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

mohd adnan

Thymus munbyanus Boiss. & Reut., is a small shrub endemic to Morocco and Algeria, and it is commonly used to treat several diseases, including digestive, circulatory, genital, skin, urinary, nervous and respiratory diseases. The extracts of this thyme are rich in a wide variety of phenolic compounds such as polyphenols and volatile phenols and exhibit numerous biological activities. The present review summarizes the literature investigations reported on Thymus munbyanus concerning various pharmacological and biological properties as well as phytochemical aspects. This species revealed a richness in phenolic compounds in its volatile oils, including thymol and carvacrol, as well as in its non-volatile extracts including phenolic acids phenolic acids (rosmarinic acid, caffeic acid, salvianolic acid, ferulic acid, etc.), flavonoids (luteolin, gallocatechin, quercetin, isorhamnetin, etc.). Moreover, powerful antioxidant and antimicrobial properties were reported for this Thymus species,...

Thymus vulgaris generally known as thyme, German thyme, garden thyme or just thyme, is a species of flowering plant belonging to mint family Lamiaceae, which is native to southern Europe from the western Mediterranean to southern Italy. It is estimated there are more than 2,50,000 flower plant species. Studying medicinal plants helps to be aware of plant toxicity and protect human and animals from natural poisons. The aim of this study was to overcome about its phytochemical and pharmacological profiles based on its active chemical constituents. Thymus vulgaris oil is a combination of monoterpenes and those will acts as antioxidative, anti-dysmenorrhea, hepatoprotective effect, antimicrobial, antitussive, antispasmodic, and antibacterial activities. It also shows anti-inflammatory effects, anti-fungal effect, antiadhesion activity, larvicidal effect.

Members of the Lamiaceae family are considered chief sources of bioactive therapeutic agents. They are important ornamental, medicinal, and aromatic plants, many of which are used in traditional and modern medicine and in the food, cosmetic, and pharmaceutical industries. In North Africa, on the Mediterranean side, there is the following particularly interesting Lamiaceous species: Thymus hirtus Willd. sp. Algeriensis Boiss. Et Reut. The populations of this endemic plant are distributed from the subhumid to the lower arid zone and are mainly employed as ethnomedicinal remedies in the following Maghreb countries: Algeria, Libya, Morocco, and Tunisia. In fact, they have been applied as antimicrobial agents, antispasmodics, astringents, expectorants, and preservatives for several food products. The species is commonly consumed as a tea or infusion and is used against hypercholesterolemia, diabetes, respiratory ailments, heart disease, and food poisoning. These medicinal uses are relate...

Journal Pre-proofs Original article Thymus musilii Velen. as a promising source of potent bioactive compounds with its pharmacological properties: In vitro and in silico analysis Khalil Mseddi, Fathi Alimi, Emira Noumi, Vajid N Veettil, Sumukh Deshpande, Mohd Adnan, Assia Hamdi, Salem Elkahoui, Alghamdi Ahmed, Adel Kadri, Mitesh Patel, Mejdi Snoussi PII: DOI: Reference: S1878-5352(20)30238-0 https://doi.org/10.1016/j.arabjc.2020.06.032 ARABJC 2725 To appear in: Arabian Journal of Chemistry Received Date: Revised Date: Accepted Date: 1 May 2020 22 June 2020 23 June 2020 Please cite this article as: K. Mseddi, F. Alimi, E. Noumi, V.N. Veettil, S. Deshpande, M. Adnan, A. Hamdi, S. Elkahoui, A. Ahmed, A. Kadri, M. Patel, M. Snoussi, Thymus musilii Velen. as a promising source of potent bioactive compounds with its pharmacological properties: In vitro and in silico analysis, Arabian Journal of Chemistry (2020), doi: https://doi.org/10.1016/j.arabjc.2020.06.032 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Published by Elsevier B.V. on behalf of King Saud University. 1 Thymus musilii Velen. as a promising source of potent bioactive compounds 2 with its pharmacological properties: In vitro and in silico analysis 3 4 Khalil Mseddia, b, Fathi Alimic,d, Emira Noumia,e, Vajid N Veettila, Sumukh Deshpandef, 5 Mohd Adnana, Assia Hamdig, Salem Elkahouia, h, Alghamdi Ahmeda, Adel Kadrii, Mitesh 6 Patelj, Mejdi Snoussia, k* 7 aDepartment 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. cDepartment of Chemistry, College of Science, University of Hail, P.O. Box 2440, 81451 Hail, Saudi Arabia. dNatural Water Treatment Laboratory, Water Researches and Technologies Centre of Borj-Cedria (CERTE), Carthage University, BP 273, 8020 Soliman, Tunisia. eLaboratory 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. 8 fCentral 9 Cardiff, CF14 4XN, Wales, United Kingdom. Biotechnology Services, College of Biomedical and Life Sciences, Cardiff University, 10 gLaboratoire 11 Faculté de Pharmacie, 5000 Monastir, Tunisia. hLaboratory de Développement Chimique Galénique et Pharmacologique des Médicaments, of Bioactive Substances, Center of Biotechnology, Ecopark of Borj-Cedria, BP 901, 2050, Hammam-Lif, Tunisia. iFaculty of Science and Arts in Baljurashi, Albaha University, P.O. Box 1988, Albaha, Saudi Arabia 12 jBapalal 13 Gujarat University, Surat, Gujarat, India. Vaidya Botanical Research Centre, Department of Biosciences, Veer Narmad South kLaboratory of Genetics, Biodiversity and Valorization of Bio-resources, Higher Institute of Biotechnology of Monastir, University of Monastir. 14 * Corresponding author (E-mail: snmejdi@yahoo.fr) 15 1 16 17 Abstract 18 For the first time, we reported the phytochemical composition of the volatile oil from Thymus 19 musiliii Velen (T. musiliii). The antioxidant and antimicrobial activities against various food-borne 20 and clinical pathogenic microorganisms were also tested. The thyme oil was particularly rich in 21 thymol (67.697±0.938 %), and thymyl acetate (12.993±0.221%). The strongest antioxidant 22 activity of the essential oil was registered with the tests: ABTS (IC50= 5.6 x10-4 mg/mL) and β- 23 carotene/linoleic acid (IC50= 3.2x10-3 mg/mL). This thymol-chemotype oil was active against all 24 microorganisms tested with an inhibition growth zone ranging from 21.33±1.52 mm for Proteus 25 mirabilis (P. mirabilis) to 37.33±1.15 mm for Candida vaginalis (C. vaginalis) strain. Overall, the 26 tested oil exhibited bactericidal and fungicidal activities and only a small quantity of the tested 27 essential oil was found to be sufficient for inhibiting the growth of the tested microorganisms. 28 Furthermore, molecular docking results implies that, among the bioactive compounds, β- 29 caryophyllene interacted strongly with the active site residues of TyrRS, GLMS and Gyrase 30 enzymes and consequently support our in vitro results with the highest inhibition potential of this 31 essential oil against tested pathogens, especially 32 Escherichia coli (E. coli). Our results suggested that essential oil of T. musiliii exhibited strong 33 biological activities with a promising source of various natural compounds. Staphylococcus aureus (S. aureus) and 34 Keywords: Thymus musiliii Velen.; GC-MS; antioxidant; antibacterial; antifungal; molecular 35 docking. 36 37 38 2 39 40 1. Introduction 41 The use of medicinal plants as a source of therapy against various disorders have been 42 practiced in Saudi Arabia since ages and many practices reported in the Prophetic Medicine are 43 currently used in folk medicine in the Arabian Peninsula (Al-Essa et al., 1998). Among this 44 category of plants, there are cultivated plants and others are spontaneous ones. These aromatic 45 plants are grown as needed for their aerial parts (flowers, seeds, leaves, stems, bark) or their 46 underground parts (bulbs, roots). Studies in the past have reported the presence of valuable 47 medicinal plants from the different regions of Saudi Arabia (El-Tawil, 1983). However, the 48 information of the indigenous medicinal plants of Saudi Arabia is scattered in a disorganized 49 manner (Al-Asmari et al., 2014). Scientific studies have proven that these plants, including garlic, 50 pomegranate, black seeds, costus, miswak, henna, ferns, Eucalyptus, ginger, and fenugreek are 51 effective for treating human diseases (Adnan 2019, Adnan et. al. 2020a, Adnan et. al. 2020b, 52 Noumi et al., 2017). These species are exploited in human food, traditional medicine as well as for 53 industrial purposes (agro-food, perfumery, cosmetics, pharmaceutical, etc.). 54 The mint family (Lamiaceae) is one of the largest and most distinctive families of flowering 55 plants, with about 220 genera and almost 4000 species worldwide (Pirbalouti et al., 2015). This 56 family has an almost cosmopolitan distribution. These plants are frequently aromatic in all parts 57 and include many widely used culinary herbs, such as thyme. The genus Thymus L. belongs to the 58 Nepetoideae subfamily of Lamiaceae family is a well-known aromatic herb and consists of about 59 330 species of herbaceous perennials and small shrubs in the world (Nickavar et al., 2005; Salehi 60 et al., 2019). 3 61 The Mediterranean region can be described as the center of the genus (Jamzad, 2010; 62 Morales, 2002; Cronquist, 1988). Thymus plants also includes many aromatic perennial and 63 herbaceous plant that are cultivated in frequency due to their wide use in the food, cosmetic, and 64 pharmaceutical industries (Nabavi et al., 2015). The genus Thymus is a taxonomically complex 65 group of aromatic plants, traditionally used for medicinal purposes because of their antiseptic, 66 antispasmodic and antitussive properties (Pina-Vaz et al., 2004, Nabavi et al., 2015). Previous 67 chemical investigation on Thymus species have shown the presence of aromatic terpenes and 68 terpenoids, flavonoids, and phenolic acid (Miri et al., 2002; Miguel et al., 2004; Ebrahimi et al., 69 2008; Tohidi et al., 2017). Thymol and carvacrol are the main phenolic compound of thyme oil. 70 The major non-phenolic compounds were linalool and p-cymene (Piccaglia and Marotti, 1991). 71 Recent studies have shown that Thymus species have antibacterial, antifungal, and 72 antioxidant activities (Bassam et al., 2004; Rahimmalek et al., 2009; Jordan et al., 2009). 73 Gedikoğlu, et al. (2019) reported that the essential oil of thyme showed antimicrobial activity 74 against Bacillus cereus NRRL (B3711), Staphylococcus aureus (ATCC 9144), Staphylococcus 75 epidermidis (ATCC 12228), Escherichia coli ATCC (25922), Salmonella enteritidis (ATCC 76 13076) and Salmonella typhimurium (ATCC 14028). The anti-bacterial characteristic of Thymus 77 spp. is due to the occurrence of thymol in this genus. This substance can be used as a disinfectant. 78 In Saudi Arabia, at least three species of Thymus (endemic and introduced) were identified: 79 T. bovei Benth., T. decussatus Benth. and T. musilii Velen. In addition, T. vulgaris was largely 80 cultivated in many regions of the kingdom. This species, T. musilii Velen. belongs to 81 division: Tracheophyta, subdivision: Spermatophytina, Class: Magnoliopsida, Superorder: 82 Asteranae, Order: Lamiales, Family: Lamiaceae Lindl., and Genus: Thymus L. It is distributed 83 mainly in Iraq, Palestine, and Saudi Arabia (World Checklist of Selected Plant Families, 2010). 4 84 Growing to 30–70 cm tall by 40-60 cm wide, it is a bushy, woody-based evergreen 85 subshrub with highly aromatic, green leaves and clusters of white flowers in early summer. 86 Preferred the dry slopes, rocks and maquis, it was always found on clay or limestone soils. It has 87 sessile leaves varying from elliptic to linear or diamond-shaped towards the apex. The flowers 88 have a tube-like calyx and tubular corolla with a three lobed lower lip, and are united in spikes at 89 the top of the branches (Figure 1). The roots are robust, and the fruit consists of a smooth, dark 90 colored nutlet. In Bedouin population of Saudi Arabia, leaves and flowering tops of T. musilii were 91 used as a garnish or added as a flavoring in cooking variety of foods, as well as in preparing 92 infusion tea. An aromatic tea is made from the fresh or dried leaves. The leaves can be used either 93 fresh or dried. If the leaves are to be dried, the plants should be harvested in early and late summer 94 just before the flowers open and the leaves should be dried quickly. 95 The in vitro antimicrobial and antioxidant activities of the essential oil and extract of T. 96 vulgaris have recently been reported. Al-Asmari et al. (2017) have studied the essential oil 97 composition, whereas, Alharbi (2017) reported that the whole plant was used in traditional 98 medicine to treat abdominal pain, and as anti-helminthic and carminative effects. Belonging this 99 genus, T. musilii is a very interesting medicinal plant closely distributed on Arabian Peninsula, 100 Iraq and Jordan landscapes (Batanouny and Sheikh, 1972; Govaerts, 2003). In the north of Saudi 101 Arabia, it is locally used as an antiseptic traditional drug. This species has also been used for curing 102 many bacterial and fungal diseases in traditional medicine in Saudi Arabia (survey, data not 103 shown). In fact, it used by local Saudi population to cure many ailments. Leaves are used in treating 104 respiratory diseases and the flowering tops are used as anti-helminthic, antiseptic and 105 antispasmodic drug. However, antimicrobial and antioxidant properties of T. musilii Velen seem 106 not to have been reported before. 5 107 To the best of our knowledge, this study is the first report on the biological properties of T. 108 musilii Velen. The aim of this work was to investigate the chemical composition of the volatile oil 109 obtained from the aerial parts of T. musilii cultivated under greenhouse conditions in Al-Gaad, 110 Hail (Saudi Arabia) by using GC-MS technique. Additionally, the antioxidant and antimicrobial 111 activities of the oil were assessed. To reach this objective, molecular docking studies of the 112 bioactive compounds were also performed against tyrosyl-tRNA synthetase TyrRS from S. aureus, 113 glucosamine 6-phosphate synthase (GLMS) from E. coli and Gyrase from S. aureus enzymes to 114 better understand their mechanism of action. 115 2. Material and Methods 116 2.1. Plant material sampling and essential oil extraction 117 The plant used in this study were collected in October 2019 from a nursery belonging to the 118 Ministry of Agriculture in the region of Hail (Al-Gaad, Ha'il, Saudi Arabia). Dr. Ahmed Alghamdi, 119 from the Department of Biology, Faculty of Science, University of Hail, Saudi Arabia identified 120 the plant at the species level. A voucher specimen (AN 001) was deposited in the Department of 121 Biology, University of Hail, Saudi Arabia. The volatile oil was collected using a clevenger-type 122 apparatus after 3 hours of hydro-distillation using 100 g from the aerial air-dried organs (flowering 123 stage). The obtained oil was dried using anhydrous sodium sulfate and stored until use at -20°C. 124 The yield of extraction was calculated after three running cycle and expressed according to the dry 125 weight. 126 2.2. Characterization of the volatile oil 127 A Hewlett–Packard 6890 chromatograph equipped with a flame ionization detector (FID) and an 128 electronic pressure control injector was used to study the chemical composition of the obtained 129 volatile oil from T. musilii aerial parts. A gas chromatography apparatus coupled to mass 6 130 spectrometry (GC–MS) on a gas chromatograph HP 7890 (II) and HP 5975 mass spectrometer 131 (Agilent Technologies, Palo Alto, CA, USA) with an electron impact ionization of 70 eV was used. 132 An HP-5MS capillary column (Agilent Technologies, Hewlett-Packard, CA, USA; 30 m×0.25 133 mm), with 0.25 m film thickness was used. Temperature was fixed to rise from 40°C to 280°C at 134 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, 135 scan time and mass range of 1s and 40–300 m/z, respectively. The identification of the bioactive 136 components in T. musilii volatile oil was based on the calculated retention index (RI) relative to 137 (C8–C22) n-alkanes and in comparison, with authentic compounds. Further identification of 138 compounds was made by matching their recorded mass spectra with those stored in the Wiley/NBS 139 mass spectral library of the GC–MS data system and other published mass spectra (Adams, 2007) 140 and data expressed as relative percentage of the total peak area as previously described by Essid 141 et al. (2015) and Salem et al. (2018). 142 2.3. Antioxidant assays 143 2.3.1. DPPH radical–scavenging activity 144 The free radical-scavenging activity of the tested essential oil was measured using the 145 protocol described by Chakraborty and Paulraj (2010) and Adnan et al. 2018. The ability to 146 scavenge the DPPH radical was calculated using the following equation (Eq.1): 147 Eq (1): DPPH scavenging activity (%) = (A0 –A1) /A0 ×100 148 Where, 149 A0 is the absorbance of the control and A1 is the absorbance of the sample. 150 151 The antioxidant activity was expressed as IC50 (mg/mL) which represented the extract 152 concentrations scavenging 50% of DPPH radicals (Nishaa et al., 2012). 153 2.3.2. ABTS radical scavenging activity assay 7 154 The radical scavenging activity against ABTS radical cations was measured using the 155 method of Chakraborty and Paulraj (2010). The inhibition percentage of ABTS radical was 156 calculated using the following equation (Eq.2): 157 Eq (2): ABTS scavenging activity (%) = (A0 –A1) /A0 ×100 158 Where, 159 A0 is the absorbance of the control and A1 is the absorbance of the sample. 160 The antiradical activity was expressed as IC50 (mg/mL) which represented the extract 161 concentrations scavenging 50% of ABTS radicals (Nishaa et al., 2012). A lower IC50 value 162 represents a stronger ABTS scavenging capacity. 163 2.3.3. Reducing Power Capability Assay 164 The reducing power was determined using the method of Bi et al. (2013). The extract concentration 165 providing 0.5 of absorbance (IC50) was calculated from the graph of absorbance at 700 nm against 166 sample concentration (Barros et al., 2008). Ascorbic acid was used as a standard. 167 2.3.4. β-carotene/linoleic acid method 168 The β-carotene method was carried out according to Ikram et al. (2009). Antioxidant activity 169 (inhibition percentage, PI %) was evaluated using the following equation (Eq 3): 170 (Eq 3): P I% = A β−carotene T120/A β−carotene t0 × 100 (Miraliakbari and Shahidi, 2008), 171 Where, 172 A 173 standard and control measured before and after incubation for 2h, respectively. All tests were 174 performed in triplicate and ascorbic acid (standard) was used for comparison. 175 2.4. Screening of antimicrobial activities β-carotene t0 and A β-carotene T120 refer to the corresponding absorbance values of the test sample, 8 176 The antimicrobial activity of the obtained essential oil was tested against four type strains 177 namely E. coli ATCC 35218, P. aeruginosa ATCC 27853, P. mirabilis ATCC 29245, and K. 178 pneumoniae ATCC 27736. Two clinical strains, S. aureus MDR (multidrug resistant bacteria), and 179 Enterobacter cloacae (E. cloacae) were used. The antifungal activity was performed using 180 Candida albicans (C. albicans) ATCC 10231, Cryptococcus neoformans (C. neoformans) ATCC 181 14116, C. vaginalis (clinical strain), and Candida sp. (clinical strain). Two fungal strains 182 (Aspergillus spp) were also tested: A. fumigatus ATCC 204305 and A. niger. 183 Two techniques were used to screen the antimicrobial effect of the obtained essential oil 184 and its main component thymol purchased from Sigma Aldrich®, Germany. The disc diffusion 185 assay was performed on Mueller-Hinton agar plates for all bacteria, Sabouraud chloramphenicol 186 agar for yeasts, and Potato Dextrose agar for the Aspergillus strains. 10 mg of essential oil and 187 thymol/ 6mm-disc were tested in triplicate. Ampicillin and Amphotericin B were used as control. 188 The minimal inhibitory concentration (MIC) and minimal bactericidal/fungicidal concentration 189 (MBC/MFC) values were determined by using the microdilution assay as previously described by 190 (Snoussi et al., 2018). MBC/MIC ratio and MFC/MIC ratio were used to interpret the activity of 191 the essential oil as described by Gatsing et al., (2009). 192 2.5. Molecular docking analysis of TyrRS, GLMS and Gyrase with phytochemicals of T. 193 musilii 194 Crystal structures of tyrosyl-tRNA synthetase TyrRS from S. aureus (PDB: 1JIJ.pdb) (Qiu et al, 195 2001), glucosamine 6-phosphate synthase (GLMS) from E. coli (PDB: 1XFF.pdb) (Isupov et al., 196 1996), and Gyrase from S. aureus (PDB: 2XCT.pdb) (Bax et al., 2010) were fetched from Protein 197 Data Bank (RCSBPDB). Following to the retrieval of crystal structures, LCMS identified 198 phytochemicals 3-dimensional structures such as α-thujene, α-pinene, β-myrcene, α-terpinene, p9 199 cymene, (1,8)-cineole, γ-terpinene, α-terpinolene, Borneol, Terpinen-4-ol, α-terpineol, 2- 200 Isopropyl-5-methylanisole, Thymol, Carvacrol, Thymyl acetate, Carvacryl acetate, and β- 201 caryophyllene were acquired from eminent database PubChem and converted to PDB format using 202 Open Babel (O'Boyle et al., 2011). These seventeen compounds were then docked separately 203 against the receptor structure (1JIJ, 1XFF and 2XCT) using molecular docking software Autodock 204 4.2.6 (Morris et al., 2009). Docking protocol was performed in a similar manner, which can be 205 related to previous analyses (Sonawane and Barage, 2015; Parulekar and Sonawane, 2018). Apart 206 from the grid centre and grid size, all other parameters used for docking with these seventeen 207 compounds were kept same. For the preparation of the grid map using a grid box, Auto Grid 208 (Morris et al., 2009) was used. The grid size was set to 126 ×126 × 126xyz points for TyrRS and 209 gyrase receptors. For GLMS, grid size was set to 96 × 122 × 126 xyz points. Grid spacing was 210 kept to 0.375 Å for all the receptors. The grid centre for TyrRS was designated at dimensions (x, 211 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 212 gyrase at (x, y and z): 7.841, 39.224 and 118.021. The grid box is cantered in such a way that it 213 encloses the entire binding site of both the receptors and provides enough space for translation and 214 rotation of ligands. The generated docked conformation was ranked by predicted binding energy 215 and topmost binding energy docked conformation was analyzed using UCSF Chimera (Pettersen 216 et al., 2004) for intermolecular hydrogen bonding of active site amino acid residues from the 217 receptors with docked ligands (Nadaf et al., 2018). 218 2.6. Statistical analysis 219 The laboratory biological assays were conducted in triplicates for each sample. The IC50 of DPPH, 220 ABTS, and β-carotene bleaching methods values were calculated by linear regression analysis. 10 221 ANOVA and Duncan tests were performed with SPSS 16.0. The means of the test’s values were 222 also evaluated with the Least Significant Differences test at 0.05 significance level. 223 3. Results and Discussion 224 3.1. Chemical composition of T. musilii Velen essential oil 225 The air-dried aerial-parts of T. musilii yielded 2.736±0.015 % (v/w) essential oil on hydro- 226 distillation. Seventeen components were identified in the obtained oil, belonging mainly to 227 oxygenated 228 (11.013±0.039%) and sesquiterpenes hydrocarbons (1.953±0.005%). These data are summarized 229 in Table 1. The chemical structure of the seventeen compounds identified in T. musilii essential 230 oil were depicted in figure 2. monoterpenes (87.010±0.279%) followed by monoterpenes hydrocarbons 231 This essential oil can be defined as thymol/thymyl acetate chemotype (67.697/12.993%) 232 as shown in the chromatogram (figure 3). Thymol (67.697±0.938%), thymyl acetate 233 (12.993±0.221%), o-cymene (4.617±0.119%), carvacrol (3.417±0.105%), and γ-terpinene 234 (2.633±0.072). 235 Numerous studies have reported that oxygenated monoterpenes were the dominant family 236 of compounds found in the Thymus genus essential oil (De Martino et al., 2009; Zarshenas and 237 Krenn, 2015). The diversity of the composition of the volatile oil obtained from different species 238 and subspecies belonging to the genus thymus can be explicated by endogenous (plant varieties, 239 vegetative state, organ tested) and exogenous factors like climatic features, soil characteristics, and 240 seasons (Tzakou et al., 1998; Cosentino et al., 1999; Pirbalouti et al., 2013a,b). It has also been 241 reported that the frequency of irrigation and salicylic acid concentration can affect the yield and 242 the content of essential oil obtained from T. daenensis Celak. and T. vulgaris L. (Khazaie et al., 243 2008, Pirbalouti et al., 2013c; Alavi-Samani et al., 2013). In addition, application of fertilizers 11 244 increases the vegetative biomass, oil yield and diversity, and antioxidant activities of T. 245 daenensis Celak. (Bistgani et al., 2018). 246 Thymol and carvacrol are the main phenolic compound of thyme oil. The major 247 nonphenolic compounds were linalool and p-cymene (Piccaglia and Marotti, 1991). Thymol was 248 the dominant phenolic compound detected in several Thymus species with different percentage as 249 reported by Tohidi et al., (2019) including: T. carmanicus (40.8%), T. daenensis (20-80.4%), T. 250 eriocalyx (5.3-66.34%), T. fallax (19.88-65.9%), T. fedtschenkoi (31.8%), T. kotschyanus (6.8- 251 66.15%), T. migricus (55.6-79.74%), T. pubescens (37.9-63.5%), T. serpyllum (52.45%), T. 252 transcaucasicus (35.83-62.92%), and T. trauveterri (24.43-63.33%). 253 It has also been reported that thymol is the main phenolic compound in the essential oil of 254 T. cappadocicus Boiss. (Albayrak and Aksoy, 2012), T. pulegioides (Pinto et al., 2006), T. 255 fontanesii (Dob et al., 2006), T. hyemalis (Rota et al., 2008), T. ciliatus Desf. Benth. (Kabouche et 256 al., 2009), T. marschallianus Willd (Cavar Zeljkovic et al., 2015), T. pannonicus (Pluhár et al., 257 2010), T. vulgaris (Asbaghian et al., 2011), T. zygis (Ballester-Costa et al., 2013), T. numidicus 258 Poiret (Mina et al., 2014), T. quinquecostatus Celak. (Kim et al., 2014) and T. lanceolatus (Khadir 259 et al., 2016). More recently, Jan et al. (2020) reported that T. afghanicus harvested from the 260 Himalayan-Afghanistan area was a thymol chemotype (27.7%). 261 In this study, thymyl acetate, which is formed after acetylation of thymol produced directly 262 by terpene synthases (Keszei et al. 2008), was found to be the second phenolic compound in T. 263 musilii oil (12.993%). This molecule has been reported in the essential oil of some Thymus species 264 with different percentage including T. longicaulis (0-12.8%) and T. pulegioides L. (0.4-0.7%) 265 from Italy (De Martino et al., 2009), T. caespititius Brot. from Portugal (11-15%, Mendes et al., 12 266 2013), T. serpyllum L. from Serbia (38.5%, Cancarevic et al., 2013), and T. lanceolatus from 267 Algeria (0.006%; Khadir et al., 2016). 268 3.2. Antioxidant activities of T. musilii essential oil 269 Because of the complex chemical compounds effect of the plants volatile oil, the 270 antioxidant capacity of T. musilii essential oil is studied by four methods, DPPH, ABTS, FRAP 271 and β-carotene bleaching methods in order to estimate the effectiveness of these compound 272 diversity. Table 4 summarizes the free radicals scavenging activities of T. musilii essential oil and 273 the commercialized standards, ascorbic acid and butylated hydroxyl-toluene (BHT). The IC50 of 274 the essential oil and the standards, which is the concentration required for scavenging half (50%) 275 of the tested radicals, showed that ABTS and peroxyl radicals were strongly significantly inhibited 276 by T. musilii (Table 4). Interestingly, T. musilii oil possess high antioxidant activities using ABTS 277 (IC50 = 5.6x10-4±2x10-5 mg/mL) and β-carotene bleaching (IC50 = 3.2x10-3±5x10-4 mg/mL) 278 methods, followed by DPPH test (IC50= 0.049±1x10-4 mg/mL). This essential oil is significantly 279 active on peroxyl radicals than the both tested standards (Table 4). 280 Literature review showed that no previous work was countered on T. musilii essential oil 281 antioxidant capacity. However, several studies were conducted on the genus Thymus essential oils 282 and on its antioxidant capacity (El-Bakkal et al., 2020; Goudjil et al., 2020). For instance, the anti- 283 radicalar essential oils from the cultivated T. carmanicus, T. kotschyanus, T. migricus, and T. 284 vulgaris collected, under various conditions, from Iran were studied by DPPH method (Tohidi et 285 al., 2020). Under red, red-blue, blue, white and greenhouse light treatments, T. carmanicus (IC50= 286 278; 259.2; 281; 467.4; 198.2 µg/mL), T. kotschyanus (IC50= 621.8; 421.1; 304.6; 557.4; 287 384.7µg/mL), T. migricus (IC50= 358; 911.6; 176.8; 1274; 631.8 µg/mL), and T. vulgaris (IC50= 288 560; 766; 400.6; 227.6; 314.3 µg/mL) inhibited DPPH radicals (Tohidi et al., 2020). Thymus 13 289 longicaulis C. Presl subsp. longicaulis var. longicaulis essential oil collected from Turkey had 290 strong radical inhibition percentage (IP= 87.69 % at 0.4 mg/mL; 93.28 % at 1 mg/mL; 94.15 % at 291 2 mg/mL) using b-carotene–linoleic acid method. The same plant species possess moderate effect 292 (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, 293 moderate effect using reducing power protocol (Absorbance =0.128 at 0.2 mg/mL, 0.241 at 0.4 294 mg/mL, 0.550 at 1 mg/mL), and it had no chelating activity till 1 mg/mL (Sarikurkcu et al., 2010). 295 Compared to the previous studies, T. musilii essential oil in the present study exhibited a 296 strong antioxidant effect. This activity can be explained by the chemical composition classes, 297 monoterpene hydrocarbons (11.01%) and oxygenated monoterpenes (87.01 %), of the volatile oil. 298 Most researchers revealed the antiradical effect of monoterpenes (Badawy et al., 2019; 299 Wojtunik‐Kulesza et al., 2019). The antioxidant capacity of thymol (IC50= 31.426 mg/mL), β- 300 cymene (IC50= 916.89 mg/mL), α-terpineol (IC50= 480.56 mg/mL), myrcene (IC50= 22.136 301 mg/mL), α-pinene (IC50= 880.74 mg/mL) were evaluated using N,N-dimethyl-1,4- 302 phenylenediamine (DMPD) reagent (Badawy et al., 2019). 303 Other study focused on the antioxidant of α-terpinene (IC50= 0.6 and 7.5 mM) and γ- 304 terpinene (IC50= 2.8 and 30.0 mM) using ABTS and DPPH methods, respectively (Li and Liu, 305 2009). Previous work demonstrated that γ-terpinene (IC50= 15.5 mg/mL) inhibited DPPH radicals 306 (Sonboli et al., 2005). This antioxidant assay may be related to a high area of thymol (67.7%). 307 Several studies confirmed the strong in vitro and in vivo biological effect of thymol (Abd El-Naby 308 et al., 2020; Arafa et al., 2020; Jafari et al., 2020). The registered effect may referred to the major 309 compound, thymol (67.7%), and/or to the synergism between main and minor compounds of the 310 essential oil (Ciesla et al., 2016). The antioxidant activities were studied, in literature, towards the 14 311 whole essential oils, to single compounds and as well as to combination (Graßmann, 2005; Tohidi 312 et al., 2020). 313 3.3. Antimicrobial activities of T. musilii essential oil 314 The antibacterial activity of T. musilii essential oil was tested against six bacteria, four 315 yeasts and two fungal strains using both disc diffusion (figure 3) and microdilution assays. 316 Obtained results showed that, the tested bacteria were resistant to ampicillin with a mean diameter 317 of growth inhibition zone ranging from 6.33±0.57 mm to 7.33±0.57 mm. In addition, the mean 318 diameter of growth inhibition zones ranged from 21.33±1.52 mm for P. mirabilis to 36.33±1.15 319 mm for K. pneumoniae. The clinical strain S. aureus MDR, resistant to ampicillin, was susceptible 320 to the oil tested (25.33±1.15 mm). Small quantities of oil (12.5 mg/mL) can inhibit the growth of 321 all tested bacteria, except for E. cloacae (MIC value=3.125 mg/mL). MBCs values were ranging 322 from 6.25 mg/mL (E. cloacae) to 100 mg/mL for P. aeruginosa. As compared to the single 323 bioactive molecule, thymol, T. musilii essential oil exhibited bactericidal activity for all tested 324 bacteria with MBC/MIC ratio inferior to 4 except for P. aeruginosa (MBC/MIC ratio =8). All 325 these data are summarized in table 2. Using the literature review, high antimicrobial activity of 326 Thymus species (chemotype thymol) was recorded against a large collection of bacterial and fungal 327 species (Table 5). 328 Similar results were obtained with the yeast and fungi strains tested. Interestingly, high 329 diameter of inhibition zone was recorded for the two clinical yeast strains: Candida sp. 330 (37.33±1.15 mm), and C. vaginalis (37.33±1.15 mm). The MIC and MFC values were 6.25 mg/mL 331 and 12.5 mg/mL, respectively for both strains. Using the MFC/MIC ratio scheme proposed by 332 Gasting et al., (2009), T. musilii seems to be more effective than thymol on the four tested yeast 333 strains as they have the lowest ratio ranging from 2 to 4. It is important to highlight also that the 15 334 tested (thymol/thymyl acetate) chemotype oil was very active on the two Aspergillus strains with 335 mean inhibition zone about 88.66±1.15 mm for A. fumigatus to 87.33±1.15 mm for A. niger. All 336 these data are summarized in table 3. 337 Using the disc diffusion test, Vladimir-Knežević and colleagues (2012) reported similar 338 results with T. longicaulis species (Chemotype thymol, 46.3%) tested against Haemophilus 339 influenzae (IZ= 42 mm), Neisseria meningitidis (IZ= 53 mm), S. aureus (IZ= 35 mm), S. 340 pneumoniae (IZ= 43 mm), and S. pyogens (IZ= 41 mm). Additionally, Bozin et al. (2006) reported 341 that T. vulgaris essential oil (chemotype thymol) was active against a wide range of Gram-positive 342 and Gram-negative bacteria, including the same species tested in our study. In fact, the highest 343 growth inhibition zones were recorded for Micrococcus flavus (IZ=48.2 mm), S. epidermidis 344 (IZ=48 mm), S. aureus (IZ=26.2 mm), B. subtilis (IZ=40.6 mm), E. coli (IZ=29.4 mm), and P. 345 aeruginosa (IZ=12 mm). 346 Previous reports have noticed the anti-C. albicans activity of different species belonging 347 to the Thymus genus. In fact, Pinto et al. (2006) reported a significant activity of T. pulegioides oil 348 (thymol 26%/carvacrol 21% chemotype) against Candida, Aspergillus and dermatophyte species 349 explained by the alteration in the cytoplasmic membrane and ergosterol content. 350 In addition, Pirbalouti et al. (2009) founded that T. daenensis Celak. essential oil effectively 351 inhibits the growth of vaginal C. albicans strains at high concentration (50-55 µl). The same oil 352 was active against E. coli O157:H7, B. cereus, L. monocytogenes, and C. albicans with a diameter 353 of growth inhibition zone and MIC values about (7 mm/>10 mg.mL-1, 25 mm/0.625 mg.mL-1, 16 354 mm/2.5 mg.mL-1, and 19mm/˂0.039 mg.mL-1 respectively (Pirbalouti et al., 2010). Thymol-rich 355 chemotype of T. daenensis Celak essential oil can inhibit the growth of S. aureus isolated from 356 milk with MIC and MBC values about 62 µg/mL and 630 µg/mL, respectively (Pirbalouti et al., 16 357 2014). Couladis et al. (2004) reported the high activity of T. striatus (Chemotype thymol, 59.5%) 358 against a large collection of Aspegillus, Cladosporium, Penicillium, Trichoderma, Tricophyton, 359 Microsporum, and Epidermophyton strains with MICs values ranging from 0.5 to 2 µl. In 2014, 360 Nikolic and colleagues reported that T. serpyllum (Thymol, 38.5%) was active against four 361 Candida species (C. albicans, C. tropicalis, C. glabrata, and C. krusei) with MICs values ranging 362 from 01. to 0.2 µl. More recently, Satyal and colleagues (2016) demonstrated that T. vulgaris 363 essential oils inhibit the growth of C. neoformans var. neoformans, and C. albicans with MICs 364 values about (313/156) µg.mL-1, and (1250/625) µg.mL-1, respectively for linalool and geraniol 365 chemotypes. 366 A brief literature review summarized the antimicrobial activity of thymol against a large 367 collection of bacteria, yeast and fungi (Table 6). High activity of the Thymus plant species can be 368 associated to the dominance of thymol with different percentage. In fact, this molecule is known 369 to exhibit antimicrobial, antioxidant, immunological, anti-inflammatory, anticancer, and 370 cardiovascular protection properties (Nagoor Meeran et al., 2017; D’agostino et al., 2019). This 371 terpenoid molecule inhibits the hyphal production in Fusarium graminearum (Gao et al., 2016), 372 decreases the membrane permeability leading to the loss of cytoplasmic membrane integrity and 373 loss of electrolytes in C. albicans species by binding to ergosterol (De Castro et al., 2015), and 374 inhibits the telomerase activity in S. cerevisiae species (Darvishi et al., 2013). It has been 375 demonstrated that thymol can kill Methicillin-resistant S. aureus strain by increasing the formation 376 of reactive oxygen species (Li et al., 2014). 377 3.4. Molecular docking analysis 378 In order to correlate the binding of isolated Thymus bioactive molecules with its biological 379 activities, the main compounds were docked to the active site of TyrRS, GLMS and Gyrase, 17 380 respectively to demonstrate their potential inhibition against S. aureus and E. coli pathogens. The 381 binding affinities of top-rated pose of different ligand-receptor complex (Table 4) revealed that 382 among all tested bioactive compounds, the best binding affinity was found with β-caryophyllene- 383 enzymes with values of -5.4 kcal/mol, -6.8 kcal/mol and -6.2 kcal/mol, respectively for β- 384 caryophyllene-TyrRS, β-caryophyllene-GLMS and β-caryophyllene-Gyrase, suggesting its 385 highest binding efficiency and therefore was selected for further investigation. 386 To get insight into the mechanism of TyrRS, GLMS and Gyrase inhibition by β- 387 caryophyllene, we elucidate their molecular interaction mode in the active site residues of 388 receptors. The outcomes compiled in table 8 showed that β-caryophyllene-TyrRS complex was 389 mainly stabilized by Alkyl interactions with Met77, Ile78 and Leu128, Pi-Alkyl interactions with 390 Leu128 and Leu173 and Pi-sigma interactions with Phe 136 residues. Alkyl and Pi-Alkyl 391 interactions were also formed between β-caryophyllene and GLMS residues of Ile7, Ala38 and 392 Pro166. However, the amino acid residues involved in stabilizing the complex caryophyllene- 393 Gyrase are Ala1374 (Pi-Alkyl), Leu1448 (Pi-Alkyl and Alkyl) and Tyr1451 (Pi-Alkyl). As shown, 394 Phe136 and Leu173 of TyrRS from S. aureus, Ala38 from Gyrase in S. aureus and Leu1448 from 395 GLMS in E. coli formed stronger Pi-Sigma, Alkyl and Pi-Alkyl interactions with the natural 396 bioactive compounds (Table 7 and 8) and therefore, could possibly inhibit the activity of enzyme 397 resulting in the neutralization of their virulence. 398 4. Conclusion 399 In the present study, the antioxidant and the antimicrobial assays of the essential oil from T. musilii 400 were evaluated. The obtained findings suggest that this cultivated species can constitute a good 401 source of antioxidant, antibacterial and antifungal compounds, namely, thymol. Nevertheless, 402 these biological results deserve further deep in vivo studies in order to use this plant as possible 18 403 bio-source in food and pharmaceutical industries. Molecular docking results together with the 404 findings of in-vitro antimicrobial potency suggest that T. musilii essential oil is a potent inhibitor 405 of S. aureus and E. coli and subsequently lead to novel discovery of plant-based therapeutic 406 products. 407 Declaration of Competing Interest 408 All the authors listed have agreed with the submission and declared no conflict of interest. 409 Funding 410 This work was supported by the University of Ha’il (grant No. 160991). 411 References 412 Abbaszadeh S., Sharifzadeh, A., Shokri, H., Khosravi, A., Abbaszadeh, A. 2014. Antifungal 413 efficacy of thymol, carvacrol, eugenol and menthol as alternative agents to control the growth 414 of food-relevant fungi. Journal of Medical Mycology, 242, e51-e56. 415 Abd El-Naby A.S., Al-Sagheer A.A., Negm S.S., Naiel M.A.E., 2020. Dietary combination of 416 chitosan nanoparticle and thymol affects feed utilization, digestive enzymes, antioxidant 417 status, and intestinal morphology of Oreochromis niloticus. Aquaculture, 515, 734577. 418 Adams, R.P., 2007. Identification of Essential Oil Components by Gas 419 Chromatography/Quadrupole Mass Spectroscopy. Allured Publishing Corporation, Carol 420 Stream, III, USA. 421 422 Adnan M., 2019. Bioactive potential of essential oil extracted from the leaves of Eucalyptus globulus (Myrtaceae). Journal of Pharmacognosy and Phytochemistry, 8(1), 213–216. 423 Reddy M., Adnan M., Alreshidi M., Saeed M., Patel M. 2020a. Evaluation of Anticancer, 424 Antibacterial and Antioxidant Properties of a Medicinally Treasured Fern Tectaria coadunata 19 425 with its Phytoconstituents Analysis by HR-LCMS. Anti-Cancer Agents Med. Chem. 20: 1– 426 12. 427 428 Adnan M., Patel M., Deshpande S., Alreshidi M., Siddiqui A.J., Reddy M.N., Emira N. and De 429 Feo V. 2020b. Effect of Adiantum philippense extract on biofilm formation, adhesion with its 430 antibacterial activities against foodborne pathogens and characterization of bioactive 431 metabolites: An in vitro-in silico approach. Frontiers in Microbiology, 11, p.823. doi: 432 10.3389/fmicb.2020.00823 433 Adnan M., Patel M., Reddy M.N., Alshammari E., 2018a. Formulation, evaluation and bioactive 434 potential of Xylaria primorskensis terpenoid nanoparticles from its major compound xylaranic 435 acid. Scientific reports, 8(1):1740. 436 Al Maqtari M.; Alghalibi S.M.; Alhamzy E.H. 2011. Chemical composition and antimicrobial 437 activity of essential oil of Thymus vulgaris from Yemen. Turk. J. Biochem., 36, 342–349. 438 Al-Asmari A. K.; Md Tanwir A., Ahmed A. Al-F., Mohammed S.A., 2017. Chemical composition 439 of essential oil of Thymus vulgaris collected from Saudi Arabian market. Asian Pac. J. Trop 440 Biomed. 2017; 7(2): 147–150. 441 Al-Asmari, A.K., Al-Elaiwi, A.M., Athar, M.T., Tariq, M., Al-Eid, A., Al-Asmary, S.M., 2014. A 442 Review of Hepatoprotective Plants Used in Saudi Traditional Medicine. Evid. Based 443 Complement. Alternat. Med., 1-22. 444 Alavi-Samani S.M., Pirbalouti A.G., Kachouei M.A., Hamedi B. , 2013. The influence of reduced 445 irrigation on herbage, essential oil yield and quality of Thymus vulgaris and Thymus daenensis. 446 Journal of Herbal Drugs, 4(3), 109-113. 20 447 Albayrak S., Aksoy A., 2012. Essential oil composition and in vitro antioxidant and antimicrobial 448 activities of Thymus cappadocicus Boiss. Journal of Food Processing and Preservation, 1-10. 449 Al-Essa, M.A., Al-Mehaidib, A., Al-Gain, S., 1998. Parental awareness of the liver disease among 450 children in Saudi Arabia. Ann Saudi Med., 18(1), 79-81. 451 Alharbi N.A., 2017. Survey of Plant Species of Medical Importance to Treat Digestive Tract 452 Diseases in Tabuk Region, Saudi Arabia. Journal of King Abdulaziz University-Science, 29 453 (1): 51-61. 454 Andrade-Ochoa, S., Nevárez-Moorillón, G.V., Sánchez-Torres, L.E., Villanueva-García, M., 455 Sánchez-Ramírez, B.E., Rodríguez-Valdez, L.M., Rivera-Chavira, B.E., 2015. Quantitative 456 structure-activity relationship of molecules constituent of different essential oils with 457 antimycobacterial activity against Mycobacterium tuberculosis and Mycobacterium bovis. 458 BMC Complement. Altern. Med. 15, 332. 459 Aouadhi, C., Ghazghazi, H., Dallali, S., Sebei, H., Hasnaoui, B., Maaroufi, A., 2013. Comparison 460 of chemical composition, antioxidant and antimicrobial activities of Thymus capitatus L. 461 essential oils from two Tunisian localities (Sousse and Bizerte). Int. J. Agron. Plant. Prod. 4 462 (8), 1772-1781. 463 Aprotosoaie, A.C., Miron, A., Ciocarlan, N., Brebu, M., Rosu, C.M., Trifan, A., Vochita, G., 464 Gherghel, D., Luca, S.V., Nita, A., Costache, I.-I., Mihai, C.T., 2019. Essential oils of 465 Moldavian Thymus species: Chemical composition, antioxidant, anti-Aspergillus and 466 antigenotoxic activities. Flavour Frag. J.; 34, 175-186. 467 Arafa W.M., Abolhadid S.M., Moawad A., Abdelaty A.S., Moawad U.K., ShokierK.A.M., 468 Shehata O., Gadelhaq S.M., 2020. Thymol efficacy against coccidiosis in pigeon (Columba 469 livia domestica). Preventive Veterinary Medicine 176, 104914. 21 470 Asbaghian S., Shafaghat A., Zarea K., Kasimov F., Salimi, F., 2011. Comparison of volatile 471 constituents, and antioxidant and antibacterial activities of the essential oils of Thymus 472 caucasicus, T. kotschyanus and T. vulgaris. Natural Product Communication, 6(1), 137–140. 473 Badawy M.E.I., Marei G.I.K., Rabea E.I., Taktak N.E.M., 2019. Antimicrobial and antioxidant 474 activities of hydrocarbon and oxygenated monoterpenes against some foodborne pathogens 475 through in vitro and in silico studies. Pesticide Biochemistry and Physiology 158, 185-200. 476 Ballester-Costa C., Sendra E., Fernández-López J., Pérez-Álvarez J.A., Viuda-Martos M., 2013. 477 Chemical composition and in vitro antibacterial properties of essential oils of 478 four Thymus species from organic growth. Ind. Crops Prod. ; 50:304–311. 479 Barros, L., Falcão, S., Baptista, P., Freire, C., Vilas-Boas, M., & Ferreira, I. C., 2008. Antioxidant 480 activity of Agaricus sp. mushrooms by chemical, biochemical and electrochemical assays. 481 Food chemistry, 111(1), 61-66. 482 483 484 485 Bassam A., Ghaleb A., Dahood A., Naser J., 2004. Antibacterial Activities of Some Plant Extracts Utilized in Popular Medicine in Palestine, Turkish Journal of Biology, 28: 99-102. Batanouny K.H., Sheikh M.Y., 1972. Ecological Observations along Baghdad‐Huseiba Road. Western Desert, Iraq With 2 figures. Feddes Repertorium, 83(4), 245-263. 486 Bax B.D., Chan P.F., Eggleston D.S., Fosberry A., Gentry D.R., Gorrec F., Giordano I., Hann 487 M.M., Hennessy A., Hibbs M., Huang J., Jones E., Jones J., Brown K.K., Lewis C.J., May 488 E.W., Saunders M.R., Singh O., 489 A.J., Wohlkonig A., Pearson N.D., Gwynn M.N., 2010. Type IIA topoisomerase inhibition by 490 a new class of antibacterial agents. Nature 466, 935–940 Spitzfaden C.E., Shen C., Shillings A., Theobald 491 Ben Jabeur M., Hamada W., 2014. Antifungal activity of chemically different essential oils from 492 wild Tunisian Thymus spp., Natural Product Research: Formerly Natural Product Letters.1-5. 22 493 Bi, H., Gao, T., Li, Z., Ji, L.. Wei Yang, B Jeff Iteku, Enxu Liu, Yifa Zhou (2013). Structural 494 Elucidation and Antioxidant Activity of a Water-Soluble Polysaccharide From the Fruit Bodies 495 of Bulgaria Inquinans (Fries). Food Chem., 138(2-3):1470-5. 496 497 Bistgani Z.E., Siadat S.A., Bakhshandeh A., Pirbalouti A.G., Hashemi M., Maggi F., Morshedloo M.R., 2018. Industrial Crops and Products, 121 (1), 434-440. 498 Borugă O., Jianu C., Mişcă C., Goleţ I., Gruia A.T., Horhat F.G., 2014. Thymus vulgaris essential 499 oil: chemical composition and antimicrobial activity Journal of Medicine and Life. 7, Special 500 Issue 3, 56-60. 501 Bozin, B., Mimica-Dukic, N., Simin, N., Anackov, G., 2006. Characterization of the volatile 502 composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant 503 activities of the entire oils. J. Agric. Food. Chem. 54(5), 1822–1828 504 505 506 507 508 509 510 Cancarevic A., Bugarski B., Savikin K., Zdunic G., 2013. Biological activity and ethnomedicinal use Thymus vulgaris and Thymus serpyllum. Medical Material, 33(33), 3–17. Cavar Zeljkovic´ S.; Maksimovic´ M., 2015. Chemical composition and bioactivity of essential oil from Thymus species in Balkan Peninsula. Phytochem. Rev., 14, 335–352. Chakraborty, Kajal, and R. Paulraj. "Sesquiterpenoids with free-radical-scavenging properties from marine macroalga Ulva fasciata Delile." Food chemistry 122.1 (2010): 31-41. Ciesla L.M., Wojtunik‐Kulesza K.A., Oniszczuk A., Waksmundzka‐Hajnos M., 2016. Antioxidant 511 synergism and antagonism between selected monoterpenes using 512 2‐diphenyl‐1‐picrylhydrazyl method. Flavour and fragrance journal 31(6), 412-419. the 2, 513 Cosentino S., Tuberoso C. I. G., Pisano B., Satta M., Arzedi E., Palmas F., 1999. In-vitro 514 antimicrobial activity and chemical composition of Sardinian Thymus essential oils. Letter in 515 Applied Microbiology, 29, 130-135. 23 516 517 518 519 Couladis M., Tzakou O., Kujundzic S., Sokovic M., Mimica-Dukic N., 2004. Chemical analysis and antifungal activity of Thymus striatus. Phytother. Res. 18(1), 40-42. Cronquist A., 1988. The Evolution and Classification of Flowering Plants. The New York Botanical Garden, New York, USA. 520 Cutillas, A.B., Carrasco, A., Martinez-Gutierrez, R., Tomas, V., Tudela, J., 2018. Thyme essential 521 oils from Spain: Aromatic profile ascertained by GC-MS, and their antioxidant, anti- 522 lipoxygenase and antimicrobial activities. J. Food Drug. Anal.; 26, 529-544. 523 D’agostino M.; Tesse N.; Frippiat J.P.; Machouart M.; Debourgogne A., 2019. Essential Oils and 524 Their Natural Active Compounds Presenting Antifungal Properties. Molecules , 24, 3713- 525 3734. 526 527 Darvishi E.; Omidi M.; Bushehri A.A.; Golshani A.; Smith M.L., 2013. Thymol antifungal mode of action involves telomerase inhibition. Med. Mycol., 51, 826–834. 528 De Castro R.D.; de Souza T.M.P.A.; Bezerra L.M.D.; Ferreira G.L.S.; Costa E.M.M.; Cavalcanti 529 A.L., 2015. Antifungal activity and mode of action of thymol and its synergism with nystatin 530 against Candida species involved with infections in the oral cavity: An in vitro study. BMC 531 Complement. Altern. Med., 15, 417. 532 De Martino L., Bruno M., Formisano C., De Feo V., Napolitano F., Rosselli S., Senatore F., 2009. 533 Chemical composition and antimicrobial activity of the essential oils from two species 534 of Thymus growing wild in southern Italy. Molecules. , 14(11):4614-2464. 535 Dob T., Dahmane D., Benabdelkader T., Chelghoum C., 2006. Composition and antimicrobial 536 activity of the essential oil of Thymus fontanesii. Pharmaceutical Biology, 44(8), 607–612. 24 537 Du, E., Gan, L., Li, Z., Wang, W., Liu, D., Guo, Y.,2015. In vitro antibacterial activity of 538 thymol and carvacrol and their effects on broiler chickens challenged with Clostridium 539 perfringens. J. Anim Sci Biotechnol. 24(6), 58-69. 540 Ebrahimi S.N., Hadian J., Mirjalili M.H., Sonboli A. and Yousefzadi M., 2008. Essential oil 541 composition and antibacterial activity of Thymus caramanicus at different phonological stages. 542 Food Chemistry, 110: 927-931. 543 El-Bakkal S.E., Zeroual S., Elouazkiti M., Mansori M., Bouamama H., Zehhar N., El-Kaoua M., 544 2020. Comparison of Yield Chemical Composition and Biological Activities of Essential Oils 545 Obtained from Thymus pallidus and Thymus satureioides Coss. Grown in Wild and Cultivated 546 Conditions in Morocco. Journal of Essential Oil Bearing Plants, 1-14. 547 El-Tawil, B.A.H., 1983. Chemical constituents of indigenous plants used in native medicine of 548 Saudi Arabia. Arab Gulf J. Sci. Res., (1), 395- 419. 549 Essid R, Rahali FZ, Msaada K, Sghair I, Hammami M, Bouratbine A, Aoun K, Limam F (2015) 550 Antileishmanial and cytotoxic potential of essential oils from medicinal plants in 551 northern Tunisia. Ind. Crop. Prod. 77, 795–802. 552 Falcone P., Speranza B., Del Nobile M. A., Corbo M. R., Sinigaglia M., 2005. A Study on the 553 Antimicrobial Activity of Thymol Intended as a Natural Preservative. Journal of Food 554 Protection, 68, (8) , 1664–1670. 555 Falsafi T., Moradi P., Mahboubi M., Rahimi E., Momtaz H., Hamedi B., 2015. Chemical 556 composition and anti-Helicobacter pylori effect of Satureja bachtiarica Bunge essential oil. 557 Phytomedicine, 221, 173-177. 558 559 Gatsing D., Tchakoute V., Ngamga D., Kuiate J.R., Tamokou J.D.D., B.F., Tchouanguep F.M., Fodouop S.P.C., 2009. In Nji Nkah vitro antibacterial activity 25 560 of Crinum purpurascens Herb. leaf extract against the Salmonella species causing 561 typhoid fever and its toxicological evaluation. Iran. J. Med. Sci. 34,126-136. 562 Gedikoğlu A., Münevver S., Ayşe Ç., 2019. "Evaluation of Thymus vulgaris and Thymbra spicata 563 essential oils and plant extracts for chemical composition, antioxidant, and antimicrobial 564 properties." Food science & nutrition 7.5 1704-1714. 565 Goudjil M.B., Zighmi S., Hamada D., Mahcene Z., Bencheikh S.E., Ladjel S., 2020. Biological 566 activities of essential oils extracted from Thymus capitatus (Lamiaceae). South African Journal 567 of Botany 128, 274-282. 568 Govaerts R., 2003. World Checklist of Selected Plant Families Database in ACCESS: 1-216203. 569 The Board of Trustees of the Royal Botanic Gardens, Kew. (Thymus musilii Velen., 570 Sitzungsber. Königl. Böhm. Ges. Wiss., Math.-Naturwiss. Cl. 11: 5 (1911 publ. 1912). 571 R.Govaerts) 572 573 Graßmann J., 2005. Terpenoids as Plant Antioxidants, in: Litwack, G. (Ed.) Vitamins & Hormones. Academic Press, pp. 505-535. 574 Hernández-Hernández E., Regalado-González C., Vázquez-Landaverde P., Guerrero-Legarreta I., 575 & García-Almendárez B. E., 2014. Microencapsulation, chemical characterization, and 576 antimicrobial activity of Mexican Lippia graveolens H.B.K. and European Origanum vulgare 577 L. oregano essential oils. The ScientificWorldJournal, 641814. 578 Ikram E. H. K., Khoo H. E., Abbe M. M. J., Amin I., Salma I., Azrina A., Halimatul S. M. N., 579 Norzatol A. M. D., Ruzaidi A. M. M., 2009. Antioxidant capacity and total phenolic content 580 of Malaysian underutilized fruits. Journal of food Composition and Analysis 22, 5 388-393. 581 Isupov M. N., Obmolova G., Butterworth S., Badet-Denisot M. A., Badet B., Polikarpov I., 582 Littlechild J. A., Teplyakov A., 1996. Substrate binding is required for assembly of the active 26 583 conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 å crystal 584 structure of the glutaminase domain of glucosamine 6-phosphate synthase. Structure, 4(7), 585 801–810. 586 Jafari A., Karimipour M., Khaksar M.R., Ghasemnejad-Berenji M., 2020. Protective effects of 587 orally administered thymol against titanium dioxide nanoparticle–induced testicular damage. 588 Environ Sci. Pollut. Res. Int., 27(2), 2353-2360. 589 590 591 592 Jamzad Z., 2010. Thymus and Satureja spp. of Iran, Research instituted of Forests and rangelands Press, 172 P. Jan, A.K., Khan, S.W., Khan, N.M., Sher, H., Khuda, F., 2020. Composition of the essential oil of Thymus afghanicus. Chem. Nat. Compd., 56(1), 156-157. 593 Jesus, F., Ferreiro, L., Bizzi, K., Loreto, É., Pilotto, M., Ludwig, A., Alves, S., Zanette, R., 594 Santurio, J., 2015. In vitro activity of carvacrol and thymol combined with antifungals or 595 antibacterials against Pythium insidiosum. Journal de Mycologie Médicale, 252, e89-e93. 596 Jordan, M.J., Martinez, R.M., Martinez, C., Martinez M.I., Sotomayor, J.A., 2009. Polyphenolic 597 extract and essential oil quality of Thymus zygis ssp. gracilis shrubs cultivated under different 598 watering levels. Ind. Crop. Prod., 29,145-153. 599 600 601 602 Kabouche, A., Ghannadi, A., Kabouche, Z., 2009. Thymus ciliatus the highest thymol containing essential oil of the genus. Nat. Prod. Commun., 4(9), 1251-1252. Keszei, A., Brubaker, C.L., Foley, W.J., 2008. A molecular perspective on terpene variation in Australian Myrtaceae. Aust. J. Bot., 56, 197–213. 603 Khadir, A., Sobeh, M., Gad, H.A., Benbelaid, F., Bendahou, M., Peixoto, H., Sporer, F., Ashour, 604 M.L., Wink, M., 2016). Chemical composition and biological activity of the essential oil from 605 Thymus lanceolatus. Z. Naturforsch., 71, 155-163. 27 606 Khadir, A., Sobeh, M., Gad, H.A., Benbelaid, F., Bendahou, M., Peixoto, H., Sporer, F., Ashour, 607 M.L., Wink, M., 2016. Chemical composition and biological activity of the essential oil from 608 Thymus lanceolatus. Z. Naturforsch C. J. Biosci., 71(5-6), 155-163. 609 Khazaie, H.R., Nadjafi, F., Bannayan, M., 2008. Effect of irrigation frequency and planting 610 density on herbage biomass and oil production of thyme (Thymus vulgaris) and hyssop 611 (Hyssopus officinalis). Ind. Crop. Prod., 27, 3, 315-321. 612 Kim, Y.S., Hwang. J.W., Kang, S.H., Kim, E.H., Jeon, Y.J., Jeong, J.H., Kim, H.R., Moon, 613 S.H., Jeon, B.T., Park, P.J., 2014. Thymol from Thymus quinquecostatus Celak. protects 614 against tert-butyl hydroperoxide-induced oxidative stress in Chang cells. J. Nat. Med. 68(1), 615 154-162. 616 Kumar, A., Kamal, A., Singh, S., Padalia, R.C., Tandon, S., Chauhan, A., Saikia, D., Verma, R.S., 617 2020. Chemical composition, antimicrobial activity, kinetics and mechanism of action of 618 Himalayan-thyme (Thymus linearis Benth.). J. Essent. Oil Res.; 32, 64-73. 619 620 Li G. X., Liu Z. Q., 2009. Unusual antioxidant behavior of α-and γ-terpinene in protecting methyl linoleate, DNA, and erythrocyte. J. Agric. Food Chem., 57(9), 3943-3948. 621 Li H., Yang T., Li F.Y., Yao Y., Sun Z.M., 2014. Antibacterial activity and mechanism of action 622 of Monarda punctate essential oil and its main components against common bacterial 623 pathogens in respiratory tract. Int. J. Clin. Exp. Pathol.; 7, 7389. 624 Mancini, E., Senatore, F., Del Monte, D., De Martino, L., Grulova, D., Scognamiglio, M., Snoussi, 625 M., De Feo, V., 2015. Studies on chemical composition, antimicrobial and antioxidant 626 activities of five Thymus vulgaris L. essential oils. Molecules. 20, 12016-12028. 28 627 Mandadi R., Mohd A., Mousa A., Mohd S., Mitesh P., 2020. Evaluation of Anticancer, 628 Antibacterial and Antioxidant Properties of a Medicinally Treasured Fern Tectaria coadunata 629 with its Phytoconstituents Analysis by HR-LCMS. Anti-Cancer Agents ME., 20, 1-12. 630 Mathela, C.S., Singh, K.K., Gupta, V.K., 2010. Synthesis and in vitro antibacterial activity of 631 thymol and carvacrol derivatives. Acta Poloniae Pharmaceutica-Drug Research, 67 (4), 375- 632 380. 633 Mathela, C.S., Singh, K.K., Gupta, V.K., 2010. Synthesis and in vitro antibacterial activity of 634 thymol and carvacrol derivatives. Acta Poloniae Pharmaceutica-Drug Research, 67 (4), 375- 635 380. 636 Mendes M.D., Figueiredo A.C., Oliveira M.M., Trindade H., 2013. Essential oil production in 637 shoot cultures versus field-grown plants of Thymus caespititius. Plant Cell Tiss. Organ Cult., 638 113, 341-351. 639 Messara, Y., Fernane, F., Meddour, R., 2016. Chemical composition, antibacterial, and antifungal 640 activities of the essential oil of Thymus numidicus Poiret from Algeria. Phytothérapie. 1-6. 641 Miguel, G., Simoes, M., Figueiredo, A.C., Barroso, J.C., Pedro, L.G. Carvalho, L., 2004. 642 Composition and antioxidant activities of the essential oils of Thymus caespititius, Thymus 643 camphoratus and Thymus mastichina. Food Chem., 86,183-188. 644 645 646 647 648 649 Mina, K., Salima, B., Abdelghani, D., Saoudi, A., 2014. Antipseudomonal activity of the essential oil of Thymus numidicus Poiret. Int. J. Pharm. Sci. Rev. Res., 25(2), 149-153. Miraliakbari H., Shahidi F., 2008. Antioxidant activity of minor components of tree nut oils. Food Chem., 111 (2), 421-427. Miri, R., Ramezan, M., Javidnia, K., Ahmadi, L., 2002. Composition of the volatile oil of Thymus transcaspicus Klokov from Iran. Flavour Frag. J., 17, 245-246. 29 650 Mkaddem, M.G., Romdhane, M., Ibrahim, H., Ennajar, M., Lebrihi, A., Mathieu, F., Bouajila J., 651 2010. Essential oil of Thymus capitatus Hoff. et Link. from Matmata, Tunisia: gas 652 chromatography-mass spectrometry analysis and antimicrobial and antioxidant activities. J. 653 Med. Food., 13(6), 1500-1504. 654 Mobaiyen, H., Dehghan, G., Elmi, F., and Talebpour, A.H. (2017). The Comparison of 655 Composition and biological activities in wild and cultivated of Thymus kotschyanus essential 656 oils and methanolic extracts from East Azarbayjan, Iran. Crescent J. Med. Biol. Sci.; 4, 17-22. 657 Morales R., 2002. The history, botany and taxonomy of the genus Thymus. In: Stahl-Biskup., Saez, 658 F. (Eds), Thyme: The Genus Thymus. Taylor & Francis, London. pp: 1-43. 659 Morris, G.M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K., Goodsell, D.S., Olson, A.J., 660 2009. AutoDock4 and AutoDockTools4: Automated docking with selective receptor 661 flexibility. J. Comput. Chem., 30(16), 2785-2791. 662 Nabavi S.M., Marchese A., Izadi M., Curti V., Daglia M., Nabavi S.F., 2015. Plants belonging to 663 the genus Thymus as antibacterial agents: from farm to pharmacy. Food Chem., 15(173), 339- 664 347. 665 Nagoor M.M.F., Javed H., Al T.H., Azimullah, S., Ojha, S.K., 2017. Pharmacological properties 666 and molecular mechanisms of Thymol: prospects for its therapeutic potential and 667 pharmaceutical development. Front. Pharmacol., 26, 8,380. 668 669 Nickavar B., Mojab F., Dolat-Abadi R., 2005. Analysis of the essential oils of two Thymus species from Iran. Food Chem., 90, 609-611. 670 Nikolic, M., Glamoclija, J., Ferreira, I., Calhelha, R.C., Fernandes, A., Markovic, T., Markovic, 671 D., Giweli, A., Sokovic, M., 2014. Chemical composition, antimicrobial, antioxidant and 30 672 antitumor activity of Thymus serpyllum L., Thymus algeriensis Boiss. and Reut. and Thymus 673 vulgaris L. essential oils. Ind. Crops Prod. 52, 183-190. 674 Nishaa. S, Vishnupriya, M., Sasikumar, J.M., Hephzibah, P.C., Gopalakrishnan, V.K., 2012. 675 Antioxidant activity of ethanolic extract of Maranta Arundinacea L. tuberous rhizomes. Asian 676 J. Pharm. Clin. Res., 5(4), 85-88. 677 Noumi, E., Snoussi, M., Merghni, A., Nazzaro, F., Quindós, G., Akdamar, G., Mastouri, M., Al- 678 Sieni, A., Ceylan, O., 2017. Phytochemical composition, anti-biofilm and anti-quorum sensing 679 potential of fruit, stem and leaves of Salvadora persica L. methanolic extracts. Microb. 680 Pathog., 109, 169-176. 681 682 O'Boyle, N.M., Banck, M., James, C.A., Morley, C., Vandermeersch, T., and Hutchison, G.R. (2011). Open Babel: An open chemical toolbox. J. Cheminform. 3, 33. 683 Ouknin, M., Romane, A., Costa, J., and Majidi, L., 2019. Comparative study of the chemical 684 profiling, antioxidant and antimicrobial activities of essential oils of different parts of Thymus 685 willdenowii Boiss & Reut. Nat. Prod. Res.; 33, 2398-2401. 686 687 Palaniappan K., Holley R. A., 2010. Use of natural antimicrobials to increase antibiotic susceptibility of drug resistant bacteria. Int. J. Food Microbiol., 1402, 164-168. 688 Parulekar, R.S., Sonawane, K.D. (2018). Molecular modeling studies to explore the binding 689 affinity of virtually screened inhibitor toward different aminoglycoside kinases from diverse 690 MDR strains. J. Cell Biochem. 119(3), 2679-2695. 691 Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., Ferrin 692 TE., 2004. UCSF Chimera--a visualization system for exploratory research and analysis. J. 693 Comput. Chem. 25(13), 1605-1612. 31 694 695 Piccaglia R., Marotti M., 1991. Composition of the essential oil of an Italian Thymus vulgaris L. ecotype. Flavour Frag. J., 6, 241-244. 696 Pina-Vaz C., Rodrigues A. G., Pinto E., Costa-de-Oliveira S., Tavares C., Salgueiro L. R., 697 Cavaleiro C., Gonçalves M. J., Martinez-de-Oliveira J., 2004 . Antifungal activity 698 of Thymus oils and their major compounds. J. Eur. Acad. Dermatol. 18, 73–78. 699 Pinto E., Pina-Vaz C., Salgueiro L., Gonçalves M.J., Costa-de-Oliveira S., Cavaleiro C., Palmeira 700 A., Rodrigues A., Martinez-de-Oliveira J., 2006. Antifungal activity of the essential oil of 701 Thymus pulegioides on Candida, Aspergillus and dermatophyte species. J. Med. Microbiol., 702 55, 1367-1373. 703 704 Pirbalouti A.G., Bahmani M., Avijgan M., 2009. Anti- Candida activity of Iranian medicinal plants. Electron. J. Biol., 5, 85-88. 705 Pirbalouti A.G., Barani M., Hamedi B., Kachouei M.A., Karimi A., 2013b. Environment effect on 706 diversity in quality and quantity of essential oil of different wild populations of kerman Thyme. 707 Genetika, 45(2), 441-450. 708 Pirbalouti A.G., Hashemi M., Ghahfarokhi F.T., 2013a. Essential oil and chemical compositions 709 of wild and cultivated Thymus daenensis Celak and Thymus vulgaris L. Ind. Crop. Prod., 48, 710 43-48. 711 712 Pirbalouti A.G., Jahanbazi P., Enteshari S., Malekpoor F., Hamedi B. 2010. Antimicrobial activity of some of the Iranian medicinal plants. Arch. Biol. Sci. Belgrade., 62, 633-642. 713 Pirbalouti A.G., Neshat S.H., Rahimi E., Hamedi B., Malekpoor F., 2014. Chemical composition 714 and antibacterial activity of essentials oils of Iranian herbs against Staphylococcus aureus 715 isolated from milk. Int. J. Food Prop., 17, 2063-2071. 32 716 Pirbalouti A.G., Samani M. R., Hashemi M., Zeinali H., 2013. Salicylic acid affects growth, 717 essential oil and chemical compositions of thyme (Thymus daenensis Celak.) under reduced 718 irrigation. Plant Growth Regul., 1-13. 719 720 Pirbalouti A.G., Zohreh E. B., Fatemeh M., 2015. An overview on genus Thymus. Journal of Herbal Drug., 6(2), 93-100. 721 Pirbalouti, A.G., Rahimmalek, M., Malekpoor, F., Karimi, A., 2011. Variation in antibacterial 722 activity, thymol and carvacrol contents of wild populations of Thymus daenensis subsp. 723 daenensis Celak. P.O.J. 4(4), 209-214 724 Pirbalouti, A.G., Rahimmalek, M., Malekpoor, F., Karimi, A., 2011. Variation in antibacterial 725 activity, thymol and carvacrol contents of wild populations of Thymus daenensis subsp. 726 daenensis Celak. P.O.J. 4(4), 209-214. 727 Pluhár Z., Sárosi S., Pintér A., Simkó H., 2010. Essential oil polymorphism of wild growing 728 Hungarian thyme (Thymus pannonicus) populations in the Carpathian Basin. Nat. Prod. 729 Commun., 5, 1681–1686. 730 Qiu X., Janson C.A., Smith W.W., Green S.M., McDevitt P., Johanson K., Carter P., Hibbs M., 731 Lewis C., Chalker A., Fosberry A., Lalonde J., Berge J., Brown P., Houge‐Frydrych C.S., 732 Jarvest, R.L., 2001. Crystal structure of Staphylococcus aureus tyrosyl‐tRNA synthetase in 733 complex with a class of potent and specific inhibitors. Protein Sci., 10, 2008-2016. 734 Rahimmalek M., Bahreininejad B., Khorrami M., Sayed T. B.E., 2009. Genetic variability and 735 geographical differentiation in Thymus daenensis subsp. daenensis Cleak, an endangered 736 aromatic and medicinal plant as revealed by Inter Simple Sequence Repeat (ISSR) markers. 737 Biochem. Genet., 47, 831-842. 33 738 Rivas L., McDonnell M. J., Burgess C. M., O'Brien M., Navarro-Villa A., Fanning S., Duffy G., 739 2010. Inhibition of verocytotoxigenic Escherichia coli in model broth and rumen systems by 740 carvacrol and thymol. Int. J. Food Microbiol., 1391, 70-78. 741 Rota M.C., Herrera A., Martínez R.M., Sotomayor J.A., Jordán M.J., 2008. Antimicrobial activity 742 and chemical composition of Thymus vulgaris, Thymus zygis and Thymus hyemalis essential 743 oils. Food Control., 19(7), 681–687. 744 Salehi B.; Abu-Darwish M.S.; Tarawneh A.H.; Cabral C.; Gadetskaya A.V.; Salgueiro L.; 745 Hosseinabadi T.; Rajabi S.; Chanda W.; Sharifi-Rad M.; et al. 2019. Thymus spp. Plants-Food 746 applications and phytopharmacy properties. Trends Food Sci. Technol., 85, 287–306. 747 Salem, N., Kefi, S, Tabben, O., Ayed, A., Jallouli, S., Feres, N., Hammami, M., Khammassi, S., 748 Hrigua, I., Nefisi, S., Sghaier, A., Limam, F., Elkahoui S., 2018. Variation in chemical 749 composition of Eucalyptus globulus essential oil under phenological stages and evidence 750 synergism with antimicrobial standards. Ind. Crop. Prod., 124, 115-125. 751 Sarikurkcu C., Sabih Ozer M., Eskici M., Tepe B., Can Ş., Mete E., 2010. Essential oil composition 752 and antioxidant activity of Thymus longicaulis C. Presl subsp. longicaulis var. longicaulis. 753 Food Chem Toxicol., 48(7), 1801-1805. 754 755 Satyal P., Murray B.L., McFeeters R.L., Setzer W.N., 2016. Essential Oil Characterization of Thymus vulgaris from Various Geographical Locations. Foods, 5, 70-81. 756 Shen Q., Zhou W., Li H., Hu L., Mo H., 2016. ROS Involves the Fungicidal Actions of Thymol 757 against Spores of Aspergillus flavus via the Induction of Nitric Oxide. PLoS 758 One. 19;11(5):e0155647. 759 Snoussi, M., Noumi, E., Devasya, P.R., Trabelsi, N., Kanekar, S., Nazzaro, F., Fratianni, F., Fla 760 mini, G., De Feo, V., Al-Sieni, A., 2018. Antioxidant properties and anti-quorum sensing 34 761 potential of Carum copticum essential oil and phenolics against Chromobacterium violaceum. 762 J. Food Sci. Technol. 55(8), 2824-2832. 763 Sonawane, K.D., Barage, S.H. (2015). Structural analysis of membrane-bound hECE-1 dimer 764 using molecular modeling techniques: insights into conformational changes and Abeta1-42 765 peptide binding. Amino Acids., 47(3), 543-559. 766 Sonboli A., Salehi P., Kanani M.R., Ebrahimi S.N., 2005. Antibacterial and antioxidant activity 767 and essential oil composition of Grammosciadium scabridum Boiss. from Iran. Z. Naturforsch 768 C. J. Biosci., 60(7-8), 534-538. 769 Tohidi B., Mehdi R., Ahmad A., (2017). Essential oil composition, total phenolic, flavonoid 770 contents, and antioxidant activity of Thymus species collected from different regions of 771 Iran. Food Chem., 220, 153-161. 772 Tohidi B., Rahimmalek M., Arzani A., Sabzalian M.R., 2020. Thymol, carvacrol, and antioxidant 773 accumulation in Thymus species in response to different light spectra emitted by light-emitting 774 diodes. Food Chem., 307, 125521. 775 Tohidi B., Rahimmalek M., Trindade H., 2019. Review on essential oil, extracts composition, 776 molecular and phytochemical properties of Thymus species in Iran. Ind. Crop. Prod., 134, 89- 777 99. 778 Tombetta D., Castelli F., Sarpietro M. G., Venuti V., Cristani M., Daniele C., Saija A., Mazzanti 779 G., Bisignano G., 2005. Mechanisms of antibacterial action of three monoterpenes. 780 Antimicrob. Agents Chemother., 496, 2474-2478. 781 Tzakou O., Verykokidou E., Rousis V., Chinou I., 1998. Chemical composition and antibacterial 782 properties of Thymus longicaulis subsp. chaoubardii oils: three chemotypes in the same 783 population. J. Essent. Oil Res., 10, 97-99. 35 784 Ulukanli, Z., Cenet, M., Ince, H., and Yilmaztekin, M., 2018. Antimicrobial and Herbicidal 785 Activities of the Essential Oil from the Mediterranean Thymus eigii. J. Essent. Oil Bear. Plants.; 786 21, 214-222. 787 Vladimir-Knežević, S., Kosalec, I., Babac, M., Petrović, M., Ralić, J., Matica, B., Blažeković, B., 788 2012, Antimicrobial activity of Thymus longicaulis C. Presl essential oil against respiratory 789 pathogens. Cent. Eur. J. Biol. 7(6), 1109-1115. 790 791 Wojtunik‐Kulesza K.A., Kasprzak K., Oniszczuk T., Oniszczuk A., 2019. Natural monoterpenes: Much more than only a scent. Chem. Biodivers., 16 (12), e1900434. 792 World Checklist of Selected Plant Families (2010), copyright © The Board of Trustees of the 793 Royal Botanic Gardens, Kew. Thymus musilii Velen.. Accessed through: Euro+Med PlantBase 794 at http://ww2.bgbm.org/euroPlusMed/PTaxonDetail.asp?UUID=B7F1730F-E243-4A96- 795 9592-12FB15BFDE1A 796 797 Zarshenas M.M., Krenn L. A., 2015. Critical overview on Thymus daenensis Celak: phytochemical and pharmacological investigations. J. Integr. Med.; 13(2), 91–98. 36 (A ) (B ) Figure 1. specimen. flowering of white leaves. (C ) T. musilii Velen (A): whole plant at stage, (B): clusters flowers, (C): green . 37 Figure 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. A b u n d a n c e T IC : Z A A T A R 1 5 . 71 37 8. 3 3 7 R E P -3 . D \ d a t a . m s 9 5 0 0 0 0 0 1 9 0 0 0 0 0 0 8 5 0 0 0 0 0 8 0 0 0 0 0 0 7 5 0 0 0 0 0 7 0 0 0 0 0 0 6 5 0 0 0 0 0 3 6 0 0 0 0 0 0 5 5 0 0 0 0 0 5 0 0 0 0 0 0 8 .3 4 5 4 5 0 0 0 0 0 2 4 0 0 0 0 0 0 Figure 3. Chromatogram obtained for T. musilii Velen essential oil. The main components identified are: 1 (Thymol), 2 (Thymol Acetate), and 3 (o-cymene). 3 5 0 0 0 0 0 1 5 .9 2 8 3 0 0 0 0 0 0 9 .2 6 5 2 5 0 0 0 0 0 2 0 0 0 0 0 0 1 9 .0 8 6 1 0 .3 6 0 1 5 0 0 0 0 0 1 0 0 0 0 0 0 5 0 0 0 0 0 7 .8 4 .4163 5 5 .9 2 8 6 .0 9 8 8 .5 2 9 5 .0 0 1 21. 2 2 .4923 2 1 2 .5 6 2 1 0 .0 0 1 5 .0 0 1 7 .7 9 4 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 38 C. albicans ATCC 10231 (B ) C. neoformans ATCC 14116 A. fumigatus ATCC 204305 (A ) (C ) K. pneumoniae ATCC 27736 Candida sp. (Clinical strain) C. vaginalis (Clinical strain) A. niger Figure 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. 39 Table 1. Chemical composition of T. musilii Velen essential oil. Peak # RI* on HP-5MS column 1 931 α-thujene C10H16 2 939 α-pinene C10H16 3 992 β-myrcene C10H16 4 1018 α-terpinene C10H16 5 1026 p-cymene C10H14 Compounds 6 1033 1,8-cineole C10H18O 7 1062 γ-terpinene C10H16 8 1087 α-terpinolene C10H16 9 1165 Borneol C10H18O 10 1174 Terpinen-4-ol C10H18O 11 1189 α-terpineol C10H18O 12 1227 2-Isopropyl-5-methylanisole C11H16O 13 1290 Thymol C10H14O 14 1292 Carvacrol C10H14O 15 1356 Thymyl acetate C12H16O2 Carvacryl acetate C12H16O2 β-caryophyllene C15H24 16 1367 17 1404 Chemical classes Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpenes hydrocarbons Total compounds Identified (%) Percentage (Mean±SD) Chemical formula 0.437±0.015 0.303±0.015 0.710±0.034 0.853±0.028 4.617±0.119 0.397±0.005 2.633±0.072 1.460±0.081 0.763±0.030 0.390±0.017 0.890±0.036 0.080±0.138 67.697±0.938 3.417±0.105 12.993±0.221 0.383±0.015 1.953±0.102 11.013±0.039 87.010±0.279 1.953±0.005 100 RI: Retention index on a HP-5MS column. The data are expressed as mean±SD (n=3); SD: Standard Deviation; 40 Table 2. Growth inhibition zone, MIC and MBC values obtained for bacterial strains tested using disc diffusion and microdilution assays. Code B1 B2 B3 Strain E. coli ATCC 35218 P. aeruginosa ATCC 27853 Proteus mirabilis ATCC 29245 B4 K. pneumoniae ATCC 27736 B9 S. aureus MDR (Clinical strain) B10 E. cloacae (Clinical strain) T. musilii Velen essential oil Mean±SD MBC/MIC MICa MBCb * ratio (mm) 35.33±1.15 12.5 50 4 Main Compound (Thymol) Mean±SD (mm) 100 >4 7±0a 2 6±0a b 21.33±1.52 25 b 36.33±1.15 12.5 25 2 12.5 25 2 6±0a 2 8.66±1.15b c 25.33±1.15 31.00±1.00 3.125 c 50 4 6.25 2 6.25 2 7.33±0.57a 0.78 6.25 2 6.66±0.57a 3.125 b 6.25 6.33±0.57a 3.125 9±1b Mean±SD (mm) 7.33±0.57a 12.5 12.5 MBC/MIC ratio 7±0a 3.125 12.5 MBC 12.66±0.57b c 35.33±1.15 MIC Ampicillin 1.56 2 6.66±0.57a 0.39 0.78 2 *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). 41 Table 3. Growth inhibition zone, MIC and MFC values obtained for fungal and yeast strains tested using disc diffusion and microdilution assays. Code Y1 Y2 Y3 Y4 M1 Strain C. albicans ATCC 10231 C. neoformans ATCC 14116 C. vaginalis (Clinical strain) Candida sp. (Clinical strain) A. fumigatus ATCC 204305 A. niger T. musilii Velen essential oil Main Compound (Thymol) Mean±SD* MFC/MIC MIC MFC (mm) ratio Mean±SD* MFC/MIC MIC MFC (mm) ratio 34.00±1.00c 36.66±1.15c 37.33±1.15c 37.33±1.15b 88.66±1.15c 6.25 25 6.25 - 12.5 12.5 - 8 22.66±1.15b 2 12±1a 6.25 Mean±SD* (mm) 4 13.66±0.57a 12.5 100 3.125 6.25 Amphotericin B (10 mg/ml) 50 100 2 15.33±0.57b 12.66±0.57b 25 100 4 6.66±0.57a 11.66±0.57a 25 b 82.66±2.31 74.33±0.57b - 100 - 4 - 12.33±0.57a 2 2 - 15.00±1.00a M2 87.33±1.15c 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). 42 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 T. musilii Velen Test System ABTS β- carotene IC50 (mg/mL) IC50 (mg/mL) 5.6x10-4 ±2x10-5 a 3.2x10-3 ±5x10-4 a DPPH IC50 (mg/mL) 0.049 ±1x10-4 b BHT 0.023±3x10-4a 0.018±4x10-4b 0.042±3.5x10-3c 0.05±3x10-3 a Ascorbic Acid 0.022± 5x10-4 a 0.021±1x10-3 b 0.017 ±1x10-3 b 0.09±7x10-3 b FRAP IC50 (mg/mL) >1 c 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. 43 Thymus species T. vulgaris L. Origin Main Components Bacteria and Fungi tested Reference Yemen B. subtilis, S. aureus, S. epidermidis, P. Thymol (51.34%), p-cymene (18.35%), ßaeruginosa, E. coli, Mycobacterium caryophyllene (4.26%). smegmatis, C. albicans and C. vaginalis. Al Maqtari et al., 2011 Romania S. aureus ATCC 25923, P. aeruginosa ATCC 27853, S. Typhimurium ATCC 14028, E. coli Thymol (47.59%), γ-terpinene (30.90%) and pATCC 25922, K. pneumoniae ATCC 13882, cymene (8.41%). E. faecalis ATCC 29212 and C. albicans ATCC 10231 Borugă et al., 2014 C. albicans ATCC 10234, C. glabrata, C. Thymol (49.1%), p-Cymene (20%), carvacrol krusei, C. tropicalis ATCC 750, P. aeruginosa, (3.5%), α-thujene (1.9%), α-pinene (1.2%), ß- E. faecalis, S. sanguinis, S. salivarius, S. Balkan mycrene (1.3%), trans-ß-ocimene (1.4%), γ- mutans, L. acidophilus, S. aureus. Peninsula Terpinene (4.2%), borneol (1.7%), terpinene-4-ol (2%), ß-caryophyllene (3.7%), δ-cadinene (2.3%). Nikolic et al., 2014 Italy S. aureus ATCC 25923, E. faecalis ATTC 29212, B. cereus ATCC 1177, B. subtilis Thymol (46.2-67.5%), caryophyllene oxide (2.2- ATCC 6633, E. coli ATCC 25922, P. 7.3%), geranyl propanoate (0-2.2%), linalool aeruginosa ATCC 27853, S. epidermidis (0.3-2.7%), trans-myrtanol (0-2.3%), citronellyl ATCC 12228, K. pneumoniae ATCC 10031, S. formate (0-2.5%), ethyl-2-octynoate (0-1.8%). typhi Ty2 ATCC 19430 and P. vulgaris ATCC 13315. Mancini et al., 2015 France Thymol (47.06%), p-cymene (20.07%), γ- C. albicans ATCC 18804, Cryptococcus terpinene (9.03%), linalool (5.00%), carvacrol neoformans 24067 (serotype D or var. (3.24%). neoformans), Aspergillus niger ATCC 16888. Satyal et al., 2016 44 T. longicaulis C. Presl Thymol (55.44±0.62%), m-Cymene Republic (11.88±0.32%), γ-Terpinene (5.74±0.20%), oCymen-5-ol (5.14±0.19%), ß-caryophyllene of Moldova (1.53±0.07%), Terpinen-4-ol (1.04±0.04%), 2Carene (1.04±0.04%). Thymyl acetate (0-12.8%), t-Cadinol (0.3-9.2 %), p-cymene (0.4-9.0 %), ß-caryophyllene (2.25.7%), γ-terpinene (0.9-5.5%), Germacrene D Italy (5.3 %), thymol (6.4-9.3%), thymol methyl ether (0.8-5.5%), carvacrol (0-12.8%), Carvacryl acetate (0-13.6%). Thymol (46.3%), δ-3-Carene (1.6%), p-Cymene (9.4%), γ-terpinene (16.2%), linalool (1.4%), Balkan Peninsula borneol (2.2%), thymyl methyl ether (11.4%), βCaryophyllene (2.1%), carvacrol (1.4%). T. pulegioides L. Italy Iran T. daenensis Celak. Iran T. capitatus L. Algeria A. flavus MUCL 19006 Aprotosoaie et al., 2019 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 H. influenzae, N. meningitidis, S. aureus, S. pneumoniae, S. pyogenes, C. albicans Vladimir-Knezˇevic et al. (2012) S. aureus ATTC 25923, S. faecalis ATTC Thymol (21.8-26.3%), p-cymene (17.6-19.9%), 29212, B. subtilis ATCC 6633, B. cereus PCI linalool (4.7-5.6 %), ß-caryophyllene (5.9-7.5%), De Martino et al., 213, P. mirabilis ATCC 12453, E. coli ATCC thymol methyl ether (6.0-10.8%), carvacrol (3.12009 25922, S. typhi Ty2 ATCC 19430 and P. 4.7%). aeruginosa ATCC 27853. Thymol (3.8-78.3%), ρ-cymene (2.7-11.6%), caryophyllene (2.1-5.6%), methyl carvacrol (2.9L. monocytogenes, S. aureus, S. iniae, E. coli, 4.9%), g-terpinene (2.5-12.9%), geraniol (0P. aeruginosa, K. pneumonia, H. pylori, A. Zarshenas and Krenn, 3.4%), α-humulene (0-3.2%), carvacrol (22015 niger, A. fumigatus, C. albicans and S. 15.2%), γ-terpinene (3.9-12.9%), cerevisiae. aromadendrene (0-3.9%), carvacrol methyl ether (3.4-4.27%), δ-terpinene (0-4.3%). α-pinene (0.51%), 1,8-cineole (0.58%), γPirbalouti et al., 2009 terpinene (5.74%), linalool (0.52%), thymol C. albicans vaginal, E. coli O157:H7; B. Pirbalouti et al., 2010 (74.32%), carvacrol (4.31%), trans-caryophyllene cereus, L. monocytogenes and S. aureus. Pirbalouti et al., 2014 (3.56%), caryophyllene oxide (0.42%). Thymol (51.22%), carvacrol (12.59%), γ- E. coli, S. typhi, S. aureus, S. pneumoniae, Goudjil et al., 2020 terpinene (10.3%), trans-13-octadecenoic acid Cladosporium herbarum, Alternaria 45 Tunisia T. cappadocicus Boiss. T. striatus T. algeriensis Boiss. and Reut T. numidicus Poiret Turkey (9.04%), linalool (2.29%), caryophyllene infectoria, A. ochraceus, and Trichophyton sp. (2.01%), pentadecanoic acid (1.92%), α-terpinene (1.78%), ß-myrcene (1.49%), caryophyllene oxide (1.21%). E. coli ATCC 8739, S. typhimurium NCTC Thymol (69.95-81.49%), α-cubebene (0-3.44%), 6017, S. aureus ATCC 29213, P. aeruginosa β-ocimene (3.09-3.16%), carvacrol (0-2.56%), α- ATCC 27853, A. hydrophila, L. Aouadhi et al., 2013 terpinene (2.25-3.83%). monocytogenes ATCC 7644, B. cereus, A. flavus, A. niger and C. albicans. S. aureus CIP7625, L. monocytogenes Scott A 724, E. coli ATCC 10536, K. pneumoniae Thymol (89.06%), p-cimene (5.04%), γ-terpinene CIP8291, S. cerevisiae ATCC 4226, C. Mkaddem et al., 2010 (3.19%). albicans IPA 200, M. ramamnianus ATCC 9314, A. westerdijkiae NRRL 3174. A. hydrophila, E. coli, M. morganii, K. pneumoniae, P. mirabilis, P. aeruginosa, S. typhimurium, Y. enterocolitica, B. brevis, B. Albayrak and Aksoy, Thymol (70.82%), cymene (9.52%), g-terpinene cereus, B. subtilis, L. monocytogenes, S. (9.27%). 2012 aureus, C. albicans and S. cerevisiae. A. alternata, A. niger, A. ochraceus, A. Thymol (59.5%), γ-terpinene (11.6%), p-cymene versicolor, A. flavus, A. terreus, C. Balkan (6.4%), carvacrol-methyl ether (5.9%), carvacrol cladosporioides, P. funiculosum, P. helianthi, Peninsula (4.9%), α-terpinene (3.3 %), E-caryophyllene T. viride, T. mentagrophytes, M. canis, and E. (2.3%). floccosum Thymol (36%), carvacrol (14%), α-pinene C. albicans ATCC 10234, C. glabrata, C. (1.1%), ß-mycrene (2.3%), p-cymene (6.3%), krusei, C. tropicalis ATCC 750, P. aeruginosa, Balkan ß-bisabolene (4%0, α-terpinene (1.6%), γ- E. faecalis, S. sanguinis, S. salivarus, S. Peninsula terpinene (4.8%), linalool (1.3%), camphor mutans, L. acidophilus, S. aureus. (1.1%), caryophyllene oxide (1%). Algeria Thymol (40.40%), carvacrol (13.37%), thymol S. aureus ATCC 25923, E. coli, P. aeruginosa methyl ether (8.30%), β-myrcene (2.37%), p- ATCC 27853, C. albicans. Couladis et al., 2004 Nikolic et al., 2014 Messara et al., 2017 46 T. zygis T. serpillum L. T. lanceolatus T. linearis Benth. cymene (7.18%), γ-terpinene (6.41%), linalool (4.06%), β-caryophyllene (2.48%), β-bisabolene (3.26%). 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 S. aureus ATCC 6538, E. coli ATCC 8739, P. (448.5±22.4-1462.8±38.2 mM), linalool aeruginosa ATCC 9027, C. albicans ATCC Spain (223.6±2.8-386.8±13.6 mM), terpinen-4-ol 10231. (8.9±0.3-45.7±0.3 mM), thymol (1923.2±27.53636.2±15.2 mM), carvacrol (34.3±1.3112.9±2.5 mM), E-β-caryophyllene (24.1±0.350.4±1.0 mM) C. albicans ATCC 10234, C. glabrata, C. Thymol (38.5%), carvacrol (4.7%), α-pinene krusei, C. tropicalis ATCC 750, P. Balkan (2%), camphene (2.4%), γ-terpinene (7.2%), aeruginosa, E. faecalis, S. sanguinis, S. Peninsula linalool (2.4%), borneol (6%), thymol methyl salivarius, S. mutans, L. acidophilus, S. ether (3.8%), thymol acetate (2.8%). aureus. S. aureus ATCC 29213, S. epidermidis ATCC 14990, S. capitis ATCC 35661, S. pyogenes Thymol (69.61%), γ-terpinene (8.38%), p- ATCC 12344, S. agalactiae ATCC 27956, cymene (5.07%), carvacrol (3.57%), α-terpinene Bacillus subtilis ATCC 6051, P. fluorescens Algeria (1.31%), linalool (1.01%), β-mycrene (1.72%), ATCC 13525, S. typhimurium ATCC 14028, S. α-thujene (1.07%), α-pinene (0.73%), d-limonene flexneri ATCC 700930, E. coli ATCC 25922, A. fumigatus ATCC 1022, Geotrichum (0.62%), β-pinene (0.43%). candidum ATCC 12784, S. racemosum ATCC 14831, C. albicans (ATCC 90028). Thymol (54.9%), γ-terpinene (16.6%), p-cymene S. aureus MRSA 33591, S. epidermidis MRSE (5.2%), α-thymol methyl ether (3.2%), terpinene 51625, S. aureus MRSA (BAA-44), S. aureus (2.6%), thymyl acetate (2.8%), β-bisabolene MTCC-96, S. epidermidis MTCC-435, E. India (2.3%), (E)-caryophyllene (2.0%), myrcene faecalis MTCC-439, C. albicans ATCC (1.8%), α-thujene (1.6%), carvacrol (1.5%), 14053, C. tropicalis ATCC 2013180, C. borneol (1.1%). glabrata ATCC-15126. Cutillas et al., 2018 Nikolic et al., 2014 Khadir et al., 2016 Kumar et al., 2020 47 T. kotschyanus Iran T. eigii Turkey T. willdenowii Boiss & Reut Morocco T. musilii Velen Saudi Arabia α-pinene (5.49-12.72%), β-Myrcene (0.801.51%), α-terpinene (1.62-1.80%), p-cymene (021.35%), m-cymene (0-8.87%), 1,8-cineole (4.57-4.79%), γ-terpinene (4.00-8.01%), 4terpineol (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 (0.153.30%), caryophyllene (1.27-2.92%). Thymol (24.77%), carvacrol (14.00%), p-cymene (10.91%), γ-terpinene (6.53%), borneol (6.48%), caryophyllene (3.92%), α-pinene (2.03%), αthujene (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%). 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. 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 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. E. coli ATCC 25922, P. mirabilis ATCC 35659, B. cereus ATCC 10876, C. albicans ATCC 10231, A. brasilliensis ATCC 16404. Ouknin et al., 2019 E. coli ATCC 35218, P. aeruginosa ATCC 27853, P. mirabilis ATCC 29245, K. Thymol (67.69±0.93%), thymyl acetate pneumoniae ATCC 27736, S. aureus MDR, E. (12.99±0.22%), p-cymene (4.61±0.11%), cloacae, C. albicans ATCC 10231, Carvacrol (3.41±0.10%), γ-terpinene Cryptococcus neoformans ATCC 14116, C. (2.63±0.07%). vaginalis, Candida sp., A. fumigatus ATCC 204305 and A. niger. This study 48 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 Bacillus subtilis 422.332 ppm Bacillus licheniformis 422.811 ppm Lactobacillus curvatus 723.45 ppm Falcone et al. 2005 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 Trombetta et al., 2005 Escherichia coli ATCC 15221 5.00 mg/mL Candida albicans ATCC 10231 0.16 µl/mL 0.32 µl/mL 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 Pinto et al., 2006 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 0.32µl/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 Escherichia coli N00-666 2.5 mM Palaniappan and Holley, 2010 Staphylococcus aureus blaZ+ 2.5 mM Streptococcus pyogenes ermB+ 0.31mM Escherichia coli O157:H7 500-1000 µg/mL 1000-2000 µg/mL 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 Rivas et al., 2010 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 49 Hafnia alvei Staphylococcus aureus Lactobacillus sakei Pseudomonas putida Bacillus thermosphacta Streptococcus mutans MTCC 890 Staphylococcus aureus MTCC 96 Bacillus subtilis MTCC 121 Staphylococcus epidermidis MTCC 435 Escherichia coli MTCC 723 Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus Bacillus cereus Micrococcus luteus Phytophthora infestans Phytophthora ultimum Botrytis cinerea Rhizoctonia solani Aspergillus niger Aspergillus fumigatus Aspergillus flavus Aspergillus ochraceus Alternaria alternata Botrytis cinerea Cladosporium spp. Penicillium citrinum Penicillium chrysogenum Fusarium oxysporum Rhizoctonia oryzae Escherichia coli Clostridium perfringens Salmonella Typhimurium Salmonella Enteritidis Salmonella Pullorum Lactobacillus acidophilus Lactobacillus reuteri Lactobacillus salivarius Pythium insidiosum Helicobacter pylori Mycobacterium tubercolosis Mycobacterium bovis Candida albicans 500 µg/mL 500 µg/mL 1000 µg/mL 1000 µg/mL 125 µg/mL 62.5 µg/mL 125 µg/mL 125 µg/mL 250 µg/mL < 0.019-0.039 mg/mL <0.019-0.039 mg/mL < 0.019-156 mg/mL < 0.019-0.156 mg/mL 500 µg/mL 500 µg/mL 2000 µg/mL 2000 µg/mL 250 µg/mL - Pirbalouti et al., 2011 Hernàndez-Hernàndez et al., 2013 1250 µg/mL 400.26 µl/l 263 µl/l >600 µl/l 64.56 µl/l 100 mg/mL 150 mg/mL 100 mg/mL 100 mg/mL 100 mg/mL 100 mg/mL 100 mg/mL 100 mg/mL 100 mg/mL 100 mg/mL 100 mg/mL 187.5 μg/mL 375 μg/mL 375 μg/mL 750 μg/mL 375 μg/mL 1500 μg/mL 1500 μg/mL 1500 μg/mL 160-320 µg/mL 0.043 ± 0.024 µl/mL 0.75 µg/mL 2.02 µg/mL 39 µg/mL Mathela et al., 2010 375 μg/mL 750 μg/mL 750 μg/mL 1500 μg/mL 750 μg/mL 3000 μg/mL 3000 μg/mL 3000 μg/mL - Ben Jabeur and Hamada, 2014 Abbaszadeh et al., 2014 Du et al., 2015 Jesus et al., 2015 Falsafi et al., 2015 Andrade-Ochoa et al., 2015 De Castro et al., 2015 50 Candida krusei Candida tropicalis Aspergillus flavus CGMCC 32890 Bacillus cereus Salmonella Typhimurium Escherichia coli Staphylococcus aureus Cronobacter sakazakii lv27 Cronobacter malonaticus lv31 Cronobacter muytjensii s50 Cronobacter turicensis lv53 Cronobacter condimenti s37 Escherichia coli ATCC 35218 Pseudomonas aeruginosa ATCC 27853 Proteus mirabilis ATCC 29245 Klebsiella pneumoniae ATCC 27736 Staphylococcus aureus MDR Enterobacter cloacae Candida albicans ATCC 10231 Cryptococcus neoformans ATCC 14116 Candida vaginalis (Clinical strain) Candida sp. (Clinical strain) 39 µg/mL 78 µg/mL 80 μg/mL 0.007 mg/mL 0.003 mg/mL 0.007 mg/mL 0.007 mg/mL 0.05 % 0.05 % 0.05 % 0.05 % 0.05 % 3.125 mg/mL 12.5 mg/mL 3.125 mg/mL 3.125 mg/mL 0.78 mg/mL 0.39 mg/mL 12.5 mg/mL 50 mg/mL 25 mg/mL 25 mg/mL 0.12 mg/mL 0.12 mg/mL 0.12 mg/mL 6.25 mg/mL 50 mg/mL 6.25 mg/mL 6.25 mg/mL 1.56 mg/mL 0.78 mg/mL 100 mg/mL 100 mg/mL 100 mg/mL 100 mg/mL Shen et al., 2016 Guimarães et al., 2019 Berthold-Pluta et al., 2019 This study 51 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. 52 Table 8. Interacting active site residues of receptors with natural bio-compounds. 2D interactions, Receptor Ligand Interactions, Distance in Angstroms 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 (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 (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°. 3D interaction Receptor–Ligand Receptor – Ligand: 1JIJ – β-Caryophyllene Receptor – Ligand: 1XFF – β-Caryophyllene Receptor – Ligand: 2XCT – β-Caryophyllene 53

RELATED PAPERS

RELATED TOPICS

Log In

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

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

Related Papers

RELATED PAPERS

RELATED TOPICS