Thymus musilii Velen. Methanolic Extract: In Vitro and In Silico Screening of Its Antimicrobial, Antioxidant, Anti-Quorum Sensing, Antibiofilm, and Anticancer Activities

Thymus musilii Velen. is a rare plant species cultivated in the Ha’il region (Saudi Arabia) under greenhouse conditions. In this work, we described, for the first time, the phytochemical composition, antimicrobial, antioxidant, anti-quorum sensing, and anticancer activities of T. musilii methanolic extract using both experimental and computational approaches. The obtained results showed the identification of eight small-like peptides and eighteen phyto-compounds by using high-resolution liquid chromatography–mass spectrometry (HR-LCMS) dominated mainly by compounds belonging to isoprenoid, fatty acyl, flavonoid, and alkaloid classes. The tested extracts exhibited high antifungal and antibacterial activity with the mean diameter of growth inhibition zones ranging from 12.33 ± 0.57 mm (Pseudomonas aeruginosa ATCC 27853) to 29.33 ± 1.15 mm (Candida albicans ATCC 10231). Low minimal inhibitory concentrations were recorded for the tested micro-organisms ranging from 0.781 mg/mL to 12.5 mg/mL. While higher doses were necessary to completely kill all tested bacterial and fungal strains. Thyme extract was able to scavenge DPPH•, ABTS•+, β-carotene, and FRAP free radicals, and the IC50 values were 0.077 ± 0.0015 mg/mL, 0.040 ± 0.011 mg/mL, 0.287 ± 0.012 mg/mL, and 0.106 ± 0.007 mg/mL, respectively. The highest percentage of swarming and swimming inhibition was recorded at 100 µg/mL with 39.73 ± 1.5% and 25.18 ± 1%, respectively. The highest percentage of biofilm inhibition was recorded at 10 mg/mL for S. typhimurium ATCC 14028 (53.96 ± 4.21%) and L. monocytogenes ATCC 7644 (49.54 ± 4.5 mg/mL). The in silico docking study revealed that the observed antimicrobial, antioxidant, and anticancer activities of the constituent compounds of T. musilii are thermodynamically feasible, notably, such as those of the tripeptides (Asn-Met-His, His-Cys-Asn, and Phe-His-Gln), isoprenoids (10-Hydroxyloganin), and diterpene glycosides (4-Ketoretinoic acid glucuronide).


Introduction
The global rise in infectious diseases due to antibiotic-resistant strains of bacteria is becoming a global health problem, and, in turn, potentially profound and complicated ailments, such as diabetes, cardiovascular diseases, and cancer, lead to significant causes of death and morbidity worldwide [1][2][3]. The growing resistance of microbes against commercial drugs is one of the main reasons of concern for both researchers and clinicians. Besides that, the side effects in the host caused by antibiotics, especially when increasing the dosage of the drug to suppress infections, induce allergic reactions, diarrhea, nausea, and drug-drug interactions [4][5][6]. Thus, to encompass the ongoing spread of antimicrobial resistance as well as the excessive and unregulated use of antibiotics, scientists have been motivated to search for new agents and alternative antibiotics to combat bacterial pathogens. One of the most ideal approaches to fight bacterial infections is to eliminate virulence factors that are often involved in pathogenicity and infections [7,8].
The genus Thymus belongs to the Lamiaceae family and is very popular due to its large use as a remedy in folk medicine and as a condiment, mainly in the Mediterranean zone. The Kingdom of Saudi Arabia has counted at least three species of Thymus, namely, T. bovei Benth., T. decussatus Benth., and T. musilii Velen. Additionally, T. vulgaris is largely cultivated in many provinces of the kingdom. In this study, we focused on the T. musilii Velen. species, which is largely distributed in Iraq, Palestine, and Saudi Arabia [9]. Traditionally, local Saudi Arabians used the leaves and flowers of this plant as a garnish, added it to a variety of foods or for preparing infusion tea, as well as to cure several microbial diseases. Thyme is widely cultivated in Mediterranean countries, where it is exported to several countries across the world, whether as dried leaves or in the form of volatile oils. Despite the economic and environmental importance of this plant, it is considered one of the endangered plants of several environments in the Ha'il region.
Medicinal plants are a rich reservoir of bioactive molecules, having specific biochemical and organoleptic characteristics with an imminent potential to promote health and to be explored as promising therapeutic drugs [10][11][12][13][14][15][16]. In fact, it was previously demonstrated that T. musilii essential oil is a rich source of oxygenated monoterpenes (87.010 ± 0.279%), monoterpene hydrocarbons (11.013 ± 0.039%), and sesquiterpene hydrocarbons (1.953 ± 0.005%). The same authors reported that the same T. musilii essential oil was dominated by thymol (67.697 ± 0.938%), thymyl acetate (12.933 ± 0.221 %), and p-cymene (4.617 ± 0.0.119 %) [16]. Bioactive plant-based products have been recognized as a useful resource that may lead to the discovery of new antimicrobial substances with possibly new mechanisms of action. Extensive work has been conducted exploring the effect of various extracts and essential oils of plants in traditional medicine, with a great interest in these compounds as alternative remedies to cure various ailments [16,17]. Additionally, infectious diseases are responsible for many diseases in humans, including cardiovascular and neurodegenerative diseases due to an imbalance of redox states, involving either the excessive generation of reactive oxygen species (ROS) or the dysfunction of the antioxidant system [18,19].
Traditionally, in natural chemistry fields, researchers used to isolate and characterize chemical constituents and evaluate them for their biological activities. Molecular docking has become an increasingly important tool for computed drug discovery [20]. The docking method could be used to simulate the interaction between a small molecule and a protein at the atomic level, allowing us to assess small molecule behavior in target protein binding sites, as well as explain fundamental biochemical processes [21,22]. The biological effect of a plant extract is recognized in phytochemical screening, but it cannot indicate which phyto-constituent exerts an action. As a result, in silico docking studies are essential for comprehending the interaction and affinity of identified phytochemicals with biological targets [16,[22][23][24]. Hence, it could be deduced that in silico analyses usually mirror or confirm the in vitro and in vivo findings and confirm the biological effects of the assessed compounds [25]. Hence, the aim of this study was to investigate, for the first time, the chemical composition of organic extracts from T. musilii Velen. cultivated in the Ha'il region by using the HR-LCMS technique to study its antioxidant activities. The ability of T. musilii plant extracts to kill pathogenic micro-organisms using disc diffusion and microdilution assays and to attenuate the secretion of some Pseudomonas aeruginosa virulence-related properties controlled by the quorum sensing system were also studied. The effect on colon, breast, and lung cancer cells were also investigated. Moreover, ADME profiles and molecular docking targeting selected proteins were also analyzed.

Plant Material Sampling
The plants used in this study were collected in October 2019 from a nursery belonging to the Ministry of Agriculture in the region of Ha'il (Al-Gaad, Ha'il, Saudi Arabia). A voucher specimen (AN 001) was deposited in the Department of Biology, at the University of Ha'il, Saudi Arabia [16]. Methanolic extract was performed by using the protocol previously described by Haddaji et al. [23]. For the experiment, the powdered flowering aerial parts of T. musilii (4 g) were mixed with pure methanol (40 mL) in a dark glass bottle with agitation for three days at room temperature. Then, the crude extract was filtrated by using Whatman filter paper (No. 1). The methanolic extract was then removed using a rotary evaporator (BUCHI Labortechnik GA, CH-9230 Flawil 1, Switzerland). Finally, the concentrated extract was stored at 4 • C until the actual experiment was conducted. The yield of extraction was calculated after three running cycles and expressed according to the dry weight.

Quantification of Total Phenols, Total Flavonoids, and Total Condensed Tannins
Total phenol content (TPC) was estimated according to the Folin-Ciocalteu method, as described by Kumar et al. [26]. An aliquot of 100 µL from T. musilii stock solution (1 mg/mL) was added to 750 µL of Folin-Ciocalteu reagent (Sigma Aldrich, Germany). The liquid mixture was allowed to stand for 5 min before 750 µL of saturated Na 2 CO 3 solution was added. After 90 min of incubation in the shade and at room temperature, absorbance was recorded at 725 nm using a UV-Vis spectrophotometer (Agilent Cary 60 UV-Vis, USA). The obtained data were expressed as milligram (mg) of gallic acid equivalents per gram of plant extract (mg EGA/g extract). Ten milligrams of gallic acid were dissolved in methanol-50% (100 µg/mL) and then further diluted to 0.78, 1.56, 3.125, 6.25, 12.5, or 25 µg/mL. The quantification was completed based on the standard calibration extinction curve of gallic acid concentrations (y = 6.9326 x − 0.0141; R 2 = 0.9981).
Total flavonoid content (TFC) was determined using the Aluminum chloride (AlCl 3 ) method developed by Benariba et al. [27]. For the experiment, 1.5 mL of T. musilii extract (1 mg/mL) was mixed with 1.5 mL of AlCl 3 -6H 2 O solution (2%). The mixture was vortexed gently and incubated for 10 min at room temperature. Absorbance was recorded at 367 nm against the blank (the same mixture without the sample). Total flavonoid amounts were expressed as milligram (mg) of quercetin equivalents per gram of plant extract (mg QE/g Ext) for each organ. The standard calibration curve of quercetin was conducted (dilutions about 1.56, 3.125, 6.25, 12.5, or 25 µg/mL). Quantification was completed based on quercetin standard curve (y = 12.234 x − 0.0894; R 2 = 0.9997).
Total tannin content (TTC) was determined by a colorimetric method using modified vanillin assay [28]. Then, 3 mL of 4% methanolic vanillin solution and 1.5 mL of concentrated H 2 SO 4 were added to 50 µL of extract (1 mg/mL). The mixture was allowed to stand for 15 min, and the absorbance was measured at 500 nm against methanol or water as blank. Total condensed tannin concentration was expressed as mg catechin/g dry weight (mg CE/mg). All samples were analyzed in three replications. The standard calibration curve of catechin was conducted (dilutions about 0.039, 0. 78 [29] for the determination of the diameter of growth inhibition zone estimated on agar medium. The optical density of all bacterial suspensions was adjusted, and bacterial cell density was about 1 × 10 7 UFC/ml (OD 600 = 0.1). While the cell density of the tested yeast strains diluted in glass tubes containing sterile saline (0.8% NaCl) was adjusted to 1 × 10 7 UFC/ml (OD 540 = 0.5) [30]. Using a sterile cotton swab, each tested micro-organism was spread on the surface of agar media (Mueller-Hinton for bacteria and Sabouraud Chloramphenicol agar for fungi). Sterile 6 mm discs (Biolife, Milan, Italy) were impregnated with 10 µL/disc from a stock solution of methanolic extract (200 mg/mL) and deposited in triplicate on the surface of each inoculated plate. All Petri dishes were incubated overnight at 37 • C, and the mean diameter of growth inhibition zone was recorded and expressed as mGIZ ± SD (mm).
To estimate the minimal inhibitory concentrations (MIC values expressed in mg/mL) and minimal bactericidal/fungicidal concentrations (MBC and MFC values), the obtained extract was serially diluted in DMSO-5% supplemented with 0.025% tween 80 (from 100 mg/mL to 0.097 mg/mL) in 96-well microtiter plates containing 95 µL of the microbial suspension and 95 µL of the enrichment broth (Lauria-Bertani for bacteria and Sabouraud dextrose broth for fungal strains). MIC concentration is estimated visually and is defined as the lowest concentration needed to inhibit the growth of each tested micro-organism in the wells. While MBC and MFC concentrations are defined as the lowest concentration needed to kill 99.99% of the tested micro-organisms and are confirmed by point-inoculating MH and SC media from wells with concentrations higher than the recorded MIC value for each micro-organism tested [30].

Antioxidant Activities
The ability of T. musilii extracts to scavenge the DPPH • stable free radicals was determined following the same method as previously reported by Mseddi et al. [16]. The method of Koleva et al. [31] was used for β-Carotene bleaching test and ferric reducing antioxidant power [32], and the Oyaizu method for the determination of reducing power was used [33]. The radical-scavenging activity of the extracts against ABTS + (2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) radical cations was measured using the same protocol described by Hamdi et al. [28].

DPPH Radical-Scavenging Activity Assay
For the experiment, a stock solution (20 mg/mL) from T. musilii extract and the standard (stock solution 1 mg/ml) at different concentrations were pipetted in separate test tubes. A volume of 0.5 mL of each sample and standard was mixed with the same volume of DPPH• methanolic solution. The mixture was shaken vigorously and allowed to stand for 30 min in darkness and at a temperature of 25 • C. The absorbance of the resulting solution was measured at 520 nm with a spectrophotometer. All measurements were performed in triplicate. A mixture of 0.5 mL of DPPH• solution and 0.5 mL of methanol were taken as a control. Pure methanol was taken as a blank. Inhibition of free radical DPPH• as a percentage (PI%) was calculated by the following equation (Equation (1)): where A Control and A Sample are the absorbances of the control solution and of a test sample or standard, respectively. The IC 50 corresponding to the concentration needed to scavenge 50% of DPPH• free radicals were estimated from the dose-response curve by using Graph-Pad 8 software (San Diego, CA, 92108-2711 USA).

ABTS Radical-Scavenging Activity Assay
The antiradical assay was performed using 2,2 -azino-bis (3-ethylbenzthiazoline-6sulphonic acid), commonly called the ABTS cation scavenging activity test. The radical monocation of ABTS was generated by reacting ABTS solution (7 mM) with 2.45 mM K 2 S 2 O 8 . The mixture was allowed to stand for 15 h in the dark at room temperature. Thyme extract was dissolved in methanol for the organic extracts and distilled water for the aqueous extract. The different concentrations of T. musilii extract and of the tocopherol (vitamin E) standard were tested. The standard was used for comparison. The measure of the antioxidant activity was realized by adding 200 µL of each standard and sample to 800 µL of diluted ABTS·+. The absorbance was measured spectrophotometrically at 734 nm after 30 min. All measurements were performed in triplicate. The antioxidant capacity of the test sample and standard were expressed as percent of inhibition (%). The percentage of scavenging of ABTS. + was calculated by Equation (1). The IC 50 corresponding to the concentration needed to scavenge 50% of ABTS. + radicals were estimated from the dose-response curve by using Graph-Pad Prism software.

B-Carotene/Linoleic Acid Method
The formation of a free radical from linoleic acid was obtained after heating the β-carotene/linoleic acid complex. A total of 2 mL of β-carotene solution (1.5 mg of βcarotene/2.5 mL of chloroform) was added to 20 µL of linoleic acid, and 200 µL tween 20 was mixed. The chloroform was removed at 40 • C under vacuum using a rotary evaporator. A volume of 50 mL of distilled water was added to the dried mixture to form a β-carotene-linoleic acid emulsion. To determine the β-carotene bleaching activity of each extract, 0.800 mL of the emulsion was added to 0.200 mL of T. musilii extract at different concentrations (stock solution 20 mg/mL) and the standard (stock solution 1 mg/ml). The mixtures were incubated in a water bath at 50 • C for 120 min, and the absorbance was estimated at 470 nm before and after incubation. Tests were carried out in triplicate. The antioxidant activity of extracts was calculated using the following equation (Equation (2)): where A 0 and A c0 refer to the absorbance values measured at zero time for the test sample or standard and the control, respectively, and At and Act refer to the corresponding absorbance values of the test sample or standard and the control measured after incubation for 120 min, respectively. The IC 50 corresponding to the concentration needed for β-carotene bleaching inhibition at 50% was estimated from the dose-response curve by using Graph-Pad Prism software.

Reducing Power
The reduction of Fe 3+ to Fe 2+ was determined by measuring absorbance of the Perls Prussian blue complex. This method is based on the reduction of (Fe 3+ ) ferricyanide in stoichiometric excess relative to the antioxidants. For this purpose, different concentrations (0.0312, 0.0625, 0.125, 0.25, 0.5, and 1 mg/mL) of T. musilii extract were mixed with 1 mL Life 2023, 13, 62 6 of 24 of 0.2 M sodium phosphate buffer (pH 6.6) and 1 mL (1%) of potassium ferricyanide [K 3 Fe(CN) 6 ]. The mixture was incubated at 50 • C for 20 min and then acidified with 1 mL of trichloroacetic acid (10%). Finally, 0.25 mL of FeCl 3 (0.1%) was added to this solution. Distilled water was used as blank and for control. Absorbance of this mixture was measured at 700 nm using a UV spectrophotometer. Increased absorbance indicates ferric reducing power capability of the sample. The IC 50 corresponding to the concentration needed for reduction of Fe 3+ to Fe 2+ at 50% was estimated from the dose-response curve by using Graph-Pad Prism software.

Effect on Pseudomonas aeruginosa PAO1 Motility
P. aeruginosa PAO1 starter strain was used to study the effect of T. musilii methanolic extract on its flagellar motility (swarming and swimming) using the same procedure described by Snoussi et al. [34]. Three different concentrations of T. musilii methanolic extracts were used (50, 75, and 100 µg/mL). All experiments were conducted in triplicate.

Effect on Biofilm Formation by Pathogenic Bacteria and Yeast
The effect of T. musilii methanolic extract to inhibit the biofilm formation on 96-well plates by S. aureus ATCC 25923, E. faecalis ATCC 19433, E. coli ATCC 25922, P. aeruginosa ATCC 27853, Salmonella typhimurium ATCC 14028, L. monocytogenes ATCC 7644, and C. albicans ATCC 10,239 was tested by crystal violet technique [35]. For the experiment, 50 µL of an overnight-grown micro-organism culture and tryptone broth was prepared at different concentrations of T. musilii extracts (1, 1/2, 1/4, 1/8, and 1/16 MIC concentration value). The percentage of biofilm inhibition (expressed in percentage) was calculated as previously described by Ceylan et al. [36].

Anticancer Activities
T. musilii methanolic extract was tested against human lung (A549), breast (MCF-7), and colon (HCT-116) cancer cells. For the experiment, cell lines were cultured upon 80% confluence, and 0.4% Trypan Blue solution (Hi-Media, India) (0.4%) was used to stain the cells. Then, cells were treated for 48 h with various T. musilii concentrations ranging from 100 µg/mL to 500 µg/mL. Afterward, 200 µL of medium containing 10% MTT reagent was added to each well to obtain a final concentration of 0.5 mg/mL, and the plates were incubated for a further 3 h at 37 • C with 5% CO 2 atmosphere. After removing the medium, 100 µL of DMSO was added to dissolve the formed formazan crystals. The absorbance was measured at 570 nm and 630 nm. The percentage growth inhibition was calculated after subtracting the background and the blank, and the concentration of the test drug needed to inhibit cell growth by 50% (IC 50 ) was calculated from the dose-response curve for the respective cell line [37,38].

ADME Study
The pharmacokinetic, drug-likeness, and medicinal properties of the identified molecules from T. musilii methanolic extract were studied using free online software (SwissADME: http://www.swissadme.ch/; accessed on 2 October 2022) [39]. the physiological environment, was used to prepare the retrieved protein structure [40,41]. To achieve low 3D structures with appropriate chiralities, the identified phyto-constituents were prepared using the LigPrep panel, which generates each ligand structure's ionization state at a physiological pH of (7.2 ± 0.2). Co-crystalized ligand was selected in the receptor grid generating window to create the glide grid file [42]. The molecular docking analysis was then completed utilizing Schrodinger's glide, which included loading the prepared ligand's structure and receptor grid file into Maestro's workspace and docking the ligands using standard precision (SP) docking techniques into the receptor-binding pocket [43].

Statistical Analysis
All experiments were performed in triplicates, and average values were calculated using the SPSS 25.0 statistical package for Windows. Differences in means were calculated using Duncan's multiple range tests for means with a 95% confidence interval (p ≤ 0.05). For anticancer activities, a significance test was carried out among the treatments by two-way ANOVA followed by Bonferroni post hoc test at p < 0.001.

Phytochemistry and Antioxidant Activities of T. musilii Methanolic Extract
The phytochemical analysis revealed that, for T. musilii methanolic extract, the total flavonoid content was about 0.278 ± 0.019 mg of quercetin equivalent per gram of dry extract. Similarly, the total tannin content was evaluated at 0.084 ± 0.002 mg of tannic acid equivalent per gram of dry extract and total phenol content at 0.420 ± 0.004 mg of gallic acid equivalent per gram of dry extract. The tested extract was able to scavenge different stable free radicals with different degrees as compared to standard molecules: DPPH • free radicals (IC 50 = 0.077 ± 0.0015 mg/mL), ABTS •+ radicals (IC 50 = 0.040 ± 0.0011 mg/mL), β-carotene radicals (IC 50 = 0.278 ± 0.0012 mg/mL), and FRAP radicals (IC 50 = 0.106 ± 0.007 mg/mL). These data are summarized in Table 3.
With the DPPH method, about a 3.34-fold difference and a 3.5-fold difference was established between T. musilii methanolic extract and BHT and ascorbic acid (AA), respectively. While with the ABTS method, a 2.22-fold difference and a 1.9-fold difference was noticed as compared to the same standard molecules. For the β-carotene assay, a significant difference (p < 0.005) between T. musilii and the standard molecules was established (a 6.83-fold difference with BHT and a 16.88-fold difference with AA). While only a 1.17-fold to 2.12-fold difference was established between T. musilii methanolic extract and BHT and AA for the FRAP assay.

Anticancer Activity of T. musilii Methanolic Extract
The results (Figure 2) of the anticancer activities of T. musilii methanolic extract tested against three cell lines (MCF-7, A549, and HCT-116) showed promising results with an increasing percentage of cell viability inhibition depending on the concentration used.
The results (Figure 2) of the anticancer activities of T. musilii methanolic extract tested against three cell lines (MCF-7, A549, and HCT-116) showed promising results with an increasing percentage of cell viability inhibition depending on the concentration used.

ADME Analysis
The ADME (absorption, distribution, metabolism, and excretion) properties of all identified compounds (24 phyto-compounds) were studied. Table 6 summarizes the ADME properties of the most interesting molecules (10, 13, 15, 17, 18, 20, 21, 22, and 23). The predicted results showed that the selected compounds did not violate Lipinski's rule and possessed a good bioavailability score ranging from 0.55 to 0.85. The highest bioavailability score (0.85) was recorded for the two compounds 4-Ketoretinoic acid glucuronide and 6-9-12-15-18-Tetracosapentaynoic-acid. In addition, all selected compounds exhibited good topological polar surface area values (TPSAs) lower than 125 Å2, suggesting that they are expected to be orally absorbed. The consensus Log Po/w value was acceptable for all selected bioactive compounds varying from 1.62 (2,5-dihydroxy-3,4-dimethoxy-6-methyl-Benzene butanoic acid) to 5.91 (6-9-12-15-18-Tetracosapentaynoic-acid). For the pharmacokinetics properties, most selected compounds had high gastrointestinal absorption. In addition, most compounds were blood-brain barrier (BBB) permeant. Only one selected molecule was a P-gp substrate (Emetine). Furthermore, the selected molecules could inhibit one or more cytochrome P450 isoenzymes. Interestingly, compound 20 (Dehydrorotenone) was able to inhibit four cytochrome P450 isoenzymes (CYP 1A2, CYP2C19, CYP2C9, and CYP3A4). All selected compounds exhibited negative Log Kp values (skin permeability) ranging from −3.79 to −6.99, highlighting their suitability as good compounds to be deliv-Life 2023, 13, 62 13 of 24 ered transdermally. Figure 3 represents the bioavailability radar showing the drug-likeness behavior of all selected compounds. The results showed that most compounds were within the pink area of the polygon.

Molecular Docking Analysis
The binding affinities and molecular interactions of various receptors involved in these biological activities were evaluated in order to obtain a better understanding of the mechanistic effects behind the biological impacts of the identified phyto-constituents, as shown in Supplementary Material Table S1. All the identified phyto-constituents displayed negative binding energies (ranging from −0.5 to −10 kcal/mol) with the different targeted receptors, among them a complex with 1JIJ, 2XCT, and 1JNX, and the short peptide Asn-Met-His scored the highest of all the receptors examined.
On the other hand, His-Cys-Asn (−5.022 kcal/mol) and Phe-His-Gln (−10.062 kcal/mol) had promising docking scores on 1HD2 and 4UYA receptors, respectively. Less affinity was observed for the identified phyto-compounds in the fungal target 2QZW, and the isoprenoid 10-Hydroxyloganin (−5.008 kcasl/mol) had the highest docking score ligand among them. In the human mitotic kinesin Eg5 receptor BBG, 4-Ketoretinoic acid glucuronide had the highest docking score of −8.794 kcal/mol, which is better than the co-crystalized inhibitor. Maestro's ligand interaction tool was used to generate the 2D graphical representations of the ligand-protein interactions shown in Supplementary Material Figure S2.

Molecular Docking Analysis
The binding affinities and molecular interactions of various receptors involved in these biological activities were evaluated in order to obtain a better understanding of the mechanistic effects behind the biological impacts of the identified phyto-constituents, as shown in Supplementary Material Table S1. All the identified phyto-constituents displayed negative binding energies (ranging from −0.5 to −10 kcal/mol) with the different targeted receptors, among them a complex with 1JIJ, 2XCT, and 1JNX, and the short peptide Asn-Met-His scored the highest of all the receptors examined.
On the other hand, His-Cys-Asn (−5.022 kcal/mol) and Phe-His-Gln (−10.062 kcal/mol) had promising docking scores on 1HD2 and 4UYA receptors, respectively. Less affinity was observed for the identified phyto-compounds in the fungal target 2QZW, and the isoprenoid 10-Hydroxyloganin (−5.008 kcasl/mol) had the highest docking score ligand among them. In the human mitotic kinesin Eg5 receptor BBG, 4-Ketoretinoic acid glucuronide had the highest docking score of −8.794 kcal/mol, which is better than the co-
Antioxidant activity is usually measured by quantifying the ability of antioxidant compounds (electron donors/reducing agents) to quench free radicals. Plant phyto-compounds are involved in different chemical pathways to quench reactive oxygen species and free radicals (the transfer of hydrogen and/or single electron transfer) and the reduction in metal ions [76,77]. In this study, we investigated the antioxidant potential of T. musilii methanolic extract by using four different assays to obtain information about the full antioxidant capacity of the tested extract. The results revealed that T. musilii methanolic extract was able to scavenge all tested free radicals at low concentrations as compared to T. musilii essential oil (DPPH • IC 50 0.049 ± 1 × 10 −4 mg/mL, ABTS •+ IC 50 5.6 × 10 −4 ± 2 × 10 −5 mg/mL, β-carotene IC 50 = 3.20 × 10 −3 ± 5 × 10 −4 , and FRAP IC 50 > 1 mg/mL) [16]. The tested single, pure standard molecules (BHT and ascorbic acid) were able to scavenge DPPH • and ABTS •+ radicals at low concentrations as compared to T. musilii extract. The same results were reported for the β-carotene bleaching activity and the reduction in ferric ions.
Moreover, it has been well documented that T. vulgaris aqueous extract containing polysaccharides (starch, homogalacturonan, and rhamnogalacturonan) was able to increase (in vitro) enzymatic antioxidant activity, such as catalase, glutathione, glutathione-S transferase, and superoxide dismutase [80]. Using Pearson's correlation coefficient (Table 7), the statistical analysis revealed positive and significant correlation (p < 0.01) between TPC and TFC (r = −1.000) and TFC and TTC (r = −1.000). Significance: ** correlation is significant at 0.01 level; * correlation is significant at 0.05 level.
The negative Pearson's correlation coefficients highlighted that TPC and TTC are the main classes that may explain the antioxidant profile of T. musilii methanolic extract. Similarly, there was a positive and significant correlation (p < 0.01) between TPC and both DPPH (r = −1.000) and β-carotene (r = −1.000) assays. Similarly, a strong correlation was noticed between TTC and all the four used assays. For TFC, a strong positive correlation was established with DPPH (r = −1.000; p < 0.01), β-carotene (r = −1.000; p < 0.01), ABTS (r = −0.996; p < 0.05), and FRAP (r = −0.995; p < 0.05) assays. A correlation between the tested antioxidant assays with TFC were positively high (0.995 < r < 1.000), indicating that T. musilii methanolic extract possesses comparable activities in all four assays. In fact, it has been well documented that TPC, TFC, and TTC found in the plant kingdom are responsible for their antioxidant activities due to their redox properties [81][82][83][84][85][86].
In addition, T. musilii methanolic extract was able to reduce the motility of the P. aeruginosa PAO1 strain at low concentrations with a percentage of inhibition ranging from 14.29 ± 1.00 (at 50 µg/mL) to 39.73 ± 1.50 (at 100 µg/mL) for swarming activity and from 15.11 ± 0.50 (at 75 µg/mL) to 25.18 ± 1.00 (at 100 µg/mL) for swimming activity. Similarly, T. musilii methanolic extract inhibited the biofilm production of S. aureus ATTC 25923, L. monocytogenes ATCC 7644, E. coli ATCC 25922, and S. Typhimurium ATCC 14,028 at sub-MIC values with different degrees. Previous reports have demonstrated that thyme extract (Thymus sp.) decreased the production of violacein by 41% in Chromobacterium violaceum CV026 and swarming motility in P. aeruginosa PAO1 and E. coli O157:H7 by 48% and 17%, respectively [87]. In addition, Mohsenipour et al. [68] demonstrated that both ethanolic (80%) and methanolic (96%) extracts from T. vulgaris were able to inhibit the biofilm formation of S. aureus, B. cereus, S. pneumoniae, P. aeruginosa, E. coli, and K. pneumoniae in a concentration-dependent manner. At 20 mg/mL, the highest inhibition of biofilm formation was observed against S. pneumoniae (88.51%) and the lowest inhibition was observed for the biofilm formation of E. coli (56.83%). Similarly, the same authors [68] showed that both ethanolic and methanolic extracts from T. vulgaris were able to disrupt mature biofilm, and S. pneumoniae was the most sensitive structure (65.82%) with a mean value of destruction of about 43.15% for all tested bacteria. In addition, thyme (T. vulgaris) ethanolic extract was able to inhibit the biofilm formation of Campylobacter jejuni on an abiotic surface (polystyrene) up to 35% at concentrations of 50-200 µg/mL [88]. More recently, Mulugeta and colleagues showed that T. vulgaris crude extract obtained using the Soxhlet method attenuated the production of pyocyanin, LasA, violacein, and swarming motility and exhibited antibiofilm activities towards E. coli, S. aureus, P. aeruginosa, and S. typhimurium foodborne pathogenic bacteria [89].
A computational approach using ADME analysis of the pharmacokinetic, physiochemical, and drug-likeness properties of the identified molecules in T. musilii methanolic extract revealed that eight molecules were depicted to have good bioavailability score, topological polar surface area values (TPSAs), consensus Log Po/w values, and high gastrointestinal properties. Moreover, in silico mathematical analysis showed that for TyrRS S. aureus (1JIJ), the docking scores of phyto-constituents ranged from −2.234 to −7.566 kcal/mol, where three ligands showed docking scores comparable to those of the co-crystalized ligand (−7.973 kcal/mol). In this target, Asn-Met-His had the best docking score, which was −7.566. The polar group of Asn-Met-His and the reference compound (co-crystalized ligand) interacted with Asp195(1.90), Asp40(2.14), Asp177(2.59), and Asp80(2.30) at the binding site via a hydrogen bond [105]. In addition, salt bridge interactions were observed between Asn-Met-His and Asp80(3.14), lys84(4.78), and Asp195 (2.99). The results of the molecular docking at the active site S. aureus DNA gyrase (2XCT) revealed that most of the docked ligands had docking scores better than the reference Ciprofloxacin (−8.521 kcal/mol) [106]. Among the screened library short peptides, namely, Asn-Met-His, Ser-Met-Arg, Ser-Met-Ser, His-Cys-Asn, Phe-His-Gln, Val-Ser-Lys, and Ser-Val-Lys, these displayed a better docking score than the reference drug. The binding interaction of Asn-Met-His showed that it interacted with the Mn+2 ion through a salt bridge, which greatly enhanced rates of enzyme-mediated DNA breakage, as reported in earlier studies. Furthermore, the Asn-Met-His polar group and nucleotide bases (DG G9 and DT G10) exhibited hydrogen bonding, π-cation interactions (DA H13 and DC H12), and hydrophobic π-π interactions (DC H13). In this complex, the protonated amino terminal shows the hydrogen as well as the salt bridge bond with the charged negative amino acid Asp437. In human peroxiredoxin 5 (1HD2), the highest docking score was shown by the co-crystalized or reference ligand (−7.245 kcal/mol). However, significant binding affinities were also observed with peptides His-Cys-Asn, Ser-Val-Lys, Asn-Met-His, and Phe-His-Gln, with docking values ranging from −5.022 to −4.780 kcal/mol. The human peroxiredoxin family contains one cysteine residue, Cys47, which is conserved in all other peroxiredoxins and was directly linked to peroxide catalysis. Cys47, a conserved cysteine residue, is found within a minor cavity at the N-terminus of the kinked helix α2 [106]. This active pocket contains conserved amino acid residues, including Thr44, Gly46, Cys47, and Arg127, which are important in docked compound recognition through hydrogen bonding and hydrophobic interactions.
A detailed examination of the docking poses of the screened phyto-compounds revealed the presence of a hydrogen bond with Cys47 with the peptides His-Cys-Asn, Ser-Val-Lys, Asn-Met-His, and Phe-His-Gln, as well as the antioxidant reference ligand Benzoic Acid, indicating the antioxidant potential of the peptide. The receptor 2QZW is an aspartic proteinase (Sap) 1 secreted by C. albicans, which reportedly plays a key role in superficial Candida infections. Sap 1 is among the most crucial virulent factors generated by C. albicans cells since these hydrolytic enzymes engage in a variety of fungal physiological processes as well as other aspects of fungal-host interactions [107]. For these reasons, Saps clearly hold promise as new potential drug targets. A docking study on this target showed that the isoprenoid 10-Hydroxyloganin (−5.008 kcal/mol) had the highest docking score ligand among the identified phyto-compounds. The binding interaction showed that it formed five hydrogen bonds with active site residues Gly102(1.77), Lys24(2.30), Thr19(1.98), and Asp17(2.05 and 2.00). In this target, pepstatin (co-crystalized ligand) interacts with Asp86, Glu193, Arg195, and Asp37, whereas none of our identified phyto-compounds showed a comparable interaction, which is the primary reason for the lower affinity towards the C. albicans target Sap 1.
To evaluate the affinity of identified phyto-compounds for cancer targets, a docking investigation on the MLK4 kinase domain (4UYA), which controls the JNK, p38, and ERK kinase signaling pathways, was performed [108]. The results of molecular docking at the active site A4UYA revealed that most of the docked ligands had docking scores better than the reference (−7.897 kcal/mol), among which Phe-His-Gln (−10.062 kcal/mol), 6-9-12-15-18-Tetracosapentaynoic-acid (−10.012 kcal/mol), and Asn-Met-His (−8.983 kcal/mol) had significant docking scores. The binding interaction of the most scored peptide Phe-His-Gln shows that at physiological pH, the deprotonated carboxylate ion makes a salt bridge with Lys265, while the protonated amine group interacts with Thr288. In this complex, a π-cationic interaction with the imidazole ring and Mg 2+ ion in the active site of the MLK4 kinase domain was also visible. The human mitotic kinesin Eg5 receptor 4BBG is essential for the formation of the mitotic spindle. Mitotic kinesins, which play an important role in mitotic cell division, are attractive anticancer therapeutic targets [109]. In this context, the kinesin Eg5 has received a lot of interest, and there are several inhibitors at various stages of clinical studies. 4-Ketoretinoic acid glucuronide had the top scoring phyto-compound in this target, with a docking score of −8.794 kcal/mol, which is more significant than co-crystalized inhibitor 3-(((2-Aminoethyl) sulfanyl) (3-ethylphenyl) phenyl methyl) phenol (−8.684 kcal/mol). The binding mode of 4-Ketoretinoic acid glucuronide revealed that it had only one hydrogen bond with Arg221, while the co-crystalized ligand interacted with the acidic amino acids Glu212 and Glu116. Additionally, a van der Waals interaction was also observed during the binding of 4-Ketoretinoic acid glucuronide with the amino acid residues Pro137, Gly134, Asp130, Trp127, Try211, Leu214, Ala218, and Ala219.
The C-terminal BRCT region of BRCA1 is the receptor for 1JNX, which is required for DNA repair, tumor suppressor activities, and transcriptional regulation [110,111]. A docking study on this target showed that Asn-Met-His (−5.979 kcal/mol), Ser-Met-Arg (−5.308 kcal/mol), and Phe-His-Gln (−4.978 kcal/mol) had the most promising docking score among the identified phyto-compounds. In the binding mode, Asn-Met-His interacted with four hydrogen bonds with Cys1768(1.50), Lys1793(1.86), Glu1781 (1.62), and 1.21), sharing many common residues with the active site of the investigated enzyme. Overall, the biological effects of the phyto-constituents in T. musilii Velen., notably, the tripeptides, seem to be thermodynamically feasible.

Conclusions
In the present work, we reported for the first time the chemical composition of T. musilii methanolic extract by using the HR-LCMS technique. Several bioactive molecules belonging to different chemical classes (isoprenoids, flavonoids, alkaloids, terpenoids, polyphenols, and fatty acyls) with promising biological activities were reported. The tested extract was able to inhibit the growth of several gram-positive, gram-negative, and yeast strains at low concentrations. Similarly, good antioxidant activities were also recorded, and T. musilii was able to scavenge various free radicals at low concentrations. Similarly, T. musilii attenuated the production of some virulence properties in P. aeruginosa (swarming and swimming) and biofilm formation by pathogenic bacteria. In silico approaches showed a good ADME profile of most identified molecules in T. musilii methanolic extract and a high binding score to the tested target proteins. More studies are needed to highlight the mechanism of action of T. musilii crude extract and its bioactive components.