidis ATCC 13076,
S. typhimurium SL 1344,
K. pneumoniae (FI),
S. boydii (CI),
C. tropicalis (CI),
E. coli (MDR), and
K. pneumoniae (MDR) when treated with OOEt. The remaining tested microorganisms displayed MIC values between 8.57 and 17.15 mg/mL, indicating a range of susceptibilities to the OOEt.
In the DPPH radical scavenging activity test, the tested concentrations ranged from 1.075 to 200 µg/mL (
Table 5). Based on the obtained results, the EC
50 value for ascorbic acid was calculated as 8.5232 µg/mL and the EC
90 value as 28.60 µg/mL. However, it is important to note that the lowest concentration of OOEt tested was 1.075 µg/mL, which exhibited a scavenging activity of 59.83%, surpassing the 50% threshold. Therefore, attempting to approximate the EC
50 based on this data would result in a calculation error. Consequently, the EC
90 calculation would also be affected by this error. Nevertheless, considering the available data, it is reasonable to suggest that the EC
90 value for OOEt could be in the vicinity of 25 µg/mL.
The biochemical composition and respective percentages of OOEt, as determined by GC-MS analysis, are displayed in
Table 6. The GC-MS chromatogram of OOEt is given in
Figure 1.
4. Discussion
In this study, the antimicrobial activity of OOEt was evaluated against a variety of microorganisms using both disk diffusion and MIC methods. OOEt demonstrated antimicrobial activity against all 30 tested strains, with high susceptibility (≥15 mm) in each instance where 200 mL of the extract was applied. In the disk diffusion test, the most susceptible Gram-positive bacterium, E. faecium, showed a 52-mm inhibition zone at 100 µL of OOEt and a MIC value of 17.15 mg/mL. Among Gram-negative bacteria, P. aeruginosa and P. fluorescens displayed the highest sensitivity, both presenting a 24 mm inhibition zone at 100 µL of OOEt in the disk diffusion test and a MIC value of 17.15 mg/mL. For multidrug-resistant bacteria, E. coli (MDR) exhibited the highest susceptibility compared to all positive controls, with a disk diffusion inhibition zone of 19 mm at 200 µL of OOEt and a MIC value of 34.3 mg/mL. In the case of fungal strains, OOEt was more effective than the positive controls, with inhibition zones observed in the disk diffusion test and corresponding MIC values.
A. baumannii is an opportunistic pathogen that colonizes hospitalized patients, leading to severe infections, septic shock, and death. These bacteria often cause urinary tract infections and pneumonia, especially in patients in intensive care units [
44]. A large-scale surveillance study conducted in the United States found that
A. baumannii is responsible for 5–10% of acquired cases of pneumonia in intensive care [
45]. Although the frequency of nosocomial pneumonia caused by
A. baumannii varies from country to country and region to region (27–50%), the mortality rate in these types of pneumonia is between 30 and 70% [
46]. Among
A. baumannii infections, urinary tract infections experienced by patients with catheters have an important place. As a result of a study, it was found that 1.6% of urinary tract infections acquired in intensive care were due to
A. baumannii [
47]. Cases of meningitis associated with
A. baumannii also occur, especially in patients undergoing brain surgery with ventricular drainage. The mortality rates (70%) of these cases are quite high [
48]. In addition, these microorganisms lead to many types of infections, such as skin and wound infections, endocarditis, peritonitis (often in patients with peritoneal dialysis), conjunctivitis, osteomyelitis, and synovitis [
44]. Bacteremia and sepsis caused by
A. baumannii are also common in patients in intensive care units [
44,
46]. The widespread use of broad-spectrum antibiotics in hospitals has led to the rapid emergence of multidrug-resistant (MDR) strains of
A. baumannii. Despite this, only a few antibiotics are effective against
A. baumannii (MDR) infections [
49]. Ozgen et al. [
50] observed a 10.5 mm inhibition zone for the ethanol extract of
O. vulgare leaves against
A. baumannii ATCC BAA-747. Canlı et al. [
51] showed that
Lavandula stoechas (Lamiaceae) caused 11 mm of inhibition zone for 35.1 mg ethanol extract against the same
A. baumannii (MDR) strain. In our study, we determined that OOEt presented a 19 mm inhibition zone for 200 µL OOEt against
A. baumannii (MDR) and a MIC value of 34.3 mg/mL. This result is proof that OOEt Is more effective than the other two plants.
In our study, the A. baumannii (MDR) strain we used demonstrated high resistance, with the largest measured zone being 16 mm for the tested antibiotics, indicating that most antibiotics were ineffective or displayed very low efficacy. However, our findings indicate that OOEt effectively inhibits the growth of A. baumannii (MDR), highlighting the potential of OOEt as a promising candidate for the development of new antimicrobial agents, especially against highly resistant strains such as the A. baumannii (MDR) strain used in our study.
Enterococci are facultative anaerobic Gram-positive bacteria that naturally inhabit the intestinal flora of animals and humans. These bacteria are typically considered to have low pathogenicity, mainly infecting immunocompromised individuals in oncology, hematology, nephrology, or transplantation units. Enterococci can cause various infections in the urinary and biliary tracts, wounds, and life-threatening diseases, such as bacteremia or endocarditis. They are the second- to third-most important bacterial group, causing approximately 12% of nosocomial infections [
52]. The
Enterococcus genus comprises over 50 species, with
E. faecalis and
E. faecium being the primary causative agents of infections in humans. Enterococci emerged in the 1970s as a leading cause of nosocomial infections [
53].
E. faecalis accounts for 85–90% of enterococcal infections, while
E. faecium accounts for 5–10% [
54]. In the last two decades,
E. faecium has rapidly evolved as a global nosocomial pathogen, successfully adapting to the nosocomial environment and acquiring resistance to glycopeptides [
53]. Sener et al. [
55] reported the antimicrobial activity of a 65% ethanol extract of
Origanum majorana against fifteen bacterial strains, including
E. faecium. The extract showed antimicrobial activity against
E. faecium with a 9-mm inhibition zone at 100 µL. In our study, we found that OOEt had a 52-mm inhibition zone at 100 µL against
E. faecium and a MIC value of 21.7 mg/mL. These results indicate that OOEt is more effective against
E. faecium than the ethanol extract of
O. majorana.
Pseudomonas aeruginosa (Pseudomonadaceae) is a Gram-negative bacterium that is ubiquitous and can survive in a wide variety of environments [
56].
P. aeruginosa, defined as an opportunistic pathogen, is the most common bacterium that causes nosocomial infections, bacteremia, ventilator-associated pneumonia, urinary tract infections, and skin and soft tissue infections [
56,
57].
P. aeruginosa causes fatal infections in immunocompromised patients in oncology, post-surgery, severe burns, or those infected by HIV. It has been described as one of the most life-threatening bacteria and was listed by the WHO as a priority pathogen in the R&D of new antibiotics in 2017. Due to the adaptability of
P. aeruginosa and high antibiotic resistance, antibiotics often show limited efficacy, and thus mortality increases [
58]. Husein et al. [
59] observed a 14.7-mm inhibition zone for a 70% ethanol extract of
Origanum syriacum against
P. aeruginosa. In our study, we found that OOEt exhibited a 24 mm inhibition zone in the disk diffusion assay using 100 µL and had a MIC value of 17.15 mg/mL against
P. aeruginosa. These results showed that OOEt has more effective results compared to the previously reported
O. syriacum extract, highlighting its potential in terms of antimicrobial efficacy against
P. aeruginosa.
Candida species are among the most deadly fungi.
Candida species cause invasive candidiasis in immunocompromised patients who have been in intensive care for a long time due to severe trauma. Among them,
C. albicans is the most common cause of life-threatening systemic candidiasis.
C. albicans is an opportunistic pathogen that exists symbiotically in most individuals and is one of the most common causes of mucosal and systemic infections.
C. albicans, unlike most fungal pathogens, is generally considered to be obligatorily associated with warm-blooded animals [
60,
61]. Kerbouche et al. [
62] discovered the antimicrobial activity of an ethanol extract of
Origanum floribundum against
C. albicans with a 9.7 mm inhibition zone. In our study, we found that OOEt exhibited a 15-mm inhibition zone in the disk diffusion assay using 200 µL against
C. albicans, demonstrating more effective results compared to the previously reported
O. floribundum extract. Furthermore, the MIC value of OOEt was found to be 4.28 mg/mL, which highlights its notably high efficacy as an antimicrobial agent against
C. albicans. Additionally, our results also revealed significant antimicrobial activity against another
Candida species,
C. tropicalis, with inhibition zones of 31 mm for both 100 and 200 µL and a MIC value of 34.3 mg/mL, emphasizing the importance of OOEt as a potential antimicrobial agent against multiple
Candida species.
Living organisms are constantly exposed to reactive oxygen species generated as a result of respiratory, metabolic, or disease stress [
63]. It is important to eliminate oxidation caused by reactive oxygen species, which cause many diseases, and to neutralize free radicals [
64]. In our study, we observed that the DPPH radical scavenging activity of OOEt was comparable to that of ascorbic acid, which served as a positive control. The EC
50 value for ascorbic acid was determined to be 8.5232 µg/mL, while the EC
90 value was found to be 28.60 µg/mL. Based on our findings, we propose that the EC
90 value for OOEt is approximately 25 µg/mL, which falls within or below the EC
90 range of ascorbic acid. In a study conducted by Kosakowska et al. [
65], the antioxidant activity of essential oils and hydroethanolic extracts from Greek oregano (
O. vulgare L. subsp.
hirtum) and common oregano (
O. vulgare L. subsp.
vulgare) was evaluated. The DPPH scavenging activities for the hydroethanolic extracts of Greek and common oregano were reported as 70.90% and 69.83%, respectively, with corresponding Trolox equivalent values of 252.10 and 242.43 µmol/g. In another study by Kaurinovic et al. [
66], the DPPH scavenging activity of various
O. basilicum and
O. vulgare extracts was investigated, and the IC
50 values for
O. vulgare water and n-BuOH extracts were found to show stronger antioxidant effects than BHT. In comparison to the findings of Kaurinovic et al., our results indicate that OOEt possesses a more potent antioxidant capacity. Our findings demonstrate that OOEt has remarkably potent antioxidant activity, even surpassing the effect of ascorbic acid, a well-known antioxidant agent. This suggests that OOEt could be a valuable natural source of antioxidants and may have potential applications in the prevention and treatment of diseases associated with oxidative stress.
The GC-MS analysis of OOEt revealed the presence of several compounds with known biological activities, which may contribute to the observed antimicrobial and antioxidant activities. The most abundant compound identified in the extract was carvacrol (82.34%), which has been previously reported to exhibit antioxidant and antimicrobial activities [
34,
35,
36]. Other notable compounds include thymoquinone (1.09%), which has demonstrated neuroprotective and anti-inflammatory effects [
32,
33], and borneol (1.00%), which has been shown to possess antibacterial activity [
30]. In addition to these major compounds, the extract also contained several other biologically active compounds, albeit in smaller quantities. These include sabinene hydrate (0.75%), which has been reported to have antioxidant activity [
29], 4-carvomenthenol (0.79%), known for its anti-inflammatory activity [
31], thymol (0.38%), which has antioxidant and antimicrobial activities [
34,
35,
36], carvacrol acetate (0.15%), which has anti-inflammatory and anti-nociceptive activities [
37], and caryophyllene (0.63%), known for its antibiofilm and anticancer activities [
38,
39]. Furthermore, β-bisabolene (0.39%) has been shown to exhibit anticancer and bactericidal activities [
40,
41], and eicosane (0.66%) has demonstrated antifungal activity [
42]. The presence of these bioactive compounds in OOEt, particularly in high quantities, such as carvacrol, may help explain the potent antimicrobial and antioxidant activities observed in our study. The synergistic effects of these compounds could also contribute to the overall efficacy of OOEt as a potential natural antimicrobial and antioxidant agent.
In summary, our study demonstrated that OOEt exhibits potent antimicrobial activity against a wide range of pathogenic microorganisms, including both bacterial and fungal strains. In some cases, the antimicrobial efficacy of OOEt was even more potent than that of synthetic antibiotics, highlighting its potential as a natural alternative for combating infections. Additionally, the antioxidant activity of OOEt was found to be stronger than that of ascorbic acid, a widely used antioxidant compound. These findings suggest that OOEt may serve as a valuable natural source of antimicrobial and antioxidant agents, which could be beneficial for various applications in medicine, food preservation, and cosmetics. Further studies are warranted to explore the potential synergistic effects of the bioactive compounds identified in OOEt as well as to investigate their safety and efficacy in vivo.