Next Article in Journal
Control of Erwinia amylovora Growth by Moringa oleifera Leaf Extracts: In Vitro and in Planta Effects
Next Article in Special Issue
Systematic Analysis of the Molecular Mechanisms of Cold and Hot Properties of Herbal Medicines
Previous Article in Journal
Entanglement of Arabidopsis Seedlings to a Mesh Substrate under Microgravity Conditions in KIBO on the ISS
Previous Article in Special Issue
Oral and Topical Anti-Inflammatory Activity of Jatropha integerrima Leaves Extract in Relation to Its Metabolite Profile
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 11
Issue 7
10.3390/plants11070954
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Chemical Composition, Antioxidant and Antibacterial Activities of Thymus broussonetii Boiss and Thymus capitatus (L.) Hoffmann and Link Essential Oils

by
Imane Tagnaout
1,*,
Hannou Zerkani
1,
Nadia Hadi
1,
Bouchra El Moumen
1,
Fadoua El Makhoukhi
1,2,
Mohamed Bouhrim
1,
Rashad Al-Salahi
3,
Fahd A. Nasr
4,
Hamza Mechchate
5,* and
Touriya Zair
1
1
Research Team of Chemistry of Bioactive Molecules and the Environment, Laboratory of Innovative Materials and Biotechnology of Natural Resources, Faculty of Sciences, Moulay Ismaïl University, B.P. 11201 Zitoune, Meknes 50070, Morocco
2
Laboratoire Centre Eau, Ressources Naturelles, Environnement Et Développement Durable, Faculty of Sciences, University Mohammed V, Rabat B.P. 8007, Rabat 10000, Morocco
3
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
4
Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
5
Laboratory of Inorganic Chemistry, Department of Chemistry, University of Helsinki, P.O. Box 55, FI-00014 Helsinki, Finland
*
Authors to whom correspondence should be addressed.
Plants 2022, 11(7), 954; https://doi.org/10.3390/plants11070954
Submission received: 31 January 2022 / Revised: 22 March 2022 / Accepted: 24 March 2022 / Published: 31 March 2022
(This article belongs to the Special Issue Pharmacological and Toxicological Study of Medicinal Plants)
Browse Figures
Versions Notes

Abstract

:
Thymus capitatus and Thymus broussonnetii are two Moroccan endemic medicinal plants used traditionally by the local population. The present study aims to investigate their essential oil chemical composition, antioxidant and antibacterial activities. The chemical composition of the essential oils was determined using the GC-MS analysis, the antioxidant activity assessed using DPPH and FRAP methods while the antimicrobial activity was evaluated against nine bacteria species tested (Enterococcus faecalis, Serratia fonticola, Acinetobacter baumannii, Klebsiella oxytoca, sensitive Klebsiella pneumoniae, sensitive Escherichia coli, resistant Escherichia coli, resistant Staphylococcus aureus and Enterobacter aerogenes). The major identified compounds of T. capitatus essential oil where carvacrol (75%) and p-cymene (10.58%) while carvacrol (60.79%), thymol (12.9%), p-cymene (6.21%) and γ-terpinene (4.47%) are the main compounds in T. broussonnetii essential oil. The bioactivity of the essential oils of the two species of thyme was explained by their richness in oxygenated monoterpenes known for their great effectiveness with an IC50 of 3.48 ± 0.05 and 4.88 ± 0.04 μL/mL and EC50 of 0.12 ± 0.01 and 0.20 ± 0.02 μL/mL in the DPPH and FRAP assays, respectively, with an important antibacterial activity. These results encourage the use of these plants as a source of natural antioxidants, and antibacterial additives, to protect food from oxidative damage and to eliminate bacteria that are responsible for nosocomial infections.

1. Introduction

Since its first appearance, antibiotic therapy has improved the health aspects of human life by fighting against infectious diseases. Despite the progress of this therapy, we are still living in an era where antibiotic-resistant infections are alarmingly increasing [1]. Antimicrobial resistance is not a new phenomenon; it is considered a way of microbes’ survival by mutation of existing genes, or by acquisition of a resistant gene from another already resistant organism. Thus, the abusive use of antibiotics causes selectiveness, allowing the single genotype most apt to develop [2]. Among the pathogenic bacteria that cause real problems for human health, we find species belonging to the families of Enterobacteriaceae, Pseudomonadaceae and Moraxellaceae, which are almost resistant to all the prescribed antibiotics [3]. Despite the importance of oxidation in the life cycle of cells, oxidative stress is the main cause in the etiology of many human diseases, such as atherosclerosis, arthritis, cardiovascular disorders, Alzheimer and cancer [4]. Meanwhile, synthetic antioxidants such as butylatedhydroxytoluene (BHT) used in the food industry for food stabilization have carcinogenic effects [5]. To solve these problems, the use of medicinal plants with antioxidant and antimicrobial properties is one of the most interesting approaches to adopt. Recently, essential oils (EOs) have received increasing attention due to their therapeutic properties, which are beneficial to health, and are generally considered as safe according to the United States Agency for Food and Drug Products [6]. The EOs are natural substances that are composed of volatile secondary metabolites. They are characterized by a strong odor, and they consist of a complex mixture of compounds including: monoterpene and sesquiterpenes hydrocarbons, their oxygenated derivatives (e.g., alcohols, aldehydes, ketones and ethers), several derivatives of phenyl propane, phenols and various volatile organic compounds [7,8]. The EOs chemical profile is always characterized by the presence of one, or a few majority compounds at high concentrations compared to others, which are minority compounds. Several studies have linked the EOs antimicrobial and antioxidant activities to both the functional groups of the majority compounds and the minority compounds possible synergistic or antagonistic effects [9,10]. The EOs are recognized to have several biological activities, such as antibacterial, antifungal and insecticide [11]. Various studies reported the antimicrobial, and antioxidant activities of the EOs of certain medicinal plants [6,12,13,14]. Consequently, the EOs antimicrobial and antioxidant activities can differ according to variations in the chemical composition [15,16]. Thus, the study of the EOs chemical composition, and the evaluation of their biological activities are necessary to confirm their use as preservatives in food, pharmaceutical and cosmetic fields. The genus Thymus is considered to be one of the eight most important genera in the Lamiaceae family comprising around 215 species, whichare native to the Mediterranean basin [17]. Thyme’s medicinal and aromatic properties have made it one of the most popular genera in the world. Besides, its essential oils are widely used to flavor and preserve several food products. The majority of these oils are characterized by their richness in oxygenated monoterpenes, in particular phenolic compounds such as thymol and its isomer carvacrol, accompanied by other more or less biologically active compounds such as: eugenol, p-cymene, terpinene, linalool, geraniol and borneol [18]. Several studies linked Thymus EO chemical composition to its antimicrobial and antioxidant activities [19]. T. capitatus (Synonym Thymbra capitata (L.) Cav. or Corido thymus capitatus) is an endemic Mediterranean plant [20]. In Morocco, this species grows in the thermo-Mediterranean and meso-Mediterranean vegetation stages. It is a much-branched subshrub of 20–50 cm height, covered with whorled leaves, sessile, deciduous if dry, glabrous but slightly ciliated at the base; the light pink flowers, spotted with mauve, grouped in dense ovoid flower heads, differ from other Thymes by the shape of the dorsally flattened calyx [21]. It is used as a food preservative for meat and fish [22]. T. broussonetii, endemic to Morocco, is an erect shrub with erect stems. It leaves are stem-like, broadly ovate lanceolate and punctuated on both sides. It flowers are wider and often purple colored, it is characterized by dense male inflorescences, its calyx is bilabiated, the upper lip not very toothed, and its pink corolla, having 2–3 times the length of the calyx, contains a narrow but clearly protruding tube [21]. The severe exploitation to which this species is exposed, can lead to its rarefaction and/or disappearance [23]. There is no study on the variation in the chemical composition of T. broussonetii EO. The Mediterranean flora in general, and Morocco in particular, gathers a diversified flora with an endemism rate equal to 878 species, including several species of plants, that are little or not studied at all [24,25]. The present study is a contribution to the chemical and biological valorization of the EOs of two endemic plant species growing in Morocco: Thymus capitatus and Thymus broussonnetii Boiss.

2. Results and Discussion

2.1. Essential Oil Yields

In industry, hydrodistillation was considered as the most practical extraction method. Average yields of essential oils were calculated based on the dry plant matter of the aerial part of each plant. Indeed, the leaves and flowering tops of T. capitatus from the Khemisset region in the center of Morocco provide an EO yield of 2.6 ± 0.1%. It is relatively high compared to that of northern Morocco (Tetouan) whose yield was 2.05% [26], as well as that of Tunisia with 1.26% [27] and 2.52 mL/g [28]. According to Bounatirou et al. the essential oil yield of T. capitatus reaches its maximum during the post-flowering phase [29]. T. broussonetii EO yield is 3.35 ± 0.05%; it appears richer in EO than T. capitatus. This result is much higher than those reported by several researchers [12,30,31]. From the results reported in various studies and those obtained in our work, we can conclude that: firstly, the EO yields of the studied T. capitatus and T. broussonetii are very high, which will allow their exploitation on an industrial scale. Secondly, several factors influence the EO yield, such as the harvest period and its place, the drying and extraction techniques as well as the age of the plant [32].

2.2. Chemical Composition of T. capitatus and T. broussonetii EOs

The chemical compositions of T. capitatus and T. broussonetii EOs were determined by gas chromatography coupled with mass spectrometry (GC-SM). The analysis of the chromatograms (Figure 1) showed that the chemical profile of our EOs varies from those of other origins, by the existence of qualitative and quantitative differences of the individual components. The identification of the chemical composition of the EOs (Table 1) revealed a determined number of compounds representing 98.48% of the total composition for T. capitatus and 98.34% of the total composition for T. broussonetii. T. capitatus EO is rich in carvacrol (75%), p-cymene (10.58%), linalool (2.91%), (E)-caryophyllene (1.61%) and epoxy caryophyllene (1.5%). As for T. broussonetii EO, it is characterized by a high percentage of carvacrol (60.79%), thymol (12.9%), p-cymene (6.21%), γ-terpinene (4.47%) and (E)-caryophyllene (4.15%). The EOs of these two species (T. capitatus and T. broussonetii), which belong to the genus Thymus, are strongly dominated by oxygenated monoterpenes (79.3% and 75.98%, respectively) and hydrocarbon monoterpenes (13.87% and 14.84% respectively). In comparison, sesquiterpenes compounds are present in broussonetii thyme oil (7.52%) more than in capitized thyme (5.31%). It should be noted that carvacrol was the main constituent of the two studied thyme oils. In addition, it was revealed that phenols were particularly abundant in T. capitatus essential oil (76.59%), which was richer in carvacrol (75%) than T. broussonetii EO (60.79%). The p-cymene content was higher in T. capitatus essential oil (10.58% against 6.21%), and the contents of thymol, (E)-caryophyllene and γ-terpinene in T. capitatus EO represent only 0.18%, 0.53% and 1.61%, respectively, while they represent 12.9%, 4.15% and 4.47% in T. broussonetii EO. T. capitatus EO from northern Morocco (Tetouan), is composed mainly of carvacrol and other compounds with relatively low contents such as, p-cymene, γ-terpinene, linalool, β-caryophyllene and β-pinene [26]. El Ouariachi et al., (2011) confirmed the same composition through their studies for the same plant from the same origin [33]. In Tunisia, several authors were reported, that the essential oil of T. capitatus was mainly composed of carvacrol (61.6–83%), p-cymene (5–17%), γ-terpinene (2-14%) and β-caryophyllene (1–4%) but this composition changes depending on the location and the growing season [29]. Similarly, the EOs of T. capitatus from Sicily [34,35], Albania [36], Greece and Portugal [37] are characterized by a high content of carvacrol. While, the T. capitatus EO of Sardinia (Italy) was dominated by thymol (29.3%) and p-cymene (26.4%); while, carvacrol represents only (10.8%) [38]. In another study, it was revealed that, T. capitatus essential oil collected from the south of Tunisia was richer in thymol with a rate of 89% [39]. Finally, the Turkish T. capitatus essential oil was characterized by a large amount of carvacrol (35.6%), p-cymene (26.4%) and thymol (18.6 %) [40]. In this work, compared to several studies that were previously conducted in Morocco, the chemical composition of the studied T. broussonetii EO shows qualitative and quantitative differences, El Ouariachi et al. showed that thyme harvested from northern Morocco (Al Hoceïma region) has borneol (27.6%) as major component, followed by p-cymene (20.9%), and carvacrol (15.7%) [41]. While Belaqziz and his collaborators, found that p-cymene (21%) is the major compound in thyme harvested from southwest Morocco (around Essaouira), followed by borneol (16.5%), α-pinene (11.8%) and thymol (11.3%) [42]. In the same region, Saad et al. identified thymol (39.64%) as the most abundant compound accompanied by carvacrol (21.31%) and borneol (20.13%) in T. broussonetii EO [43]. Another study conducted by Alaoui Jamali et al. revealed that the main constituents of the essential oil of T. broussonetii are carvacrol (43.4%), thymol (12.3%), γ-terpinene (8.9%), borneol (8.5%) and p-cymene (5.2%) [12]. El Bouzidi et al., in the EO of wild broussonetii thyme identified the same composition [14]. While the oil obtained from cultivated broussonetii thyme is characterized by a higher level of carvacrol (60.8%), p-cymene (7.2%) and α-pinene (6.5%). In addition, Tantaoui et al. showed that the major element in the essential oil of T. broussonetii was carvacrol (47.8%), accompanied by considerable quantities of hydrocarbons [44]. Based on the results set out in Figure 2, we find that the essential oils of these two species (T. capitatus and T. broussonetii) are rich in phenols (76.59% and 73.69%) and hydrocarbons (16.55% and 21.97%). We also note the presence, in small proportion, of non-aromatic alcohols (3.08%, 2.09%), ethers (1.5%, 0.59%). As for esters (0.7%) and ketones (0.06%), they are only present in T. capitatus EO. However, the level of compounds identified in the analyzed EOs confirms previous literature findings. The difference in chemical composition is caused by several factors, such as species, origin, growth stage, environmental influences and genetic background. Generally, the influence of these factors on the biosynthetic pathways induces changes in qualitative and quantitative terms of the characteristic majority compounds, which lead to the existence of different chemotypes distinguishing EOs from different origins [19,45].

2.3. Antioxidant Activity of T. capitatus and T. broussonetii Essential Oils

The antioxidant activities of T. capitatus and T. broussonetii EOs and of standard (BHA) were evaluated using three methods, DPPH, FRAP and TCA. The results were illustrated in Figure 3 and Figure 4. The EOs are qualified as natural antioxidants, because of their ability to reduce and/or prevent the formation of free radicals. In the food industry, EOs are considered potential substitutes to synthetic antioxidants for food preservation [46]. In this work, the antioxidant activity of the studied EOs is determined by two different methods, DPPH and FRAP. In the presence of antioxidants, the purple DPPH radical transforms into a stable yellow molecule. The degree of discoloration may indicate the ability to trap free radicals. According to the DPPH method, it turns out that the studied EOs have the capacity to reduce the free radicals of DPPH. At a concentration of 0.12 μL/mL, the BHA standard presented a percentage of DPPH free radical inhibition (89.5%) that is higher than the studied EOs (T. broussonetii (78.4%), T. capitatus (75%)). Indeed, the studied essential EOs showed a dose-dependent anti-free radical activity, the more we increase the concentration, the more the percentage of inhibition increases until reaching a plateau. This phenomenon is explained by the transfer of single electrons located in the external orbital of DPPH. At a certain concentration, the antioxidant reacts with the radical, when we increase the concentration; the antioxidant activity remains constant since this is accompanied by the saturation of the electronic layers of the radical. According to the IC50 values presented in Figure 3, the antioxidant power of BHA (IC50 = 0.82 ± 0.01 mg/mL) is greater than that of the studied thyme. Among the tested EOs, the most active is T. broussonetii essential oil with an IC50 of around 3.48 ± 0.05 μL/mL. The high activity of T. broussonetii essential oil has already been indicated in several works including El Ouariachi et al., Alaoui Jamali et al. and El Bouzidi et al., [14,31,41,47]. T. capitatus EO also demonstrated a high antioxidant power with an IC50 of 4.88 ± 0.04 μL/mL. This observation was reported by, Amarti et al., Bounatirou et al. and Zaïri et al. [29,48,49], the latter showed that this essential oil harvested during the flowering phase and after flowering, has more antioxidant power compared to that of the vegetative period before flowering. For the FRAP method, the presence of reducing agents in the medium results in the reduction of the complex Fe3+(ferric cyanide) of yellow color, to the ferrous form of greenish blue color, by the donation of an electron. The increase in absorbance at 700 nm indicates an increase in the iron reduction capacity. According to the results of the FRAP tests illustrated in Figure 3, it appears that the EO of T. broussonetii has a higher antioxidant power (EC50 = 0.13 ± 0.01 mg/mL) than that of standard BHA (EC50 = 0.5 ± 0.01 mg/mL). It is a very promising result for food preservation. since the undesirable side effects of synthetic antioxidants are widely known, namely liver damage or carcinogenesis [50]. While, the essential oil of T. capitatus that has an EC50 of about 0.20 ± 0.02 μL/mL is considered close to BHA standard. The results obtained by the FRAP method confirm the antioxidant potential of the studied EOs. In addition, tests show that the oils have a good affinity with Fe3+ ions. The phosphomolybdenum method is quantitative since the total antioxidant activity is expressed as the number of equivalents of ascorbic acid. EOs of tested species of genera Thymus revealed good total antioxidant capacity. Results showed that T. broussonetii EO exhibited highest TAC with 12.54 mg AAE/g of EO) followed by T. capitatus EO (11.74 mg AAE/g of EO). As compared to the synthetic antioxidant BHA (14.33 mg AAE/g), all the oils exhibited approximate total antioxidant capacity (Figure 4). Furthermore, the results suggest that essential oils from Thymus species could be used as natural antioxidants in food systems. Generally, the antioxidant activity is the result of the interaction between all the chemical components of the EO (alcohols, phenols, and terpene and ketone compounds), acting in an antagonistic or synergistic way. In fact, several studies have highlighted the correlation between the phenol content and the antioxidant capacity of plants [12,16]. According to this study, it is evident that the great antioxidant capacity of the studied EOs of Thymus (capitatus and broussonetii) is linked respectively to their richness in oxygenated monoterpenes (79.3% and 75.98%) and in monoterpene hydrocarbons (13.87% and 14.84%), which have a large percentage of phenols (76.59%, 73.79%) and hydrocarbons (16.55, 21.97%). In addition, the presence of minority compounds such as aromatic alcohols, ethers, ketones and esters in the studied EOs, can influence the antioxidant activity. According to the literature, essential oils composed of monoterpenes and/or oxygenated sesquiterpenes are known for their great antioxidant properties [51]. These compounds are capable of trapping free radicals by their phenolic hydroxyl groups. In the case of the studied thyme species, their antioxidant potential is linked to the high content of phenolic compounds such as carvacrol and thymol [16,52].

2.4. Antibacterial Activities of T. capitatus and T. broussonetii Essential Oils

2.4.1. Diffusion Method

The antibacterial activity of the EOs can be classified into three levels: (i) weak activity (zone of inhibition ≤ 12 mm), (ii) moderate activity (12 mm < zone of inhibition < 20 mm) and (iii) strong activity (inhibition zone ≥ 20 mm). The results of the antibacterial activity of T. capitatus and T. broussonetii EOs were presented in Table 2. Diffusion tests carried out with a volume of 5 μL, showed that the tested EOs have an antibacterial activity, against most of the examined bacteria with diameters of inhibition zones ranging from 8.55 ± 0.21 to 49.9 ± 0.14 mm. The EOs isolated from the aerial parts of the studied thyme, showed a strong antibacterial activity against all the tested strains, in particular against K. oxytoca, S. fonticola, sensitive E. coli, E. aerogenes, E. faecalis and A. baumannii, having inhibition zones that vary from 20.70 ± 1.20 mm to 49.9 ± 0.14 mm. While moderate activity was observed against sensitive K. pneumoniae and resistant E. coli, with inhibition zones ranging from 14.15 ± 0.15 mm to 19.95 ± 0.07 mm. S. aureus was more resistant to the tested EOs. Compared with the antibiotics timentin TIM85, cefoxitin FOX30 and piperacillin PRL100 used as controls, the EOs of the studied thymes showed a stronger inhibitory action. The strains that showed resistance to the action of antibiotics are vulnerable to the action of the two types of essences of T. capitatus and T. broussonetii; this is the case of E. faecalis, S. fonticola, K. oxytoca, and susceptible E. coli and E. aerogenes. Similar results were reported previously for the EOs of T. capitatus from different origins [53,54,55,56]. According to Bounatirou et al., T. capitatus EOs harvested in the flowering and post-flowering phase have better antibacterial activity against S. aureus with an inhibition zone of 17.4 mm. While the thyme essential oil harvested in the vegetative phase before flowering, shows no antibacterial activity against the tested microorganisms [29]. Thus, the essential oil of Tunisian T. capitatus revealed an important antibacterial potential against Gram-negative bacteria in particular E. coli and K. pneumoniae giving inhibition zones of 30 mm and 20 mm, respectively [39]. Regarding T. broussonetii, its EO also showed an interesting antibacterial power with a dose of 10 μL against E. coli and S. aureus with inhibition zones of 21 mm and 19 mm [42]. El Bouzidi et al., noted that the EO of broussonetii thyme with the same dose gave inhibition zones of 30.17 mm and 35 mm against E. coli and S. aureus, respectively [14]. Also, Fadli and his collaborators’ study, showed an antibacterial effect of the EO of T. broussonetii with inhibition zones of 22.6 mm, 25.3 mm and 22.7 mm against the S. aureus, E. coli and K. pneumoniae strains respectively [30].

2.4.2. Determination of Minimum Inhibitory (MIC) and Bactericidal (MBC) Concentrations in μL/mL for Bacterial Strains

The antimicrobial activities of the studied EOs were evaluated according to the diameter of inhibition, which indicates the power of inhibition of the EO against the tested strains. The results of the tests were presented in Table 3. According to the obtained results, the MIC values confirm the results of the diffusion method. In these tests, the essential oil of T. capitatus was more active than that of T. broussonetii. The lowest MIC manifested by the tested EOs was 2 μL/mL against sensitive E. coli and sensitive K. pneumoniae. The most resistant strain was S. aureus, inhibited at a MIC of 16 μL/mL by T. capitatus EO and 32 μL/mL by T. broussonetii EO, while E. coli resistant was inhibited at a MIC of 8 μL/mL for T. capitatus and 16 μL/mL for T. broussonetii. The results of T. capitatus and T. broussonetii were the same against the E. faecalis, S. fonticola, K. oxytoca and A. baumannii strains (MIC = 4 μL/mL). While, the E. aerogenes strain was inhibited at 4 μL/mL by T. capitatus EO and at 8 μL/mL by T. broussonetii EO. Based on the MBC/MIC report, the EOs of the studied Thymes reported a bactericidal effect towards the bacteria of E. aerogenes, K. pneumoniae sensitive, E. faecalis, S. fonticola, K. oxytoca, A. baumannii, Staphylococcus aureus, Resistant and wild Escherichia coli. These results show the bactericidal power of T. capitatus and T. broussonetii EOs against the studied bacteria. Based on these results, the essential oils of T. capitatus and T. broussonetii could potentially be used as natural preservatives in food against the well-known causative agents of foodborne illness such as S. aureus and E. coli [57]. In the literature, the essential oils from the leaves of the thymus genus are known for their antibacterial power, which is linked to the origin of the plants and to the essential oil composition [14,30,48,49]. In this work, the EOs inhibitory activities of the studied Thymes are probably due mainly to the phenolic action of carvacrol and thymol (two oxygenated monoterpenes) [58,59]. Several authors explained the antimicrobial activity of carvacrol and thymol against E. coli in vitro experiments [60,61]. Carvacrol is considered a biocide, causing disruption of the bacterial membrane, which leads to leaks of intracellular ATP and potassium ions and finally, death of the cell [62]. However, the dominance of oxygenated monoterpene compounds does not necessarily mean, the presence of better antibacterial effects for most of the analyzed strains, because possible synergistic and/or antagonistic effects of the minority compounds present in the oil should also be taken into consideration.

3. Material and Methods

3.1. Materials and Reagents

Timentin (TIM85), Cefoxitin (FOX30) and Piperacillin (PRL100), were purchased from Sigma Aldrich (St-Quentin Fallavier, France). All chemicals and solvents were of highly analytical grade and were used as received from the supplier without further purification.

3.2. Plant Material

Samples of Thymus capitatus, and Thymus broussonetii Boiss aerial parts (stems, leaves and flowers) were collected in May 2018, in the Khemisset region in the center of Morocco. The species identification was carried out at the plant ecology laboratory in the Scientific Institute of Rabat. The different parts of the plants were dried in the shade for 13 days (Figure 5).

3.3. Bacterial Strains

The antibacterial activity in this study was tested on nine bacterial strains that are frequent in human pathology. They belong to the Gram-negative category. The bacterial strains used were the sensitive Escherichia coli, resistant Escherichia coli, Enterobacter aerogenes, sensitive Klebsiella pneumonia, Enterococcus faecalis, Serratia fonticola, Acinetobacter baumannii, Klebsiella oxytoca and Staphylococcus aureus. They were isolated from the patients’ biological fluids, and then preserved by subculturing on specific agar medium in the laboratory of bacteriology in Mohammed V hospital in Meknes.

3.4. Extraction of Essential Oils

The essential oils were extracted by hydrodistillation using a Clevenger type apparatus (VWR, Radnor, PA, USA) for three hours. The process was repeated three times for each 100 g of the plant sample. The obtained essential oils were dried over anhydrous sodium sulfate. They were then stored at a temperature of 4 °C in the dark until they were used.

3.5. Chromatographic Analysis of Essential Oils

The chromatographic analysis of T. capitatus and T. broussonetii essential oils was carried out using a gas chromatograph of the Thermo Electron type (Trace GC Ultra) (conquerscientific, Poway, CA, USA) coupled to a mass spectrometer system of the Thermo Electron Trace MS type. (Thermo Electron: Trace Ultra GC, Polaris Q MS) (conquerscientific, Poway, CA, USA), the fragmentation was achieved by an electronic impact intensity of 70 eV. The chromatograph was equipped with a DB-5 column (5% phenyl-methyl-siloxane) (30 m × 0.25 mm × 0.25 μm film thickness), a flame ionization detector (FID) supplied by a mixture of He gas /Air. The column temperature was programmed at a speed of 4 °C/min from 50 to 200 °C for 5 min. The injection mode is split (leak rate: 1/70, flow rate mL/min), the carrier gas used was nitrogen with a flow rate of 1 mL/min. The identification of the essential oil compounds was carried out by comparison of their Kováts index (KI) and Adams with those of the reference products known in the literature [63,64]. The mass spectra and indices of each of these compounds were also compared with those of the databases cited above [65,66]. The Kováts index compares the retention time of any product with that of a linear alkane of the same carbon number.

3.6. Essential Oils Antioxidant Activity

3.6.1. DPPH Anti-Free Radical Method

The T. capitatus and T. broussonetii EOs antioxidant activity was established by the method using DPPH (1,1-diphenyl-2-picrylhydrazyl) as a relatively stable radical [67]. The DPPH solution was prepared by dissolving 2.4 mg of DPPH powder in 100 mL of ethanol. Different concentrations of T. capitatus and T. broussonetii EOs were prepared by dilution in absolute ethanol (1–20 μL/mL). The test was carried out by mixing 200 μL of each concentration with 2.8 mL of DPPH solution. These same concentrations were prepared with butylhydroxyanisole (BHA) to serve as positive controls. In addition, a blank was made with absolute ethanol alone. The samples were then left in the dark for 30 min. The absorbance of the mixtures was measured at 517 nm. The results were expressed as an inhibition percentage of DPPH free radical (IP %):
IP (%) = (Acontrol − ASample)/Acontrol × 100
IP%: inhibition percentage of DPPH free radical.
Acontrol: Absorbance of the solution without the samples.
ASample: Absorbance of the solution in presence of the samples (essential oils or BHA).
The graph representing the inhibition percentage as a function of the samples concentrations made it possible to determine IC50, which is the required concentration to inhibit or reduce 50% of the initial concentration of DPPH. It was determined graphically by linear regression. Since there is no absolute measure of the antioxidant capacity of a compound, the results were often reported in relation to a reference antioxidant, such as BHA.

3.6.2. Ferric Reducing Antioxidant Power Method

The power of phenolic extracts to reduce ferric iron (Fe3+) present in the potassium ferricyanide complex into ferrous iron (Fe2+) was determined according to the method described by Zovko Koncic et al. [68]. In test tubes, 1 mL of each essential oil of plant was diluted in absolute ethanol at different concentrations (1–20 μL/mL). Then, each tube was mixed with 2.5 mL of a phosphate buffer solution (0.2 M, pH = 6.6) and 2.5 mL of a potassium ferricyanide solution K3Fe(CN)6 at 1%. The obtained solution was incubated in a water bath at 50 °C for 20 min. Then 2.5 mL of 10% trichloroacetic acid was added to stop the reaction processes. The whole solution was centrifuged at 3000 revolutions per minute for 10 min. After, 2.5 mL of the supernatant of each concentration was mixed with 2.5 mL of distilled water, and 0.5 mL of the aqueous FeCl3 solution (0.1%). The absorbance of the reaction medium was measured at 700 nm against a similarly prepared blank, replacing the essential oil with absolute ethanol in order to calibrate the apparatus (UV-VIS spectrophotometer (VWR, Radnor, PA, USA). The positive control was represented by a solution of standard antioxidants; the absorbance of BHA was measured under the same conditions as the samples. An increase in absorbance corresponds to an increase in the reducing power of the tested EOs.

3.6.3. Total Antioxidant Capacity

The total antioxidant capacity (TAC) of the essential oils was evaluated according to the method described by Prieto et al. [68]. An aliquot of 100 μL of each essential oil diluted in ethanol was combined with 1 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The tubes were incubated in a boiling water bath at 95 °C for 90 min. After the samples were cooled at room temperature, the absorbance of the aqueous solution of each sample was measured at 695 nm in the spectrophotometer (Thermo scientific, UV1-V7.09 (VWR, Radnor, PA, USA)). The results were expressed as equivalents of ascorbic acid (mg/g) of oil. Moreover, the BHA was used as a control positive.

3.7. Essential Oils Antibacterial Activity

The antibacterial activity was determined using the agar disk diffusion method (Mueller Hinton Agar). The microorganisms resulting from the growth on the nutritive agar incubated at 37 °C for 18 h were suspended in a saline solution (NaCl) at 0.9%, and adjusted to a turbidity of 0.5 Mac Farland standard (108CFU/mL). The suspension was inoculated in 90 mm diameter Petri dishes using a sterile, non-toxic wooden cotton swab. The EOs were dissolved in 0.5% (v/v) of dimethyl sulfoxide (DMSO), which does not affect the growth of bacteria according to our experiences. Sterile paper disks of 6 mm in diameter impregnated with 5 μL of the studied EOs were placed in the petri dishes and incubated for 24 h at 37 °C. Commercial antibiotics timentin TIM85, cefoxitin FOX30 and piperacillin PRL100 were used as a positive reference to determine the sensitivity of the tested strains. Finally, the diameters of the inhibition zones (DIZ) were measured in order to determine the studied EOs antibacterial activities.

3.8. Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) in Liquid Medium

A bacterial suspension with a density that is equivalent to the 0.5 Mac Farland standard (108 CFU/mL) was prepared from an 18 to 24 h bacterial culture. A stock solution was prepared by mixing (v/v) essential oil and DMSO (the chosen emulsifier). The dilution of the essential oil used is 70% (70% EO, 30% DMSO). A volume of 40 μL of the bacterial suspension was deposited in test tubes containing 4 mL of liquid culture medium. Then, we aseptically added the different volumes of the emulated essential oils, in order to obtain an increasing range of final concentrations of the essential oils from C1 to C9 that correspond respectively to 1, 4, 8, 16, 32, 64, 128, 256 and 512 μL/mL. A negative control tube containing only the suspension and the culture medium was prepared. Three repetitions were made for each test. The whole was vortexed for homogenization, and was then incubated in an oven at a temperature of 37 °C for 24 h. The MIC is the lowest essential oil concentration that can inhibit any growth that is visible to the naked eye after 24 h of incubation at 37 °C. In addition, the MBC was determined by inoculating 100 mL of each tube, which has a concentration that is greater than or equal to the MIC, into solid medium (MHA). The lowest essential oil concentration at which 99.99% of bacteria were eliminated after 24 h of incubation at 37 °C was the MBC. The calculation of the MBC/MIC ratio made it possible to determine the bactericidal (MBC/MIC < 4) or bacteriostatic (MBC/MIC ≥ 4) effect of the studied essential oils.

4. Statistical Analysis

The statistical analysis was performed by OriginPro 8.5 software. All data were expressed as means ± SD of a minimum of three replicates or measurements and werecompared by one-way ANOVA test, followed by the Tukey test. P-values less than or equal to 0.05 wereconsidered statistically significant.

5. Conclusions

The aim of this work was to characterize the chemical composition of the aerial part essential oil of T. capitatus and T. broussonetii Boiss from Morocco and to evaluate the antioxidant and antibacterial properties of the essential oils. From the results obtained, it seems that these two plants have virtues that can justify their important use in traditional medicine. The composition of the EOs partially explains the higher potential observed (antioxidant and antibacterial), especially the role of the carvacrol/thymol major compounds in the overall bioactivity. The antibacterial results require more attention as they showed excellent activities even against bacteria. From all of the above, we confirm the traditional use of those plants EO against infectious disease and we also suggest more advanced tests for more personalized applications either in the clinical as antibiotics or the industries fields as preservatives.

Author Contributions

Conceptualization, I.T. and T.Z.; methodology, H.Z., N.H.; validation, B.E.M.; formal analysis, F.E.M.; data curation, H.M.; writing—original draft preparation, I.T. and M.B.; writing—review and editing R.A.-S. and F.A.N.; supervision, T.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by Researchers Supporting Project, King Saud University, Riyadh, Saudi Arabia, through grant no. RSP-2021/353. Open access funding provided by University of Helsinki.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available upon request.

Acknowledgments

The authors extend their appreciation to the Researchers Supporting Project, King Saud University, Riyadh, Saudi Arabia, for funding this work through grant no. RSP-2021/353.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. World Health Organization, Antimicrobial resistance, WHO Media Center. Available online: https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance (accessed on 12 December 2021).
  2. Swamy, M.K.; Akhtar, M.S.; Sinniah, U.R. Antimicrobial properties of plant essential oils against human pathogens and their mode of action: An updated review. Evid. Based Complement. Altern. Med. 2016, 2016, 3012462. [Google Scholar] [CrossRef] [PubMed]
  3. Chaudhary, A.S. A review of global initiatives to fight antibiotic resistance and recent antibiotics’ discovery. Acta Pharm. Sin. B 2016, 6, 552–556. [Google Scholar] [CrossRef] [Green Version]
  4. Es-safi, I.; Mechchate, H.; Amaghnouje, A.; Kamaly, O.M.A.; Jawhari, F.Z.; Imtara, H.; Grafov, A.; Bousta, D. The Potential of Parsley Polyphenols and Their Antioxidant Capacity to Help in the Treatment of Depression and Anxiety: An In Vivo Subacute Study. Molecules 2021, 26, 2009. [Google Scholar] [CrossRef]
  5. Ito, N.; Hirose, M.; Fukushima, G.; Tauda, H.; Shira, T.; Tatematsu, M. Studies on activity of conventional antibiotics. Ind. Crop. Prod. 1986, 71, 37–43. [Google Scholar]
  6. Fancello, F.; Petretto, G.L.; Zara, S.; Sanna, M.L.; Addis, R.; Maldini, M.; Foddai, M.; Rourke, J.P.; Chessa, M.; Pintore, G. Chemical characterization, antioxidant capacity and antimicrobial activity against food related microor-ganisms of Citrus limon var. pompia leaf essential oil. LWT-Food Sci. Technol. 2016, 69, 579–585. [Google Scholar] [CrossRef]
  7. Viuda-Martos, M.; El Gendy, A.E.-N.G.S.; Sendra, E.; Fernández-López, J.; El Razik, K.A.A.; Omer, E.A.; Pérez-Alvarez, J.A. Chemical Composition and Antioxidant and Anti-Listeria Activities of Essential Oils Obtained from Some Egyptian Plants. J. Agric. Food Chem. 2010, 58, 9063–9070. [Google Scholar] [CrossRef] [PubMed]
  8. Hammer, K.A.; Carson, C.F. Antibacterial and Antifungal Activities of Essential Oils. Lipids Essent. Oils Antimicrob. Agents 2010, 6, 255–306. [Google Scholar] [CrossRef]
  9. Tongnuanchan, P.; Benjakul, S. Essential Oils: Extraction, Bioactivities, and Their Uses for Food Preservation. J. Food Sci. 2014, 79, R1231–R1249. [Google Scholar] [CrossRef]
  10. Bakkali, F.; Averbeck, S.; Averbeck, D.; Idaomar, M. Biological effects of essential oils-A review. Food Chem. Toxicol. 2008, 46, 446. [Google Scholar] [CrossRef]
  11. Celiktas, O.Y.; Kocabas, E.E.H.; Bedir, E.; Sukan, F.V.; Ozek, T.; Baser, K.H.C. Antimicrobial activities of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and seasonal variations. Food Chem. 2007, 100, 553–559. [Google Scholar] [CrossRef]
  12. Es-safi, I.; Mechchate, H.; Amaghnouje, A.; Elbouzidi, A.; Bouhrim, M.; Bencheikh, N.; Hano, C.; Bousta, D. Assessment of Antidepressant-Like, Anxiolytic Effects and Impact on Memory of Pimpinella Anisum L. Total Extract on Swiss Albino Mice. Plants 2021, 10, 1573. [Google Scholar] [CrossRef] [PubMed]
  13. Bouhaddouda, R.; Aouadi, N.; Labiod, S. Evaluation of chemical composition and biological activities of essential oil and methanolic extract of Origanum vulgare L. ssp. glandulosum (DESF.) Ietswaart from Algeria. Int. J. Pharmacogn. Phytochem. Res. 2016, 8, 104–112. [Google Scholar]
  14. El Bouzidi, L.; Jamali, C.A.; Bekkouche, K.; Hassani, L.; Wohlmuth, H.; Leach, D.; Abbad, A. Chemical composition, antioxidant and antimicrobial activities of essential oils obtained from wild and cultivated Moroccan Thymus species. Ind. Crop. Prod. 2013, 43, 450–456. [Google Scholar] [CrossRef]
  15. Gachkar, L.; Yadegari, D.; Rezaei, M.; Taghizadeh, M.; Astaneh, S.D.; Rasooli, I. Chemical and biological characteristics of Cuminum cyminum and Rosmarinus officinalis essential oils. Food Chem. 2007, 102, 898–904. [Google Scholar] [CrossRef]
  16. Hazzit, M.; Baaliouamer, A.; Veríssimo, A.; Faleiro, M.L.; Miguel, M. Chemical composition and biological activities of Algerian Thymus oils. Food Chem. 2009, 116, 714–721. [Google Scholar] [CrossRef]
  17. Pirbalouti, A.G.; Bistghani, Z.E.; Malekpoor, F. An overview on genus Thymus. J. Herb. Drugs 2015, 6, 93–100. [Google Scholar]
  18. Nikolić, M.; Glamočlija, J.; Ferreira, I.C.F.R.; Calhelha, R.C.; Fernandes, Â.; Marković, T.; Marković, D.; Giweli, A.; Soković, M. Chemical composition, antimicrobial, antioxidant and antitumor activity of Thymus serpyllum L., Thymus algeriensis Boiss. and Reut and Thymus vulgaris L. essential oils. Ind. Crops Prod. 2014, 52, 183–190. [Google Scholar] [CrossRef]
  19. Ballester-Costa, C.; Sendra, E.; Fernández-López, J.; Pérez-Álvarez, J.A.; Viuda-Martos, M. Assessment of Antioxidant and Antibacterial Properties on Meat Homogenates of Essential Oils Obtained from Four Thymus Species Achieved from Organic Growth. Foods 2017, 6, 59. [Google Scholar] [CrossRef] [Green Version]
  20. Figueiredo, A.; Barroso, J.; Pedro, L.; Salgueiro, L.; Miguel, M.; Faleiro, M. Portuguese Thymbra and Thymus Species Volatiles: Chemical Composition and Biological Activities. Curr. Pharm. Des. 2008, 14, 3120–3140. [Google Scholar] [CrossRef]
  21. Benabid, A. Flore et Ecosystèmes du Maroc: Evaluation et Préservation de la Biodiversité; Food and Agriculture Organization of the United Nations: Paris, France, 2000. [Google Scholar]
  22. Mohammed, A.; Miguel, M.; Antunes, M.; Figueiredo, A.; Pedro, L.; Barroso, J. Antioxidant Activity of Thymbra Capitata Essential Oil in Meat-Treated Oil. In International Symposium on Medicinal and Aromatic Plants-SIPAM2009 853; International Society for Horticultural Science: Leuven, Belgium, 2010; pp. 319–322. [Google Scholar] [CrossRef] [Green Version]
  23. Mehdioui, R.; Kahouadji, A. Etude ethnobotaniqueauprès de la population riveraine de la forêtd’Amsittène: Cas de la Commune d’Imin’Tlit (Province d’Essaouira). Bull. L’institut Sci. Rabat Sect. Sci. La Vie 2007, 29, 11–20. [Google Scholar]
  24. Fennane, M.; Tattou, M.I. Statistiques et commentaires sur l’inventaireactuel de la florevasculaire du Maroc. Flora 2008, 1986, 1989. [Google Scholar]
  25. Rankou, H.; Culham, A.; Jury, S.L.; Christenhusz, M.J. The endemic flora of Morocco. Phytotaxa 2013, 78, 1–69. [Google Scholar] [CrossRef]
  26. Amarti, F.; Satrani, B.; Aafi, A.; Ghanmi, M.; Farah, A.; Aberchane, M.; El Ajjouri, M.; El Antry, S.; Chaouch, A. Composition chimique et activitéantimicrobienne des huilesessentielles de Thymus capitatus et de Thymus bleicherianus du Maroc. Phy-totherapie 2008, 6, 342–347. [Google Scholar]
  27. Achour, S.; Khelifi, E.; Attia, Y.; Ferjani, E.; Hellal, A.N. Concentration of Antioxidant Polyphenols from Thymus capitatus extracts by Membrane Process Technology. J. Food Sci. 2012, 77, C703–C709. [Google Scholar] [CrossRef] [PubMed]
  28. Bouchikhi, T. Luttecontre la Bruche du Haricot Acanthoscelidesobtectus (Coleoptera, Bruchidae) et la mite Tineolabisselliella( Lepidoptera, Tineidae) par des plantesaromatiques et leurshuilesessentielles; Université AboubakrBelkaid: Chetouane, Algérie, 2011. [Google Scholar]
  29. Bounatirou, S.; Smiti, S.; Miguel, M.G.; Faleiro, L.; Rejeb, M.N.; Neffati, M.; Costa, M.M.; Figueiredo, A.C.; Barroso, J.G.; Pedro, L.G. Chemical composition, antioxidant and antibacterial activities of the essential oils isolated from Tunisian Thymus capitatus Hoff. et Link. Food Chem. 2007, 105, 146–155. [Google Scholar] [CrossRef]
  30. Fadli, M.; Saad, A.; Sayadi, S.; Chevalier, J.; Mezrioui, N.-E.; Pagès, J.-M.; Hassani, L. Antibacterial activity of Thymus maroccanus and Thymus broussonetii essential oils against nosocomial in-fection–bacteria and their synergistic potential with antibiotics. Phytomedicine 2012, 19, 464–471. [Google Scholar] [CrossRef]
  31. Jamali, C.A.; El Bouzidi, L.; Bekkouche, K.; Lahcen, H.; Markouk, M.; Wohlmuth, H.; Leach, D.; Abbad, A. Chemical Composition and Antioxidant and Anticandidal Activities of Essential Oils from Different Wild Moroccan Thymus Species. Chem. Biodivers. 2012, 9, 1188–1197. [Google Scholar] [CrossRef] [PubMed]
  32. Moghaddam, M.; Mehdizadeh, L. Chemistry of Essential Oils and Factors Influencing Their Constituents. In Soft Chemistry and Food Fermentation; Academic Press: Cambridge, MA, USA, 2017; Chapter 13; pp. 379–419. [Google Scholar] [CrossRef]
  33. El Ouariachi, E.M.; Paolini, J.; Bouyanzer, A.; Tomi, P.; Hammouti, B.; Salghi, R.; Majidi, L.; Costa, J. Chemical composition and antioxidant activity of essential oils and solvent extracts of Thymus capitatus (L.) Hoffmanns and link from Morocco. J. Med. Plants Res. 2011, 5, 5773–5778. [Google Scholar]
  34. Casiglia, S.; Bruno, M.; Scandolera, E.; Senatore, F.; Senatore, F. Influence of harvesting time on composition of the essential oil of Thymus capitatus (L.) Hoffmanns. & Link. growing wild in northern Sicily and its activity on microorganisms affecting historical art crafts. Arab. J. Chem. 2019, 12, 2704–2712. [Google Scholar] [CrossRef] [Green Version]
  35. Napoli, E.; Curcuruto, G.; Ruberto, G. Screening of the essential oil composition of wild Sicilian thyme. Biochem. Syst. Ecol. 2010, 38, 816–822. [Google Scholar] [CrossRef]
  36. Ibraliu, A.; Mi, X.; Ristić, M.; Stefanovic, Z.D.; Shehu, J. Analysis of essential oils of three wild medicinal plants in Albania. J. Med. Plants Res. 2011, 5, 58–62. [Google Scholar]
  37. Bentes, J.; Miguel, M.G.; Monteiro, I.; Costa, M.; Figueired, A.C.; Barroso, J.G.; Pedro, L.G. Antioxidant activities of the essential oils and extracts of Portuguese Thymbra capitata and Thymus masti-china. Ital. J. food Sci. 2009, 21, 183–195. [Google Scholar]
  38. Cosentino, S.; Tuberoso, C.; Pisano, M.B.; Satta, M.; Mascia, V.; Arzedi, E.; Palmas, F. In-vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils. Lett. Appl. Microbiol. 1999, 29, 130–135. [Google Scholar] [CrossRef]
  39. Mkaddem, M.G.; Romdhane, M.; Ibrahim, H.; Ennajar, M.; Lebrihi, A.; Mathieu, F.; Bouajila, J. Essential Oil of Thymus capitatus Hoff. et Link. from Matmata, Tunisia: Gas Chromatography-Mass Spectrometry Analysis and Antimicrobial and Antioxidant Activities. J. Med. Food 2010, 13, 1500–1504. [Google Scholar] [CrossRef] [PubMed]
  40. Goren, A.C.; Bilsel, G.; Bilsel, M.; Demir, H.; Kocabaş, E.E. Analysis of essential oil of Coridothymus capitatus (L.) and its antibacterial and antifungal activity. Z. Für Naturforsch. C 2003, 58, 687–690. [Google Scholar] [CrossRef] [PubMed]
  41. Hamdani, I.; Bouyanzer, A.; Hammouti, B.; Majidi, L.; Costa, J.; Paolini, J.; Chetouani, A. Chemical Composition and Antioxidant Activity of Essential Oils of Thymus Broussonetii Boiss. and Thymus Algeriensis Boiss. from Morocco. Asian Pacific J. Trop. Dis. 2014, 4, 281–286. [Google Scholar] [CrossRef]
  42. Belaqziz, R.; Harrak, R.; Romane, A.; Oufdou, K.; ElFels, M.A.E.A. Antimicrobial and Insecticidal Activities of the Endemic Thymus broussonettiBoiss. and Thymus maroccanus Ball. Rec. Nat. Prod. 2010, 4, 230. [Google Scholar]
  43. Saad, A.; Fadli, M.; Bouaziz, M.; Benharref, A.; Mezrioui, N.-E.; Hassani, L. Anticandidal activity of the essential oils of Thymus maroccanus and Thymus broussonetii and their synergism with amphotericin B and fluconazol. Phytomedicine 2010, 17, 1057–1060. [Google Scholar] [CrossRef] [PubMed]
  44. Tantaoui-Elaraki, A.; Errifi, A.; Benjilali, B. Antimicrobial activity of four chemically different essential oils. Riv. Ital. Eppos Sesto 1992, 92, 13–22. [Google Scholar]
  45. El Karkouri, J.; Bouhrim, M.; Al Kamaly, O.M.; Mechchate, H.; Kchibale, A.; Adadi, I.; Amine, S.; Alaoui Ismaili, S.; Zair, T. Chemical Composition, Antibacterial and Antifungal Activity of the Essential Oil from Cistus ladanifer L. Plants 2021, 10, 2068. [Google Scholar] [CrossRef] [PubMed]
  46. Horvathova, E.; Navarova, J.; Galova, E.; Sevcovicova, A.; Chodakova, L.; Snahnicanova, Z.; Melusova, M.; Kozics, K.; Slamenova, D. Assessment of Antioxidative, Chelating, and DNA-Protective Effects of Selected Essential Oil Components (Eugenol, Carvacrol, Thymol, Borneol, Eucalyptol) of Plants and Intact Rosmarinus officinalis Oil. J. Agric. Food Chem. 2014, 62, 6632–6639. [Google Scholar] [CrossRef] [PubMed]
  47. Radi, F.Z.; Bouhrim, M.; Mechchate, H.; Al-zahrani, M.; Qurtam, A.A.; Aleissa, A.M.; Drioiche, A.; Handaq, N.; Zair, T. Phytochemical Analysis, Antimicrobial and Antioxidant Properties of Thymus zygis L. and Thymus Willdenowii Boiss. Essential Oils. Plants 2021, 11, 15. [Google Scholar] [CrossRef]
  48. Zaïri, A.; Nouir, S.; Zarrouk, A.; Haddad, H.; Khélifa, A.; Achour, L.; Tangy, F.; Chaouachi, M.; Trabelsi, M. Chemical composition, Fatty acids profile and Biological properties of Thymus capitatus (L.) Hoffmanns, essential Oil. Sci. Rep. 2019, 9, 20134–20138. [Google Scholar] [CrossRef] [PubMed]
  49. Amarti, F.; Satrani, B.; Ghanmi, M.; Aafi, A.; Farah, A.; Aarab, L.; El Ajjouri, M.; Guedira, A.; Chaouch, A. Activitéantioxydante et composition chimique des huiles essentielles de quatre espèces de thym du Maroc. Acta Bot. Gallica 2011, 158, 513–523. [Google Scholar] [CrossRef]
  50. Xu, W.; Zhang, F.; Luo, Y.; Ma, L.; Kou, X.; Huang, K. Antioxidant activity of a water-soluble polysaccharide purified from Pteridium aquilinum. Carbohydr. Res. 2009, 344, 217–222. [Google Scholar] [CrossRef]
  51. Miladi, H.; Ben Slama, R.; Mili, D.; Zouari, S.; Bakhrouf, A.; Ammar, E. Essential oil of Thymus vulgaris L. and Rosmarinus officinalis L.: Gas chromatography-mass spectrometry analysis, cytotoxicity and antioxidant properties and antibacterial ac-tivities against foodborne pathogens. Nat. Sci. 2013, 5, 11. [Google Scholar]
  52. EL Abed, N.; Kaabi, B.; Smaali, I.; Chabbouh, M.; Habibi, K.; Mejri, M.; Marzouki, M.N.; Ahmed, S.B.H. Chemical Composition, Antioxidant and Antimicrobial Activities of Thymus capitata Essential Oil with Its Preservative Effect against Listeria monocytogenes Inoculated in Minced Beef Meat. Evidence-Based Complement. Altern. Med. 2014, 2014, 152487. [Google Scholar] [CrossRef] [Green Version]
  53. Jayari, A.; El Abed, N.; Jouini, A.; Abdul-Wahab, O.M.S.; Maaroufi, A.; Ahmed, S.B.H. Antibacterial activity of Thymus capitatus and Thymus algeriensis essential oils against four food-borne pathogens inoculated in minced beef meat. J. Food Saf. 2017, 38, e12409. [Google Scholar] [CrossRef]
  54. El Moussaoui, N.; Sanchez, G.; Khay, E.O.; Idaomar, M.; Ibn Mansour, A.; Abrini, J.; Aznar, R. Antibacterial and Antiviral Activities of Essential Oils of Northern Moroccan Plants. Br. Biotechnol. J. 2013, 3, 318–331. [Google Scholar] [CrossRef]
  55. Gutierrez, J.; Barry-Ryan, C.; Bourke, P. Antimicrobial activity of plant essential oils using food model media: Efficacy, synergistic potential and interactions with food components. Food Microbiol. 2009, 26, 142–150. [Google Scholar] [CrossRef]
  56. Qaralleh, H.; Abboud, M.M.; Khleifat, K.M.; Tarawneh, K.A.; Althunibat, O. Antibacterial activity in vitro of Thymus capitatus from Jordan. Pak. J. Pharm. Sci. 2009, 22, 247–251. [Google Scholar] [PubMed]
  57. Burt, S. Essential oils: Their antibacterial properties and potential applications in foods—A review. Int. J. Food Microbiol. 2004, 94, 223–253. [Google Scholar] [CrossRef] [PubMed]
  58. Castilho, P.C.; Savluchinske-Feio, S.; Weinhold, T.S.; Gouveia, S.C. Evaluation of the antimicrobial and antioxidant activities of essential oils, extracts and their main components from oregano from Madeira Island, Portugal. Food Control 2012, 23, 552–558. [Google Scholar] [CrossRef]
  59. Mothana, R.A.A.; Hasson, S.S.; Schultze, W.; Mowitz, A.; Lindequist, U. Phytochemical composition and in vitro antimicrobial and antioxidant activities of essential oils of three endemic SoqotraenBoswellia species. Food Chem. 2011, 126, 1149–1154. [Google Scholar] [CrossRef]
  60. Di Pasqua, R.; Hoskins, N.; Betts, G.; Mauriello, G. Changes in Membrane Fatty Acids Composition of Microbial Cells Induced by Addiction of Thymol, Carvacrol, Limonene, Cinnamaldehyde, and Eugenol in the Growing Media. J. Agric. Food Chem. 2006, 54, 2745–2749. [Google Scholar] [CrossRef] [PubMed]
  61. Burt, S.A.; Vlielander, R.; Haagsman, H.P.; Veldhuizen, E.J.A. Increase in Activity of Essential Oil Components Carvacrol and Thymol against Escherichia coliO157:H7 by Addition of Food Stabilizers. J. Food Protect. 2005, 68, 919–926. [Google Scholar] [CrossRef] [PubMed]
  62. Kachoie, M.A.; Pirbalouti, A.G.; Hamedi, B.; Borojeni, H.A.R.; Pishkar, G.R. Effect of drought stress on antibacterial activity of Thymus daenensis subsp. daenensisCelak. J. Med. Plants Res. 2013, 7, 2923–2927. [Google Scholar]
  63. Clevenger, J.F. Apparatus for the Determination of Volatile Oil. J. Am. Pharm. Assoc. 1928, 17, 345–349. [Google Scholar] [CrossRef]
  64. Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry; Allured Publishing Corporation: Carol Stream, IL, USA, 2007; Volume 456. [Google Scholar]
  65. Kovats, E.S. Gas Chromatographic Characterization of Organic Substances in the Retention Index System. Adv. Chromatogr. 1965, 1, 229–247. [Google Scholar]
  66. National Institute of Standards and Technology. Recherche des Données D’espèce par le Nom Chimique. Available online: http://webbook.nist.gov/chemistry (accessed on 15 July 2021).
  67. Prieto, P.; Pineda, M.; Aguilar, M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of vitamin E. Anal. Biochem. 1999, 269, 337–341. [Google Scholar] [CrossRef]
  68. Zovko Koncic, M.; Kremer, D.; Karlović, K.; Kosalec, I. Evaluation of antioxidant activities and phenolic content of Berberis vulgaris L. and Berberis croatica Horvat. Food Chem. Toxicol. 2010, 48, 2176–2180. [Google Scholar] [CrossRef]
Plants 11 00954 g001 550
Figure 1. GC-MS chromatogram of T. capitatus (1) and T.broussonetii (2) EOs.
Figure 1. GC-MS chromatogram of T. capitatus (1) and T.broussonetii (2) EOs.
Plants 11 00954 g001
Plants 11 00954 g002 550
Figure 2. Distribution of the main chemical families of T. capitatus and T. broussonetii EOs.
Figure 2. Distribution of the main chemical families of T. capitatus and T. broussonetii EOs.
Plants 11 00954 g002
Plants 11 00954 g003 550
Figure 3. IC50 and EC50 of T. capitatus and T. broussonetii EOs and of BHA standard measured by DPPH (1) and FRAP (2) methods. Data are expressed as mean ± SD. The experiment was performed in a minimum of three replicates. *** p < 0.001 compared to the samples (1); *** p < 0.001 compared to the control BHA (2).
Figure 3. IC50 and EC50 of T. capitatus and T. broussonetii EOs and of BHA standard measured by DPPH (1) and FRAP (2) methods. Data are expressed as mean ± SD. The experiment was performed in a minimum of three replicates. *** p < 0.001 compared to the samples (1); *** p < 0.001 compared to the control BHA (2).
Plants 11 00954 g003
Plants 11 00954 g004 550
Figure 4. Total antioxidant capacity (Abs. at 695 nm) of T. capitatus and T. broussonetii essential oils; with the regression curve of ascorbic acid. Data are expressed as mean ± SD. The experiment was performed in a minimum of three replicates.
Figure 4. Total antioxidant capacity (Abs. at 695 nm) of T. capitatus and T. broussonetii essential oils; with the regression curve of ascorbic acid. Data are expressed as mean ± SD. The experiment was performed in a minimum of three replicates.
Plants 11 00954 g004
Plants 11 00954 g005 550
Figure 5. Morphological profile of T. capitatus (Left) and T. broussonetii (Right).
Figure 5. Morphological profile of T. capitatus (Left) and T. broussonetii (Right).
Plants 11 00954 g005
Table
Table 1. Chemical composition of T. capitatus and T. broussonetii EOs.
Table 1. Chemical composition of T. capitatus and T. broussonetii EOs.
N°CompoundsIK
(Adams)		Thymus
capitatus		Thymus broussonetiiFormulaM
1α-Thujene930-0.38C10H16136
2α-pinene9390.540.54C10H16136
3Camphene9540.14-C10H16136
4Octen-3-ol 9790.490.71C8H16 O128
5β-Pinene979-0.09C10H16136
6Myrcene9900.880.97C10H16136
7α-Phellandrene1002-0.15C10H16136
8p-Mentha-1(7),8-diene 10040.25-C10H16136
9δ-3-Carene10110.09-C10H16136
10α–Terpinene10170.36-C10H16136
11p-Cymene102610.586.21C10H14134
12Limonene10290.390.31C10H16136
13β-Phellandrene1029-0.19C10H16136
14γ–Terpinene10590.534.47C10H16136
15(Z)-Sabinene hydrate1070-0.33C10H18138
16m-Cymene10850.11-C10H12132
17Terpinolene1088-1.2C10H16136
18Linalool10962.290.14C10H18O154
19(E)-Sabinene hydrate1098-0.16C10H18O2170
20Terpinen-4-ol 11770.200.74C10H18O154
21Borneol1169-0.25C10H18O154
23α-terpineol1188-0.09C10H18O154
24Carvacrol, methyl ether1244-0.2C11H16O164
25Thymol12900.1812.9C10H14O150
26Carvacrol12997560.79C10H14O150
27Terpinylisobutyrate14730.14-C14H24O2224
28Piperitenone13430.06-C10H14O150
29Eugenol13590.15-C10H12O2164
30Trans-sobrerol13740.1-C10H18O2170
31(E)-Caryophyllene14191.614.15C15H24204
32Aromadendrene1441-0.90C15H24204
33α-Humulene1454-0.13C15H24204
349-epi-(e)-caryophyllene1466-0.19C15H24204
35Muurolene<γ->1479-0.19C15H24204
36Viridiflorene1496-0.76C15H24204
37β–Bisabolene15050.790.47C15H24204
38δ-Cadinene1523-0.34C15H24204
39(E)-iso-γ-Bisabolene15240.28-C15H24204
40Thymohydroquinone15550.69-C10H14O2166
41Epoxy caryophyllene15831.500.16C15H24O220
42Caryophylla-4(12),8(13)-dien-5α-ol16400.10-C15H24O220
43Selina-3,11-dien-6-α-ol16440.25-C15H24O220
44Germacra-4(15),5,10(14)-trien-1α-ol16860.22-C15H24O220
45Geranyl benzoate19590.56-C17H22O2258
46cis-Totarol, methyl ether2237-0.23C21H32O300
Percentage of monoterpenes hydrocarbons13.8714.84
Percentage of oxygenated monoterpenes79.375.98
Percentage of sesquiterpenes hydrocarbons2.687.13
Percentage of oxygenated sesquiterpenes2.630.39
Total identified (%)98.4898.34
Table
Table 2. Antibacterial activities of T. capitatus and T. broussonetii EOs and of antibiotics (FOX30, TIM85, PRL100) using the disk diffusion method (mm).
Table 2. Antibacterial activities of T. capitatus and T. broussonetii EOs and of antibiotics (FOX30, TIM85, PRL100) using the disk diffusion method (mm).
StrainsT. capitatusT. broussonetiiAntibiotics
FOX30TIM85PRL100
Enterococcus faecalis23.6 ± 0.85 abc20.85 ± 0.75 abc0 09 ± 0.00
S. aureus9.3 ± 0.35 abc8.55 ± 0.2 abc000
Serratia fonticola49.8 ± 0.28 abc45.35 ± 0.65 abc0 00
Acinetobacter baumannii22.6 ± 0.57 c20.7 ± 1.20 c21 ± 0.0015 ± 0.0010.5 ± 0.00
Klebsiella oxytoca49.9 ± 0.14 abc48.05 ± 0.95 abc0011 ± 0.00
Klebsiella pneumoniae sensitive19.95 ± 0.07 bc14.15 ± 0.15 bc12 ± 0.000 ± 0.000
E. coli sensitive45.2 ± 1.13 abc45.6 ± 0.4 abc22 ± 0.009 ± 0.000
E. coli resistant15.5 ± 0.7 abc14.65 ± 0.35 abc000
Enterobacter aerogenes28.7 ± 0.42 bc28.9 ± 0.3 bc20 ± 0.0014 ± 0.0010 ± 0.00
Data are expressed as mean ± SD. The experiment was performed in a minimum of three replicates. Significance level of at least p < 0.05 compared to FOX30 (a), TIM85 (b), PRL100 (c).
Table
Table 3. Minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and the MBC/MIC report values (mg/mL) of T. capitatus and T. broussonetii EOs.
Table 3. Minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and the MBC/MIC report values (mg/mL) of T. capitatus and T. broussonetii EOs.
StrainsEssential Oils
T. capitatusT. broussonetii
MICMBCMBC/MICMICMBCMBC/MIC
E. aerogenes4 ± 0.00080 ± 0.0020 ± 0.0038 ± 0.0028 ± 0.0032 ± 0.000
S. aureus16 ± 0.00532 ± 0.0091 ± 0.00132 ± 0.00132 ± 0.001 ± 0.004
E. faecalis4 ± 0.0018 ± 0.0012 ± 0.0064 ± 0.0014 ± 0.0011 ± 0.001
E. coli sensitive2 ± 0.0054 ± 0.002 ± 0.002 ± 0.0014 ± 0.0022 ± 0.001
E. coli resistant8 ± 0.00232 ± 0.0044 ± 0.00116 ± 0.00332 ± 0.0071 ± 0.001
K. oxytoca4 ± 0.0038 ± 0.0011 ± 0.0024 ± 0.0018 ± 0.0021 ± 0.000
S. fonticola4 ± 0.0068 ± 0.0031 ± 0.0064 ± 0.0078 ± 0.0011 ± 0.000
K. pneumoniae sensitive2 ± 0.004 ± 0.00410 ± 0.0012 ± 0.0054 ± 0.0081 ± 0.000
A. baumannii4 ± 0.0024 ± 0.0020 ± 0.0024 ± 0.0088 ± 0.0022 ± 0.003
Data are expressed as mean ± SD. The experiment was performed in a minimum of three replicates.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Tagnaout, I.; Zerkani, H.; Hadi, N.; El Moumen, B.; El Makhoukhi, F.; Bouhrim, M.; Al-Salahi, R.; Nasr, F.A.; Mechchate, H.; Zair, T. Chemical Composition, Antioxidant and Antibacterial Activities of Thymus broussonetii Boiss and Thymus capitatus (L.) Hoffmann and Link Essential Oils. Plants 2022, 11, 954. https://doi.org/10.3390/plants11070954

AMA Style

Tagnaout I, Zerkani H, Hadi N, El Moumen B, El Makhoukhi F, Bouhrim M, Al-Salahi R, Nasr FA, Mechchate H, Zair T. Chemical Composition, Antioxidant and Antibacterial Activities of Thymus broussonetii Boiss and Thymus capitatus (L.) Hoffmann and Link Essential Oils. Plants. 2022; 11(7):954. https://doi.org/10.3390/plants11070954

Chicago/Turabian Style

Tagnaout, Imane, Hannou Zerkani, Nadia Hadi, Bouchra El Moumen, Fadoua El Makhoukhi, Mohamed Bouhrim, Rashad Al-Salahi, Fahd A. Nasr, Hamza Mechchate, and Touriya Zair. 2022. "Chemical Composition, Antioxidant and Antibacterial Activities of Thymus broussonetii Boiss and Thymus capitatus (L.) Hoffmann and Link Essential Oils" Plants 11, no. 7: 954. https://doi.org/10.3390/plants11070954

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop