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Original article
13 (
8
); 6782-6801
doi:
10.1016/j.arabjc.2020.06.032

Thymus musilii Velen. as a promising source of potent bioactive compounds with its pharmacological properties: In vitro and in silico analysis

, , , , , , , , , , ,
a Department of Biology, University of Hail, College of Science, P.O. Box 2440, 81451 Ha’il, Saudi Arabia
b Department of Biology, Sfax University, Faculty of Science of Sfax, 3000 Sfax, Tunisia
c Department of Chemistry, College of Science, University of Hail, P.O. Box 2440, 81451 Hail, Saudi Arabia
d Natural Water Treatment Laboratory, Water Researches and Technologies Centre of Borj-Cedria (CERTE), Carthage University, BP 273, 8020 Soliman, Tunisia
e Laboratory of Bioresources: Integrative Biology and Valorization, (LR14-ES06), University of Monastir, Higher Institute of Biotechnology of Monastir, Avenue Tahar Haddad, BP 74, 5000 Monastir, Tunisia
f Central Biotechnology Services, College of Biomedical and Life Sciences, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom
g Laboratoire de Développement Chimique Galénique et Pharmacologique des Médicaments, Faculté de Pharmacie, 5000 Monastir, Tunisia
h Laboratory of Bioactive Substances, Centre of Biotechnology of Borj Cedria, BP 901 Hammam lif 2050, Tunisia
i Faculty of Science and Arts in Baljurashi, Albaha University, P.O. Box 1988, Albaha, Saudi Arabia
j Bapalal Vaidya Botanical Research Centre, Department of Biosciences, Veer Narmad South Gujarat University, Surat, Gujarat, India
k Laboratory of Genetics, Biodiversity and Valorization of Bio-resources, Higher Institute of Biotechnology of Monastir, University of Monastir, Tunisia

⁎Corresponding author at: Department of Biology, University of Hail, College of Science, P.O. Box 2440, 81451 Ha’il, Saudi Arabia. snmejdi@yahoo.fr (Mejdi Snoussi)

Received: , Accepted: ,
© The Author(s) 2020
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

For the first time, we reported the phytochemical composition of the volatile oil from Thymus musilii Velen (T. musilii). The antioxidant and antimicrobial activities against various food-borne and clinical pathogenic microorganisms were also tested. The thyme oil was particularly rich in thymol (67.697 ± 0.938%), and thymyl acetate (12.993 ± 0.221%). The strongest antioxidant activity of the essential oil was registered with the tests: ABTS (IC50 = 5.6 × 10−4 mg/mL) and β-carotene/linoleic acid (IC50 = 3.2 × 10−3 mg/mL). This thymol-chemotype oil was active against all microorganisms tested with an inhibition growth zone ranging from 21.33 ± 1.52 mm for Proteus mirabilis (P. mirabilis) to 37.33 ± 1.15 mm for Candida vaginalis (C. vaginalis) strain. Overall, the tested oil exhibited bactericidal and fungicidal activities and only a small quantity of the tested essential oil was found to be sufficient for inhibiting the growth of the tested microorganisms. Furthermore, molecular docking results implies that, among the bioactive compounds, β-caryophyllene interacted strongly with the active site residues of TyrRS, GLMS and Gyrase enzymes and consequently support our in vitro results with the highest inhibition potential of this essential oil against tested pathogens, especially Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Our results suggested that essential oil of T. musiliii exhibited strong biological activities with a promising source of various natural compounds.

Keywords

Thymus musilii Velen.
GC–MS
Antioxidant
Antibacterial
Antifungal
Molecular docking
1

1 Introduction

The use of medicinal plants as a source of therapy against various disorders have been practiced in Saudi Arabia since ages and many practices reported in the Prophetic Medicine are currently used in folk medicine in the Arabian Peninsula (Al-Essa et al., 1998). Among this category of plants, there are cultivated plants and others are spontaneous ones. These aromatic plants are grown as needed for their aerial parts (flowers, seeds, leaves, stems, bark) or their underground parts (bulbs, roots). Studies in the past have reported the presence of valuable medicinal plants from the different regions of Saudi Arabia (El-Tawil, 1983). However, the information of the indigenous medicinal plants of Saudi Arabia is scattered in a disorganized manner (Al-Asmari et al., 2014). Scientific studies have proven that these plants, including garlic, pomegranate, black seeds, costus, miswak, henna, ferns, Eucalyptus, ginger, and fenugreek are effective for treating human diseases (Noumi et al., 2017; Adnan, 2019; Reddy et al., 2020; Adnan et al., 2020). These species are exploited in human food, traditional medicine as well as for industrial purposes (agro-food, perfumery, cosmetics, pharmaceutical, etc.).

The mint family (Lamiaceae) is one of the largest and most distinctive families of flowering plants, with about 220 genera and almost 4000 species worldwide (Pirbalouti et al., 2015). This family has an almost cosmopolitan distribution. These plants are frequently aromatic in all parts and include many widely used culinary herbs, such as thyme. The genus Thymus L. belongs to the Nepetoideae subfamily of Lamiaceae family is a well-known aromatic herb and consists of about 330 species of herbaceous perennials and small shrubs in the world (Nickavar et al., 2005; Salehi et al., 2019).

The Mediterranean region can be described as the center of the genus (Cronquist, 1988; Morales, 2002; Jamzad, 2010). Thymus plants also includes many aromatic perennial and herbaceous plant that are cultivated in frequency due to their wide use in the food, cosmetic, and pharmaceutical industries (Nabavi et al., 2015). The genus Thymus is a taxonomically complex group of aromatic plants, traditionally used for medicinal purposes because of their antiseptic, antispasmodic and antitussive properties (Pina-Vaz et al., 2004, Nabavi et al., 2015). Previous chemical investigation on Thymus species have shown the presence of aromatic terpenes and terpenoids, flavonoids, and phenolic acid (Miri et al., 2002; Miguel et al., 2004; Ebrahimi et al., 2008; Tohidi et al., 2017). Thymol and carvacrol are the main phenolic compound of thyme oil. The major non-phenolic compounds were linalool and p-cymene (Piccaglia and Marotti, 1991).

Recent studies have shown that Thymus species have antibacterial, antifungal, and antioxidant activities (Bassam et al., 2004; Rahimmalek et al., 2009; Jordan et al., 2009). Gedikoğlu, et al. (2019) reported that the essential oil of thyme showed antimicrobial activity against Bacillus cereus NRRL (B3711), Staphylococcus aureus (ATCC 9144), Staphylococcus epidermidis (ATCC 12228), Escherichia coli ATCC (25922), Salmonella enteritidis (ATCC 13076) and Salmonella typhimurium (ATCC 14028). The anti-bacterial characteristic of Thymus spp. is due to the occurrence of thymol in this genus. This substance can be used as a disinfectant.

In Saudi Arabia, at least three species of Thymus (endemic and introduced) were identified: T. bovei Benth., T. decussatus Benth. and T. musilii Velen. In addition, T. vulgaris was largely cultivated in many regions of the kingdom. This species, T. musilii Velen. belongs to division: Tracheophyta, subdivision: Spermatophytina, Class: Magnoliopsida, Superorder: Asteranae, Order: Lamiales, Family: Lamiaceae Lindl., and Genus: Thymus L. It is distributed mainly in Iraq, Palestine, and Saudi Arabia (World Checklist of Selected Plant Families, 2010).

Growing to 30–70 cm tall by 40–60 cm wide, it is a bushy, woody-based evergreen subshrub with highly aromatic, green leaves and clusters of white flowers in early summer. Preferred the dry slopes, rocks and maquis, it was always found on clay or limestone soils. It has sessile leaves varying from elliptic to linear or diamond-shaped towards the apex. The flowers have a tube-like calyx and tubular corolla with a three lobed lower lip, and are united in spikes at the top of the branches (Fig. 1). The roots are robust, and the fruit consists of a smooth, dark colored nutlet. In Bedouin population of Saudi Arabia, leaves and flowering tops of T. musilii were used as a garnish or added as a flavoring in cooking variety of foods, as well as in preparing infusion tea. An aromatic tea is made from the fresh or dried leaves. The leaves can be used either fresh or dried. If the leaves are to be dried, the plants should be harvested in early and late summer just before the flowers open and the leaves should be dried quickly.

T. musilii Velen specimen. (A): whole plant at flowering stage, (B): clusters of white flowers, (C): green leaves.
Fig. 1
T. musilii Velen specimen. (A): whole plant at flowering stage, (B): clusters of white flowers, (C): green leaves.

The in vitro antimicrobial and antioxidant activities of the essential oil and extract of T. vulgaris have recently been reported. Al-Asmari et al. (2017) have studied the essential oil composition, whereas, Alharbi (2017) reported that the whole plant was used in traditional medicine to treat abdominal pain, and as anti-helminthic and carminative effects. Belonging this genus, T. musilii is a very interesting medicinal plant closely distributed on Arabian Peninsula, Iraq and Jordan landscapes (Batanouny and Sheikh, 1972; Govaerts, 2003). In the north of Saudi Arabia, it is locally used as an antiseptic traditional drug. This species has also been used for curing many bacterial and fungal diseases in traditional medicine in Saudi Arabia (survey, data not shown). In fact, it used by local Saudi population to cure many ailments. Leaves are used in treating respiratory diseases and the flowering tops are used as anti-helminthic, antiseptic and antispasmodic drug. However, antimicrobial and antioxidant properties of T. musilii Velen seem not to have been reported before.

To the best of our knowledge, this study is the first report on the biological properties of T. musilii Velen. The aim of this work was to investigate the chemical composition of the volatile oil obtained from the aerial parts of T. musilii cultivated under greenhouse conditions in Al-Gaad, Hail (Saudi Arabia) by using GC–MS technique. Additionally, the antioxidant and antimicrobial activities of the oil were assessed. To reach this objective, molecular docking studies of the bioactive compounds were also performed against tyrosyl-tRNA synthetase TyrRS from S. aureus, glucosamine 6-phosphate synthase (GLMS) from E. coli and Gyrase from S. aureus enzymes to better understand their mechanism of action.

2

2 Material and methods

2.1

2.1 Plant material sampling and essential oil extraction

The plant used in this study were collected in October 2019 from a nursery belonging to the Ministry of Agriculture in the region of Hail (Al-Gaad, Ha'il, Saudi Arabia). Dr. Ahmed Alghamdi, from the Department of Biology, Faculty of Science, University of Hail, Saudi Arabia identified the plant at the species level. A voucher specimen (AN 001) was deposited in the Department of Biology, University of Hail, Saudi Arabia. The volatile oil was collected using a clevenger-type apparatus after 3 h of hydro-distillation using 100 g from the aerial air-dried organs (flowering stage). The obtained oil was dried using anhydrous sodium sulfate and stored until use at −20 °C. The yield of extraction was calculated after three running cycle and expressed according to the dry weight.

2.2

2.2 Characterization of the volatile oil

A Hewlett–Packard 6890 chromatograph equipped with a flame ionization detector (FID) and an electronic pressure control injector was used to study the chemical composition of the obtained volatile oil from T. musilii aerial parts. A gas chromatography apparatus coupled to mass spectrometry (GC–MS) on a gas chromatograph HP 7890 (II) and HP 5975 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) with an electron impact ionization of 70 eV was used. An HP-5MS capillary column (Agilent Technologies, Hewlett-Packard, CA, USA; 30 m × 0.25 mm), with 0.25 m film thickness was used. Temperature was fixed to rise from 40 °C to 280 °C at a rate of 5 °C/min. The carrier gas was helium with a flow rate of 1.2 mL/min, a split ratio of 60:1, scan time and mass range of 1 s and 40–300 m/z, respectively. The identification of the bioactive components in T. musilii volatile oil was based on the calculated retention index (RI) relative to (C8–C22) n-alkanes and in comparison, with authentic compounds. Further identification of compounds was made by matching their recorded mass spectra with those stored in the Wiley/NBS mass spectral library of the GC–MS data system and other published mass spectra (Adams, 2007) and data expressed as relative percentage of the total peak area as previously described by Essid et al. (2015) and Salem et al. (2018).

2.3

2.3 Antioxidant assays

2.3.1

2.3.1 DPPH radical–scavenging activity

The free radical-scavenging activity of the tested essential oil was measured using the protocol described by Chakraborty and Paulraj (2010) and Adnan et al. 2018. The ability to scavenge the DPPH radical was calculated using the following equation (Eq. (1)):

(1)
DPPH scavenging activity ( % ) = A 0 - A 1 / A 0 × 100 where

  • A0 is the absorbance of the control and A1 is the absorbance of the sample.

The antioxidant activity was expressed as IC50 (mg/mL) which represented the extract concentrations scavenging 50% of DPPH radicals (Nishaa et al., 2012).

2.3.2

2.3.2 ABTS radical scavenging activity assay

The radical scavenging activity against ABTS radical cations was measured using the method of Chakraborty and Paulraj (2010). The inhibition percentage of ABTS radical was calculated using the following equation (Eq. (2)):

(2)
ABTS scavenging activity ( % ) = A 0 - - A 1 / A 0 × 100

where

  • A0 is the absorbance of the control and A1 is the absorbance of the sample.

The antiradical activity was expressed as IC50 (mg/mL) which represented the extract concentrations scavenging 50% of ABTS radicals (Nishaa et al., 2012). A lower IC50 value represents a stronger ABTS scavenging capacity.

2.3.3

2.3.3 Reducing power capability assay

The reducing power was determined using the method of Bi et al. (2013). The extract concentration providing 0.5 of absorbance (IC50) was calculated from the graph of absorbance at 700 nm against sample concentration (Barros et al., 2008). Ascorbic acid was used as a standard.

2.3.4

2.3.4 β-carotene/linoleic acid method

The β-carotene method was carried out according to Ikram et al. (2009). Antioxidant activity (inhibition percentage, PI %) was evaluated using the following equation (Eq. (3), Miraliakbari and Shahidi, 2008):

(3)
P I % = A β - c a r o t e n e T 120 / A β - c a r o t e n e t 0 × 100

where

  • Aβ-carotene t0 and Aβ-carotene T120 refer to the corresponding absorbance values of the test sample, standard and control measured before and after incubation for 2 h, respectively. All tests were performed in triplicate and ascorbic acid (standard) was used for comparison.

2.4

2.4 Screening of antimicrobial activities

The antimicrobial activity of the obtained essential oil was tested against four type strains namely E. coli ATCC 35218, P. aeruginosa ATCC 27853, P. mirabilis ATCC 29245, and K. pneumoniae ATCC 27736. Two clinical strains, S. aureus MDR (multidrug resistant bacteria), and Enterobacter cloacae (E. cloacae) were used. The antifungal activity was performed using Candida albicans (C. albicans) ATCC 10231, Cryptococcus neoformans (C. neoformans) ATCC 14116, C. vaginalis (clinical strain), and Candida sp. (clinical strain). Two fungal strains (Aspergillus spp.) were also tested: A. fumigatus ATCC 204305 and A. niger.

Two techniques were used to screen the antimicrobial effect of the obtained essential oil and its main component thymol purchased from Sigma Aldrich®, Germany. The disc diffusion assay was performed on Mueller-Hinton agar plates for all bacteria, Sabouraud chloramphenicol agar for yeasts, and Potato Dextrose agar for the Aspergillus strains. 10 mg of essential oil and thymol/6 mm-disc were tested in triplicate. Ampicillin and Amphotericin B were used as control. The minimal inhibitory concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC) values were determined by using the microdilution assay as previously described by Snoussi et al. (2018). MBC/MIC ratio and MFC/MIC ratio were used to interpret the activity of the essential oil as described by Gatsing et al. (2009).

2.5

2.5 Molecular docking analysis of TyrRS, GLMS and Gyrase with phytochemicals of T. musilii

Crystal structures of tyrosyl-tRNA synthetase TyrRS from S. aureus (PDB: 1JIJ.pdb) (Qiu et al., 2001), glucosamine 6-phosphate synthase (GLMS) from E. coli (PDB: 1XFF.pdb) (Isupov et al., 1996), and Gyrase from S. aureus (PDB: 2XCT.pdb) (Bax et al., 2010) were fetched from Protein Data Bank (RCSBPDB). Following to the retrieval of crystal structures, LCMS identified phytochemicals 3-dimensional structures such as α-thujene, α-pinene, β-myrcene, α-terpinene, p-cymene, (1,8)-cineole, γ-terpinene, α-terpinolene, Borneol, Terpinen-4-ol, α-terpineol, 2-Isopropyl-5-methylanisole, Thymol, Carvacrol, Thymyl acetate, Carvacryl acetate, and β-caryophyllene were acquired from eminent database PubChem and converted to PDB format using Open Babel (O'Boyle et al., 2011). These seventeen compounds were then docked separately against the receptor structure (1JIJ, 1XFF and 2XCT) using molecular docking software Autodock 4.2.6 (Morris et al., 2009). Docking protocol was performed in a similar manner, which can be related to previous analyses (Sonawane and Barage, 2015; Parulekar and Sonawane, 2018). Apart from the grid centre and grid size, all other parameters used for docking with these seventeen compounds were kept same. For the preparation of the grid map using a grid box, Auto Grid (Morris et al., 2009) was used. The grid size was set to 126 × 126 × 126xyz points for TyrRS and gyrase receptors. For GLMS, grid size was set to 96 × 122 × 126 xyz points. Grid spacing was kept to 0.375 Å for all the receptors. The grid centre for TyrRS was designated at dimensions (x, y and z): −11.897, 17.862 and 91.741, for GLMS at (x, y and z): 1.979, 37.952 and 20.512, and for gyrase at (x, y and z): 7.841, 39.224 and 118.021. The grid box is cantered in such a way that it encloses the entire binding site of both the receptors and provides enough space for translation and rotation of ligands. The generated docked conformation was ranked by predicted binding energy and topmost binding energy docked conformation was analyzed using UCSF Chimera (Pettersen et al., 2004) for intermolecular hydrogen bonding of active site amino acid residues from the receptors with docked ligands.

2.6

2.6 Statistical analysis

The laboratory biological assays were conducted in triplicates for each sample. The IC50 of DPPH, ABTS, and β-carotene bleaching methods values were calculated by linear regression analysis. ANOVA and Duncan tests were performed with SPSS 16.0. The means of the test’s values were also evaluated with the Least Significant Differences test at 0.05 significance level.

3

3 Results and discussion

3.1

3.1 Chemical composition of T. musilii Velen. essential oil

The air-dried aerial-parts of T. musilii yielded 2.736 ± 0.015% (v/w) essential oil on hydro-distillation. Seventeen components were identified in the obtained oil, belonging mainly to oxygenated monoterpenes (87.010 ± 0.279%) followed by monoterpenes hydrocarbons (11.013 ± 0.039%) and sesquiterpenes hydrocarbons (1.953 ± 0.005%). These data are summarized in Table 1. The chemical structure of the seventeen compounds identified in T. musilii essential oil were depicted in Fig. 2.

Table 1 Chemical composition of T. musilii Velen. essential oil.
Peak # RI* on HP-5MS column Compounds Chemical formula Percentage (Mean ± SD)
1 931 α-Thujene C10H16 0.437 ± 0.015
2 939 α-Pinene C10H16 0.303 ± 0.015
3 992 β-Myrcene C10H16 0.710 ± 0.034
4 1018 α-Terpinene C10H16 0.853 ± 0.028
5 1026 p-Cymene C10H14 4.617 ± 0.119
6 1033 1,8-Cineole C10H18O 0.397 ± 0.005
7 1062 γ-Terpinene C10H16 2.633 ± 0.072
8 1087 α-Terpinolene C10H16 1.460 ± 0.081
9 1165 Borneol C10H18O 0.763 ± 0.030
10 1174 Terpinen-4-ol C10H18O 0.390 ± 0.017
11 1189 α-Terpineol C10H18O 0.890 ± 0.036
12 1227 2-Isopropyl-5-methylanisole C11H16O 0.080 ± 0.138
13 1290 Thymol C10H14O 67.697 ± 0.938
14 1292 Carvacrol C10H14O 3.417 ± 0.105
15 1356 Thymyl acetate C12H16O2 12.993 ± 0.221
16 1367 Carvacryl acetate C12H16O2 0.383 ± 0.015
17 1404 β-caryophyllene C15H24 1.953 ± 0.102
Chemical classes
Monoterpene hydrocarbons 11.013 ± 0.039
Oxygenated monoterpenes 87.010 ± 0.279
Sesquiterpenes hydrocarbons 1.953 ± 0.005
Total compounds Identified (%) 100

RI: Retention index on a HP-5MS column. The data are expressed as mean ± SD (n = 3); SD: Standard Deviation.

Chemical structures of 17 bioactive molecules identified in T. musilii essential oil using GC–MS technique. Numbers in the figure correspond to the codes in Table 1.
Fig. 2
Chemical structures of 17 bioactive molecules identified in T. musilii essential oil using GC–MS technique. Numbers in the figure correspond to the codes in Table 1.

This essential oil can be defined as thymol/thymyl acetate chemotype (67.697/12.993%) as shown in the chromatogram (Fig. 3). Thymol (67.697 ± 0.938%), thymyl acetate (12.993 ± 0.221%), o-cymene (4.617 ± 0.119%), carvacrol (3.417 ± 0.105%), and γ-terpinene (2.633 ± 0.072).

Chromatogram obtained for T. musilii Velen essential oil. The main components identified are: 1 (Thymol), 2 (Thymol Acetate), and 3 (o-cymene).
Fig. 3
Chromatogram obtained for T. musilii Velen essential oil. The main components identified are: 1 (Thymol), 2 (Thymol Acetate), and 3 (o-cymene).

Numerous studies have reported that oxygenated monoterpenes were the dominant family of compounds found in the Thymus genus essential oil (De Martino et al., 2009; Zarshenas and Krenn, 2015). The diversity of the composition of the volatile oil obtained from different species and subspecies belonging to the genus thymus can be explicated by endogenous (plant varieties, vegetative state, organ tested) and exogenous factors like climatic features, soil characteristics, and seasons (Tzakou et al., 1998; Cosentino et al., 1999; Pirbalouti et al., 2013a,b). It has also been reported that the frequency of irrigation and salicylic acid concentration can affect the yield and the content of essential oil obtained from T. daenensis Celak. and T. vulgaris L. (Khazaie et al., 2008, Pirbalouti et al., 2013c; Alavi-Samani et al., 2013). In addition, application of fertilizers increases the vegetative biomass, oil yield and diversity, and antioxidant activities of T. daenensis Celak. (Bistgani et al., 2018).

Thymol and carvacrol are the main phenolic compound of thyme oil. The major nonphenolic compounds were linalool and p-cymene (Piccaglia and Marotti, 1991). Thymol was the dominant phenolic compound detected in several Thymus species with different percentage as reported by Tohidi et al. (2019) including: T. carmanicus (40.8%), T. daenensis (20–80.4%), T. eriocalyx (5.3–66.34%), T. fallax (19.88–65.9%), T. fedtschenkoi (31.8%), T. kotschyanus (6.8–66.15%), T. migricus (55.6–79.74%), T. pubescens (37.9–63.5%), T. serpyllum (52.45%), T. transcaucasicus (35.83–62.92%), and T. trauveterri (24.43–63.33%).

It has also been reported that thymol is the main phenolic compound in the essential oil of T. cappadocicus Boiss. (Albayrak and Aksoy, 2012), T. pulegioides (Pinto et al., 2006), T. fontanesii (Dob et al., 2006), T. hyemalis (Rota et al., 2008), T. ciliatus Desf. Benth. (Kabouche et al., 2009), T. marschallianus Willd (Cavar Zeljkovic et al., 2015), T. pannonicus (Pluhár et al., 2010), T. vulgaris (Asbaghian et al., 2011), T. zygis (Ballester-Costa et al., 2013), T. numidicus Poiret (Mina et al., 2014), T. quinquecostatus Celak. (Kim et al., 2014) and T. lanceolatus (Khadir et al., 2016a,b). More recently, Jan et al. (2020) reported that T. afghanicus harvested from the Himalayan-Afghanistan area was a thymol chemotype (27.7%).

In this study, thymyl acetate, which is formed after acetylation of thymol produced directly by terpene synthases (Keszei et al. 2008), was found to be the second phenolic compound in T. musilii oil (12.993%). This molecule has been reported in the essential oil of some Thymus species with different percentage including T. longicaulis (0–12.8%) and T. pulegioides L. (0.4–0.7%) from Italy (De Martino et al., 2009), T. caespititius Brot. from Portugal (11–15%, Mendes et al., 2013), T. serpyllum L. from Serbia (38.5%, Cancarevic et al., 2013), and T. lanceolatus from Algeria (0.006%; Khadir et al., 2016a,b).

3.2

3.2 Antioxidant activities of T. musilii essential oil

Because of the complex chemical compounds effect of the plants volatile oil, the antioxidant capacity of T. musilii essential oil is studied by four methods, DPPH, ABTS, FRAP and β-carotene bleaching methods in order to estimate the effectiveness of these compound diversity. Table 4 summarizes the free radicals scavenging activities of T. musilii essential oil and the commercialized standards, ascorbic acid and butylated hydroxyl-toluene (BHT). The IC50 of the essential oil and the standards, which is the concentration required for scavenging half (50%) of the tested radicals, showed that ABTS and peroxyl radicals were strongly significantly inhibited by T. musilii (Table 4). Interestingly, T. musilii oil possess high antioxidant activities using ABTS (IC50 = 5.6 × 10−4 ± 2 × 10−5 mg/mL) and β-carotene bleaching (IC50 = 3.2 × 10−3 ± 5 × 10−4 mg/mL) methods, followed by DPPH test (IC50 = 0.049 ± 1 × 10−4 mg/mL). This essential oil is significantly active on peroxyl radicals than the both tested standards (Table 4).

Literature review showed that no previous work was countered on T. musilii essential oil antioxidant capacity. However, several studies were conducted on the genus Thymus essential oils and on its antioxidant capacity (El-Bakkal et al., 2020; Goudjil et al., 2020). For instance, the anti-radicalar essential oils from the cultivated T. carmanicus, T. kotschyanus, T. migricus, and T. vulgaris collected, under various conditions, from Iran were studied by DPPH method (Tohidi et al., 2020). Under red, red-blue, blue, white and greenhouse light treatments, T. carmanicus (IC50 = 278; 259.2; 281; 467.4; 198.2 µg/mL), T. kotschyanus (IC50 = 621.8; 421.1; 304.6; 557.4; 384.7 µg/mL), T. migricus (IC50 = 358; 911.6; 176.8; 1274; 631.8 µg/mL), and T. vulgaris (IC50 = 560; 766; 400.6; 227.6; 314.3 µg/mL) inhibited DPPH radicals (Tohidi et al., 2020). Thymus longicaulis C. Presl subsp. longicaulis var. longicaulis essential oil collected from Turkey had strong radical inhibition percentage (IP = 87.69% at 0.4 mg/mL; 93.28% at 1 mg/mL; 94.15% at 2 mg/mL) using b-carotene–linoleic acid method. The same plant species possess moderate effect (IP = 28.17% at 0.1 mg/mL; 46.32% at 0.2 mg/mL, 63.26% at 0.5 mg/mL) using DPPH method, moderate effect using reducing power protocol (Absorbance = 0.128 at 0.2 mg/mL, 0.241 at 0.4 mg/mL, 0.550 at 1 mg/mL), and it had no chelating activity till 1 mg/mL (Sarikurkcu et al., 2010).

Compared to the previous studies, T. musilii essential oil in the present study exhibited a strong antioxidant effect. This activity can be explained by the chemical composition classes, monoterpene hydrocarbons (11.01%) and oxygenated monoterpenes (87.01%), of the volatile oil. Most researchers revealed the antiradical effect of monoterpenes (Badawy et al., 2019; Wojtunik‐Kulesza et al., 2019). The antioxidant capacity of thymol (IC50 = 31.426 mg/mL), β-cymene (IC50 = 916.89 mg/mL), α-terpineol (IC50 = 480.56 mg/mL), myrcene (IC50 = 22.136 mg/mL), α-pinene (IC50 = 880.74 mg/mL) were evaluated using N,N-dimethyl-1,4-phenylenediamine (DMPD) reagent (Badawy et al., 2019).

Other study focused on the antioxidant of α-terpinene (IC50 = 0.6 and 7.5 mM) and γ-terpinene (IC50 = 2.8 and 30.0 mM) using ABTS and DPPH methods, respectively (Li and Liu, 2009). Previous work demonstrated that γ-terpinene (IC50 = 15.5 mg/mL) inhibited DPPH radicals (Sonboli et al., 2005). This antioxidant assay may be related to a high area of thymol (67.7%). Several studies confirmed the strong in vitro and in vivo biological effect of thymol (Abd El-Naby et al., 2020; Arafa et al., 2020; Jafari et al., 2020). The registered effect may referred to the major compound, thymol (67.7%), and/or to the synergism between main and minor compounds of the essential oil (Ciesla et al., 2016). The antioxidant activities were studied, in literature, towards the whole essential oils, to single compounds and as well as to combination (Graßmann, 2005; Tohidi et al., 2020).

3.3

3.3 Antimicrobial activities of T. musilii essential oil

The antibacterial activity of T. musilii essential oil was tested against six bacteria, four yeasts and two fungal strains using both disc diffusion (Fig. 4) and microdilution assays. Obtained results showed that, the tested bacteria were resistant to ampicillin with a mean diameter of growth inhibition zone ranging from 6.33 ± 0.57 mm to 7.33 ± 0.57 mm. In addition, the mean diameter of growth inhibition zones ranged from 21.33 ± 1.52 mm for P. mirabilis to 36.33 ± 1.15 mm for K. pneumoniae. The clinical strain S. aureus MDR, resistant to ampicillin, was susceptible to the oil tested (25.33 ± 1.15 mm). Small quantities of oil (12.5 mg/mL) can inhibit the growth of all tested bacteria, except for E. cloacae (MIC value = 3.125 mg/mL). MBCs values were ranging from 6.25 mg/mL (E. cloacae) to 100 mg/mL for P. aeruginosa. As compared to the single bioactive molecule, thymol, T. musilii essential oil exhibited bactericidal activity for all tested bacteria with MBC/MIC ratio inferior to 4 except for P. aeruginosa (MBC/MIC ratio = 8). All these data are summarized in Table 2. Using the literature review, high antimicrobial activity of Thymus species (chemotype thymol) was recorded against a large collection of bacterial and fungal species (Table 5).

Selected photos showing the antibacterial (A), anti-Candida spp. (B), anti-Aspergillus spp. (C) activity of the tested essential oil and its main component thymol.
Fig. 4
Selected photos showing the antibacterial (A), anti-Candida spp. (B), anti-Aspergillus spp. (C) activity of the tested essential oil and its main component thymol.
Table 2 Growth inhibition zone, MIC and MBC values obtained for bacterial strains tested using disc diffusion and microdilution assays.
Code Strain T. musilii Velen essential oil Main Compound (Thymol) Ampicillin
Mean ± SD*
(mm)		 MICa MBCb MBC/MIC
ratio		 Mean ± SD
(mm)		 MIC MBC MBC/MIC
ratio		 Mean ± SD
(mm)
B1 E. coli ATCC 35218 35.33 ± 1.15c 12.5 50 4 12.66 ± 0.57b 3.125 6.25 2 7 ± 0a
B2 P. aeruginosa ATCC 27853 35.33 ± 1.15b 12.5 100 >4 7 ± 0a 12.5 50 4 7.33 ± 0.57a
B3 Proteus mirabilis ATCC 29245 21.33 ± 1.52b 12.5 25 2 6 ± 0a 3.125 6.25 2 6.33 ± 0.57a
B4 K. pneumoniae ATCC 27736 36.33 ± 1.15c 12.5 25 2 9 ± 1b 3.125 6.25 2 6.66 ± 0.57a
B9 S. aureus MDR (Clinical strain) 25.33 ± 1.15b 12.5 25 2 6 ± 0a 0.78 1.56 2 7.33 ± 0.57a
B10 E. cloacae (Clinical strain) 31.00 ± 1.00c 3.125 6.25 2 8.66 ± 1.15b 0.39 0.78 2 6.66 ± 0.57a

*Inhibition zone around the discs impregnated with the essential oil (10 mg/disk) expressed as mean of three replicates (mm ± SD). SD: standard deviation. a: Minimal Inhibitory Concentration (mg/ml). b: Minimal Bactericidal Concentration (mg/ml). c: MBC/MIC ratio interpreted using the scheme of antimicrobial substances are considered as bacteriostatic agents when the ratio MBC/MIC>4 and bactericidal agents when the ratio MBC/MIC≤4 (Gatsing et al., 2009). The letters (a–c) indicate a significant difference between the inhibition zones of essential oil, thymol and ampicillin against the tested bacteria according to the Duncan test (p < 0.05).

Table 3 Growth inhibition zone, MIC and MFC values obtained for fungal and yeast strains tested using disc diffusion and microdilution assays.
Code Strain T. musilii Velen essential oil Main Compound (Thymol) Amphotericin B (10 mg/ml)
Mean ± SD* (mm) MIC MFC MFC/MIC ratio Mean ± SD* (mm) MIC MFC MFC/MIC ratio Mean ± SD* (mm)
Y1 C. albicans ATCC 10231 34.00 ± 1.00c 6.25 25 4 13.66 ± 0.57a 12.5 100 8 22.66 ± 1.15b
Y2 C. neoformans ATCC 14116 36.66 ± 1.15c 3.125 6.25 2 12 ± 1a 50 100 2 15.33 ± 0.57b
Y3 C. vaginalis (Clinical strain) 37.33 ± 1.15c 6.25 12.5 2 12.66 ± 0.57b 25 100 4 6.66 ± 0.57a
Y4 Candida sp. (Clinical strain) 37.33 ± 1.15b 6.25 12.5 2 11.66 ± 0.57a 25 100 4 12.33 ± 0.57a
M1 A. fumigatus ATCC 204305 88.66 ± 1.15c – – – 82.66 ± 2.31b – – – 15.00 ± 1.00a
M2 A. niger 87.33 ± 1.15c – – – 74.33 ± 0.57b – – – 6.00 ± 0.00a
* Inhibition zone around the discs impregnated with the essential oil (10 mg/disk) expressed as mean of three replicates (mm ± SD). SD: standard deviation.
a Minimal Inhibitory Concentration (mg/ml).
b Minimal Fungicidal Concentration (mg/ml).
c MBC/MIC ratio interpreted using the scheme of antimicrobial substances are considered as fungistatic agents when the ratio MFC/MIC > 4 and fungicidal agents when the ratio MFC/MIC ≤ 4 (Gatsing et al., 2009). The letters (a–c) indicate a significant difference between the inhibition zones of essential oil, thymol and amphotericin B against fungi according to the Duncan test (p < 0.05).
Table 4 Antioxidant activities of T. musilii essential oil against DPPH, ABTS, FRAP and β-carotene/linoleic acid scavenging tests as compared to ascorbic acid and BHT.
Essential oil and standards tested Test System
DPPH
IC50 (mg/mL)		 ABTS
IC50 (mg/mL)		 β- carotene
IC50 (mg/mL)		 FRAP
IC50 (mg/mL)
T. musilii Velen 0.049 ± 1 × 10−4b 5.6 × 10−4 ± 2 × 10−5 a 3.2 × 10−3 ± 5 × 10−4 a >1c
BHT 0.023 ± 3 × 10−4a 0.018 ± 4 × 10−4b 0.042 ± 3.5 × 10−3c 0.05 ± 3 × 10−3 a
Ascorbic Acid 0.022 ± 5 × 10−4 a 0.021 ± 1 × 10−3b 0.017 ± 1 × 10−3b 0.09 ± 7 × 10−3b

BHT: Butylated hydroxytoluene. The letters (a–c) indicate a significant difference between the different antioxidant methods according to the Duncan test (p < 0.05).

Table 5 Literature review of some Thymus species thymol-chemotype and microorganisms used for the antimicrobial activities.
Thymus species Origin Main Components Bacteria and Fungi tested Reference
T. vulgaris L. Yemen Thymol (51.34%), p-cymene (18.35%), ß-caryophyllene (4.26%). B. subtilis, S. aureus, S. epidermidis, P. aeruginosa, E. coli, Mycobacterium smegmatis, C. albicans and C. vaginalis. Al Maqtari et al., 2011
Romania Thymol (47.59%), γ-terpinene (30.90%) and p-cymene (8.41%). S. aureus ATCC 25923, P. aeruginosa ATCC 27853, S. Typhimurium ATCC 14028, E. coli ATCC 25922, K. pneumoniae ATCC 13882, E. faecalis ATCC 29212 and C. albicans ATCC 10231 Borugă et al., 2014
Balkan Peninsula Thymol (49.1%), p-Cymene (20%), carvacrol (3.5%), α-thujene (1.9%), α-pinene (1.2%), ß-mycrene (1.3%), trans-ß-ocimene (1.4%), γ-Terpinene (4.2%), borneol (1.7%), terpinene-4-ol (2%), ß-caryophyllene (3.7%), δ-cadinene (2.3%). C. albicans ATCC 10234, C. glabrata, C. krusei, C. tropicalis ATCC 750, P. aeruginosa, E. faecalis, S. sanguinis, S. salivarius, S. mutans, L. acidophilus, S. aureus. Nikolic et al., 2014
Italy Thymol (46.2–67.5%), caryophyllene oxide (2.2–7.3%), geranyl propanoate (0–2.2%), linalool (0.3–2.7%), trans-myrtanol (0–2.3%), citronellyl formate (0–2.5%), ethyl-2-octynoate (0–1.8%). S. aureus ATCC 25923, E. faecalis ATTC 29212, B. cereus ATCC 1177, B. subtilis ATCC 6633, E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. epidermidis ATCC 12228, K. pneumoniae ATCC 10031, S. typhi Ty2 ATCC 19430 and P. vulgaris ATCC 13315. Mancini et al., 2015
France Thymol (47.06%), p-cymene (20.07%), γ-terpinene (9.03%), linalool (5.00%), carvacrol (3.24%). C. albicans ATCC 18804, Cryptococcus neoformans 24067 (serotype D or var. neoformans), Aspergillus niger ATCC 16888. Satyal et al., 2016
Republic of Moldova Thymol (55.44 ± 0.62%), m-Cymene (11.88 ± 0.32%), γ-Terpinene (5.74 ± 0.20%), o-Cymen-5-ol (5.14 ± 0.19%), ß-caryophyllene (1.53 ± 0.07%), Terpinen-4-ol (1.04 ± 0.04%), 2-Carene (1.04 ± 0.04%). A. flavus MUCL 19006 Aprotosoaie et al., 2019
T. longicaulis C. Presl Italy Thymyl acetate (0–12.8%), t-Cadinol (0.3–9.2%), p-cymene (0.4–9.0%), ß-caryophyllene (2.2–5.7%), γ-terpinene (0.9–5.5%), Germacrene D (5.3%), thymol (6.4–9.3%), thymol methyl ether (0.8–5.5%), carvacrol (0–12.8%), Carvacryl acetate (0–13.6%). S. aureus ATTC 25923, S. faecalis ATTC 29212, B. subtilis ATCC 6633, B. cereus PCI 213, P. mirabilis ATCC 12453, E. coli ATCC 25922, S. typhi Ty2 ATCC 19430, P. aeruginosa (ATCC 27853). De Martino et al., 2009
Balkan Peninsula Thymol (46.3%), δ-3-Carene (1.6%), p-Cymene (9.4%), γ-terpinene (16.2%), linalool (1.4%), borneol (2.2%), thymyl methyl ether (11.4%), β-Caryophyllene (2.1%), carvacrol (1.4%). H. influenzae, N. meningitidis, S. aureus, S. pneumoniae, S. pyogenes, C. albicans Vladimir-Knežević et al., 2012
T. pulegioides L. Italy Thymol (21.8–26.3%), p-cymene (17.6–19.9%), linalool (4.7–5.6%), ß-caryophyllene (5.9–7.5%), thymol methyl ether (6.0–10.8%), carvacrol (3.1–4.7%). S. aureus ATTC 25923, S. faecalis ATTC 29212, B. subtilis ATCC 6633, B. cereus PCI 213, P. mirabilis ATCC 12453, E. coli ATCC 25922, S. typhi Ty2 ATCC 19,430 and P. aeruginosa ATCC 27853. De Martino et al., 2009
T. daenensis Celak. Iran Thymol (3.8–78.3%), ρ-cymene (2.7–11.6%), caryophyllene (2.1–5.6%), methyl carvacrol (2.9–4.9%), g-terpinene (2.5–12.9%), geraniol (0–3.4%), α-humulene (0–3.2%), carvacrol (2–15.2%), γ-terpinene (3.9–12.9%), aromadendrene (0–3.9%), carvacrol methyl ether (3.4–4.27%), δ-terpinene (0–4.3%). L. monocytogenes, S. aureus, S. iniae, E. coli, P. aeruginosa, K. pneumonia, H. pylori, A. niger, A. fumigatus, C. albicans and S. cerevisiae. Zarshenas and Krenn, 2015
Iran α-pinene (0.51%), 1,8-cineole (0.58%), γ-terpinene (5.74%), linalool (0.52%), thymol (74.32%), carvacrol (4.31%), trans-caryophyllene (3.56%), caryophyllene oxide (0.42%). C. albicans vaginal, E. coli O157:H7; B. cereus, L. monocytogenes and S. aureus. Pirbalouti et al., 2009 Pirbalouti et al., 2010 Pirbalouti et al., 2014
T. capitatus L. Algeria Thymol (51.22%), carvacrol (12.59%), γ-terpinene (10.3%), trans-13-octadecenoic acid (9.04%), linalool (2.29%), caryophyllene (2.01%), pentadecanoic acid (1.92%), α-terpinene (1.78%), ß-myrcene (1.49%), caryophyllene oxide (1.21%). E. coli, S. typhi, S. aureus, S. pneumoniae, Cladosporium herbarum, Alternaria infectoria, A. ochraceus, and Trichophyton sp. Goudjil et al., 2020
Tunisia Thymol (69.95–81.49%), α-cubebene (0–3.44%), β-ocimene (3.09–3.16%), carvacrol (0–2.56%), α-terpinene (2.25–3.83%). E. coli ATCC 8739, S. typhimurium NCTC 6017, S. aureus ATCC 29213, P. aeruginosa ATCC 27853, A. hydrophila, L. monocytogenes ATCC 7644, B. cereus, A. flavus, A. niger and C. albicans. Aouadhi et al., 2013
Thymol (89.06%), p-cimene (5.04%), γ-terpinene (3.19%). S. aureus CIP7625, L. monocytogenes Scott A 724, E. coli ATCC 10536, K. pneumoniae CIP8291, S. cerevisiae ATCC 4226, C. albicans IPA 200, M. ramamnianus ATCC 9314, A. westerdijkiae NRRL 3174. Mkaddem et al., 2010
T. cappadocicus Boiss. Turkey Thymol (70.82%), cymene (9.52%), g-terpinene (9.27%). A. hydrophila, E. coli, M. morganii, K. pneumoniae, P. mirabilis, P. aeruginosa, S. typhimurium, Y. enterocolitica, B. brevis, B. cereus, B. subtilis, L. monocytogenes, S. aureus, C. albicans and S. cerevisiae. Albayrak and Aksoy, 2012
T. striatus Balkan Peninsula Thymol (59.5%), γ-terpinene (11.6%), p-cymene (6.4%), carvacrol-methyl ether (5.9%), carvacrol (4.9%), α-terpinene (3.3%), E-caryophyllene (2.3%). A. alternata, A. niger, A. ochraceus, A. versicolor, A. flavus, A. terreus, C. cladosporioides, P. funiculosum, P. helianthi, T. viride, T. mentagrophytes, M. canis, and E. floccosum Couladis et al., 2004
T. algeriensis Boiss. and Reut Balkan Peninsula Thymol (36%), carvacrol (14%), α-pinene (1.1%), ß-mycrene (2.3%), p-cymene (6.3%), ß-bisabolene (4%0, α-terpinene (1.6%), γ-terpinene (4.8%), linalool (1.3%), camphor (1.1%), caryophyllene oxide (1%). C. albicans ATCC 10234, C. glabrata, C. krusei, C. tropicalis ATCC 750, P. aeruginosa, E. faecalis, S. sanguinis, S. salivarus, S. mutans, L. acidophilus, S. aureus. Nikolic et al., 2014
T. numidicus Poiret Algeria Thymol (40.40%), carvacrol (13.37%), thymol methyl ether (8.30%), β-myrcene (2.37%), p-cymene (7.18%), γ-terpinene (6.41%), linalool (4.06%), β-caryophyllene (2.48%), β-bisabolene (3.26%). S. aureus ATCC 25923, E. coli, P. aeruginosa ATCC 27853, C. albicans. Messara et al., 2016
T. zygis Spain α-pinene (36.8 ± 1.7–93.9 ± 4.8 mM), myrcene (32.7 ± 0.6–145.6 ± 6.4 mM), α-terpinene (14.6 ± 0.5–102.1 ± 5.5 mM), p-cymene (705.7 ± 22.9–1212.8 ± 13.0 mM), γ-terpinene (448.5 ± 22.4–1462.8 ± 38.2 mM), linalool (223.6 ± 2.8–386.8 ± 13.6 mM), terpinen-4-ol (8.9 ± 0.3–45.7 ± 0.3 mM), thymol (1923.2 ± 27.5–3636.2 ± 15.2 mM), carvacrol (34.3 ± 1.3–112.9 ± 2.5 mM), E-β-caryophyllene (24.1 ± 0.3–50.4 ± 1.0 mM) S. aureus ATCC 6538, E. coli ATCC 8739, P. aeruginosa ATCC 9027, C. albicans ATCC 10231. Cutillas et al., 2018
T. serpillum L. Balkan Peninsula Thymol (38.5%), carvacrol (4.7%), α-pinene (2%), camphene (2.4%), γ-terpinene (7.2%), linalool (2.4%), borneol (6%), thymol methyl ether (3.8%), thymol acetate (2.8%). C. albicans ATCC 10234, C. glabrata, C. krusei, C. tropicalis ATCC 750, P. aeruginosa, E. faecalis, S. sanguinis, S. salivarius, S. mutans, L. acidophilus, S. aureus. Nikolic et al., 2014
T. lanceolatus Algeria Thymol (69.61%), γ-terpinene (8.38%), p-cymene (5.07%), carvacrol (3.57%), α-terpinene (1.31%), linalool (1.01%), β-mycrene (1.72%), α-thujene (1.07%), α-pinene (0.73%), d-limonene (0.62%), β-pinene (0.43%). S. aureus ATCC 29213, S. epidermidis ATCC 14990, S. capitis ATCC 35661, S. pyogenes ATCC 12344, S. agalactiae ATCC 27956, Bacillus subtilis ATCC 6051, P. fluorescens ATCC 13525, S. typhimurium ATCC 14028, S. flexneri ATCC 700930, E. coli ATCC 25922, A. fumigatus ATCC 1022, Geotrichum candidum ATCC 12784, S. racemosum ATCC 14831, C. albicans (ATCC 90028). Khadir et al., 2016a,b
T. linearis Benth. India Thymol (54.9%), γ-terpinene (16.6%), p-cymene (5.2%), α-thymol methyl ether (3.2%), terpinene (2.6%), thymyl acetate (2.8%), β-bisabolene (2.3%), (E)-caryophyllene (2.0%), myrcene (1.8%), α-thujene (1.6%), carvacrol (1.5%), borneol (1.1%). S. aureus MRSA 33591, S. epidermidis MRSE 51625, S. aureus MRSA (BAA-44), S. aureus MTCC-96, S. epidermidis MTCC-435, E. faecalis MTCC-439, C. albicans ATCC 14053, C. tropicalis ATCC 2013180, C. glabrata ATCC-15126. Kumar et al., 2020
T. kotschyanus Iran α-pinene (5.49–12.72%), β-Myrcene (0.80–1.51%), α-terpinene (1.62–1.80%), p-cymene (0–21.35%), m-cymene (0–8.87%), 1,8-cineole (4.57–4.79%), γ-terpinene (4.00–8.01%), 4-terpineol (0–2.19%), α-terpineol (0.92–1.08%), thymol methyl ether (2.10–2.44%), carvacrol methyl ether (0–4.14%), thymol (29.96–47.48%), carvacrol (0.62–3.79%), β-bourbonene (0.15–3.30%), caryophyllene (1.27–2.92%). E. faecalis ATCC 29212, S. aureus ATCC 25952, S. aureus ATCC 33591, S. aureus ATCC 29213, S. sanguis PTCC 1449, E. aerogenes ATCC 13048, K. pneumoniae ATCC 700603, P. mirabilis ATCC 43071, E. coli O157:H7 Mobaiyen et al., 2017
T. eigii Turkey Thymol (24.77%), carvacrol (14.00%), p-cymene (10.91%), γ-terpinene (6.53%), borneol (6.48%), caryophyllene (3.92%), α-pinene (2.03%), α-thujene (2.34%), β-myrcene (2.68%), α-terpinene (2.28%), 1-octen-3-ol (2.94%), 17 trans-sabinene hydrate (2.19%), 4-terpineol (2.55%), (-)-caryophyllene oxide (2.01%). E. faecalis ATCC 29212, E. casseliflavus ATCC 700327, S. aureus ATCC 29213, S. aureus ATCC BAA 977, E. hormaechei ATCC 700323, K. pneumoniae ATCC 700603, P. aeruginosa ATCC 27853, E. coli ATCC 25922, C. parapsilosis ATCC 22019, C. albicans ATCC 14053. Ulukanli et al., 2018.
T. willdenowii Boiss & Reut Morocco Thymol (35.5–47.3%), p-cymene (13.9–23.8%), γ-terpinene (8.9–20.3%), carvacrol (3–5.6%), linalool (3–3.5%), camphor (0.9–3.7%), borneol (0.7–4.7%). E. coli ATCC 25922, P. mirabilis ATCC 35659, B. cereus ATCC 10876, C. albicans ATCC 10231, A. brasilliensis ATCC 16404. Ouknin et al., 2019
T. musilii Velen Saudi Arabia Thymol (67.69 ± 0.93%), thymyl acetate (12.99 ± 0.22%), p-cymene (4.61 ± 0.11%), Carvacrol (3.41 ± 0.10%), γ-terpinene (2.63 ± 0.07%). E. coli ATCC 35218, P. aeruginosa ATCC 27853, P. mirabilis ATCC 29245, K. pneumoniae ATCC 27736, S. aureus MDR, E. cloacae, C. albicans ATCC 10231, Cryptococcus neoformans ATCC 14116, C. vaginalis, Candida sp., A. fumigatus ATCC 204305 and A. niger. This study

Similar results were obtained with the yeast and fungi strains tested. Interestingly, high diameter of inhibition zone was recorded for the two clinical yeast strains: Candida sp. (37.33 ± 1.15 mm), and C. vaginalis (37.33 ± 1.15 mm). The MIC and MFC values were 6.25 mg/mL and 12.5 mg/mL, respectively for both strains. Using the MFC/MIC ratio scheme proposed by Gatsing et al. (2009), T. musilii seems to be more effective than thymol on the four tested yeast strains as they have the lowest ratio ranging from 2 to 4. It is important to highlight also that the tested (thymol/thymyl acetate) chemotype oil was very active on the two Aspergillus strains with mean inhibition zone about 88.66 ± 1.15 mm for A. fumigatus to 87.33 ± 1.15 mm for A. niger. All these data are summarized in Table 3.

Using the disc diffusion test, Vladimir-Knežević and colleagues (2012) reported similar results with T. longicaulis species (Chemotype thymol, 46.3%) tested against Haemophilus influenzae (IZ = 42 mm), Neisseria meningitidis (IZ = 53 mm), S. aureus (IZ = 35 mm), S. pneumoniae (IZ = 43 mm), and S. pyogens (IZ = 41 mm). Additionally, Bozin et al. (2006) reported that T. vulgaris essential oil (chemotype thymol) was active against a wide range of Gram-positive and Gram-negative bacteria, including the same species tested in our study. In fact, the highest growth inhibition zones were recorded for Micrococcus flavus (IZ = 48.2 mm), S. epidermidis (IZ = 48 mm), S. aureus (IZ = 26.2 mm), B. subtilis (IZ = 40.6 mm), E. coli (IZ = 29.4 mm), and P. aeruginosa (IZ = 12 mm).

Previous reports have noticed the anti-C. albicans activity of different species belonging to the Thymus genus. In fact, Pinto et al. (2006) reported a significant activity of T. pulegioides oil (thymol 26%/carvacrol 21% chemotype) against Candida, Aspergillus and dermatophyte species explained by the alteration in the cytoplasmic membrane and ergosterol content.

In addition, Pirbalouti et al. (2009) founded that T. daenensis Celak. essential oil effectively inhibits the growth of vaginal C. albicans strains at high concentration (50–55 µl). The same oil was active against E. coli O157:H7, B. cereus, L. monocytogenes, and C. albicans with a diameter of growth inhibition zone and MIC values about (7 mm/>10 mg·mL−1, 25 mm/0.625 mg·mL−1, 16 mm/2.5 mg·mL−1, and 19 mm/<0.039 mg·mL−1 respectively (Pirbalouti et al., 2010). Thymol-rich chemotype of T. daenensis Celak essential oil can inhibit the growth of S. aureus isolated from milk with MIC and MBC values about 62 µg/mL and 630 µg/mL, respectively (Pirbalouti et al., 2014). Couladis et al. (2004) reported the high activity of T. striatus (Chemotype thymol, 59.5%) against a large collection of Aspegillus, Cladosporium, Penicillium, Trichoderma, Tricophyton, Microsporum, and Epidermophyton strains with MICs values ranging from 0.5 to 2 µl. In 2014, Nikolic and colleagues reported that T. serpyllum (Thymol, 38.5%) was active against four Candida species (C. albicans, C. tropicalis, C. glabrata, and C. krusei) with MICs values ranging from 01. to 0.2 µl. More recently, Satyal et al. (2016) demonstrated that T. vulgaris essential oils inhibit the growth of C. neoformans var. neoformans, and C. albicans with MICs values about (313/156) µg·mL−1, and (1250/625) µg·mL−1, respectively for linalool and geraniol chemotypes.

A brief literature review summarized the antimicrobial activity of thymol against a large collection of bacteria, yeast and fungi (Table 6). High activity of the Thymus plant species can be associated to the dominance of thymol with different percentage. In fact, this molecule is known to exhibit antimicrobial, antioxidant, immunological, anti-inflammatory, anticancer, and cardiovascular protection properties (Nagoor et al., 2017; D’agostino et al., 2019). This terpenoid molecule inhibits the hyphal production in Fusarium graminearum (Gao et al., 2016), decreases the membrane permeability leading to the loss of cytoplasmic membrane integrity and loss of electrolytes in C. albicans species by binding to ergosterol (De Castro et al., 2015), and inhibits the telomerase activity in S. cerevisiae species (Darvishi et al., 2013). It has been demonstrated that thymol can kill Methicillin-resistant S. aureus strain by increasing the formation of reactive oxygen species (Li et al., 2014).

Table 6 Literature review of the antimicrobial activity of the main component identified in T. musilii Velen essential oil: thymol.
Strains Tested MIC MBC/MFC Reference
Bacillus cereus 327.581 ppm – Falcone et al. 2005
Bacillus subtilis 422.332 ppm –
Bacillus licheniformis 422.811 ppm –
Lactobacillus curvatus 723.45 ppm –
Lactobacillus plantarum 941.01 ppm –
Candida lusitaniae 307.901 ppm –
Pichia subpelliculosa 422.781 ppm –
Saccharomyces cerevisiae 337.761 ppm –
Staphylococcus aureus ATCC 68380 0.31 mg/mL – Tombetta et al., 2005
Escherichia coli ATCC 15221 5.00 mg/mL –
Candida albicans ATCC 10231 0.16 µl/mL 0.32 µl/mL Pinto et al., 2006
Candida guilliermondii MAT23 0.16 µl/mL 0.16 µl/mL
Candida parapsilosis ATCC 90018 0.32 µl/mL 0.32 µl/mL
Candida krusei ATCC 6258 0.16 µl/mL 0.32 µl/mL
Candida tropicalis ATCC 13803 0.16 µl/mL 0.32 µl/mL
Candida albicans 0.16 µl/mL 0.32 µl/mL
Candida tropicalis 0.16 µl/mL 0.32 µl/mL
Candida glabrata (0.16–0.32) µl/mL 0.32 µl/mL
Candida krusei 0.16 µl/mL 0.32 µl/mL
Trichophyton rubrum 0.16 µl/mL 0.16 µl/mL
Trichophyton mentagrophyte 0.16 µl/mL 0.32 µl/mL
Epidermophyton floccosum 0.16 µl/mL 0.16 µl/mL
Microsporum gypseum 0.16 µl/mL 0.32 µl/mL
Microsporum canis 0.08 µl/mL 0.16 µl/mL
Aspergillus niger ATCC 16404 0.16 µl/mL 0.64 µl/mL
Aspergillus niger CECT 2574 0.16 µl/mL 0.64 µl/mL
Aspergillus fumigatus CECT 2071 0.16 µl/mL 0.64 µl/mL
Aspergillus fumigatus ATCC 46645 0.16 µl/mL 0.64 µl/mL
Aspergillus flavus 0.32 µl/mL 0.64 µl/mL
Aspergillus niger 0.16 µl/mL 0.64 µl/mL
Aspergillus fumigatus 0.16 µl/mL 0.64 µl/mL
Salmonella typhimurium SGI1 2.5 mM – Palaniappan and Holley, 2010
Escherichia coli N00-666 2.5 mM –
Staphylococcus aureus blaZ+ 2.5 mM –
Streptococcus pyogenes ermB+ 0.31 mM –
Escherichia coli O157:H7 500–1000 µg/mL 1000–2000 µg/mL Rivas et al., 2010
Escherichia coli O26 1000 µg/mL 1000 µg/mL
Escherichia coli O111 1000 µg/mL 2000 µg/mL
Escherichia coli O103 1000 µg/mL 1000 µg/mL
Escherichia coli O145 1000 µg/mL >2000 µg/mL
Salmonella Typhimurium 2000 µg/mL 2000 µg/mL
Listeria monocytogenes 1000 µg/mL 1000 µg/mL
Hafnia alvei 500 µg/mL 500 µg/mL
Staphylococcus aureus 500 µg/mL 500 µg/mL
Lactobacillus sakei 1000 µg/mL 2000 µg/mL
Pseudomonas putida 1000 µg/mL 2000 µg/mL
Bacillus thermosphacta – 250 µg/mL
Streptococcus mutans MTCC 890 125 µg/mL – Mathela et al., 2010
Staphylococcus aureus MTCC 96 62.5 µg/mL –
Bacillus subtilis MTCC 121 125 µg/mL –
Staphylococcus epidermidis MTCC 435 125 µg/mL –
Escherichia coli MTCC 723 250 µg/mL –
Escherichia coli < 0.019–0.039 mg/mL – Pirbalouti et al., 2011
Pseudomonas aeruginosa <0.019–0.039 mg/mL –
Staphylococcus aureus < 0.019–156 mg/mL
Bacillus cereus < 0.019–0.156 mg/mL
Micrococcus luteus 1250 µg/mL Hernández-Hernández et al., 2014
Phytophthora infestans 400.26 µl/l – Ben and Hamada, 2014
Phytophthora ultimum 263 µl/l –
Botrytis cinerea >600 µl/l –
Rhizoctonia solani 64.56 µl/l –
Aspergillus niger 100 mg/mL – Abbaszadeh et al., 2014
Aspergillus fumigatus 150 mg/mL –
Aspergillus flavus 100 mg/mL –
Aspergillus ochraceus 100 mg/mL –
Alternaria alternata 100 mg/mL –
Botrytis cinerea 100 mg/mL –
Cladosporium spp. 100 mg/mL –
Penicillium citrinum 100 mg/mL –
Penicillium chrysogenum 100 mg/mL –
Fusarium oxysporum 100 mg/mL –
Rhizoctonia oryzae 100 mg/mL –
Escherichia coli 187.5 μg/mL 375 μg/mL Du et al., 2015
Clostridium perfringens 375 μg/mL 750 μg/mL
Salmonella Typhimurium 375 μg/mL 750 μg/mL
Salmonella Enteritidis 750 μg/mL 1500 μg/mL
Salmonella Pullorum 375 μg/mL 750 μg/mL
Lactobacillus acidophilus 1500 μg/mL 3000 μg/mL
Lactobacillus reuteri 1500 μg/mL 3000 μg/mL
Lactobacillus salivarius 1500 μg/mL 3000 μg/mL
Pythium insidiosum 160–320 µg/mL Jesus et al., 2015
Helicobacter pylori 0.043 ± 0.024 µl/mL Falsafi et al., 2015
Mycobacterium tubercolosis 0.75 µg/mL Andrade-Ochoa et al., 2015
Mycobacterium bovis 2.02 µg/mL
Candida albicans 39 µg/mL – De Castro et al., 2015
Candida krusei 39 µg/mL –
Candida tropicalis 78 µg/mL –
Aspergillus flavus CGMCC 32890 80 μg/mL – Shen et al., 2016
Bacillus cereus 0.007 mg/mL – Guimarães et al., 2019
Salmonella Typhimurium 0.003 mg/mL 0.12 mg/mL
Escherichia coli 0.007 mg/mL 0.12 mg/mL
Staphylococcus aureus 0.007 mg/mL 0.12 mg/mL
Cronobacter sakazakii lv27 0.05% – Berthold-Pluta et al., 2019
Cronobacter malonaticus lv31 0.05% –
Cronobacter muytjensii s50 0.05% –
Cronobacter turicensis lv53 0.05% –
Cronobacter condimenti s37 0.05% –
Escherichia coli ATCC 35218 3.125 mg/mL 6.25 mg/mL This study
Pseudomonas aeruginosa ATCC 27853 12.5 mg/mL 50 mg/mL
Proteus mirabilis ATCC 29245 3.125 mg/mL 6.25 mg/mL
Klebsiella pneumoniae ATCC 27736 3.125 mg/mL 6.25 mg/mL
Staphylococcus aureus MDR 0.78 mg/mL 1.56 mg/mL
Enterobacter cloacae 0.39 mg/mL 0.78 mg/mL
Candida albicans ATCC 10231 12.5 mg/mL 100 mg/mL
Cryptococcus neoformans ATCC 14116 50 mg/mL 100 mg/mL
Candida vaginalis (Clinical strain) 25 mg/mL 100 mg/mL
Candida sp. (Clinical strain) 25 mg/mL 100 mg/mL

3.4

3.4 Molecular docking analysis

In order to correlate the binding of isolated Thymus bioactive molecules with its biological activities, the main compounds were docked to the active site of TyrRS, GLMS and Gyrase, respectively to demonstrate their potential inhibition against S. aureus and E. coli pathogens. The binding affinities of top-rated pose of different ligand-receptor complex (Table 4) revealed that among all tested bioactive compounds, the best binding affinity was found with β-caryophyllene-enzymes with values of −5.4 kcal/mol, −6.8 kcal/mol and −6.2 kcal/mol, respectively for β-caryophyllene-TyrRS, β-caryophyllene-GLMS and β-caryophyllene-Gyrase, suggesting its highest binding efficiency and therefore was selected for further investigation.

To get insight into the mechanism of TyrRS, GLMS and Gyrase inhibition by β-caryophyllene, we elucidate their molecular interaction mode in the active site residues of receptors. The outcomes compiled in Table 8 showed that β-caryophyllene-TyrRS complex was mainly stabilized by Alkyl interactions with Met77, Ile78 and Leu128, Pi-Alkyl interactions with Leu128 and Leu173 and Pi-sigma interactions with Phe 136 residues. Alkyl and Pi-Alkyl interactions were also formed between β-caryophyllene and GLMS residues of Ile7, Ala38 and Pro166. However, the amino acid residues involved in stabilizing the complex caryophyllene-Gyrase are Ala1374 (Pi-Alkyl), Leu1448 (Pi-Alkyl and Alkyl) and Tyr1451 (Pi-Alkyl). As shown, Phe136 and Leu173 of TyrRS from S. aureus, Ala38 from Gyrase in S. aureus and Leu1448 from GLMS in E. coli formed stronger Pi-Sigma, Alkyl and Pi-Alkyl interactions with the natural bioactive compounds (Tables 7 and 8) and therefore, could possibly inhibit the activity of enzyme resulting in the neutralization of their virulence.

Table 7 Binding affinities of top-rated pose of ligand-receptor complex. Binding affinity measured in kcal/mol.
Compounds 1XFF 1JIJ 2XCT
α-Thujene −4.5 −5.5 −4.9
α-Pinene −4.4 −5.6 −4.5
β-Myrcene −3.3 −5.1 −4
α-Terpinene −4.5 −6 −4.9
p-Cymene −4.3 −5.7 −5
1,8-Cineole −4.8 −5.1 −4.8
γ-Terpinene −4.5 −6 −4.9
α-Terpinolene −4.4 −5.9 −5.3
Borneol −4.9 −5.4 −4.8
Terpinen-4-ol −4.6 −5.8 −5
α-Terpineol −4.9 −6.1 −5.1
2-Isopropyl-5-methylanisole −4.4 −4.9 −5.1
Thymol −4.5 −5.9 −5.4
Carvacrol −5.2 −6.3 −5.4
Thymyl acetate −5.1 −6.1 −4.8
Carvacryl acetate −5 −6.1 −5.6
β-Caryophyllene −5.4 −6.8 −6.2

1XFF: glucosamine 6-phosphate synthase (GLMS) from E. coli, 1JIJ: tyrosyl-tRNA synthetase TyrRS from S. aureus, 2XCT: Gyrase from S. aureus.

Table 8 Interacting active site residues of receptors with natural bio-compounds.
2D interactions, Receptor Ligand Interactions, Distance in Angstroms 3D interaction Receptor–Ligand
Receptor – Ligand: 1JIJ – β-Caryophyllene Receptor – Ligand: 1JIJ – β-Caryophyllene
(MET77) S---S (Ligand) Alkyl interaction: 4.75 A°; (ILE78) C---C (Ligand) Alkyl interaction: 4.55 A°; (ILE78) C---C (Ligand) Alkyl interaction: 5.13 A°; (LEU128) C---C (Ligand) Alkyl interaction: 4.63 A°; (LEU128) C---C (Ligand) Pi-Alkyl interaction 4.91A°;(PHE136) phenyl ring---C (Ligand) Pi-sigma interaction: 3.71 A°; (LEU173) C---C (Ligand) Alkyl interaction: 3.85 A°; (LEU173) C---C (Ligand) Pi-Alkyl interaction: 4.69 A°; (LEU173) C---C (Ligand) Pi-Alkyl interaction: 4.76 A°.
Receptor – Ligand: 1XFF – β-Caryophyllene Receptor – Ligand: 1XFF – β-Caryophyllene
(ILE7) CC---CH (Ligand): 5.37 A°; (ALA38) C---Phenyl ring (Ligand) Pi-Alkyl interaction: 3.64 A°; (ALA38) C---Alkyl ring (Ligand) Pi-Alkyl interaction: 4.40 A°; (ALA38) C---C (Ligand) Alkyl interaction: 4.07 A°; (PRO166) phenyl ring---Alkyl ring (Ligand) Pi-Alkyl interaction: 4.76 A°.
Receptor – Ligand: 2XCT – β-Caryophyllene Receptor – Ligand: 2XCT – β-Caryophyllene
(ALA1374)C---phenyl ring (Ligand) – Pi-alkyl interaction: 4.74 A°; (LEU1448) C---phenyl ring (Ligand)–Pi-alkyl interaction: 4.84 A°; (LEU1448) C---alkyl ring (Ligand)–Alkyl interaction: 4.58 A°; (LEU1448) C---C (Ligand)–Alkyl interaction: 4.11 A°; (TYR1451) phenyl ring---C (Ligand) Pi-alkyl interaction: 4.71 A°; (TYR1451) phenyl ring---phenyl ring (Ligand) Pi-alkyl interaction 4.82 A°.

4

4 Conclusion

In the present study, the antioxidant and the antimicrobial assays of the essential oil from T. musilii were evaluated. The obtained findings suggest that this cultivated species can constitute a good source of antioxidant, antibacterial and antifungal compounds, namely, thymol. Nevertheless, these biological results deserve further deep in vivo studies in order to use this plant as possible bio-source in food and pharmaceutical industries. Molecular docking results together with the findings of in-vitro antimicrobial potency suggest that T. musilii essential oil is a potent inhibitor of S. aureus and E. coli and subsequently lead to novel discovery of plant-based therapeutic products.

Funding

This research has been funded by Scientific Research Deanship at University of Ha'il - Saudi Arabia through project number 160991.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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