# Phytochemistry and Pharmacology ofThymus broussonetii Boiss

Authors: Hanae Naceiri Mrabti; Latifa Doudach; Naoual El Menyiy; Mohammed Bourhia; Ahmad Mohammad Salamatullah; Mohamed Reda Kachmar; Omar Belmehdi; Hamza El Moudden; Nidal Naceiri Mrabti; Hicham Harhar; Nasreddine El Omari; Abdelhakim Bouyahya
Journal: Evidence-Based Complementary and Alternative Medicine
 (2021)

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## Abstract

Thymus broussonetii Boiss (T. broussonetii) is a rare medicinal and aromatic plant. It is widely used in traditional medicine to treat several diseases, including diarrhea, fever, cough, irritation, skin diseases, rheumatism, respiratory ailments, influenza, and digestion problems. In this review, we have critically summarized previous data on T. broussonetii about its phytochemistry, botanical and geographical distribution, toxicological investigation, and pharmacological properties. Using scientific research databases such as Wiley Online, SciFinder, ScienceDirect, PubMed, SpringerLink, Web of Science, Scopus Wiley Online, and Google Scholar, the data on T. broussonetii were collected and discussed. The presented data regrouped bioactive compounds and biological activities of T. broussonetii. The findings of this work showed that essential oils and extracts of T. broussonetii exhibited numerous pharmacological activities (in vitro and in vivo), particularly antibacterial, antifungal, antioxidant, anticancer, anti-inflammatory, insecticidal, antipyretic, antinociceptive, and immunological and behavioral effects. While toxicological studies of T. broussonetii essential oils and extracts are lacking, modern scientific tools revealed the presence of different classes of secondary metabolites such as terpenoids, alkaloids, flavonoids, tannins, coumarins, quinones, carotenoids, and steroids. T. broussonetii essential oils, especially from the aerial parts, exhibited potent antibacterial, antifungal, and antioxidant effects. An in-depth toxicological investigation is needed to validate the efficacy and safety of T. broussonetii extracts and essential oils and their secondary metabolites. However, further pharmacokinetic and pharmacodynamic studies should be performed to validate its bioavailability.


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## Body

## 1. Introduction

Thymus broussonetii Boiss (Thymus broussonetii) belongs to the Lamiaceae family and the genus of Thymus. It is a small shrub of 40 cm in height and is endemic to Morocco, Algeria, and Tunisia [1]. It is known locally in Morocco as “Zaitra,” “Tazouknnit,” or “Azukni” [2, 3]. T. broussonetii is distributed on the Atlantic coast between 20 and 400 m altitude and is mainly located in arid and semiarid bioclimatic zones [4].It is among the plants most used in Moroccan folk medicine against various illnesses such as urinary, nervous, genital, circulatory, skin, digestive, and respiratory diseases [2, 3]. It is also used to treat diabetes [3, 5, 6], cold, cough, fever, digestive disorders, and dolorous processes [7]. Other researchers have reported the use of this plant in food as a seasoning of traditional recipes (seasoning) and to flavor tea or milk [8]. Ethnobotanical surveys are the first step to identify the plant uses for each disorder. It provides information on the part used, the method of preparation, etc. However, the lack of plant information given by researchers in many surveys was repeatedly noticed. This is the case of several researchers who reported the use of T. broussonetii in folk medicine without mentioning the part used, the method of preparation, or/and the traditional use [9, 10].Several classes of bioactive compounds, including flavonoids, alkaloids, terpenoids, tannins, coumarins, quinones, steroids, and carotenoids, have been identified in essential oils (EOs) and extracts ofT. broussonetii, which explains its biological activities [11–25].Usingin vitro and in vivo pharmacological approaches, researchers reported the potential activity of T. broussonetii extracts and EOs. Essential oils from the aerial parts of T. broussonetii showed antibacterial effects against different pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Salmonella sp., Bacillus sp., Micrococcus luteus, etc. Moreover, the antifungal effects of T. broussonetii EOs against numerous pathogenic fungi, including Candida sp., Aspergillus brasiliensis, and Saccharomyces cerevisiae were reported by Jamali et al. [20] and Smahane et al. [16, 20, 26]. T. broussonetii extracts and EOs exhibited antioxidant effects using well-known techniques such as DPPH and FRAP assays [11, 13, 20, 25, 27]. The anticancer properties of T. broussonetii EOs have also been reported against various tumor cell lines like P815 mastocytoma, CEM, and K-562 [12, 15, 21]. Moreover, T. broussonetii was revealed to exhibit anti-inflammatory activity [28], anticorrosive potential [23], insecticidal [19, 27, 29], antiparasitic [30], antipyretic [22], antinociceptive [31], immunological, and behavioral effects [31]. In addition, the acute toxicological investigations of T. broussonetii EOs have shown death cases and some signs of toxicity [22]. However, the mechanism of action by which the bioactive compounds of T. broussonetii extracts and EOs exhibited these pharmacological effects is lacking.Due to the intensification of research on the pharmacological effects ofT. broussonetii and its compounds in recent years, we have reviewed all studies on this plant; botanical description, geographical distribution, chemical composition, all pharmacological effects, and the prospects of T. broussonetii. To the best of our knowledge, this review is the first report providing a scientific database that highlighted several aspects related to T. broussonetii and suggested the future potential clinical applications of this plant.


## 2. Research MethodologyThymus broussonetii Boiss

In this work, data conacring botanical description, taxonomy, destruction, phytochemistry, and pharmacological activities ofT. broussonetii were collected using different databases (Google Scholar, Web of Science, PubMed, Scopus, ScienceDirect, SpringerLink, SciFinder, and Wiley Online). The collected data were organized in several areas and highlighted. The chemical structures of T. broussonetii were drawn using ChemDraw Pro 8.0 software.


## 3. Results and Discussion

### 3.1. Botanical Description and Geographical Distribution

Thymus broussonetii is an evergreen plant that grows to a height of around 5 centimeters. Its flowers clustered toward the top of the stems in a dense ovate-cylindrical inflorescence with floral leaves broader than the leaves, often purple-colored, attenuate-sharp at the tip, ciliated at the margins and concealing the calyces, these 2-lipped, the upper shallowly toothed; pink corolla 2-3 times the length of the calyx, with a distinctly protruding narrow tube. It differs from subsp. hannonis (Maire) Morales by the subpetiolate leaves and bracts hairy only on the inner side [1]. T. broussonetii is an endemic plant to Morocco, Tunisia, and Algeria [1]. In Morocco, it is found in the Middle Atlantic, the High Atlas, and in the north of the kingdom [32].


### 3.2. Chemical Composition

The secondary metabolites produced byT. broussonetii were the subject of numerous studies, almost all of which have been carried out on the aerial parts of this plant. The phytochemical screening of T. broussonetii extracts and EOs revealed its richness in phenolic compounds, in particular terpenoids, flavonoids, and phenolic acids. Analysis of T. broussonetii EOs by gas chromatography (GC) identified more than sixty terpenoids (Table 1; Figure 1).Table 1
Chemical composition ofT. broussonetii.
PartsExtracts/EOsCompounds groupsCompoundsReferencesLeavesEssential oilTerpenoidsBorneol,p-cymene, carvacrol, camphene, α -terpinene, α-Pinene, trans-sabinene hydrate, caryophyllene oxide, (E)-β-caryophyllene, Bornyl acetate, carvacrol methyl ether, camphor, Linalool, cis-sabinene hydrate, 4-terpineol, p-cymen-8-ol, Thymol, trans-verbenol, 1-octen-3ol, 1,8-cineol, β-pinene[11]Flowers and leavesEssential oilTerpenoids(E)-β-caryophyllene, γ-terpinene, p-cymene, carvacrol, thymol, 4-terpineol, β-pinene, terpendiol, borneol, caryophyllene oxide, geraniol formate, p-menth-1,4(8)-diene, linalyl propionate, β-cadrene, thujol, cinerone, 4-isopropy-lM-2-cyclohexane-1-ol, 1-octen-3-ol[12]Aerial partsEssential oilTerpenoidsα-pinene, p-cymene, carvacrol, viridiflorene, borneol, γ-terpinene, myrcene, camphene, α–thujene, aromadendrene, caryophyllene oxide, α–terpinene, β–pinene, thymol, germacrene D, δ-cadinene, linalool[13]Aerial partsEssential oilTerpenoidsCamphor,α–terpineol, eucalyptol, germacrene D, borneol, terpinen-4-ol, bicyclogermacrene, β–caryophyllene, β-bourbonene, spathulenol, δ–terpineol, bornyl acetate, caryophyllene oxide, T-muurolol, γ–cadinene, thymol, trans-sabinene hydrate, linalool, cis-sabinne-hydrate, limonene, p-cymene, dihydrocarvone, trans-carveol, δ–cadinene, alloaromadendrene, carvacrol[14]LeavesEssential oilTerpenoidsCarvacrol, p-cymene,γ -terpinene, thymol, β--pinene, 4-terpineol, borneol, linalyl propionate, p-menth-1,4(8)-diene, geraniol formate, cinerone, carvacrol methyl ether, 4-isopropyl-1M-2cyclohexane-1-ol,1-octen-3-ol[15]Essential oilTerpenoidsCarvacrol, p-cymene,α-pinene, α-terpinene, 3-octanol, myrcene, α-terpineol, borneol, linalyl acetate, linalool, β--pinene, methyl carvacrol, p-cymen-8-ol, p-mentha-1,4(8)-diene, limonene, camphene, γ-terpinene, thymol[16]Aerial partsEssential oilTerpenoidsCarvacrol, thymol,γ-terpinene, borneol, p-cymene, α-pinene, camphene, myrcene, α-terpinene, α-thujene, limonene, β-pinene, linalool[17]Aerial partsEssential oilTerpenoidsThymol, carvacrol, borneol, viridiflorene, spathulenol, aromadendrene, camphene,α-terpineol, O-cymene, terpinene-4-ol, γ-terpinene, alloaromadendrene, γ-cadinene, α-pinene, cis-dihydrocarvone, trans-sabinene hydrate, α-amorphene, β-patchoulene, β-cubebene, isospathulenol[18]LeavesEssential oilTerpenoidsp-cymene, borneol, α-pinene, thymol, camphene, γ-terpinene, carvacrol, Ledene, Limonene, Myrcene, Aromadendrene, β-pinene, α-thujene,α-terpinene, terpinen-4-ol, dihydrocarvone, allo-Aromadendrene, β-caryophyllene, cis-sabinene hydrate, tricyclene, sabinene, α-phellandrene, p-Mentha-1,4(8)-diene, linalool, γ-muurolene, spathulenol[19]Aerial partsEssential oilTerpenoidsCarvacrol, thymol, borneol,γ-terpinene, p-cymene, camphene, α-pinene, myrcene, viridiflorene, α-terpinene, α-thujene, aromadendrene, β-pinene, limonene, caryophyllene oxide, tricyclene, δ-cadinene, alloaromadendrene, germacrene D, linalool, and limonene[20]Aerial partsEssential oilTerpenoidsBorneol, thymol,p-cymene, γ-terpinene, carvacrol, 4-terpineol, linalyl propionate, camphor, δ-3-carene, camphene, β-pinene, geraniol formate, p-menth-1,4(8)-diene, p-mentha-1,8-diene, 4-isopropyl-1M-2 cyclohexane-1-ol, terpinene-1-ol, carvenone, bornyl acetate, cinerone, alloaromadendrene, (E)-β-caryophyllene, α-muurolene, β-cedrene, α-cadinene, caryophyllene oxide, germacrene D[21]Essential oilTerpenoidsThymol, borneol, carvacrol,p-cymene, δ- terpinene, camphene, spathulenol, myrcene, α-terpineol, aromadendrene, limonene, β--pinene, α-terpinene[22]Aerial partsEssential oilTerpenoidsCarvacrol, o-cymene,γ.-terpinene, α-pinene, thymol, (+)-4-carene, 4-terpineol, α-thujene, tau-cadinol, spathulenol, limonene, β-caryophyllene, and camphene[23]Aerial partsEssential oilTerpenoidsThymol,α-pinene, β-caryophyllene, carvacrol, γ-terpinene, borneol[24]LeavesMethanolic extractAlkaloids−[25]Alcohol extractFlavonoids+Aqueous extractTannins+Ethanolic extractCoumarins+Methanolic extractTerpenoids+Petroleum extractQuinones+Steroids+Aqueous extractCarotenoids+StemsMethanolic extractAlkaloids−Alcohol extractFlavonoids+Aqueous extractTannins+Ethanolic extractCoumarins+Methanolic extractTerpenoids+Petroleum extractQuinones+Steroids+Aqueous extractCarotenoids+Figure 1
Chemical composition of T.Thymus broussonetii.
(a)(b)(c)The essential oil ofT. broussonetii is mainly composed of spathulenol, eucalyptol, 1,8-cineole, β-caryophyllene, terpinolene, camphene, limonene, myrcene, sabinene, terpineol, terpinene, p-cymene, o-cymene, α-thujene, α-pinene, camphor, bornyl acetate, borneol, thymol, linalool, and carvacrol [11–24].Chemical variability was observed in the composition ofT. broussonetii extracted by different methods. Zerrifi et al. [17] have found that T. broussonetii EOs are rich in oxygenated monoterpenes (64.5%), monoterpene hydrocarbons (29.0%), sesquiterpene hydrocarbons (5.8%), and oxygenated sesquiterpenes (0.4%), while oxygenated sesquiterpenes had the lowest percentage. The carvacrol was the main compound [17].The same results were found by Jamali et al. [20]. For the T. broussonetii essential oil from Essaouira (Morocco), it consisted mainly of oxygenated monoterpenes (64.5%), while the oxygenated sesquiterpenes were poorly represented (0.4%). The main component was carvacrol (43.4%), followed by thymol (12.3%) [20].Carvacrol (39.51%) as the main constituent was also found by Chebli et al. [23]. The other components were o-cymene (14.80%), γ-terpinene (10.32%), α-pinene (9.7%), thymol (7.9%), and 4-terpineol (3.22%) [23].In another study, camphor (46.17%) was found to be the major component followed byα-terpineol (7.69%), eucalyptol (5.76), germacrene D (5.21%), and borneol (4.42%) of T. broussonnetii essential oil in Tamri region (Western high Atlas), Morocco [14]. In addition, linalool, γ-terpinene, cis-sabinene hydrate, β-caryophyllene, p-Menth-1,4(8)-diene, caryophyllene oxide, and carvenoneare were the main compounds identified in the essential oil of T. broussonnetii aerial parts [21].In comparison with wild-harvested and cultivatedT. broussonnetii, chromatographic analysis of their essential oil revealed the presence of 19 compounds, namely α-pinene (5.0%), p-cymene (5.2%), borneol (8.5%), γ-terpinene (8.9%), thymol (12.3%), and carvacrol (43.4%) for wild-harvested plants in Morocco, whereas the oil obtained from cultivated plants was characterized by a higher content of α-pinene (6.5%), p-cymene (7.2%), and carvacrol (60.8%) [13].The chemical analysis of polar fraction fromT. broussonnetii leaf extracts indicated the presence of flavonoids, tannins, coumarins, terpenoids, quinones, steroids, and carotenoids in the various extracts (aqueous extract, alcohol extract, and petroleum extract). Alkaloid compounds were not detected in the methanolic extract of plant leaves. In addition, flavonoids, tannins, coumarins, terpenoids, quinones, steroids, and carotenoids were the main compounds identified in the T. broussonnetii stem extracts [25].


### 3.3. Pharmacological Properties

#### 3.3.1. Antibacterial Activity

Several studies have shown the antibacterial effectiveness of different essential oils from the aerial part ofThymus broussonetii [28, 39, 40, 29, 41]. Table 2 summarizes all the studies which evaluated this activity in Thymus broussonetii, including the plant part used, type of extract, the antibacterial test, the strains studied, and the key results. The literature screening indicated that scientists had investigated the effect of Thymus broussonetii against the most critical pathogenic agents belonging to Gram-negative and Gram-positive bacteria. Indeed, Lattaoui and Tantaoui-elaraki, [34] assessed the antibacterial activity of the essential oil of T. broussonetii aerial part against three bacteria (Staphylococcus aureus, Escherichia coli, and Bacillus megaterium). The result of this study showed that T. broussonetii essential oils inhibited the growth of all bacterial strains with MIC values of 1, 3, and 4% (v/v) against S. aureus, E. coli, and B. megaterium, respectively. Belaqziz et al. [33] reported the antibacterial activity of T. broussonetti leaf EOs using agar disc diffusion against two Gram-positive bacteria, including S. aureus and Bacillus subtilis, and four Gram-negative bacteria, namely E. coli, Salmonella sp, Vibrio cholerae, and Pseudomonas aeruginosa. The results showed that the essential oil exhibited promising antibacterial power against the strains tested; Bacillus subtilis (Ф = 33 ± 0.4 mm), S. aureus (Ф = 19 ± 0.8 mm), Salmonella sp. (Ф = 9 ± 0.9 mm), Escherichia coli (Ф = 21 ± 0.1 mm), Vibrio cholerae (Ф = 40 ± 0.4 mm) and P. aeruginosa (Ф = 9 ± 0.1 mm). In another study, El Bouzidi et al. [13] tested the antibacterial activity of essential oils obtained from both wild and cultivated T. broussonetii using agar disc diffusion and macrodilution methods against Salmonella sp. (CCMM B17), E. coli (CCMM B4), E. coli (ATCC 25922), Bacillus cereus (ATCC 14579), Bacillus subtilis (ATCC 9524), Micrococcus luteus (ATCC10240), S. aureus (CCMM B3), and the clinically isolated strain, Enterobacter cloacae. Both EOs obtained from T. broussonetii (wild and cultivated) exhibited inhibitory activity on all the selected microorganisms, with inhibitory zones ranging between 23.33 and 53.67 mm and MIC values varied from 0.12 to 1.82 mg/mL. In fact, Micrococcus luteus was the most sensitive strain with MIC values of 53.50 and 53.67 mg/mL for wild and cultivated T. broussonetii, respectively, followed by B. subtilis, B. cereus, and S. aureus. However, Smahane et al. [26] investigated the inhibitory effect of T. broussonetii aerial part EOs against S. aureus, E. coli, and P. aeruginosa using disk diffusion and broth microdilution methods. The results revealed that all microorganisms tested were inhibited by essential oils with inhibitory zones ranging between 8.67 and 42.67 mm and MIC values ranged between 0.2 and 20 μg/mL.Table 2
Antibacterial effects ofT. broussonetii.
Used partsExtractsUsed methodsTested strainsKey resultsReferencesAerial partEssential oilAgar disk diffusion methodBroth microdilution methodStaphylococcus aureusФ = 42.67 ± 1.45 mm[26]MIC = 0.2μl/mLMBC = 0.6μl/mLEscherichia coliФ = 29.33 ± 0.54 mmMIC = 1.3μl/mLMBC = 1.3μl/mLPseudomonas aeruginosaФ = 8.67 ± 1.20 mmMIC = 20μl/mLMBC ≥ 80μl/mLAerial partEssential oilAgar disc diffusionBroth macrodilution methodStaphylococcus aureusФ = 35.00 ± 1.00 mm[13]MIC = 0.9 mg/mLMMC = 0.9 mg/mLBacillus subtilisФ = 49.67 ± 1.53 mmMIC = 0.23 mg/mLMMC = 0.23 mg/mLBacillus cereusФ = 48.67 ± 1.15 mmMIC = 0.23 mg/mLMMC = 0.23 mg/mLMicrococcus luteusФ = 53.50 ± 1.00 mmMIC = 0.12 mg/mLMMC = 0.12 mg/mLEscherichia coli 1 ATCC 25922Ф = 30.17 ± 1.00 mmMIC = 0.90 mg/mLMMC = 0.90 mg/mLE. coli 2 CCMM B4Ф = 29.67 ± 1.53 mmMIC = 0.90 mg/mLMMC = 0.90 mg/mLEnterobacter cloacaeФ = 27.33 ± 0.58 mmMIC = 0.90 mg/mLMMC = 0.90 mg/mLSalmonella sp.Ф = 31.67 ± 1.53 mmMIC = 0.90 mg/mLMMC = 0.90 mg/mLStaphylococcus aureusФ = 34.83 ± 1.04 mmMIC = 0.91 mg/mLMMC = 0.91 mg/mLBacillus subtilisФ = 49.00 ± 1.00 mmMIC = 0.23 mg/mLMMC = 0.23 mg/mLBacillus cereusФ = 47.33 ± 1.15 mmMIC = 0.23 mg/mLMMC = 0.23 mg/mLMicrococcus luteusФ = 53.67 ± 1.15 mmMIC = 0.12 mg/mLMMC = 0.12 mg/mLEscherichia coli 1 ATCC 25922Ф = 27.5 ± 1.53 mmMIC = 0.91 mg/mLMMC = 0.91 mg/mLE. coli 2 CCMM B4Ф = 29.33 ± 1.53 mmMIC = 0.91 mg/mLMMC = 0.91 mg/mLEnterobacter cloacaeФ = 23.33 ± 1.53 mmMIC = 1.82 mg/mLMMC = 1.82 mg/mLSalmonella sp.Ф = 31.33 ± 1.53 mmMIC = 0.91 mg/mLMMC = 0.91 mg/mLAerial partEssential oilAgar diffusion methodBacillus subtilisФ = 33 ± 0.4 mm[33]Staphylococcus aureusФ = 19 ± 0.8 mmSalmonella sp.Ф = 19 ± 0.9 mmEscherichia coliФ = 21 ± 0.1 mmVibrio choleraeФ = 40 ± 0.4 mmPseudomonas aeruginosaФ = 9 ± 0.1 mmAerial partEssential oilDisc diffusion methodMicrocystis aeruginosaФ = 90 ± 0.00 mm[17]MIC = 0.047 mg/mLMBC = 0.095 mg/mlAerial partEssential oilAgar diffusion methodStaphylococcus aureusNo measurable zone of inhibition[34]Aerial partEssential oilAgar diffusion methodEscherichia coliNo measurable zone of inhibition[16]MIC = 1%Staphylococcus aureusMIC = 3%Bacillus megateriumMIC = 4%Recently, Zerrifi and collaborators determined thein vitro antibacterial activity of T. broussonetii aerial part EOs using paper disk diffusion and microdilution methods against Microcystis aeruginosa. According to this study, the essential oils exhibited promising antibacterial power against the strain tested with an inhibitory zone of 90 mm, and MIC and MBC values of 0.047 and 0.095 mg/mL, respectively [17].


#### 3.3.2. Antifungal Activity

The antifungal activity ofT. broussonetii EOs against many fungal strains was reported in several works [13, 16, 18, 20, 26, 34]. The previous publications on the antifungal activity that studied the essential oils from aerial parts of T. broussonetii by different methods are summarized in Table 3.Table 3
Antifungal activity ofT. broussonetii.
Used partsExtractsUsed methodsTested strainsKey resultsReferencesAerial partsEssential oilAgar disc diffusionBroth microdilution methodCandida albicansФ = 50.00 ± 1.00 mm[20]MIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida kruseiФ = 49.67 ± 1.53 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida glabrataФ = 49.33 ± 1.53 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida parapsilosisФ = 51.17 ± 0.76 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLEssential oilAgar disc diffusionBroth microdilution methodCandida albicansФ = 50.00 ± 1.00 mm[13]MIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida kruseiФ = 49.67 ± 1.53 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida glabrataФ = 49.33 ± 1.53 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida parapsilosisФ = 51.17 ± 0.76 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida albicansФ = 49.67 ± 1.15 mmMIC = 0.46 mg/mLMMC = 0.46 mg/mLCandida kruseiФ = 47.33 ± 1.53 mmMIC = 0.46 mg/mLMMC = 0.46 mg/mLCandida glabrataФ = 48.50 ± 0.50 mmMIC = 0.46 mg/mLMMC = 0.46 mg/mLCandida parapsilosisФ = 50.00 ± 1.00 mmMIC = 0.46 mg/mLMMC = 0.46 mg/mLAerial partsEssential oilAgar diffusion methodmacrodilution broth methodCandida albicansФ = 38.5 ± 0.70 mm[18]MIC = 0.25μg/mLAerial partsEssential oilAgar diffusion methodCandida albicansSlightly more sensitive in presence of 0.2% oil[34]Aspergillus nigerLower sensitivity relatively resistantAerial partsEssential oilAgar diffusion methodSaccharomyces cerevisiaeMIC = 3%[16]Candida albicansMIC = 3%ZygorrhynchusspMIC = 4%Aspergillus nigerMIC = 3%Aerial partEssential oilAgar disk diffusion methodBroth microdilution methodCandida albicansФ = 35.67 ± 0.33 mm[26]MIC = NDMBC = NDAspergillus brasiliensisФ = NDMIC = NDMBC = NDSaad et al. [18] determined the in vitro antifungal efficacy of the essential oil from the aerial part against Candida albicans using the agar diffusion and macrodilution broth methods. Consequently, the zones of inhibition and MIC value were 38.5 mm and 0.25 μg/mL, respectively. Moreover, Jamali et al. [20] evaluated the EOs from aerial parts of the studied plant for their antifungal action against Candida albicans, Candida krusei, Candida glabrata, and Candida parapsilosis using agar disc diffusion and microdilution methods. The results revealed a strong antifungal activity against all the fungi tested with zones of inhibition ranging from 49.33 to 51.17 mm and MIC value of 0.45 mg/mL. Using the same methods and the same fungal strains, El Bouzidi et al. [13] investigated the antifungal activity of EOs obtained from wild and cultivated T. broussonetii. Therefore, these oils inhibited the growth of all fungal species with MIC values of 0.45 and 0.45 mg/mL for wild and cultivated Thymus broussonetii, respectively. In another study, the essential oil of T. broussonetii was tested against two fungal strains (Candida albicans and Aspergillus brasiliensis). The results revealed a strong antifungal inhibition against Candida albicans with zones of inhibition of 35.67 ± 0.33 mm [26].


#### 3.3.3. Antioxidant Activity

Different studies have evaluated the antioxidant activity of extracts and EOs from different parts ofT. broussonetii using well-known techniques such as DPPH and FRAP assays [11, 13, 20, 25, 27] (Table 4). Indeed, Jamali et al. [20] investigated the antioxidant activity of the essential oils from aerial parts of T. broussonetii, and the results showed that the essential oil exhibited an interesting anti-DPPH (IC50 = 97.48 ± 2.24 μg/mL) and a high reducing power (EC50 = 167.86 ± 1.46 μg/ml) compared with the standard antioxidants, quercetin, and BHT with IC50 values of 1.07 ± 0.01 and4.21 ± 0.08 μg/mL, respectively, for DPPH and with EC50 values of 2.29 ± 0.1 and 7.09 ± 0.1 μg/mL, respectively, for FRAP. In another study, the wild and cultivated T. broussonetii EOs were tested for their antioxidant activity by DPPH and ferric ion reduction assays. The results showed an interesting antioxidant effect of the wild and cultivated T. broussonetii EOs with IC50 values of 132.23 ± 3.09 and 145.83 ± 3.47 μg/mL, respectively, for DPPH and with EC50 values of 167.87 ± 1.46 and 169.355 ± 2.04 μg/mL, respectively, for FRAP [13]. Moreover, Ouariachi et al. [11] demonstrated that the essential oils from T. broussonetii possessed high antioxidant activity using DPPH (IC50 = 90 μg/mL). On the other hand, Ahlam et al. [25] reported the antioxidant activity of the aqueous and methanol extracts from leaves and stems of T. broussonetii using FRAP and DPPH methods. The results revealed that both extracts exhibited a good antioxidant activity with FRAP capacity values ranging between 0.105 ± 0.021 and 1.579 ± 0.014 mg/mL and anti-DPPH power with IC50 values ranging between 0.132 ± 0.034 and 7.665 ± 0.411 mg/mL. The highest activity was observed in methanol extract from stems with EC50 and IC50 values of 0.105 ± 0.021 and 0.132 ± 0.034 mg/mL, respectively. On the other hand, essential oil showed a DPPH-radical-scavenging activity with IC50 = 13.24 ± 0.06 mg/mL [27].Table 4
Antioxidant effects ofT. broussonetii.
Used partsExtractsUsed methodsKey resultsReferencesLeavesAqueous extractDPPHIC50 = 22.61 ± 1.022 mg/mL[25]Methanol extractIC50 = 6.484 ± 0.190 mg/mLStemsAqueous extractIC50 = 7.665 ± 0.411 mg/mLMethanol extractIC50 = 0.132 ± 0.034 mg/mLLeavesAqueous extractFRAPEC50 = 0.597 ± 0.013 mg/mLMethanol extractEC50 = 1.579 ± 0.014 mg/mLStemsAqueous extractEC50 = 0.489 ± 0.011 mg/mLMethanol extractEC50 = 0.105 ± 0.021 mg/mLAerial partsEssential oilDPPHIC50 = 13.24 ± 0.06 mg/mL[27]Aerial partsEssential oilDPPHIC50 = 90 μg/mL[11]Aerial parts (wild)Essential oilDPPHIC50 = 132.23 ± 3.09 μg/mL[13]FRAPEC50 = 167.87 ± 1.46 μg/mLAerial parts (cultivated)Essential oilDPPHIC50 = 145.83 ± 3.47 μg/mLFRAPEC50 = 169.355 ± 2.04 μg/mLAerial parts (wild)Essential oilDPPHIC50 = 97.48 ± 2.24 μg/mL[20]FRAPEC50 = 167.86 ± 1.46 μg/mL


#### 3.3.4. Anticancer Activity

The anticancer properties ofT. broussonetii have also been studied. Indeed, some investigations tested the efficiency of T. broussonetii essential oils on many cell lines [12, 15, 21] (Table 5). Ait M’Barek et al. [15] evaluated the antiproliferative effect of T. broussonetii EOs from stem and leaves on human ovarian adenocarcinoma IGR-OV1 parental cell line OV1/P. The results showed that the EOs tested inhibited the proliferation of this adenocarcinoma with an IC50 value of 0.40 ± 0.02 (%v/v).Table 5
Anticancer effects ofT. broussonetii.
Parts usedExtractsUsed methodsCell linesKey resultsReferencesLeaves and stemsEssential oilsCrystal violet assayThe parental human ovarian adenocarcinoma cell line IGR-OV1 (OV1/P)IC50 = 0.40 ± 0.02%(v/v)[15]Flowers and leavesEssential oils (variety: TbA)MTT assayP815 mastocytoma cell lineIC50 = 4.7%(v/v)[12]CEMIC50 = 3.6%(v/v)K-562IC50 = 10%(v/v)MCF -7IC50 = 10%(v/v)MCF -7 gemIC50 = 8.9%(v/v)Essential oils (variety: TbB)MTT assayP815 mastocytoma cell lineIC50 = 8.5%(v/v)CEMIC50 = 3.1%(v/v)K-562IC50 = 13.5%(v/v)MCF -7IC50 = 15.4%(v/v)MCF -7 gemIC50 = 17.5%(v/v)Flowers and leavesEssential oilsMTT assayP815 mastocytoma cell lineIC50 = 0.016%(v/v)[21]Moreover,Thymus broussonetii EOs extracted from flowers and leaves have been tested by Jaafari et al. [21] on the P815 mastocytoma cell line using MTT assay. In this study, the essential oils exhibited an important dose-dependent cytotoxic effect against the P815 cell line (IC50 = 0.016%).In another study, the authors evaluated the cytotoxic activity of essential oils from two chemotypes ofT. broussonetii against five tumor cell lines, namely P-815 (murine mastocytoma), K-562 (human chronic myelogenous leukemia), CEM (acuteT lymphoblastoid leukemia), and MCF 7 (human breast adenocarcinoma) and its counterpart resistant to gemcitabine (MCF -7 gem) using MTT assay. Consequently, cell viability showed a cell proliferation inhibition by the tested products in a dose-dependent manner with IC50 values ranging between 3.1 and 17.5% (v/v). Additionally, cell cycle analysis detected cell cycle arrest at S and G0/G1 phases in cells. This considerable activity might be due to the high content of thymol and carvacrol known for their promising anticancer effects via numerous mechanisms of action such as angiogenesis, inhibition of cell migration, autophagy, apoptosis, and cell cycle arrest [35, 36].


#### 3.3.5. Anti-Inflammatory Activity

The antiedema effects of hexane, chloroform, and methanol extracts ofT. broussonettii were evaluated on croton oil-induced ear edema in mice. The chloroform extract showed the highest activity, reducing the oedematous response by 47%, the ID50 value of the indomethacin used as the reference drug (286 g/cm2) is three times higher than that of the chloroform extract 93 g/cm2. The chloroform extract of T. broussonettii possesses an anti-inflammatory activity ascribable to its triterpenic acid content; in fact, ursolic and oleanolic acid justify the edema inhibition observed. Ursolic acid was more potent than oleanolic acid with ID50 values of 56 and 132 g/cm2 corresponding to 0.12 and 0.29 mol/cm2, respectively [28] (Table 6).Table 6
Other pharmacological activities ofT. broussonetii.
ActivitiesUsed partsExtractsExperimental approachesKey resultsReferencesAnti-inflammatory activityLeavesn-hexaneCroton oil ear test in miceinhibition of the croton oil-induced ear edema in miceEdema reduction = 9%[28]ChloroformEdema reduction = 47%Chloroform + methanolEdema reduction = 16%MethanolEdema reduction = -5%Anticorrosive activityAerial partsEssential oilsLoss measurements and electrochemical techniques82.35% inhibition efficiency at a dose of 2 g/L[23]Insecticidal activityAerial partsEssential oilsFourth instar larvae ofCulex pipiensLC50 = 0.23[19]Antiparasitic activityAerial partsEssential oilsOral administration (20 g/animal) at the time of infection and thereafter for several daysAbsence of intracerebral cystsNo anomalies[30]Antipyretic activityStemWater, butanol, and ethyl acetateYeast-induced fever in ratsSignificantly reduced the temperature in febrile rats[37]Acute toxicityAerial partsEssential oilsSwiss mice (25–35 g)LD50 = 2.66 g/kg[22]Antinociceptive activityLeaves and stemWaterChemical and thermal models (in vivo)Writhing inhibition = 88.9%[31]Ethyl acetate and butanolWrithing inhibition = 69%Writhing inhibition = 62.8%Insecticidal activityAerial partsEssential oilsEffect against adults ofTribolium castaneum herbstLD50 = 0.08 μl/cm2LD90 = 0.19 μl/cm2[29]Insecticidal activityAerial partsEssential oilsEffect againstTribolium castanum pest foodstuffsTL50 = 1.5 μl/cm2[27]Immunological and behavioral activitiesLeaves and stemWater, butanol, and ethyl acetateTested the neurostimulant effects of the extractsIncreased (in vivo) the number of leukocyte categories studied[31]


#### 3.3.6. Anticorrosive Potential

The essential oils ofT. broussonnetii at different concentrations (ranging from 0.05 to 2 g/L) were tested against corrosion on C38 steel in 1 M medium, HCl, using electrochemical impedance spectroscopy (EIS), potentiodynamic polarization, and weight loss methods. The essential oil was found to be rich in bioactive substances, mainly carvacrol (39.51%) followed by benzene, 1-methyl-2-(1-methylethyl) (14.80%), gammaterpinene (10.32%), alpha-pinene (9.7%), thymol (7.9%), and 3-cyclohexen-1-ol, 4-methyl-1-(1-methylethyl) (3.22%). Using the EIS test, the essential oil (2 g/L) inhibited the corrosion of metals and alloys in acid solutions with a percentage of 82.35% of the inhibition efficiency. The polarization studies showed that T. broussonnetii EOs inhibit both anodic metal dissolution and cathodic hydrogen reduction reactions. At the highest inhibition concentration, the maximum inhibition efficiency observed indicates that many molecules were adsorbed on the metal surface. At 2 g/L, the best efficiency obtained in the presence of essential oil was 81.63%. It has been noted that the inhibition efficiency increases with increasing temperature. The highest efficiency was 90% and reached 328 K. The inhibitory mechanism was probably achieved by chemical adsorption (chemisorption) of TBS molecules on the surface of carbon steel and this indeed increases with rising temperature [23] (Table 6).


#### 3.3.7. Insecticidal Activities

TheT. broussonetti EOs were investigated for their insecticidal activity, using the larvae test sensibility technique. The chemical analysis by GC-MS showed that the major compounds of T. broussonetii essential oil were p-cymene (21.0%), borneol (16.5%), α-pinene (11.8%), and thymol (11.3%). The EOs of this plant proved larvicidal effectiveness against the fourth instar larvae of Culex pipiens and were significantly higher at the dose of 0.125 ppm compared to the control. The lethal concentration 50 (LC50) during exposure of the insect population to EOs at 24 hours was 0.23, and the effective toxicity on C. pipiens larvae was associated with the thymol compound of thyme oil [19] (Table 6).


#### 3.3.8. Antipyretic Activity

At a dose of 200 mg/kg b.w.,T. broussonetii aqueous, butanol, and ethyl acetate extracts were investigated in vivo for their antipyretic effect on yeast-induced fever. In normothermic rats, the extracts were tested to determine whether the antipyretic activity is related to a hypothermic effect. Indeed, all extracts significantly reduced rectal temperature in febrile animals. However, they did not induce hypothermia in normal rats. Besides, an inhibition of platelet aggregation has been observed by acting in the same way as NSAI drugs. Furthermore, extracts of T. broussonetii contain many types of compounds such as triterpenes, saponins, tannins, flavonoids, and several salicylates. The presence of these compounds can enhance this antipyretic activity [22] (Table 6).


#### 3.3.9. Antinociceptive

The immunostimulatory and neurotropic antistress effects of extracts (aqueous, ethyl acetate, and butanolic extracts) and EOs ofT. broussonetii were evaluated at three doses. Therefore, the aqueous and ethyl acetate extracts showed the best results. In fact, thyme extracts increased the number of leucocyte categories studied, in particular polynuclear cells, total lymphocytes, TCD4+, TCD8+, and NK cells. It has been suggested that intraperitoneal administration of T. broussonetii extracts has a potent direct effect on leucocytes in vivo. In contrast, this assumes that the two extracts partially prevent stress-induced disturbances in the rate of leukocytes. The ethyl acetate extract inhibited the increase in polynuclear cells caused by stress, increased lymphocytes, and decreased polynuclear counts in the stressed mice treated with the aqueous extract compared to the stressed mice [31].T. broussonetii was investigated to study the behavioral effects using the light/dark box test. At 12 mg/kg, the aqueous extract increased the number of transitions and the number of traversed squares and decreased the time spent in the dark compartment. The ethyl acetate extract increased both the number of traversed squares and the number of transitions without affecting the time spent in the dark compartment. The aqueous extract exerted an anxiolytic effect on the animals, while it could rather enhance locomotor and exploratory activities. The improvement in animal activity observed in the light/dark box after treatment with the aqueous extract is rather due to its anxiolytic-like effect and the ethyl acetate extract improved exploratory and locomotor activities in mice (Table 6).


#### 3.3.10. Antiparasitic Activity

In another work, the effect ofT. broussonetii EOs was assessed on the experimental transmission of Toxoplasma gondii cysts in mice. These oils were administered orally (20 μg/animal) at the infection time and thereafter for several days. In mice given the essential oils, no cyst was observed. In addition, no disorder was noted in the control animals given the thyme EOs [30] (Table 6).


#### 3.3.11. Insecticidal Activity

The insecticidal activity ofT. broussonetii EO was screened using the contact toxicity assay. The oil proved insecticidal effectiveness against Tribolium castaneum Herbst. After 24 h of treatment, the LD50 and LD90 were 0.08 and 0.19 μl/cm2, respectively. These results suggest that the contents of thyme EOs, in particular those obtained from the genus Thymus, have a good botanical bioinsecticide potential against Tribolium castaneum Herbst [29].The insecticidal activity of the EO of this plant was examined againstTribolium castanum by the contact toxicity assay. The essential oil exhibited the highest insecticidal activity with a median lethal time (TL50) of 1.5 μL/cm2 with LT50 (lethal time required to kill 50% of the exposed insects) values of 30,36 (24,62–38,48) at a dose of 1 μl/cm2 and 4,81(3,8–5,99) at a dose of 1,5 μl/cm2, respectively and a LT90 (lethal time required to kill 90% of the exposed insects) of 222,78(138,62–475,59) at a dose of 1 μl/cm2 and 16,07 (11,4–30,08), respectively. The Thymus broussonnetii Boiss EO could act as a substitute for biopesticide and reduce the harmful impact of chemical insecticides on the environment and humans [27] (Table 6).


#### 3.3.12. Immunological and Behavioral Effects

The antinociceptive effect of aqueous, butanol, and ethyl acetate extracts ofT. broussonetii was studied using thermal and chemical nociception models and naloxone (a nonselective opioid antagonist) to determine the role of the opioid system in the antinociceptive activity of these extracts. To determine the phytoconstituents of the extracts tested, phytochemical screening was carried out, which revealed the presence of tannins in all the extracts. Quinones, saponins, and flavonoids were detected in butanol and ethyl acetate extracts, while terpenes were only identified in the ethyl acetate extract [31].The butanol and aqueous extracts showed an antinociceptive effect in both phases of formalin (50–300 mg/kg), tail immersion, and writing tests. At the same time, only the nociceptive response of the second phase was significantly reduced by the ethyl acetate extract (100–300 mg/kg). In the first and second phases, the aqueous extract was the most effective, with ED50 values of 177 (147–200) and 134 (95–170) mg/kg, respectively. The aqueous extract (200 mg/kg) showed a potent effect and significantly reduced the number of writhes induced by acetic acid, with 88.9% of writhes inhibition compared to those of ethyl acetate (69%) and butanol (63%) extracts. These obtained proved that T. broussonetii contains active compounds (polar and nonpolar) having antinociceptive activity with distinct mechanisms of action [31] (Table 6).


### 3.4. Toxicological Investigations

An acute toxicity screening was carried out forT. broussonetii EOs in order to verify their harmlessness to avoid a possible overdose and to properly determine the toxicological profile of the T. broussonetii species. This was assessed using the Leitchfield and Wilcoxon method, and the effective lethal dose (LD50) was measured. Subsequently, signs of toxicity such as diarrhea, convulsion, piloerection, motor coordination, and behavioral changes (excitation and twitches) were determined. For the groups receiving the dose of 1 g/kg, the change in body weight was also determined. On the other hand, thymol (36.7%) and borneol (21.9%) were the two major compounds, followed by p-cymene (7.6%) and β-pinene (0.7%). At a dose of 2 mg/kg, some cases of death and signs of toxicity were recorded. The LD90s and LD50s were estimated to be 7.31 (5.64–13.54) and 4.47 (3.6–6.72) g/kg, respectively [22].


## 3.1. Botanical Description and Geographical Distribution

Thymus broussonetii is an evergreen plant that grows to a height of around 5 centimeters. Its flowers clustered toward the top of the stems in a dense ovate-cylindrical inflorescence with floral leaves broader than the leaves, often purple-colored, attenuate-sharp at the tip, ciliated at the margins and concealing the calyces, these 2-lipped, the upper shallowly toothed; pink corolla 2-3 times the length of the calyx, with a distinctly protruding narrow tube. It differs from subsp. hannonis (Maire) Morales by the subpetiolate leaves and bracts hairy only on the inner side [1]. T. broussonetii is an endemic plant to Morocco, Tunisia, and Algeria [1]. In Morocco, it is found in the Middle Atlantic, the High Atlas, and in the north of the kingdom [32].


## 3.2. Chemical Composition

The secondary metabolites produced byT. broussonetii were the subject of numerous studies, almost all of which have been carried out on the aerial parts of this plant. The phytochemical screening of T. broussonetii extracts and EOs revealed its richness in phenolic compounds, in particular terpenoids, flavonoids, and phenolic acids. Analysis of T. broussonetii EOs by gas chromatography (GC) identified more than sixty terpenoids (Table 1; Figure 1).Table 1
Chemical composition ofT. broussonetii.
PartsExtracts/EOsCompounds groupsCompoundsReferencesLeavesEssential oilTerpenoidsBorneol,p-cymene, carvacrol, camphene, α -terpinene, α-Pinene, trans-sabinene hydrate, caryophyllene oxide, (E)-β-caryophyllene, Bornyl acetate, carvacrol methyl ether, camphor, Linalool, cis-sabinene hydrate, 4-terpineol, p-cymen-8-ol, Thymol, trans-verbenol, 1-octen-3ol, 1,8-cineol, β-pinene[11]Flowers and leavesEssential oilTerpenoids(E)-β-caryophyllene, γ-terpinene, p-cymene, carvacrol, thymol, 4-terpineol, β-pinene, terpendiol, borneol, caryophyllene oxide, geraniol formate, p-menth-1,4(8)-diene, linalyl propionate, β-cadrene, thujol, cinerone, 4-isopropy-lM-2-cyclohexane-1-ol, 1-octen-3-ol[12]Aerial partsEssential oilTerpenoidsα-pinene, p-cymene, carvacrol, viridiflorene, borneol, γ-terpinene, myrcene, camphene, α–thujene, aromadendrene, caryophyllene oxide, α–terpinene, β–pinene, thymol, germacrene D, δ-cadinene, linalool[13]Aerial partsEssential oilTerpenoidsCamphor,α–terpineol, eucalyptol, germacrene D, borneol, terpinen-4-ol, bicyclogermacrene, β–caryophyllene, β-bourbonene, spathulenol, δ–terpineol, bornyl acetate, caryophyllene oxide, T-muurolol, γ–cadinene, thymol, trans-sabinene hydrate, linalool, cis-sabinne-hydrate, limonene, p-cymene, dihydrocarvone, trans-carveol, δ–cadinene, alloaromadendrene, carvacrol[14]LeavesEssential oilTerpenoidsCarvacrol, p-cymene,γ -terpinene, thymol, β--pinene, 4-terpineol, borneol, linalyl propionate, p-menth-1,4(8)-diene, geraniol formate, cinerone, carvacrol methyl ether, 4-isopropyl-1M-2cyclohexane-1-ol,1-octen-3-ol[15]Essential oilTerpenoidsCarvacrol, p-cymene,α-pinene, α-terpinene, 3-octanol, myrcene, α-terpineol, borneol, linalyl acetate, linalool, β--pinene, methyl carvacrol, p-cymen-8-ol, p-mentha-1,4(8)-diene, limonene, camphene, γ-terpinene, thymol[16]Aerial partsEssential oilTerpenoidsCarvacrol, thymol,γ-terpinene, borneol, p-cymene, α-pinene, camphene, myrcene, α-terpinene, α-thujene, limonene, β-pinene, linalool[17]Aerial partsEssential oilTerpenoidsThymol, carvacrol, borneol, viridiflorene, spathulenol, aromadendrene, camphene,α-terpineol, O-cymene, terpinene-4-ol, γ-terpinene, alloaromadendrene, γ-cadinene, α-pinene, cis-dihydrocarvone, trans-sabinene hydrate, α-amorphene, β-patchoulene, β-cubebene, isospathulenol[18]LeavesEssential oilTerpenoidsp-cymene, borneol, α-pinene, thymol, camphene, γ-terpinene, carvacrol, Ledene, Limonene, Myrcene, Aromadendrene, β-pinene, α-thujene,α-terpinene, terpinen-4-ol, dihydrocarvone, allo-Aromadendrene, β-caryophyllene, cis-sabinene hydrate, tricyclene, sabinene, α-phellandrene, p-Mentha-1,4(8)-diene, linalool, γ-muurolene, spathulenol[19]Aerial partsEssential oilTerpenoidsCarvacrol, thymol, borneol,γ-terpinene, p-cymene, camphene, α-pinene, myrcene, viridiflorene, α-terpinene, α-thujene, aromadendrene, β-pinene, limonene, caryophyllene oxide, tricyclene, δ-cadinene, alloaromadendrene, germacrene D, linalool, and limonene[20]Aerial partsEssential oilTerpenoidsBorneol, thymol,p-cymene, γ-terpinene, carvacrol, 4-terpineol, linalyl propionate, camphor, δ-3-carene, camphene, β-pinene, geraniol formate, p-menth-1,4(8)-diene, p-mentha-1,8-diene, 4-isopropyl-1M-2 cyclohexane-1-ol, terpinene-1-ol, carvenone, bornyl acetate, cinerone, alloaromadendrene, (E)-β-caryophyllene, α-muurolene, β-cedrene, α-cadinene, caryophyllene oxide, germacrene D[21]Essential oilTerpenoidsThymol, borneol, carvacrol,p-cymene, δ- terpinene, camphene, spathulenol, myrcene, α-terpineol, aromadendrene, limonene, β--pinene, α-terpinene[22]Aerial partsEssential oilTerpenoidsCarvacrol, o-cymene,γ.-terpinene, α-pinene, thymol, (+)-4-carene, 4-terpineol, α-thujene, tau-cadinol, spathulenol, limonene, β-caryophyllene, and camphene[23]Aerial partsEssential oilTerpenoidsThymol,α-pinene, β-caryophyllene, carvacrol, γ-terpinene, borneol[24]LeavesMethanolic extractAlkaloids−[25]Alcohol extractFlavonoids+Aqueous extractTannins+Ethanolic extractCoumarins+Methanolic extractTerpenoids+Petroleum extractQuinones+Steroids+Aqueous extractCarotenoids+StemsMethanolic extractAlkaloids−Alcohol extractFlavonoids+Aqueous extractTannins+Ethanolic extractCoumarins+Methanolic extractTerpenoids+Petroleum extractQuinones+Steroids+Aqueous extractCarotenoids+Figure 1
Chemical composition of T.Thymus broussonetii.
(a)(b)(c)The essential oil ofT. broussonetii is mainly composed of spathulenol, eucalyptol, 1,8-cineole, β-caryophyllene, terpinolene, camphene, limonene, myrcene, sabinene, terpineol, terpinene, p-cymene, o-cymene, α-thujene, α-pinene, camphor, bornyl acetate, borneol, thymol, linalool, and carvacrol [11–24].Chemical variability was observed in the composition ofT. broussonetii extracted by different methods. Zerrifi et al. [17] have found that T. broussonetii EOs are rich in oxygenated monoterpenes (64.5%), monoterpene hydrocarbons (29.0%), sesquiterpene hydrocarbons (5.8%), and oxygenated sesquiterpenes (0.4%), while oxygenated sesquiterpenes had the lowest percentage. The carvacrol was the main compound [17].The same results were found by Jamali et al. [20]. For the T. broussonetii essential oil from Essaouira (Morocco), it consisted mainly of oxygenated monoterpenes (64.5%), while the oxygenated sesquiterpenes were poorly represented (0.4%). The main component was carvacrol (43.4%), followed by thymol (12.3%) [20].Carvacrol (39.51%) as the main constituent was also found by Chebli et al. [23]. The other components were o-cymene (14.80%), γ-terpinene (10.32%), α-pinene (9.7%), thymol (7.9%), and 4-terpineol (3.22%) [23].In another study, camphor (46.17%) was found to be the major component followed byα-terpineol (7.69%), eucalyptol (5.76), germacrene D (5.21%), and borneol (4.42%) of T. broussonnetii essential oil in Tamri region (Western high Atlas), Morocco [14]. In addition, linalool, γ-terpinene, cis-sabinene hydrate, β-caryophyllene, p-Menth-1,4(8)-diene, caryophyllene oxide, and carvenoneare were the main compounds identified in the essential oil of T. broussonnetii aerial parts [21].In comparison with wild-harvested and cultivatedT. broussonnetii, chromatographic analysis of their essential oil revealed the presence of 19 compounds, namely α-pinene (5.0%), p-cymene (5.2%), borneol (8.5%), γ-terpinene (8.9%), thymol (12.3%), and carvacrol (43.4%) for wild-harvested plants in Morocco, whereas the oil obtained from cultivated plants was characterized by a higher content of α-pinene (6.5%), p-cymene (7.2%), and carvacrol (60.8%) [13].The chemical analysis of polar fraction fromT. broussonnetii leaf extracts indicated the presence of flavonoids, tannins, coumarins, terpenoids, quinones, steroids, and carotenoids in the various extracts (aqueous extract, alcohol extract, and petroleum extract). Alkaloid compounds were not detected in the methanolic extract of plant leaves. In addition, flavonoids, tannins, coumarins, terpenoids, quinones, steroids, and carotenoids were the main compounds identified in the T. broussonnetii stem extracts [25].


## 3.3. Pharmacological Properties

### 3.3.1. Antibacterial Activity

Several studies have shown the antibacterial effectiveness of different essential oils from the aerial part ofThymus broussonetii [28, 39, 40, 29, 41]. Table 2 summarizes all the studies which evaluated this activity in Thymus broussonetii, including the plant part used, type of extract, the antibacterial test, the strains studied, and the key results. The literature screening indicated that scientists had investigated the effect of Thymus broussonetii against the most critical pathogenic agents belonging to Gram-negative and Gram-positive bacteria. Indeed, Lattaoui and Tantaoui-elaraki, [34] assessed the antibacterial activity of the essential oil of T. broussonetii aerial part against three bacteria (Staphylococcus aureus, Escherichia coli, and Bacillus megaterium). The result of this study showed that T. broussonetii essential oils inhibited the growth of all bacterial strains with MIC values of 1, 3, and 4% (v/v) against S. aureus, E. coli, and B. megaterium, respectively. Belaqziz et al. [33] reported the antibacterial activity of T. broussonetti leaf EOs using agar disc diffusion against two Gram-positive bacteria, including S. aureus and Bacillus subtilis, and four Gram-negative bacteria, namely E. coli, Salmonella sp, Vibrio cholerae, and Pseudomonas aeruginosa. The results showed that the essential oil exhibited promising antibacterial power against the strains tested; Bacillus subtilis (Ф = 33 ± 0.4 mm), S. aureus (Ф = 19 ± 0.8 mm), Salmonella sp. (Ф = 9 ± 0.9 mm), Escherichia coli (Ф = 21 ± 0.1 mm), Vibrio cholerae (Ф = 40 ± 0.4 mm) and P. aeruginosa (Ф = 9 ± 0.1 mm). In another study, El Bouzidi et al. [13] tested the antibacterial activity of essential oils obtained from both wild and cultivated T. broussonetii using agar disc diffusion and macrodilution methods against Salmonella sp. (CCMM B17), E. coli (CCMM B4), E. coli (ATCC 25922), Bacillus cereus (ATCC 14579), Bacillus subtilis (ATCC 9524), Micrococcus luteus (ATCC10240), S. aureus (CCMM B3), and the clinically isolated strain, Enterobacter cloacae. Both EOs obtained from T. broussonetii (wild and cultivated) exhibited inhibitory activity on all the selected microorganisms, with inhibitory zones ranging between 23.33 and 53.67 mm and MIC values varied from 0.12 to 1.82 mg/mL. In fact, Micrococcus luteus was the most sensitive strain with MIC values of 53.50 and 53.67 mg/mL for wild and cultivated T. broussonetii, respectively, followed by B. subtilis, B. cereus, and S. aureus. However, Smahane et al. [26] investigated the inhibitory effect of T. broussonetii aerial part EOs against S. aureus, E. coli, and P. aeruginosa using disk diffusion and broth microdilution methods. The results revealed that all microorganisms tested were inhibited by essential oils with inhibitory zones ranging between 8.67 and 42.67 mm and MIC values ranged between 0.2 and 20 μg/mL.Table 2
Antibacterial effects ofT. broussonetii.
Used partsExtractsUsed methodsTested strainsKey resultsReferencesAerial partEssential oilAgar disk diffusion methodBroth microdilution methodStaphylococcus aureusФ = 42.67 ± 1.45 mm[26]MIC = 0.2μl/mLMBC = 0.6μl/mLEscherichia coliФ = 29.33 ± 0.54 mmMIC = 1.3μl/mLMBC = 1.3μl/mLPseudomonas aeruginosaФ = 8.67 ± 1.20 mmMIC = 20μl/mLMBC ≥ 80μl/mLAerial partEssential oilAgar disc diffusionBroth macrodilution methodStaphylococcus aureusФ = 35.00 ± 1.00 mm[13]MIC = 0.9 mg/mLMMC = 0.9 mg/mLBacillus subtilisФ = 49.67 ± 1.53 mmMIC = 0.23 mg/mLMMC = 0.23 mg/mLBacillus cereusФ = 48.67 ± 1.15 mmMIC = 0.23 mg/mLMMC = 0.23 mg/mLMicrococcus luteusФ = 53.50 ± 1.00 mmMIC = 0.12 mg/mLMMC = 0.12 mg/mLEscherichia coli 1 ATCC 25922Ф = 30.17 ± 1.00 mmMIC = 0.90 mg/mLMMC = 0.90 mg/mLE. coli 2 CCMM B4Ф = 29.67 ± 1.53 mmMIC = 0.90 mg/mLMMC = 0.90 mg/mLEnterobacter cloacaeФ = 27.33 ± 0.58 mmMIC = 0.90 mg/mLMMC = 0.90 mg/mLSalmonella sp.Ф = 31.67 ± 1.53 mmMIC = 0.90 mg/mLMMC = 0.90 mg/mLStaphylococcus aureusФ = 34.83 ± 1.04 mmMIC = 0.91 mg/mLMMC = 0.91 mg/mLBacillus subtilisФ = 49.00 ± 1.00 mmMIC = 0.23 mg/mLMMC = 0.23 mg/mLBacillus cereusФ = 47.33 ± 1.15 mmMIC = 0.23 mg/mLMMC = 0.23 mg/mLMicrococcus luteusФ = 53.67 ± 1.15 mmMIC = 0.12 mg/mLMMC = 0.12 mg/mLEscherichia coli 1 ATCC 25922Ф = 27.5 ± 1.53 mmMIC = 0.91 mg/mLMMC = 0.91 mg/mLE. coli 2 CCMM B4Ф = 29.33 ± 1.53 mmMIC = 0.91 mg/mLMMC = 0.91 mg/mLEnterobacter cloacaeФ = 23.33 ± 1.53 mmMIC = 1.82 mg/mLMMC = 1.82 mg/mLSalmonella sp.Ф = 31.33 ± 1.53 mmMIC = 0.91 mg/mLMMC = 0.91 mg/mLAerial partEssential oilAgar diffusion methodBacillus subtilisФ = 33 ± 0.4 mm[33]Staphylococcus aureusФ = 19 ± 0.8 mmSalmonella sp.Ф = 19 ± 0.9 mmEscherichia coliФ = 21 ± 0.1 mmVibrio choleraeФ = 40 ± 0.4 mmPseudomonas aeruginosaФ = 9 ± 0.1 mmAerial partEssential oilDisc diffusion methodMicrocystis aeruginosaФ = 90 ± 0.00 mm[17]MIC = 0.047 mg/mLMBC = 0.095 mg/mlAerial partEssential oilAgar diffusion methodStaphylococcus aureusNo measurable zone of inhibition[34]Aerial partEssential oilAgar diffusion methodEscherichia coliNo measurable zone of inhibition[16]MIC = 1%Staphylococcus aureusMIC = 3%Bacillus megateriumMIC = 4%Recently, Zerrifi and collaborators determined thein vitro antibacterial activity of T. broussonetii aerial part EOs using paper disk diffusion and microdilution methods against Microcystis aeruginosa. According to this study, the essential oils exhibited promising antibacterial power against the strain tested with an inhibitory zone of 90 mm, and MIC and MBC values of 0.047 and 0.095 mg/mL, respectively [17].


### 3.3.2. Antifungal Activity

The antifungal activity ofT. broussonetii EOs against many fungal strains was reported in several works [13, 16, 18, 20, 26, 34]. The previous publications on the antifungal activity that studied the essential oils from aerial parts of T. broussonetii by different methods are summarized in Table 3.Table 3
Antifungal activity ofT. broussonetii.
Used partsExtractsUsed methodsTested strainsKey resultsReferencesAerial partsEssential oilAgar disc diffusionBroth microdilution methodCandida albicansФ = 50.00 ± 1.00 mm[20]MIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida kruseiФ = 49.67 ± 1.53 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida glabrataФ = 49.33 ± 1.53 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida parapsilosisФ = 51.17 ± 0.76 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLEssential oilAgar disc diffusionBroth microdilution methodCandida albicansФ = 50.00 ± 1.00 mm[13]MIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida kruseiФ = 49.67 ± 1.53 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida glabrataФ = 49.33 ± 1.53 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida parapsilosisФ = 51.17 ± 0.76 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida albicansФ = 49.67 ± 1.15 mmMIC = 0.46 mg/mLMMC = 0.46 mg/mLCandida kruseiФ = 47.33 ± 1.53 mmMIC = 0.46 mg/mLMMC = 0.46 mg/mLCandida glabrataФ = 48.50 ± 0.50 mmMIC = 0.46 mg/mLMMC = 0.46 mg/mLCandida parapsilosisФ = 50.00 ± 1.00 mmMIC = 0.46 mg/mLMMC = 0.46 mg/mLAerial partsEssential oilAgar diffusion methodmacrodilution broth methodCandida albicansФ = 38.5 ± 0.70 mm[18]MIC = 0.25μg/mLAerial partsEssential oilAgar diffusion methodCandida albicansSlightly more sensitive in presence of 0.2% oil[34]Aspergillus nigerLower sensitivity relatively resistantAerial partsEssential oilAgar diffusion methodSaccharomyces cerevisiaeMIC = 3%[16]Candida albicansMIC = 3%ZygorrhynchusspMIC = 4%Aspergillus nigerMIC = 3%Aerial partEssential oilAgar disk diffusion methodBroth microdilution methodCandida albicansФ = 35.67 ± 0.33 mm[26]MIC = NDMBC = NDAspergillus brasiliensisФ = NDMIC = NDMBC = NDSaad et al. [18] determined the in vitro antifungal efficacy of the essential oil from the aerial part against Candida albicans using the agar diffusion and macrodilution broth methods. Consequently, the zones of inhibition and MIC value were 38.5 mm and 0.25 μg/mL, respectively. Moreover, Jamali et al. [20] evaluated the EOs from aerial parts of the studied plant for their antifungal action against Candida albicans, Candida krusei, Candida glabrata, and Candida parapsilosis using agar disc diffusion and microdilution methods. The results revealed a strong antifungal activity against all the fungi tested with zones of inhibition ranging from 49.33 to 51.17 mm and MIC value of 0.45 mg/mL. Using the same methods and the same fungal strains, El Bouzidi et al. [13] investigated the antifungal activity of EOs obtained from wild and cultivated T. broussonetii. Therefore, these oils inhibited the growth of all fungal species with MIC values of 0.45 and 0.45 mg/mL for wild and cultivated Thymus broussonetii, respectively. In another study, the essential oil of T. broussonetii was tested against two fungal strains (Candida albicans and Aspergillus brasiliensis). The results revealed a strong antifungal inhibition against Candida albicans with zones of inhibition of 35.67 ± 0.33 mm [26].


### 3.3.3. Antioxidant Activity

Different studies have evaluated the antioxidant activity of extracts and EOs from different parts ofT. broussonetii using well-known techniques such as DPPH and FRAP assays [11, 13, 20, 25, 27] (Table 4). Indeed, Jamali et al. [20] investigated the antioxidant activity of the essential oils from aerial parts of T. broussonetii, and the results showed that the essential oil exhibited an interesting anti-DPPH (IC50 = 97.48 ± 2.24 μg/mL) and a high reducing power (EC50 = 167.86 ± 1.46 μg/ml) compared with the standard antioxidants, quercetin, and BHT with IC50 values of 1.07 ± 0.01 and4.21 ± 0.08 μg/mL, respectively, for DPPH and with EC50 values of 2.29 ± 0.1 and 7.09 ± 0.1 μg/mL, respectively, for FRAP. In another study, the wild and cultivated T. broussonetii EOs were tested for their antioxidant activity by DPPH and ferric ion reduction assays. The results showed an interesting antioxidant effect of the wild and cultivated T. broussonetii EOs with IC50 values of 132.23 ± 3.09 and 145.83 ± 3.47 μg/mL, respectively, for DPPH and with EC50 values of 167.87 ± 1.46 and 169.355 ± 2.04 μg/mL, respectively, for FRAP [13]. Moreover, Ouariachi et al. [11] demonstrated that the essential oils from T. broussonetii possessed high antioxidant activity using DPPH (IC50 = 90 μg/mL). On the other hand, Ahlam et al. [25] reported the antioxidant activity of the aqueous and methanol extracts from leaves and stems of T. broussonetii using FRAP and DPPH methods. The results revealed that both extracts exhibited a good antioxidant activity with FRAP capacity values ranging between 0.105 ± 0.021 and 1.579 ± 0.014 mg/mL and anti-DPPH power with IC50 values ranging between 0.132 ± 0.034 and 7.665 ± 0.411 mg/mL. The highest activity was observed in methanol extract from stems with EC50 and IC50 values of 0.105 ± 0.021 and 0.132 ± 0.034 mg/mL, respectively. On the other hand, essential oil showed a DPPH-radical-scavenging activity with IC50 = 13.24 ± 0.06 mg/mL [27].Table 4
Antioxidant effects ofT. broussonetii.
Used partsExtractsUsed methodsKey resultsReferencesLeavesAqueous extractDPPHIC50 = 22.61 ± 1.022 mg/mL[25]Methanol extractIC50 = 6.484 ± 0.190 mg/mLStemsAqueous extractIC50 = 7.665 ± 0.411 mg/mLMethanol extractIC50 = 0.132 ± 0.034 mg/mLLeavesAqueous extractFRAPEC50 = 0.597 ± 0.013 mg/mLMethanol extractEC50 = 1.579 ± 0.014 mg/mLStemsAqueous extractEC50 = 0.489 ± 0.011 mg/mLMethanol extractEC50 = 0.105 ± 0.021 mg/mLAerial partsEssential oilDPPHIC50 = 13.24 ± 0.06 mg/mL[27]Aerial partsEssential oilDPPHIC50 = 90 μg/mL[11]Aerial parts (wild)Essential oilDPPHIC50 = 132.23 ± 3.09 μg/mL[13]FRAPEC50 = 167.87 ± 1.46 μg/mLAerial parts (cultivated)Essential oilDPPHIC50 = 145.83 ± 3.47 μg/mLFRAPEC50 = 169.355 ± 2.04 μg/mLAerial parts (wild)Essential oilDPPHIC50 = 97.48 ± 2.24 μg/mL[20]FRAPEC50 = 167.86 ± 1.46 μg/mL


### 3.3.4. Anticancer Activity

The anticancer properties ofT. broussonetii have also been studied. Indeed, some investigations tested the efficiency of T. broussonetii essential oils on many cell lines [12, 15, 21] (Table 5). Ait M’Barek et al. [15] evaluated the antiproliferative effect of T. broussonetii EOs from stem and leaves on human ovarian adenocarcinoma IGR-OV1 parental cell line OV1/P. The results showed that the EOs tested inhibited the proliferation of this adenocarcinoma with an IC50 value of 0.40 ± 0.02 (%v/v).Table 5
Anticancer effects ofT. broussonetii.
Parts usedExtractsUsed methodsCell linesKey resultsReferencesLeaves and stemsEssential oilsCrystal violet assayThe parental human ovarian adenocarcinoma cell line IGR-OV1 (OV1/P)IC50 = 0.40 ± 0.02%(v/v)[15]Flowers and leavesEssential oils (variety: TbA)MTT assayP815 mastocytoma cell lineIC50 = 4.7%(v/v)[12]CEMIC50 = 3.6%(v/v)K-562IC50 = 10%(v/v)MCF -7IC50 = 10%(v/v)MCF -7 gemIC50 = 8.9%(v/v)Essential oils (variety: TbB)MTT assayP815 mastocytoma cell lineIC50 = 8.5%(v/v)CEMIC50 = 3.1%(v/v)K-562IC50 = 13.5%(v/v)MCF -7IC50 = 15.4%(v/v)MCF -7 gemIC50 = 17.5%(v/v)Flowers and leavesEssential oilsMTT assayP815 mastocytoma cell lineIC50 = 0.016%(v/v)[21]Moreover,Thymus broussonetii EOs extracted from flowers and leaves have been tested by Jaafari et al. [21] on the P815 mastocytoma cell line using MTT assay. In this study, the essential oils exhibited an important dose-dependent cytotoxic effect against the P815 cell line (IC50 = 0.016%).In another study, the authors evaluated the cytotoxic activity of essential oils from two chemotypes ofT. broussonetii against five tumor cell lines, namely P-815 (murine mastocytoma), K-562 (human chronic myelogenous leukemia), CEM (acuteT lymphoblastoid leukemia), and MCF 7 (human breast adenocarcinoma) and its counterpart resistant to gemcitabine (MCF -7 gem) using MTT assay. Consequently, cell viability showed a cell proliferation inhibition by the tested products in a dose-dependent manner with IC50 values ranging between 3.1 and 17.5% (v/v). Additionally, cell cycle analysis detected cell cycle arrest at S and G0/G1 phases in cells. This considerable activity might be due to the high content of thymol and carvacrol known for their promising anticancer effects via numerous mechanisms of action such as angiogenesis, inhibition of cell migration, autophagy, apoptosis, and cell cycle arrest [35, 36].


### 3.3.5. Anti-Inflammatory Activity

The antiedema effects of hexane, chloroform, and methanol extracts ofT. broussonettii were evaluated on croton oil-induced ear edema in mice. The chloroform extract showed the highest activity, reducing the oedematous response by 47%, the ID50 value of the indomethacin used as the reference drug (286 g/cm2) is three times higher than that of the chloroform extract 93 g/cm2. The chloroform extract of T. broussonettii possesses an anti-inflammatory activity ascribable to its triterpenic acid content; in fact, ursolic and oleanolic acid justify the edema inhibition observed. Ursolic acid was more potent than oleanolic acid with ID50 values of 56 and 132 g/cm2 corresponding to 0.12 and 0.29 mol/cm2, respectively [28] (Table 6).Table 6
Other pharmacological activities ofT. broussonetii.
ActivitiesUsed partsExtractsExperimental approachesKey resultsReferencesAnti-inflammatory activityLeavesn-hexaneCroton oil ear test in miceinhibition of the croton oil-induced ear edema in miceEdema reduction = 9%[28]ChloroformEdema reduction = 47%Chloroform + methanolEdema reduction = 16%MethanolEdema reduction = -5%Anticorrosive activityAerial partsEssential oilsLoss measurements and electrochemical techniques82.35% inhibition efficiency at a dose of 2 g/L[23]Insecticidal activityAerial partsEssential oilsFourth instar larvae ofCulex pipiensLC50 = 0.23[19]Antiparasitic activityAerial partsEssential oilsOral administration (20 g/animal) at the time of infection and thereafter for several daysAbsence of intracerebral cystsNo anomalies[30]Antipyretic activityStemWater, butanol, and ethyl acetateYeast-induced fever in ratsSignificantly reduced the temperature in febrile rats[37]Acute toxicityAerial partsEssential oilsSwiss mice (25–35 g)LD50 = 2.66 g/kg[22]Antinociceptive activityLeaves and stemWaterChemical and thermal models (in vivo)Writhing inhibition = 88.9%[31]Ethyl acetate and butanolWrithing inhibition = 69%Writhing inhibition = 62.8%Insecticidal activityAerial partsEssential oilsEffect against adults ofTribolium castaneum herbstLD50 = 0.08 μl/cm2LD90 = 0.19 μl/cm2[29]Insecticidal activityAerial partsEssential oilsEffect againstTribolium castanum pest foodstuffsTL50 = 1.5 μl/cm2[27]Immunological and behavioral activitiesLeaves and stemWater, butanol, and ethyl acetateTested the neurostimulant effects of the extractsIncreased (in vivo) the number of leukocyte categories studied[31]


### 3.3.6. Anticorrosive Potential

The essential oils ofT. broussonnetii at different concentrations (ranging from 0.05 to 2 g/L) were tested against corrosion on C38 steel in 1 M medium, HCl, using electrochemical impedance spectroscopy (EIS), potentiodynamic polarization, and weight loss methods. The essential oil was found to be rich in bioactive substances, mainly carvacrol (39.51%) followed by benzene, 1-methyl-2-(1-methylethyl) (14.80%), gammaterpinene (10.32%), alpha-pinene (9.7%), thymol (7.9%), and 3-cyclohexen-1-ol, 4-methyl-1-(1-methylethyl) (3.22%). Using the EIS test, the essential oil (2 g/L) inhibited the corrosion of metals and alloys in acid solutions with a percentage of 82.35% of the inhibition efficiency. The polarization studies showed that T. broussonnetii EOs inhibit both anodic metal dissolution and cathodic hydrogen reduction reactions. At the highest inhibition concentration, the maximum inhibition efficiency observed indicates that many molecules were adsorbed on the metal surface. At 2 g/L, the best efficiency obtained in the presence of essential oil was 81.63%. It has been noted that the inhibition efficiency increases with increasing temperature. The highest efficiency was 90% and reached 328 K. The inhibitory mechanism was probably achieved by chemical adsorption (chemisorption) of TBS molecules on the surface of carbon steel and this indeed increases with rising temperature [23] (Table 6).


### 3.3.7. Insecticidal Activities

TheT. broussonetti EOs were investigated for their insecticidal activity, using the larvae test sensibility technique. The chemical analysis by GC-MS showed that the major compounds of T. broussonetii essential oil were p-cymene (21.0%), borneol (16.5%), α-pinene (11.8%), and thymol (11.3%). The EOs of this plant proved larvicidal effectiveness against the fourth instar larvae of Culex pipiens and were significantly higher at the dose of 0.125 ppm compared to the control. The lethal concentration 50 (LC50) during exposure of the insect population to EOs at 24 hours was 0.23, and the effective toxicity on C. pipiens larvae was associated with the thymol compound of thyme oil [19] (Table 6).


### 3.3.8. Antipyretic Activity

At a dose of 200 mg/kg b.w.,T. broussonetii aqueous, butanol, and ethyl acetate extracts were investigated in vivo for their antipyretic effect on yeast-induced fever. In normothermic rats, the extracts were tested to determine whether the antipyretic activity is related to a hypothermic effect. Indeed, all extracts significantly reduced rectal temperature in febrile animals. However, they did not induce hypothermia in normal rats. Besides, an inhibition of platelet aggregation has been observed by acting in the same way as NSAI drugs. Furthermore, extracts of T. broussonetii contain many types of compounds such as triterpenes, saponins, tannins, flavonoids, and several salicylates. The presence of these compounds can enhance this antipyretic activity [22] (Table 6).


### 3.3.9. Antinociceptive

The immunostimulatory and neurotropic antistress effects of extracts (aqueous, ethyl acetate, and butanolic extracts) and EOs ofT. broussonetii were evaluated at three doses. Therefore, the aqueous and ethyl acetate extracts showed the best results. In fact, thyme extracts increased the number of leucocyte categories studied, in particular polynuclear cells, total lymphocytes, TCD4+, TCD8+, and NK cells. It has been suggested that intraperitoneal administration of T. broussonetii extracts has a potent direct effect on leucocytes in vivo. In contrast, this assumes that the two extracts partially prevent stress-induced disturbances in the rate of leukocytes. The ethyl acetate extract inhibited the increase in polynuclear cells caused by stress, increased lymphocytes, and decreased polynuclear counts in the stressed mice treated with the aqueous extract compared to the stressed mice [31].T. broussonetii was investigated to study the behavioral effects using the light/dark box test. At 12 mg/kg, the aqueous extract increased the number of transitions and the number of traversed squares and decreased the time spent in the dark compartment. The ethyl acetate extract increased both the number of traversed squares and the number of transitions without affecting the time spent in the dark compartment. The aqueous extract exerted an anxiolytic effect on the animals, while it could rather enhance locomotor and exploratory activities. The improvement in animal activity observed in the light/dark box after treatment with the aqueous extract is rather due to its anxiolytic-like effect and the ethyl acetate extract improved exploratory and locomotor activities in mice (Table 6).


### 3.3.10. Antiparasitic Activity

In another work, the effect ofT. broussonetii EOs was assessed on the experimental transmission of Toxoplasma gondii cysts in mice. These oils were administered orally (20 μg/animal) at the infection time and thereafter for several days. In mice given the essential oils, no cyst was observed. In addition, no disorder was noted in the control animals given the thyme EOs [30] (Table 6).


### 3.3.11. Insecticidal Activity

The insecticidal activity ofT. broussonetii EO was screened using the contact toxicity assay. The oil proved insecticidal effectiveness against Tribolium castaneum Herbst. After 24 h of treatment, the LD50 and LD90 were 0.08 and 0.19 μl/cm2, respectively. These results suggest that the contents of thyme EOs, in particular those obtained from the genus Thymus, have a good botanical bioinsecticide potential against Tribolium castaneum Herbst [29].The insecticidal activity of the EO of this plant was examined againstTribolium castanum by the contact toxicity assay. The essential oil exhibited the highest insecticidal activity with a median lethal time (TL50) of 1.5 μL/cm2 with LT50 (lethal time required to kill 50% of the exposed insects) values of 30,36 (24,62–38,48) at a dose of 1 μl/cm2 and 4,81(3,8–5,99) at a dose of 1,5 μl/cm2, respectively and a LT90 (lethal time required to kill 90% of the exposed insects) of 222,78(138,62–475,59) at a dose of 1 μl/cm2 and 16,07 (11,4–30,08), respectively. The Thymus broussonnetii Boiss EO could act as a substitute for biopesticide and reduce the harmful impact of chemical insecticides on the environment and humans [27] (Table 6).


### 3.3.12. Immunological and Behavioral Effects

The antinociceptive effect of aqueous, butanol, and ethyl acetate extracts ofT. broussonetii was studied using thermal and chemical nociception models and naloxone (a nonselective opioid antagonist) to determine the role of the opioid system in the antinociceptive activity of these extracts. To determine the phytoconstituents of the extracts tested, phytochemical screening was carried out, which revealed the presence of tannins in all the extracts. Quinones, saponins, and flavonoids were detected in butanol and ethyl acetate extracts, while terpenes were only identified in the ethyl acetate extract [31].The butanol and aqueous extracts showed an antinociceptive effect in both phases of formalin (50–300 mg/kg), tail immersion, and writing tests. At the same time, only the nociceptive response of the second phase was significantly reduced by the ethyl acetate extract (100–300 mg/kg). In the first and second phases, the aqueous extract was the most effective, with ED50 values of 177 (147–200) and 134 (95–170) mg/kg, respectively. The aqueous extract (200 mg/kg) showed a potent effect and significantly reduced the number of writhes induced by acetic acid, with 88.9% of writhes inhibition compared to those of ethyl acetate (69%) and butanol (63%) extracts. These obtained proved that T. broussonetii contains active compounds (polar and nonpolar) having antinociceptive activity with distinct mechanisms of action [31] (Table 6).


## 3.3.1. Antibacterial Activity

Several studies have shown the antibacterial effectiveness of different essential oils from the aerial part ofThymus broussonetii [28, 39, 40, 29, 41]. Table 2 summarizes all the studies which evaluated this activity in Thymus broussonetii, including the plant part used, type of extract, the antibacterial test, the strains studied, and the key results. The literature screening indicated that scientists had investigated the effect of Thymus broussonetii against the most critical pathogenic agents belonging to Gram-negative and Gram-positive bacteria. Indeed, Lattaoui and Tantaoui-elaraki, [34] assessed the antibacterial activity of the essential oil of T. broussonetii aerial part against three bacteria (Staphylococcus aureus, Escherichia coli, and Bacillus megaterium). The result of this study showed that T. broussonetii essential oils inhibited the growth of all bacterial strains with MIC values of 1, 3, and 4% (v/v) against S. aureus, E. coli, and B. megaterium, respectively. Belaqziz et al. [33] reported the antibacterial activity of T. broussonetti leaf EOs using agar disc diffusion against two Gram-positive bacteria, including S. aureus and Bacillus subtilis, and four Gram-negative bacteria, namely E. coli, Salmonella sp, Vibrio cholerae, and Pseudomonas aeruginosa. The results showed that the essential oil exhibited promising antibacterial power against the strains tested; Bacillus subtilis (Ф = 33 ± 0.4 mm), S. aureus (Ф = 19 ± 0.8 mm), Salmonella sp. (Ф = 9 ± 0.9 mm), Escherichia coli (Ф = 21 ± 0.1 mm), Vibrio cholerae (Ф = 40 ± 0.4 mm) and P. aeruginosa (Ф = 9 ± 0.1 mm). In another study, El Bouzidi et al. [13] tested the antibacterial activity of essential oils obtained from both wild and cultivated T. broussonetii using agar disc diffusion and macrodilution methods against Salmonella sp. (CCMM B17), E. coli (CCMM B4), E. coli (ATCC 25922), Bacillus cereus (ATCC 14579), Bacillus subtilis (ATCC 9524), Micrococcus luteus (ATCC10240), S. aureus (CCMM B3), and the clinically isolated strain, Enterobacter cloacae. Both EOs obtained from T. broussonetii (wild and cultivated) exhibited inhibitory activity on all the selected microorganisms, with inhibitory zones ranging between 23.33 and 53.67 mm and MIC values varied from 0.12 to 1.82 mg/mL. In fact, Micrococcus luteus was the most sensitive strain with MIC values of 53.50 and 53.67 mg/mL for wild and cultivated T. broussonetii, respectively, followed by B. subtilis, B. cereus, and S. aureus. However, Smahane et al. [26] investigated the inhibitory effect of T. broussonetii aerial part EOs against S. aureus, E. coli, and P. aeruginosa using disk diffusion and broth microdilution methods. The results revealed that all microorganisms tested were inhibited by essential oils with inhibitory zones ranging between 8.67 and 42.67 mm and MIC values ranged between 0.2 and 20 μg/mL.Table 2
Antibacterial effects ofT. broussonetii.
Used partsExtractsUsed methodsTested strainsKey resultsReferencesAerial partEssential oilAgar disk diffusion methodBroth microdilution methodStaphylococcus aureusФ = 42.67 ± 1.45 mm[26]MIC = 0.2μl/mLMBC = 0.6μl/mLEscherichia coliФ = 29.33 ± 0.54 mmMIC = 1.3μl/mLMBC = 1.3μl/mLPseudomonas aeruginosaФ = 8.67 ± 1.20 mmMIC = 20μl/mLMBC ≥ 80μl/mLAerial partEssential oilAgar disc diffusionBroth macrodilution methodStaphylococcus aureusФ = 35.00 ± 1.00 mm[13]MIC = 0.9 mg/mLMMC = 0.9 mg/mLBacillus subtilisФ = 49.67 ± 1.53 mmMIC = 0.23 mg/mLMMC = 0.23 mg/mLBacillus cereusФ = 48.67 ± 1.15 mmMIC = 0.23 mg/mLMMC = 0.23 mg/mLMicrococcus luteusФ = 53.50 ± 1.00 mmMIC = 0.12 mg/mLMMC = 0.12 mg/mLEscherichia coli 1 ATCC 25922Ф = 30.17 ± 1.00 mmMIC = 0.90 mg/mLMMC = 0.90 mg/mLE. coli 2 CCMM B4Ф = 29.67 ± 1.53 mmMIC = 0.90 mg/mLMMC = 0.90 mg/mLEnterobacter cloacaeФ = 27.33 ± 0.58 mmMIC = 0.90 mg/mLMMC = 0.90 mg/mLSalmonella sp.Ф = 31.67 ± 1.53 mmMIC = 0.90 mg/mLMMC = 0.90 mg/mLStaphylococcus aureusФ = 34.83 ± 1.04 mmMIC = 0.91 mg/mLMMC = 0.91 mg/mLBacillus subtilisФ = 49.00 ± 1.00 mmMIC = 0.23 mg/mLMMC = 0.23 mg/mLBacillus cereusФ = 47.33 ± 1.15 mmMIC = 0.23 mg/mLMMC = 0.23 mg/mLMicrococcus luteusФ = 53.67 ± 1.15 mmMIC = 0.12 mg/mLMMC = 0.12 mg/mLEscherichia coli 1 ATCC 25922Ф = 27.5 ± 1.53 mmMIC = 0.91 mg/mLMMC = 0.91 mg/mLE. coli 2 CCMM B4Ф = 29.33 ± 1.53 mmMIC = 0.91 mg/mLMMC = 0.91 mg/mLEnterobacter cloacaeФ = 23.33 ± 1.53 mmMIC = 1.82 mg/mLMMC = 1.82 mg/mLSalmonella sp.Ф = 31.33 ± 1.53 mmMIC = 0.91 mg/mLMMC = 0.91 mg/mLAerial partEssential oilAgar diffusion methodBacillus subtilisФ = 33 ± 0.4 mm[33]Staphylococcus aureusФ = 19 ± 0.8 mmSalmonella sp.Ф = 19 ± 0.9 mmEscherichia coliФ = 21 ± 0.1 mmVibrio choleraeФ = 40 ± 0.4 mmPseudomonas aeruginosaФ = 9 ± 0.1 mmAerial partEssential oilDisc diffusion methodMicrocystis aeruginosaФ = 90 ± 0.00 mm[17]MIC = 0.047 mg/mLMBC = 0.095 mg/mlAerial partEssential oilAgar diffusion methodStaphylococcus aureusNo measurable zone of inhibition[34]Aerial partEssential oilAgar diffusion methodEscherichia coliNo measurable zone of inhibition[16]MIC = 1%Staphylococcus aureusMIC = 3%Bacillus megateriumMIC = 4%Recently, Zerrifi and collaborators determined thein vitro antibacterial activity of T. broussonetii aerial part EOs using paper disk diffusion and microdilution methods against Microcystis aeruginosa. According to this study, the essential oils exhibited promising antibacterial power against the strain tested with an inhibitory zone of 90 mm, and MIC and MBC values of 0.047 and 0.095 mg/mL, respectively [17].


## 3.3.2. Antifungal Activity

The antifungal activity ofT. broussonetii EOs against many fungal strains was reported in several works [13, 16, 18, 20, 26, 34]. The previous publications on the antifungal activity that studied the essential oils from aerial parts of T. broussonetii by different methods are summarized in Table 3.Table 3
Antifungal activity ofT. broussonetii.
Used partsExtractsUsed methodsTested strainsKey resultsReferencesAerial partsEssential oilAgar disc diffusionBroth microdilution methodCandida albicansФ = 50.00 ± 1.00 mm[20]MIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida kruseiФ = 49.67 ± 1.53 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida glabrataФ = 49.33 ± 1.53 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida parapsilosisФ = 51.17 ± 0.76 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLEssential oilAgar disc diffusionBroth microdilution methodCandida albicansФ = 50.00 ± 1.00 mm[13]MIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida kruseiФ = 49.67 ± 1.53 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida glabrataФ = 49.33 ± 1.53 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida parapsilosisФ = 51.17 ± 0.76 mmMIC = 0.45 mg/mLMMC = 0.45 mg/mLCandida albicansФ = 49.67 ± 1.15 mmMIC = 0.46 mg/mLMMC = 0.46 mg/mLCandida kruseiФ = 47.33 ± 1.53 mmMIC = 0.46 mg/mLMMC = 0.46 mg/mLCandida glabrataФ = 48.50 ± 0.50 mmMIC = 0.46 mg/mLMMC = 0.46 mg/mLCandida parapsilosisФ = 50.00 ± 1.00 mmMIC = 0.46 mg/mLMMC = 0.46 mg/mLAerial partsEssential oilAgar diffusion methodmacrodilution broth methodCandida albicansФ = 38.5 ± 0.70 mm[18]MIC = 0.25μg/mLAerial partsEssential oilAgar diffusion methodCandida albicansSlightly more sensitive in presence of 0.2% oil[34]Aspergillus nigerLower sensitivity relatively resistantAerial partsEssential oilAgar diffusion methodSaccharomyces cerevisiaeMIC = 3%[16]Candida albicansMIC = 3%ZygorrhynchusspMIC = 4%Aspergillus nigerMIC = 3%Aerial partEssential oilAgar disk diffusion methodBroth microdilution methodCandida albicansФ = 35.67 ± 0.33 mm[26]MIC = NDMBC = NDAspergillus brasiliensisФ = NDMIC = NDMBC = NDSaad et al. [18] determined the in vitro antifungal efficacy of the essential oil from the aerial part against Candida albicans using the agar diffusion and macrodilution broth methods. Consequently, the zones of inhibition and MIC value were 38.5 mm and 0.25 μg/mL, respectively. Moreover, Jamali et al. [20] evaluated the EOs from aerial parts of the studied plant for their antifungal action against Candida albicans, Candida krusei, Candida glabrata, and Candida parapsilosis using agar disc diffusion and microdilution methods. The results revealed a strong antifungal activity against all the fungi tested with zones of inhibition ranging from 49.33 to 51.17 mm and MIC value of 0.45 mg/mL. Using the same methods and the same fungal strains, El Bouzidi et al. [13] investigated the antifungal activity of EOs obtained from wild and cultivated T. broussonetii. Therefore, these oils inhibited the growth of all fungal species with MIC values of 0.45 and 0.45 mg/mL for wild and cultivated Thymus broussonetii, respectively. In another study, the essential oil of T. broussonetii was tested against two fungal strains (Candida albicans and Aspergillus brasiliensis). The results revealed a strong antifungal inhibition against Candida albicans with zones of inhibition of 35.67 ± 0.33 mm [26].


## 3.3.3. Antioxidant Activity

Different studies have evaluated the antioxidant activity of extracts and EOs from different parts ofT. broussonetii using well-known techniques such as DPPH and FRAP assays [11, 13, 20, 25, 27] (Table 4). Indeed, Jamali et al. [20] investigated the antioxidant activity of the essential oils from aerial parts of T. broussonetii, and the results showed that the essential oil exhibited an interesting anti-DPPH (IC50 = 97.48 ± 2.24 μg/mL) and a high reducing power (EC50 = 167.86 ± 1.46 μg/ml) compared with the standard antioxidants, quercetin, and BHT with IC50 values of 1.07 ± 0.01 and4.21 ± 0.08 μg/mL, respectively, for DPPH and with EC50 values of 2.29 ± 0.1 and 7.09 ± 0.1 μg/mL, respectively, for FRAP. In another study, the wild and cultivated T. broussonetii EOs were tested for their antioxidant activity by DPPH and ferric ion reduction assays. The results showed an interesting antioxidant effect of the wild and cultivated T. broussonetii EOs with IC50 values of 132.23 ± 3.09 and 145.83 ± 3.47 μg/mL, respectively, for DPPH and with EC50 values of 167.87 ± 1.46 and 169.355 ± 2.04 μg/mL, respectively, for FRAP [13]. Moreover, Ouariachi et al. [11] demonstrated that the essential oils from T. broussonetii possessed high antioxidant activity using DPPH (IC50 = 90 μg/mL). On the other hand, Ahlam et al. [25] reported the antioxidant activity of the aqueous and methanol extracts from leaves and stems of T. broussonetii using FRAP and DPPH methods. The results revealed that both extracts exhibited a good antioxidant activity with FRAP capacity values ranging between 0.105 ± 0.021 and 1.579 ± 0.014 mg/mL and anti-DPPH power with IC50 values ranging between 0.132 ± 0.034 and 7.665 ± 0.411 mg/mL. The highest activity was observed in methanol extract from stems with EC50 and IC50 values of 0.105 ± 0.021 and 0.132 ± 0.034 mg/mL, respectively. On the other hand, essential oil showed a DPPH-radical-scavenging activity with IC50 = 13.24 ± 0.06 mg/mL [27].Table 4
Antioxidant effects ofT. broussonetii.
Used partsExtractsUsed methodsKey resultsReferencesLeavesAqueous extractDPPHIC50 = 22.61 ± 1.022 mg/mL[25]Methanol extractIC50 = 6.484 ± 0.190 mg/mLStemsAqueous extractIC50 = 7.665 ± 0.411 mg/mLMethanol extractIC50 = 0.132 ± 0.034 mg/mLLeavesAqueous extractFRAPEC50 = 0.597 ± 0.013 mg/mLMethanol extractEC50 = 1.579 ± 0.014 mg/mLStemsAqueous extractEC50 = 0.489 ± 0.011 mg/mLMethanol extractEC50 = 0.105 ± 0.021 mg/mLAerial partsEssential oilDPPHIC50 = 13.24 ± 0.06 mg/mL[27]Aerial partsEssential oilDPPHIC50 = 90 μg/mL[11]Aerial parts (wild)Essential oilDPPHIC50 = 132.23 ± 3.09 μg/mL[13]FRAPEC50 = 167.87 ± 1.46 μg/mLAerial parts (cultivated)Essential oilDPPHIC50 = 145.83 ± 3.47 μg/mLFRAPEC50 = 169.355 ± 2.04 μg/mLAerial parts (wild)Essential oilDPPHIC50 = 97.48 ± 2.24 μg/mL[20]FRAPEC50 = 167.86 ± 1.46 μg/mL


## 3.3.4. Anticancer Activity

The anticancer properties ofT. broussonetii have also been studied. Indeed, some investigations tested the efficiency of T. broussonetii essential oils on many cell lines [12, 15, 21] (Table 5). Ait M’Barek et al. [15] evaluated the antiproliferative effect of T. broussonetii EOs from stem and leaves on human ovarian adenocarcinoma IGR-OV1 parental cell line OV1/P. The results showed that the EOs tested inhibited the proliferation of this adenocarcinoma with an IC50 value of 0.40 ± 0.02 (%v/v).Table 5
Anticancer effects ofT. broussonetii.
Parts usedExtractsUsed methodsCell linesKey resultsReferencesLeaves and stemsEssential oilsCrystal violet assayThe parental human ovarian adenocarcinoma cell line IGR-OV1 (OV1/P)IC50 = 0.40 ± 0.02%(v/v)[15]Flowers and leavesEssential oils (variety: TbA)MTT assayP815 mastocytoma cell lineIC50 = 4.7%(v/v)[12]CEMIC50 = 3.6%(v/v)K-562IC50 = 10%(v/v)MCF -7IC50 = 10%(v/v)MCF -7 gemIC50 = 8.9%(v/v)Essential oils (variety: TbB)MTT assayP815 mastocytoma cell lineIC50 = 8.5%(v/v)CEMIC50 = 3.1%(v/v)K-562IC50 = 13.5%(v/v)MCF -7IC50 = 15.4%(v/v)MCF -7 gemIC50 = 17.5%(v/v)Flowers and leavesEssential oilsMTT assayP815 mastocytoma cell lineIC50 = 0.016%(v/v)[21]Moreover,Thymus broussonetii EOs extracted from flowers and leaves have been tested by Jaafari et al. [21] on the P815 mastocytoma cell line using MTT assay. In this study, the essential oils exhibited an important dose-dependent cytotoxic effect against the P815 cell line (IC50 = 0.016%).In another study, the authors evaluated the cytotoxic activity of essential oils from two chemotypes ofT. broussonetii against five tumor cell lines, namely P-815 (murine mastocytoma), K-562 (human chronic myelogenous leukemia), CEM (acuteT lymphoblastoid leukemia), and MCF 7 (human breast adenocarcinoma) and its counterpart resistant to gemcitabine (MCF -7 gem) using MTT assay. Consequently, cell viability showed a cell proliferation inhibition by the tested products in a dose-dependent manner with IC50 values ranging between 3.1 and 17.5% (v/v). Additionally, cell cycle analysis detected cell cycle arrest at S and G0/G1 phases in cells. This considerable activity might be due to the high content of thymol and carvacrol known for their promising anticancer effects via numerous mechanisms of action such as angiogenesis, inhibition of cell migration, autophagy, apoptosis, and cell cycle arrest [35, 36].


## 3.3.5. Anti-Inflammatory Activity

The antiedema effects of hexane, chloroform, and methanol extracts ofT. broussonettii were evaluated on croton oil-induced ear edema in mice. The chloroform extract showed the highest activity, reducing the oedematous response by 47%, the ID50 value of the indomethacin used as the reference drug (286 g/cm2) is three times higher than that of the chloroform extract 93 g/cm2. The chloroform extract of T. broussonettii possesses an anti-inflammatory activity ascribable to its triterpenic acid content; in fact, ursolic and oleanolic acid justify the edema inhibition observed. Ursolic acid was more potent than oleanolic acid with ID50 values of 56 and 132 g/cm2 corresponding to 0.12 and 0.29 mol/cm2, respectively [28] (Table 6).Table 6
Other pharmacological activities ofT. broussonetii.
ActivitiesUsed partsExtractsExperimental approachesKey resultsReferencesAnti-inflammatory activityLeavesn-hexaneCroton oil ear test in miceinhibition of the croton oil-induced ear edema in miceEdema reduction = 9%[28]ChloroformEdema reduction = 47%Chloroform + methanolEdema reduction = 16%MethanolEdema reduction = -5%Anticorrosive activityAerial partsEssential oilsLoss measurements and electrochemical techniques82.35% inhibition efficiency at a dose of 2 g/L[23]Insecticidal activityAerial partsEssential oilsFourth instar larvae ofCulex pipiensLC50 = 0.23[19]Antiparasitic activityAerial partsEssential oilsOral administration (20 g/animal) at the time of infection and thereafter for several daysAbsence of intracerebral cystsNo anomalies[30]Antipyretic activityStemWater, butanol, and ethyl acetateYeast-induced fever in ratsSignificantly reduced the temperature in febrile rats[37]Acute toxicityAerial partsEssential oilsSwiss mice (25–35 g)LD50 = 2.66 g/kg[22]Antinociceptive activityLeaves and stemWaterChemical and thermal models (in vivo)Writhing inhibition = 88.9%[31]Ethyl acetate and butanolWrithing inhibition = 69%Writhing inhibition = 62.8%Insecticidal activityAerial partsEssential oilsEffect against adults ofTribolium castaneum herbstLD50 = 0.08 μl/cm2LD90 = 0.19 μl/cm2[29]Insecticidal activityAerial partsEssential oilsEffect againstTribolium castanum pest foodstuffsTL50 = 1.5 μl/cm2[27]Immunological and behavioral activitiesLeaves and stemWater, butanol, and ethyl acetateTested the neurostimulant effects of the extractsIncreased (in vivo) the number of leukocyte categories studied[31]


## 3.3.6. Anticorrosive Potential

The essential oils ofT. broussonnetii at different concentrations (ranging from 0.05 to 2 g/L) were tested against corrosion on C38 steel in 1 M medium, HCl, using electrochemical impedance spectroscopy (EIS), potentiodynamic polarization, and weight loss methods. The essential oil was found to be rich in bioactive substances, mainly carvacrol (39.51%) followed by benzene, 1-methyl-2-(1-methylethyl) (14.80%), gammaterpinene (10.32%), alpha-pinene (9.7%), thymol (7.9%), and 3-cyclohexen-1-ol, 4-methyl-1-(1-methylethyl) (3.22%). Using the EIS test, the essential oil (2 g/L) inhibited the corrosion of metals and alloys in acid solutions with a percentage of 82.35% of the inhibition efficiency. The polarization studies showed that T. broussonnetii EOs inhibit both anodic metal dissolution and cathodic hydrogen reduction reactions. At the highest inhibition concentration, the maximum inhibition efficiency observed indicates that many molecules were adsorbed on the metal surface. At 2 g/L, the best efficiency obtained in the presence of essential oil was 81.63%. It has been noted that the inhibition efficiency increases with increasing temperature. The highest efficiency was 90% and reached 328 K. The inhibitory mechanism was probably achieved by chemical adsorption (chemisorption) of TBS molecules on the surface of carbon steel and this indeed increases with rising temperature [23] (Table 6).


## 3.3.7. Insecticidal Activities

TheT. broussonetti EOs were investigated for their insecticidal activity, using the larvae test sensibility technique. The chemical analysis by GC-MS showed that the major compounds of T. broussonetii essential oil were p-cymene (21.0%), borneol (16.5%), α-pinene (11.8%), and thymol (11.3%). The EOs of this plant proved larvicidal effectiveness against the fourth instar larvae of Culex pipiens and were significantly higher at the dose of 0.125 ppm compared to the control. The lethal concentration 50 (LC50) during exposure of the insect population to EOs at 24 hours was 0.23, and the effective toxicity on C. pipiens larvae was associated with the thymol compound of thyme oil [19] (Table 6).


## 3.3.8. Antipyretic Activity

At a dose of 200 mg/kg b.w.,T. broussonetii aqueous, butanol, and ethyl acetate extracts were investigated in vivo for their antipyretic effect on yeast-induced fever. In normothermic rats, the extracts were tested to determine whether the antipyretic activity is related to a hypothermic effect. Indeed, all extracts significantly reduced rectal temperature in febrile animals. However, they did not induce hypothermia in normal rats. Besides, an inhibition of platelet aggregation has been observed by acting in the same way as NSAI drugs. Furthermore, extracts of T. broussonetii contain many types of compounds such as triterpenes, saponins, tannins, flavonoids, and several salicylates. The presence of these compounds can enhance this antipyretic activity [22] (Table 6).


## 3.3.9. Antinociceptive

The immunostimulatory and neurotropic antistress effects of extracts (aqueous, ethyl acetate, and butanolic extracts) and EOs ofT. broussonetii were evaluated at three doses. Therefore, the aqueous and ethyl acetate extracts showed the best results. In fact, thyme extracts increased the number of leucocyte categories studied, in particular polynuclear cells, total lymphocytes, TCD4+, TCD8+, and NK cells. It has been suggested that intraperitoneal administration of T. broussonetii extracts has a potent direct effect on leucocytes in vivo. In contrast, this assumes that the two extracts partially prevent stress-induced disturbances in the rate of leukocytes. The ethyl acetate extract inhibited the increase in polynuclear cells caused by stress, increased lymphocytes, and decreased polynuclear counts in the stressed mice treated with the aqueous extract compared to the stressed mice [31].T. broussonetii was investigated to study the behavioral effects using the light/dark box test. At 12 mg/kg, the aqueous extract increased the number of transitions and the number of traversed squares and decreased the time spent in the dark compartment. The ethyl acetate extract increased both the number of traversed squares and the number of transitions without affecting the time spent in the dark compartment. The aqueous extract exerted an anxiolytic effect on the animals, while it could rather enhance locomotor and exploratory activities. The improvement in animal activity observed in the light/dark box after treatment with the aqueous extract is rather due to its anxiolytic-like effect and the ethyl acetate extract improved exploratory and locomotor activities in mice (Table 6).


## 3.3.10. Antiparasitic Activity

In another work, the effect ofT. broussonetii EOs was assessed on the experimental transmission of Toxoplasma gondii cysts in mice. These oils were administered orally (20 μg/animal) at the infection time and thereafter for several days. In mice given the essential oils, no cyst was observed. In addition, no disorder was noted in the control animals given the thyme EOs [30] (Table 6).


## 3.3.11. Insecticidal Activity

The insecticidal activity ofT. broussonetii EO was screened using the contact toxicity assay. The oil proved insecticidal effectiveness against Tribolium castaneum Herbst. After 24 h of treatment, the LD50 and LD90 were 0.08 and 0.19 μl/cm2, respectively. These results suggest that the contents of thyme EOs, in particular those obtained from the genus Thymus, have a good botanical bioinsecticide potential against Tribolium castaneum Herbst [29].The insecticidal activity of the EO of this plant was examined againstTribolium castanum by the contact toxicity assay. The essential oil exhibited the highest insecticidal activity with a median lethal time (TL50) of 1.5 μL/cm2 with LT50 (lethal time required to kill 50% of the exposed insects) values of 30,36 (24,62–38,48) at a dose of 1 μl/cm2 and 4,81(3,8–5,99) at a dose of 1,5 μl/cm2, respectively and a LT90 (lethal time required to kill 90% of the exposed insects) of 222,78(138,62–475,59) at a dose of 1 μl/cm2 and 16,07 (11,4–30,08), respectively. The Thymus broussonnetii Boiss EO could act as a substitute for biopesticide and reduce the harmful impact of chemical insecticides on the environment and humans [27] (Table 6).


## 3.3.12. Immunological and Behavioral Effects

The antinociceptive effect of aqueous, butanol, and ethyl acetate extracts ofT. broussonetii was studied using thermal and chemical nociception models and naloxone (a nonselective opioid antagonist) to determine the role of the opioid system in the antinociceptive activity of these extracts. To determine the phytoconstituents of the extracts tested, phytochemical screening was carried out, which revealed the presence of tannins in all the extracts. Quinones, saponins, and flavonoids were detected in butanol and ethyl acetate extracts, while terpenes were only identified in the ethyl acetate extract [31].The butanol and aqueous extracts showed an antinociceptive effect in both phases of formalin (50–300 mg/kg), tail immersion, and writing tests. At the same time, only the nociceptive response of the second phase was significantly reduced by the ethyl acetate extract (100–300 mg/kg). In the first and second phases, the aqueous extract was the most effective, with ED50 values of 177 (147–200) and 134 (95–170) mg/kg, respectively. The aqueous extract (200 mg/kg) showed a potent effect and significantly reduced the number of writhes induced by acetic acid, with 88.9% of writhes inhibition compared to those of ethyl acetate (69%) and butanol (63%) extracts. These obtained proved that T. broussonetii contains active compounds (polar and nonpolar) having antinociceptive activity with distinct mechanisms of action [31] (Table 6).


## 3.4. Toxicological Investigations

An acute toxicity screening was carried out forT. broussonetii EOs in order to verify their harmlessness to avoid a possible overdose and to properly determine the toxicological profile of the T. broussonetii species. This was assessed using the Leitchfield and Wilcoxon method, and the effective lethal dose (LD50) was measured. Subsequently, signs of toxicity such as diarrhea, convulsion, piloerection, motor coordination, and behavioral changes (excitation and twitches) were determined. For the groups receiving the dose of 1 g/kg, the change in body weight was also determined. On the other hand, thymol (36.7%) and borneol (21.9%) were the two major compounds, followed by p-cymene (7.6%) and β-pinene (0.7%). At a dose of 2 mg/kg, some cases of death and signs of toxicity were recorded. The LD90s and LD50s were estimated to be 7.31 (5.64–13.54) and 4.47 (3.6–6.72) g/kg, respectively [22].


## 4. Conclusion and Perspectives

Here, the phytochemistry, toxicology, and pharmacological properties ofT. broussonetii were highlighted. Phytochemical studies of this species showed its richness in numerous bioactive compounds, exhibiting important biological effects. Pharmacological investigations confirmed the safety of this plant. However, these investigations must be further investigated using several toxicological reports at several different doses and time periods. Pharmacological biology explorations demonstrated that T. broussonetii essential oils and extracts exhibit important and remarkably antimicrobial, anticancer and, anti-inflammatory properties. These investigations were conducted using in vitro approaches, and therefore, further in vivo examinations should be performed to explore the pharmacological properties of T. broussonetii importantly. Moreover, mechanisms related to the biological effects of T. broussonetii and its bioactive compounds should also be explored to validate their pharmacodynamic actions.



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*Source: 6350035-2021-09-06.xml*