Exploring the Essential Oil of Ononis natrix : A Detailed Analysis of Its Chemical Composition,Antioxidant Benefits,and Effectiveness Against Both Antibiotic-Sensitive and Resistant Bacteria

Abstract

Background/Objectives

Bacterial infections caused by resistant strains such as Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus spp. and Escherichia coli represent a growing threat to human and animal health. In the face of rising resistance, essential oils derived from medicinal plants offer a promising natural alternative. The present study aims to characterize the phytochemical composition and evaluate the antioxidant and antimicrobial activities of the essential oil extracted from the aerial part of Ononis natrix.

Methods

The phytochemical composition, antioxidant activity and antimicrobial efficacy of the essential oil were assessed using gas chromatography-mass spectrometry (GC-MS), the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and Ferric-Reducing Antioxidant Power (FRAP) assays, as well as the disk diffusion method, supplemented by determining of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), respectively.

Results

Our findings revealed that Ononis natrix essential oil contains a high level of oxygenated monoterpenes (45.6%) and oxygenated sesquiterpenes (35.35%). The lowest IC₅₀ value obtained, of the order of 339 μg/mL, was observed with the reducing power test, thus reflecting a reducing capacity of this oil towards the ferric ion Fe³⁺. In addition, the essential oil showed marked antimicrobial effects against the Candida strains and multi-resistant bacteria tested.

Conclusion

The findings of the current study demonstrated strong antioxidant and antimicrobial properties of O. natrix, which could potentially provide novel treatments, thus, addressing antibiotic resistance issues in the clinical setting.

Graphical abstract

This is a visual representation of the abstract.

Keywords

antimicrobial properties antioxidant activity chemical composition chromatography-mass spectrometry resistant bacterial strains

Introduction

Antibiotic resistance is one of the most critical health challenges worldwide, causing around 25 000 deaths every year.¹ This phenomenon is due in particular to the excessive and inappropriate use of antibiotics, as well as the poor renewal of the molecules available for several decades.² Infections caused by multi-resistant bacteria, such as Salmonella spp., Enterococcus spp., Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli, have become a particular cause for concern in both humans and animals.³ Moreover, the possibility of resistance transfer through the food chain, in particular via products of animal origin, constitutes a major public health risk.⁴

In this context, essential oils (EOs) derived from medicinal and aromatic plants represent promising natural alternatives. Rich in secondary metabolites such as monoterpenes and sesquiterpenes, they possess recognized antimicrobial and antioxidant properties.⁵ Several plant species have already been studied in this context, but the majority of work focuses on well-known plants such as Origanum vulgare, Thymus vulgaris or Rosmarinus officinalis. To date, very little data is available on the detailed chemical composition and biological activities of the essential oil of Ononis natrix, a species widely used in traditional medicine in Morocco.

Ononis natrix, a species of the Fabaceae family (Leguminosae), is widespread in several regions of Morocco, notably Bou-Azzer, the Oued Tensift basin, the Rif, the Anti-Atlas (Taznakht), and the Mamora forest. Known by the Berber vernacular names âfzzaz, âfesdad or fezzaz. Traditionally, its roots and flowers are used in infusions to treat urinary and liver disorders.⁶ Recent studies have highlighted the richness of its essential oil in volatile bioactive compounds.^7–10 However, to our knowledge, no in-depth research has yet evaluated its antioxidant and antimicrobial activities against sensitive and resistant pathogenic strains.

In light of these findings, the present study has been produced to fill these gaps. It aims to:

✓ Characterize the phytochemical composition of the essential oil extracted from the aerial part of Ononis natrix using GC-MS;

✓ Evaluate its antioxidant activity using in vitro methods (DPPH and FRAP);

✓ Examine its antimicrobial efficacy against a wide range of pathogenic strains, including multi-resistant strains.

The aim of this research is to scientifically valorize a little-researched medicinal species and explore its potential as a natural source of therapeutic agents.

Material and Methods

Plant Material

In spring 2022, aerial parts (stems, leaves and flowers) of Ononis natrix were collected in their natural habitat in February 2019 in the region of Ouarzazate (Southeastern Morocco).¹¹ Identification was made by Professor Ouhammou Ahmed, and a reference specimen was deposited at the “MARK” Regional Herbarium of the Semlalia Faculty of Sciences, Cadi Ayyad University, Marrakech, Morocco, under reference specimen code MARK-13792.¹¹ The material was air-dried at room temperature and stored until use.

Extraction of Essential Oil

EO from air-dried Ononis natrix aerial parts was extracted by hydrodistillation using a Clevenger apparatus. Briefly, 150 g of dried plant material were placed in the Clevenger apparatus and hydrodistilled with 2000 mL of distilled water for four hours at boiling temperature. This process was performed in triplicate. The essential oil was collected from the Clevenger column and stored at 4 °C until chemical composition analysis and biological tests. The essential oil yield was determined using the subsequent formula:

R H E (%) = (V H E / M S) \times 100

- RHE: Essential oil yield (mL/100 g dry matter).

- VHE: Volume of essential oil recovered (mL).

- MS: Quantity of dry plant matter used for extraction (g).

Analysis of the Essential Oil's Chemical Composition Using Gas Chromatography-Mass Spectrometry

Separation of the constituents of the essential oil extracted from Ononis natrix aerial parts was performed using GC/MS. This analysis was carried out at the Center for Analysis and Characterization, Faculty of Sciences Semlalia, Cadi Ayyad University. The essential oil was analyzed using a gas chromatograph with a TG-5MS column (30 m × 0.25 mm × 0.25 μm). A temperature of 230 °C was used for the injector and 250 °C for the detector, and an electron impact ionization energy of 70 eV was used. The column temperature was ramped at 3 °C/min from 60 °C to 230 °C, where it was held for 10 min. A 1 μL aliquot of the diluted sample (1:100 in acetone) was injected into the GC/MS system. Helium served as the carrier gas at a flow rate of 1 mL/min.¹²

The chemical components of the essential oil were identified by comparing their mass spectra with those in the NIST library and by calculating their retention indices (RI), which were determined using a homologous series of n-alkanes (C₇–C₄₀) under the same experimental conditions of the essential oils. The components were quantified using the external standard method, based on calibration curves generated from routine GC analyses of authentic standard compounds.

Antioxidant Activity

DPPH Free Radical Scavenging Test

The 2,2′-diphenyl-1-picrylhydrazyl (DPPH) test was used to assess free radical scavenging activity according to the protocol described by.¹³

Concentration series of the essential oil (EO) were previously prepared in methanol. Then, 50 μL of each concentration was added to 2 mL of a methanolic solution of DPPH at a concentration of 60 μM. For twenty minutes, the mixture was allowed to sit at room temperature in the dark. Parallel preparation of a negative control involved combining 50 μL of methanol with 2 mL of the DPPH• methanolic solution. Quercetin was used as a reference antioxidant. After incubation, absorbances were measured at 517 nm and converted to percentage DPPH• radical inhibition using the following formula:

D P P H i n h i b i t i o n % = [(O D_{c} - O D_{S}) / O D_{c}] \times 100

where OD_C denoted the absorbance of the negative control and OD_S represented the absorbance of the tested samples.

The IC50, indicating the concentration of EO or reference antioxidant needed to decrease DPPH radical activity by 50%, was determined from the graph showing the reduction of DPPH (%) as a function of concentration. IC₅₀ values were expressed as the mean of three replicates.

Reducing Activity on Potassium Ferricyanide

In this study, the reducing power of the essential oil (EO) was determined according to the protocol described by.¹⁴ Briefly, 1 mL of different concentrations of EO or quercetin (standard) prepared in methanol, was mixed with 2.5 mL of PBS (0.2 M; pH 6.6) and 2.5 mL of 1% potassium ferricyanide solution (K₃Fe(CN)₆). Tubes were incubated at 50 °C for 20 min. After incubation and cooling, 2.5 mL of 10% trichloroacetic acid was added to stop the reaction. Tubes were centrifuged at 3000 g for 10 min. Subsequently, 2.5 mL of the supernatant from each concentration was mixed with 2.5 mL distilled water and 0.5 mL 0.1% FeCl₃. Absorbance was measured at 700 nm against a blank prepared in parallel, containing methanol instead of samples. The concentration of EO with an absorbance of 0.5 is considered the IC₅₀.

Antimicrobial Assays

Microbiological tests were carried out at the Laboratory of microbiology and virology, Department of medical biology, Faculty of medicine and pharmacy, Cadi Ayyad University Marrakech, Morocco.

Strains Tested

EO extracted from Ononis natrix was evaluated for its antimicrobial activity against 9 human pathogenic microorganisms. This collection included five susceptible bacteria (Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27653, Enterococcus faecium ATCC 4602, Staphylococcus aureus ATCC 25923), Two multi-resistant bacteria (methicillin-resistant Staphylococcus aureus NCTCC 12493, vancomycin-resistant Enterococcus spp) and two yeasts (Candida albicans BU 7098 and Candida tropicalis BU 1430).

Agar Diffusion Method

The antimicrobial activity of Ononis natrix essential oil was assessed using the agar disc diffusion method, following the guidelines set by the Clinical and Laboratory Standards Institute (CLSI) M07-A10.¹⁵

A microbial suspension inoculated with one or two colonies isolated from an 18-24 h culture was adjusted to approximately 10⁶ CFU/mL for bacteria and 1-2 × 103 cells/mL for yeast. The suspension was then spread evenly onto Mueller-Hinton (MH) agar plates for bacteria or Sabouraud agar plates for yeast. The plates were dried in a laminar flow hood for 15 min, then 6 mm diameter discs were soaked with 10 μL of pure essential oil (10 μL/disc). The disks were deposited on the surface of the agar previously seeded with the test microorganisms.

In each Petri dish, discs soaked with essential oil were placed alongside discs impregnated with positive controls (ciprofloxacin 5 μg/disc, gentamicin 15 μg/disc for bacteria and fluconazole 40 μg/disc for yeast). The dishes were placed at 4 °C for 4 h to allow diffusion of any bioactive molecules that might be produced, while stopping the growth of the test microorganisms. Finally, the plates were incubated at 37 °C for 18 to 24 h for bacteria and at 28 °C for 48 h for yeasts. After incubation, the absence of microbial growth is reflected by a zone of inhibition around the disks, whose diameter was measured in mm with a graduated ruler applied in contact with the agar surface. Each test was repeated three times in two replicates.¹⁵

Microdilution Method

Quantitative evaluation EO antimicrobial activity was performed through the determination of Minimum Bactericidal Concentration (MBC) and Minimum Inhibitory Concentration (MIC) or Fungicidal Concentration (MFC) on liquid medium, by the standard liquid microdilution method using 96-well plates.

Determination of Minimum Inhibitory Concentration (MIC)

A series of ½ dilutions of essential oils were prepared at different concentrations in dimethyl sulfoxide (2% DMSO). In each microplate, the essential oil dilutions were distributed at 100 μL/well. Subsequent, 100 μL of suitable freshly prepared new liquid culture medium (MH broth for bacteria and Sabouraud broth for yeast) previously seeded with 50 μL of the microbial suspension were added to the wells. The mixture was then thoroughly mixed and the microplates incubated at 37 °C (for 18-24 h for bacteria) and at 28 °C (for 48 h for yeast). Positive controls (ciprofloxacin, gentamycin and fluconazole) were also tested. The Minimum Inhibitory Concentration (MIC) was defined as the lowest concentration of the essential oil that inhibits the visible growth of the strain tested.¹⁶

Determination of Minimum Fungicidal Concentration (MFC) and Minimum Bactericidal Concentration (MBC)

Wells showing no visible growth after incubation were inoculated onto Petri dishes containing nutrient agar for bacteria or Sabouraud agar for yeasts. Readings were taken after incubation under the same conditions as above. The minimum bactericidal concentration (MBC) or fungicidal concentration (MFC) was defined as the lowest concentration of the sample tested for which there was a total absence of colonies after incubation.¹⁶

Data Availability

The data underlying this article are available in the article and in its online supplementary material.

Statistical Analysis

All antioxidant activity assays and antimicrobial activity tests were performed in triplicate (n = 3). In contrast, the GC-MS analysis of the essential oil was each conducted once. The data were statistically analysed using SPSS Statistics software (version 24.0 for Windows, SPSS Inc.). Differences between means were assessed using the least significant difference test at a significance level of P < .05. The results are expressed as the mean value ± standard deviation.

Results and Discussions

Chemical Composition

The yield of the extracted EO was noted as 0.3%. The results of the identification of the constituents of O. natrix essential oil are presented in Table 1. A total of 18 compounds were identified by GC-MS (Figure 1), representing 99.3% of all constituents present in the oil. This EO is mainly composed of oxygenated monoterpenes (45.6%), oxygenated sesquiterpenes (35.35%), hydrocarbon monoterpenes (13.27%) and hydrocarbon sesquiterpenes (5.08%). The main compound present is hexahydrofarnesyl acetone (31.24%). In addition, other compounds are also present in significant quantities, including (+)-2-Bornanone (14.82%), endo-Borneol (6.91%) and carvone (6.15%).

Figure 1.

Chromatographic proﬁle of Ononis natrix essential oil.

Table 1.

Chemical Composition of the Essential oil Isolated from Ononis natrix Aerial Parts.

N	RT	Name	Area %	RI _Exp	RI _Lit
1	6.45	α -Pinene	2.53	933	939
2	6.71	Camphene	4.14	935	946
3	7.90	para-cymene	1.56	1027	1026
4	7.96	D-Limonene	3.31	1033	1029
5	8.41	γ-Terpinene	1.73	1060	1054
6	8.87	L-Fenchone	2.10	1083	1085
7	8.96	L-Linalool	1.89	1097	1090
8	9.70	(+)-2-Bornanone	14.82	1137	1140
9	9.98	Endo-Borneol	6.91	1139	1143
10	10.28	(-)-α-Terpineol	3.51	1185	1186
11	10.91	Methyl carvacrol	2.22	1241	1245
12	10.98	Carvone	6.15	1244	1249
13	11.49	Thymol	4.77	1291	1289
14	11.63	Carvacrol	3.23	1298	1308
16	11.92	Caryophyllene	2.82	1426	1417
15	13.11	γ-Cadinene	2.26	1513	1518
17	15.11	1,10-di-epi-Cubenol	4.11	1615	1618
18	26.86	Hexahydrofarnesyl acetone	31.24	1844	1841
Monoterpene hydrocarbons			13.27
Oxygenated monoterpenes			45.6
Sesquiterpenes hydrocarbons			5.08
Oxygenated sesquiterpenes			35.35
Total			99.3

RT: Retention times on DB-5 column; RI exp: Retention index relative to C8-C25 n-alkanes on DB-5 column; RI lit: Retention indices reported in the literature taken from NIST.

The EO yields obtained in our study are higher than those reported in other research. For instance, a study of Ononis natrix EO in Jordan reported a yield of 0.04%.⁸ However, the yield obtained in our study remain lower than those of O. natrix harvested in Guelmim, Morocco.⁷

Our study corroborates the results obtained by,⁷ who reported oxygenated monoterpenes as the majority compounds in the EO of Ononis natrix collected in Guelmim, with farnesyl acetone as the main compound (27.3%). However, the chemical profile of O. natrix EO in our study differs from that found for the same species collected in Jordan by.⁹ In this latter study, the composition was dominated by hydrocarbon sesquiterpenes, with α-copaene and germacrene D as the main constituents. Nevertheless, common constituents are observed between the two samples, notably α-pinene, α-terpinene and γ-cadinene, although their respective contents vary considerably. Furthermore, our results show significant differences from those reported by.⁸ This study on O. natrix in Jordan identified a preponderance of oxygenated sesquiterpenes (66.29%) and sesquiterpene hydrocarbons (20.10%), with (2E,6E)-farnesol (18.83%) as the major constituent.

It is worth mentioning that some factors might affect the essential oil's chemical composition within the same species which include plant material, environment, soil, harvesting period, and genetic factors as well as preservation techniques, and extraction methods.¹⁷ Consequently, these variability factors play a decisive role in shaping the chemical profile of essential oils, and, by extension, their therapeutic properties.

Antioxidant Activity

The antioxidant activity of Ononis natrix essential oil (EO) was evaluated using DPPH radical scavenging and FRAP (ferric reducing antioxidant power) assays. The IC₅₀ values, summarized in Table 2, indicate that the EO showed stronger antioxidant potential in the FRAP assay (IC₅₀ = 339 ± 0.15 μg/mL) than in the DPPH assay (IC₅₀ = 597 ± 0.25 μg/mL). In both assays, the EO exhibited significantly lower antioxidant activity compared to the reference compound quercetin (P < .001), as confirmed by one-way ANOVA followed by Tukey's post hoc test. Different letters (a for quercetin, b for EO) indicate statistically distinct groups. While quercetin remains a more potent antioxidant, this comparison does not exclude the notable antioxidant capacity of O. natrix essential oil.

Table 2.

Antioxidant Activities of Ononis natrix EO Expressed as IC₅₀ Values (μg/mL).

	DPPH	FRAP
Essential oil	597 ± 0.25	339 ± 0.15
Quercetin	1,98 ± 0,12	2,62 ± 0,21

Essential oils are complex mixtures of volatile compounds, each contributing, alone or in synergy, to the biological activities observed. It has now been established that the antioxidant properties of essential oils do not depend solely on the majority compounds, but can also be influenced by the presence of minor compounds with complementary or synergistic effects.

In Ononis natrix essential oil, the antioxidant activity observed could be attributed to the notable presence of hexahydrofarnesyl acetone and (+)-2-Bornanone, two compounds for which previous studies have reported marked antioxidant effects.^18,19 In addition, minority constituents such as thymol and carvacrol, well known for their antioxidant power, could also enhance this activity through synergistic mechanisms.^20,21

Our research confirmed DPPH free radical scavenging activity, in agreement with the results previously reported by Al-Qudah et al⁸ However, it should be emphasised that our study is, to our knowledge, the first to investigate the ability of O. natrix essential oil to chelate ferric ions, which broadens our understanding of its antioxidant potential.

In comparative terms, other species in the Ononis genus have also shown antioxidant capacities, albeit to varying degrees. For example, the essential oil of Ononis angustissima subsp. filifolia showed moderate activity against DPPH radicals (IC₅₀ = 102.30 µg/mL) and strong inhibition in the β-carotene test (91.35%).²² For its part, Ononis aurasiaca, a species endemic to Algeria, showed significant activity in the β-carotene assay (IC₅₀ = 87.5 µg/mL), but less activity towards DPPH (IC₅₀ = 231.87 µg/mL) and a low capacity to reduce ferric ions (IC₅₀ = 751.3 µg/mL).²³

These comparisons highlight the variability in antioxidant potential between species of the Ononis genus, probably linked to differences in chemical composition, influenced by genetic, ecological and geographical factors. The results obtained with O. natrix therefore show a better overall antioxidant profile, particularly in terms of chelation of ferric ions, and merit further study, particularly in vivo, in order to better define their potential biological and therapeutic implications.

Antimicrobial Activity

Qualitative Assessment

The results presented in Table 3 show the mean values for the zones of inhibition of the essential oil and the antimicrobials tested on various strains of Candida and bacteria, both antibiotic-sensitive and antibiotic-resistant.

Table 3.

Inhibition Zone Values of Ononis natrix EO and Antimicrobials Tested on Candida Strains, Antibiotic-Sensitive and Antibiotic-Resistant bacteria.

	Test microorganismes	Essential oil	Gentamicin	Ciprofloxacin	Fluconazole
Sensitive strains	Escherichia coli	11	26 ± 0.4	29.23 ± 0.58	NT
	Klebsiella pneumoniae	12	15 ± 0.73	10.83 ± 0.73	NT
	Pseudomonas aeruginosa	12	21 ± 0.5	29.67 ± 0.88	NT
	Enterococcus faecium	13	23	18.88 ± 0.75	NT
	Staphylococcus aureus	25	24 ± 1	27.67 ± 0.67	NT
Resistant strains	Enterococcus spp vancomycin resistant	11	NI	NI	NT
Resistant strains	Staphylococcus aureus methicillin resistant	20	NI	NI	NT
Yeasts	Candida albicans	85	NT	NT	26.50 ± 0.35
Yeasts	Candida tropicalis	25	NT	NT	24.17 ± 0.73

Values are expressed as mean ± standard deviation; NT: not tested; NI: no inhibition

These results show that the essential oil studied has a growth inhibiting effect on all the microorganisms tested. Notably, a particularly marked inhibition was observed on Candida strains, with inhibition diameters ranging from 25 ± 1.2 mm to 85 ± 2.45 mm. As for bacteria, zones of inhibition ranging from 11 ± 0.17 mm to 20 ± 0.7 mm were recorded for antibiotic-resistant strains, while for sensitive strains, these values were between 11 ± 0.35 mm and 25 ± 0.88 mm. It is worth noting that the Gram-negative E. coli strain proved to be the least sensitive to the essential oil studied, while the S. anteuphorbium strain showed the greatest zone of inhibition, reaching 25 ± 0.88 mm.

It is also remarkable to note that the diameters of inhibition observed with the essential oil tested are sometimes comparable to, or even greater than, those of standard antibiotics (ciprofloxacin, gentamicin and fluconazole). This finding is particularly notable in the case of S. aureus bacteria (Gram-positive), K. pneumoniae (Gram-negative) and Candida strains (C. tropicalis and C. albicans).

Quantitative Evaluation

The antibacterial activity of Ononis natrix EO was evaluated by measuring the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) using the liquid microdilution technique. The findings, shown in Table 4, indicate that the EO antimicrobial effectiveness varied depending on the strain tested.

Table 4.

Values of Minimum Inhibitory Concentrations (MIC) and Minimum Microbicidal Concentrations (MMC) of Essential oil, Antibacterial Agents and Reference Antifungal Agent Tested in mg/mL.

	Test microorganismes	Essential oil		Gentamicin		Ciprofloxacin		Fluconazole
	Test microorganismes	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MFC
Sensitive strains	Escherichia coli	1.56	1.56	0.031	0.031	0.008	0.016	NT	NT
	Klebsiella pneumoniae	3.12	3.12	0.5	0.5	0.25	0.25	NT	NT
	Pseudomonas aeruginosa	6.24	12.48	0.125	0.125	0.002	0.002	NT	NT
	Enterococcus faecium	6.24	6.24	0.031	0.031	0.008	0.016	NT	NT
	Staphylococcus aureus	1.56	1.56	0.008	0.008	0.015	0.031	NT	NT
Resistant strains	Enterococcus spp vancomycin resistant	1.56	3.12	NI	NI	NI	NI	NT	NT
Resistant strains	Staphylococcus aureus methicillin resistant	1.56	1.56	NI	NI	NI	NI	NT	NT
Yeasts	Candida albicans	1.56	3.12	NT	NT	NT	NT	0.031	0.031
Yeasts	Candida tropicalis	3.12	3.12	NT	NT	NT	NT	0.07	0.07

Abbreviations: MBC, minimum bactericidal concentration; MFC, minimum fungicidal concentration.

MIC values showed that antibiotic-resistant bacteria were inhibited at a relatively low concentration of 3.12 mg/mL. In contrast, sensitive strains required higher concentrations, ranging from 1.56 to 6.24 mg/mL, to observe growth inhibition. The essential oil also demonstrated antifungal properties, with MIC values ranging from 1.56 mg/mL to 3.12 mg/mL. Notably, the Gram-negative P. aeruginosa strain proved to be the most resistant to the essential oil tested, with an MIC of 6.24 mg/mL.

For some strains, the MIC value corresponded to the MBC or MFC, indicating a microbicidal action of the essential oil, notably against E. coli, K. pneumoniae, S. aureus and C. tropicalis. Comparatively, the reference antibacterial and antifungal agents showed lower MIC values than the essential oil (Table 4).

Ononis natrix essential oil (EO) has demonstrated significant antimicrobial activity against a range of pathogenic strains, including Gram-positive and Gram-negative bacteria and Candida strains. Our study is the first, to our knowledge, to evaluate the effect of this essential oil against multi-resistant strains, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus spp. (VRE) and Candida strains.

When compared to the previous study carried out on O. natrix from Morocco by,⁷ which reported MICs of 7.81 mg/mL against E. coli ATCC 25922 and 15.60 mg/mL against S. aureus ATCC 25923, our results reveal a more marked antimicrobial activity, with MICs/MBCs of 1.56 mg/mL for these two strains. For P. aeruginosa ATCC 27853, the same study reported high resistance (MIC = 60.96 mg/mL), which is in line with our results: this strain was also the most resistant in our study, with an MIC of 6.24 mg/mL. This confirms the data in the literature highlighting the high intrinsic resistance of this bacterium to essential oils.^24,25

Other species of the Ononis genus have also been studied recently. The essential oil of Ononis angustissima subsp. filifolia has shown efficacy against Gram-positive strains such as S. aureus (ATCC 25923 and 43300), but limited efficacy against Gram-negative strains.²² Likewise, Ononis aurasiaca, a species endemic to Algeria, showed marked activity against S. aureus (ATCC 25923 and 43300), but little effect on E. coli or P. aeruginosa.²³ These comparisons suggest that, although antimicrobial activity seems more marked against Gram-positive bacteria in the Ononis genus, O. natrix essential oil stands out for its more balanced efficacy against both types of bacteria, as well as for its antifungal activity.

The variations in efficacy observed between sensitive and resistant strains could be linked to the structure of the bacterial wall (more complex in Gram-negatives) and the ability of certain bacteria to express defence mechanisms against lipophilic agents, such as efflux pumps or degrading enzymes.²⁶

The antimicrobial activity of O. natrix EO can be attributed to its wealth of bioactive compounds such as oxygenated monoterpenes (thymol, carvacrol, (+)-2-bornanone), oxygenated sesquiterpenes (hexahydrofarnesyl acetone, 1,10-di-epi-cubenol) and sesquiterpene hydrocarbons (caryophyllene, γ-cadinene). These molecules are known to alter the integrity of cell membranes, disrupt permeability, inhibit bacterial enzyme systems, and affect the biosynthesis of essential compounds such as ergosterol in fungi.^27–30 Compounds such as camphene and D-limonene, also detected in our sample, have shown significant antimicrobial and antifungal properties in other studies.³¹

Despite these promising results, our study has several limitations. It is based exclusively on in vitro tests, and the absence of in vivo tests limits the transposability of the results to clinical contexts. In addition, the exact mechanism of the essential oil's antimicrobial action remains to be elucidated, particularly in terms of the specific interactions between the compounds and the microbial targets. No tests have been carried out to assess a possible synergy between the essential oil and traditional antibiotics, although this is a promising avenue of research in view of the increase in microbial resistance. Future studies could therefore explore the combination of O. natrix EO with conventional antibiotics in order to determine potential synergistic effects and reduce effective concentrations.

Conclusion

Our study highlighted the antioxidant and antimicrobial properties of Ononis natrix essential oil. Antioxidant activity, illustrated by its ability to scavenge free radicals and chelate ferric ions, suggests a potential for protection against oxidative stress. Furthermore, the antimicrobial activity observed, notably against resistant pathogenic strains such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus spp. (VRE), testifies to potential therapeutic interest. The presence of bioactive compounds such as oxygenated monoterpenes and sesquiterpenes reinforces these observations.

However, it should be emphasized that these results are preliminary and based exclusively on in vitro tests. In vivo studies are essential to confirm these biological effects, particularly with regard to safety, bioavailability and therapeutic efficacy. Consequently, further research, including tests on animal models and clinical studies, is required before proposing medical or industrial applications for this essential oil.

Footnotes

Abbreviations

Acknowledgements

The authors would like to thank Mrs. Arifi Rabia and Mrs. El Khiat Kbira for collecting the plant material. They also express their gratitude to the Analysis and Characterization Centre of Cadi Ayyad University for conducting the essential oil analyses.

ORCID iD

Abdelaati El Khiat

Ethical Approval

This study does not require ethical approval since it does not involve human participants, animal subjects, or any other elements requiring ethical approval. All procedures and analyses carried out complies with applicable guidelines and requirements for research integrity.

Author Contributions

Conceptualization, A.E.K., H.G.; methodology, A.E.K., S.O. and I.D.; software, Y.B. and A.A.; validation, A.E.K. and H.G.; formal analysis, S.O., I.D. and M.A.T.; investigation, S.O. and I.D.; data curation, S.O., I.D.; writing—original draft preparation, A.E.K., S.O.; and M.A.T.; writing—review and editing, A.E.K., S.O. and B.E.M .; supervision, A.E.K. and H.G. All authors have read and agreed to the published version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

Manus

J-M

. Antibiorésistance, phénomène inquiétant. Rev Francoph des Lab. 2019;2019(511):18-19.

Sabtu

Enoch

Brown

. Antibiotic resistance: what, why, where, when and how? Br Med Bull. 2015;116(1):1-21.

Chouhan

Sharma

Guleria

. Antimicrobial activity of some essential oils—present status and future perspectives. Medicines. 2017;4(3):58.

Ambrosio

CMS

de Alencar

de Sousa

RLM

Moreno

Da Gloria

. Antimicrobial activity of several essential oils on pathogenic and beneficial bacteria. Ind Crops Prod. 2017;97:128-136.

Cerbu

Kah

White

Astete

Sabliov

. Fate of biodegradable engineered nanoparticles used in veterinary medicine as delivery systems from a one health perspective. Molecules. 2021;26(3):523.

Bellakhdar

. La pharmacopée marocaine traditionnelle: Médecine arabe ancienne et savoirs populaires. 1997:189.

Elamrani

Benaissa

. Chemical composition and antibacterial activity of the essential oil of Ononis natrix from Morocco. J Essent Oil Bear Plants. 2010;13(4):477-488.

Al-Qudah

Al-Ghoul

Trawenh

, et al. Antioxidant activity and chemical composition of essential oils from Jordanian Ononis Natrix L. and Ononis Sicula guss. J Biol Act Prod from Nat. 2014;4(1):52-61.

Al-Mterin

Aboalhaija

Abaza

Kailani

Zihlif

Afifi

. Chromatographic analysis (LC-MS and GC-MS), antioxidant activity, total phenol and total flavonoid determination of Ononis natrix L. grown in Jordan. Jordan J Chem. 2021;16(1):31-39.

10.

Haj-Khlifa

Aziz

Tastift

, et al. Ameliorative effect of Ononis natrix against chronic lead poisoning in mice: neurobehavioral, biochemical, and histological study. Biol Trace Elem Res. 2025;203(1):229-242.

11.

El Khiat

Bouftini

Lafhal

, et al. Evaluation of the anti-hyperglycemic activity of Ononis natrix aqueous extract against alloxan-induced experimental model of insulinopenic diabetes in albino Swiss mice. Acad J. 2024;39(4):57-64.

12.

Ouhaddou

Ibrahimi

Loqman

, et al. Essential oil from aerial parts of Andryala pinnatifida subsp. mogadorensis: chemical composition, antioxidant and aantimicrobial synergistic effect against multidrug-resistant Bacteria. J Essent Oil Bear Plants. 2022;25(1):147-159.

13.

Şahin

Güllüce

Daferera

, et al. Biological activities of the essential oils and methanol extract of Origanum vulgare ssp. vulgare in the eastern anatolia region of Turkey. Food Control. 2004;15(7):549-557.

14.

Thaipong

Boonprakob

Crosby

Cisneros-Zevallos

Byrne

. Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. J Food Compos Anal. 2006;19(6–7):669-675.

15.

Wayne

. Clinical and laboratory standards institute (CLSI). Perform Stand Antimicrob susceptibility Test. 2015.

16.

Wayne

. Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated fastidious bacteria. CLSI Guidel M45 3rd ed Clin Lab Stand Inst. 2015.

17.

Ložienė

Venskutonis

. Influence of environmental and genetic factors on the stability of essential oil composition of Thymus pulegioides. Biochem Syst Ecol. 2005;33(5):517-525.

18.

Abd-ElGawad

El Gendy

AE-N

El-Amier

, et al. Essential oil of Bassia muricata: chemical characterization, antioxidant activity, and allelopathic effect on the weed Chenopodium murale. Saudi J Biol Sci. 2020;27(7):1900-1906.

19.

Chen

Zhang

Huang

. Comparison of chemical constituents and antioxidant activity of Achillea alpina L. and Achillea wilsoniana L. Asian J Res Biochem. 2022;10(4):15-21.

20.

Tepe

Sokmen

Akpulat

Daferera

Polissiou

Sokmen

. Antioxidative activity of the essential oils of Thymus sipyleus subsp. sipyleus var. sipyleus and Thymus sipyleus subsp. sipyleus var. rosulans. J Food Eng. 2005;66(4):447-454.

21.

de Carvalho

Silva

ÉR

Gomes

, et al. Anti-inflammatory and antioxidant activity of carvacrol in the respiratory system: a systematic review and meta-analysis. Phyther Res. 2020;34(9):2214-2229.

22.

Benmeddour

Messaoudi

Flamini

. First investigation of the chemical composition, antioxidant, antimicrobial and larvicidal activities of the essential oil of the subspecies Ononis angustissima lam. subsp. filifolia Murb. Nat Prod Res. 2024:1-16.

23.

Messaoudi

Benmeddour

Flamini

. First report on the chemical composition and the free radical scavenging and antimicrobial activities of the essential oil of Ononis aurasiaca, an endemic plant of Algeria. Nat Prod Res. 2024;38(24):4355-4367.

24.

Dra

Brahim

MAS

Boualy

, et al. Chemical composition, antioxidant and evidence antimicrobial synergistic effects of Periploca laevigata essential oil with conventional antibiotics. Ind Crops Prod. 2017;109:746-752.

25.

Zine

Ibrahimi

Loqman

, et al. Chemical composition, antioxidant, and antibacterial activities of essential oil of Atriplex semibaccata R. Br. aerial parts: first assessment against multidrug-resistant bacteria. Agronomy. 2021;11(2):362.

26.

Sissi

Ouhaddou

Dilagui

, et al. Chemical composition, antioxidant and antimicrobial activities of Anacyclus maroccanus Ball. and Anacyclus radiatus Loisel. essential oils against multidrug resistant microbial pathogens. Nat Prod Commun. 2024;19(10):1934578X-241289844.

27.

Pinto

Pina-Vaz

Salgueiro

, et al. Antifungal activity of the essential oil of Thymus pulegioides on Candida, Aspergillus and dermatophyte species. J Med Microbiol. 2006;55(10):1367-1373.

28.

Jiangseubchatveera

Liawruangrath

, et al. The chemical constituents and the cytotoxicity, antioxidant and antibacterial activities of the essential oil of Graptophyllum pictum (L.) griff. J Essent Oil Bear Plants. 2015;18(1):11-17.

29.

Shagun

Sujata

Manjul

. Chemical profiling, antioxidant and antibacterial properties of Cotinus coggygria essential oil from Western Himalaya. Int J Pharmacog Phytochem Res. 2016;8:1183-1186.

30.

Lai

Rao

Gao

. Chemical composition, cytotoxic, antimicrobial and antioxidant activities of essential oil from Anthriscus caucalis M. Bieb grown in China. Rec Nat Prod. 2018;12(3):290-294.

31.

Oloche

Gabriel

Ubwa

Amokaha

. Chemical composition and antibacterial activity of essential oil extracted from Citrus sinensis L., Citrus limon L. and Zingiber officinale roscoe. West J Med Biomed Sci. 2021;2(1):5-16.