Antioxidant and Antimicrobial Activities of Thymus vulgaris L. Essential Oil Growing Wild in Tunisia

a Research Unit Valorization and Optimization of Resource Exploitation (UR16ES04), Faculty of Science and Technology of Sidi Bouzid, University of Kairouan, Campus University agricultural city Sidi Bouzid 9100 Tunisia. b Regional Center of Agricultural Research (CRRA) Sidi Bouzid, Gafsa Road Km 6, PB 357, Sidi Bouzid 9100, Tunisia. c Laboratory of Valorization of unconventional waters, INRGREF, University of Carthage, Tunisia. d Range Ecology Laboratory, Arid Region Institute, University of Gabes, El-Jorf Road Km 22.5, 4119 Medenine, Tunisia. e Department of Chemistry, College of Science, Qassim University, Buraidah 51452, Saudi Arabia. f University of Monastir, Faculty of Sciences of Monastir, Avenue of the Environment, Monastir 5019, Tunisia. g Laboratory of Genetic, Biodiversity and Valorization of Bioressources, Higher Institute of Biotechnology of Monastir, University of Monastir, Avenue Taher Hadded BP 74, 5000 Monastir, Tunisia. h Department of Biology, Hail University, College of Science, P.O. Box 2440, 81451 Ha’il, Kingdom of Saudi Arabia. i Laboratory of Bioressources: Integrative Biology and Recovery, High Institute of BiotechnologyUniversity of Monastir, Monastir 5000, Tunisia. j Faculty of Science and Arts in Baljurashi, Albaha University, P.O. Box (1988), Albaha, Saudi Arabia. k Department of Chemistry, Faculty of Science of Sfax, University of Sfax, B.P. 1171, 3000, Sfax, Tunisia.


INTRODUCTION
Medicinal herbs and aromatic plants are considered as an important reservoir of bioactive molecules, widely used to treat various diseases [1][2][3]. They play a notable role in allelopathic communication and exhibited effective and significant biological activities [4][5][6][7]. Plantextracted phytochemicals, and essential oils (EOs) have long been a source of therapeutic compounds, and for a long time, been recognized to displayed various biological effects [8-10]. Among potential new drug sources, aromatic plants rich in EOs have received great attention among scientists, and pharmaceuticals industry due to their economic viability, low toxicity, and their potential as alternatives to synthetic agents [11,12]. EOs are important due to their application as antioxidant agents against the phenomenon of oxidative that causing many health problems, like inflammations, cancer, neurodegeneration and cardiovascular diseases. In addition, plants can produce a large variety of secondary metabolites that affect the oxidative stability of EOs and have good antioxidant properties [13]. Potential antioxidants break down the radical chain reaction and act as radical scavengers. Additionally, the high potency of natural antimicrobials linked to their hydrophobic nature, allowed them the property to hamper the spread of multidrug-resistant (MDR) bacteria. Therefore, it is urgent to identify new classes of antimicrobials that inhibit resistance mechanisms [14][15][16].
EOs are in fact an attractive choice to replace synthetic preservatives which can provide flavouring and preservation [17]. A significant number of EOs have shown their significant effects in food packaging systems, inhibition of bacterial growth and oxidative stability [18,19]. On the other hand, EOs are known for their important in vitro antimicrobial and antioxidant activities [20][21][22], but less studies were shown the exploitation of their proprieties in seafood conservation. In consequence, the purpose of this study is to provide the chemical composition by GC-MS, and evaluate the in vitro antioxidant and antimicrobial activities of Tunisian Thymus vulgaris essential oil (TVEO).

Scavenging ability on DPPH radical
DPPH . quenching ability of essential oil was measured according to the previously our work [23,24]. The antiradical activity was expressed as IC 50 (µg/ml), the extract dose required to cause a 50% inhibition.

Superoxide anion radical-scavenging activity
Superoxide anion scavenging activity was assessed as described previously [25]. Evaluating the antioxidant activity was based on IC 50 .

Reducing power
The ability of the EO to reduce Fe 3+ was assayed as cited previously [26].

Chelating effect on ferrous ions
The use of the ferrozine method assessed to evaluate in vitro chelating power. Indeed, free iron in the medium will be stabilized by ferrozine forming a complex ferrozine-Fe 2+ purple through the same protocol as done by our team [23,24].

-Carotene-linoleic acid model system (-CLAMS)
The -CLAMS method by the peroxides generated during the oxidation of linoleic acid at elevated temperature. In this study the -CLAMS was modified for the 96-well micro-plate reader as described elsewhere [27]. The results are expressed as IC 50 values (µg/ml). All samples were prepared and analyzed in triplicate.

Microorganisms
In this study, the microorganisms tested belonging to 24 reference bacterial strains and 16 fungal strains that are presented respectively in Tables 3 and 4. Bacterial strains are divided into 6 Gram-positive and 18 Gram-negative bacteria including 14 strains belonging to the genus Vibrio.

Disc-diffusion assay
Antimicrobial activity testing was done according to the protocol described previously [28,21] for Vibrio spp. strains. After incubation at 37°C for 18 to 24 h, the diameter of inhibition zone was measured with 1 mm flat rule and the diameters were interpreted according to the Committee of the French society of the antibiogram [29].

Micro-well determination of MIC, MBC and MFC
Minimal inhibition concentration (MIC), minimal bactericidal concentration (MBC) and minimal fungicidal concentration (MFC) values were determined for all bacterial and fungal strains as done previously [20].

Statistical Analysis
All the experiments were conducted in triplicate and average values were calculated using the SPSS 26.0 statistics package for Windows.

DPPH radical scavenging activity
The free radical scavenging activities of TVEO measured by DPPH assay were shown in Table  2. The oil was able to reduce the stable free radical DPPH with an IC 50 value of 0.7±0.25g/ml. This oil has a significant ability to neutralize the DPPH radial and therefore an important antioxidant activity significantly higher than the BHT standard used as positive control (11.5±0.62g/ml).
This strong activity is comparable with those exhibited by various oils of Tunisian Thymus chemotypes [21]. The richness of this oil in oxygenated monoterpenes (72.15%) reinforce its antioxidative properties [21,22] and especially, the presence of carvacrol as major components in (67,33%) which may act as radical scavenging agent [21].

Superoxide anion radical-scavenging activity
As shown in Table 2, the Duncan statistically test revealed that activity of TVEO (IC 50 =1.9 ± 0.3µg/mL) is found to be more effective than synthetic antioxidant BHT (IC 50 =1.5 ± 0.2µg/mL). This important activity is strongly linked to the chemical composition of the oil and their wealth was mainly monoterpene compounds such as majority carvacrol, α-Terpinolene, -Terpinene, -Phellandrene. In line with our findings, few studies reported that EOs containing phenolic compounds also have interesting antioxidant potentials [22]. Table 2 showed reductive potential of the studied oil whenever the measured value EC 50 =0.28±0.02 µg/mL. The value showed a strong ferric ion reducing capacity more efficiently than positive controls BHT (EC 50 =23± 1µg/mL) and vitamin C (EC 50 =37±2µg/ml). Studying the reductive capacity of T. capitatus EO harvested from different Tunisian provenance showed that the extracted oils during the postflowering stage had a reductive potential similar to BHA and BHT [33]. This antioxidant activity is also attributed to the presence of natural antioxidants such as phenolic compounds [33].

Chelating effect on ferrous ions
As shown in Table 2, the TVEO has an important chelating ability (EC 50 = 1.36 ± 0.3µg/mL). This ability is twenty times larger than the positive control EDTA (32.5 ± 1.32 µg/ml). In fact, several studies focus on Thymus genus essential oils showed that these oils have a stronger chelating power as compared to vitamin C, BHT and BHA [33,35,34,36,37]. Generally, the high ferrous ion chelating abilities of the EOs from Thymus genus would be beneficial in numerous fields such as food and pharmaceutical industry.

-Carotene-linoleic acid model system
The obtained IC 50 value (

Antibacterial activity
The antibacterial activity of TVEO was assayed in vitro by following the diffusion in agar disc method using twenty-four bacteria associated with human pathogenic. As can be seen in Table  3, TVEO had an excellent inhibitory effect on all bacteria strains. Inhibition halos was ranged from 19±1mm (E. feacalis ATCC 29212) to 37.33±0.57mm (B. cereus ATCC 11778) for Gram positive bacteria and was ranged from 10.33±0.57 mm (P. aeruginosa ATCC 27853) to 35.66±0.57mm (V. furnisii ATCC 35016) for Gram negative bacteria ( Fig. 1) with higher potency than the commercial antibiotics, gentamicin and tetracycline against the major strains. As shown previously, the antibacterial activity of several oils obtained from other thyme varieties has been studied [39]. Moreover, focus on antibacterial activity of T. sipyleus subsp. Sipyleus var. rosulans EO from Turkey revealed a highest inhibitory effect on Pseudomonas pseudoalkaligenes (59 mm) and S. aureus (56 mm), followed by B. subtilis, P. aeruginosa, S. pyogenes, and P. vulgaris and a lowest inhibitory effect was marked on Enterobacter cloacae.  [40,41]. These authors were also reported that this important activity was related to chemical composition of thyme oil rich in carvacrol (60.27%), -terpinene (11.20%), pcymene (7.58%) and bornyl acetate (4.93%). Furthermore, anti-Vibrio alginolyticus activity of TVEO (from Sidi Bouzid, Tunisia).
Several studies underline evaluated that thyme oil harvested in different Mediterranean regions displayed potent antimicrobial activity of. TVEO collected from the cultivated fields of the Botanical Gardens, University of Agriculture; Faisalabad, Pakistan showed an important antibacterial activity with a MIC ranged from 0.07 to 1.25 mg/mL [42].    This study indicates that TVEO exhibited a significant antibacterial activity against all tested bacteria which can be explained by the richness of this oil on oxygen monoterpene group (72.15%). Several studies have shown the importance of the fraction oil in inhibiting microorganism's expansion [21,42].
Studying the correlation between the chemical composition of T. maroccanus and T. broussonetii EOs and their antimicrobial effect, it was affirmed that the activity level could be attributed to the presence of high concentrations of carvacrol [42].

Antifungal activity
The inhibitory effects of the EO isolated from T. vulgaris on the growth of 16 pathogenic fungal species are shown in Table 4. Results revealed an important inhibitory effect of the oil against tested fungi. In fact, obtained values of IZ, MIC and MFC were respectively ranged from 24.33±1.53 to 48.33±1.53 mm (Fig. 1), 0.004 to 0.078mg/mL and > 0.009 to 0.15mg/mL. These values showed that the oil exhibit a more important antifungal activity than synthetic antifungal amphotericin-B. IZ, MIC and MFC obtained values of amphotericin-B were ranged from 11-24±0.57 mm, 0.009-0.078mg/mL and 0.019-0.31mg/mL, respectively. Similarly, it has been demonstrated that the anticandidal activity of T. maroccanus and T. broussonetii EOs was lower than amphotericin-B and fluconazole [42]. Majority of previous studies showed that Thymus genus Eos have a great antifungal potential, thanks to their wealth of oxygenated monoterpene and particularly phenol compounds such as carvacrol [21,42].
According to this study, fungal strains (yeasts and moulds) were more sensitive than bacteria (Gram + and Gram -) against TVEO. Indeed, the values of IZ, MIC and MFC were lowest for fungi (Table 3 and Table 4). In addition, other studies have confirmed the sensitivity of fungal tested strains by other oils. The antimicrobial activity of cumin EO showed effectively fungi sensitivity by MIC and MFC values which were ranged between 0.009-0.078 mg/mL and 0.019 to 0.31 mg/mL, in front MIC and MBC values for bacteria which ranged between 0.039 to 0.31 mg/mL and 0.31 to 1.25 mg/mL [21].
In most tested strains, MFC/MIC Ratio values showed that studied TVEO have a fungicidal effect because they were ≤ 4 (Table 4). In exception, TVEO has a fungistatic effect against Candida tropicalis and Trichophyton rubrum (MFC/MIC equal to 5 and 9, respectively).

CONCLUSION
The present investigation showed that TVEO is characterized by the abundance of carvacrol (67.33%). For all antioxidant tests activities, this EO showed a more important activities comparing to standards synthetic antioxidant. The studied EO showed high antibacterial and antifungal activities against a wide range of microorganisms known to cause serious infections. Antibacterial activity of this EO seems to be more efficient against Vibrio strains, but antifungal ones have a fungicidal effect for the majority of fungal strains. This might be related to its chemical profile.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

COMPETING INTERESTS
Authors have declared that no competing interests exist.