Phytochemistry and Pharmacology of Thymus broussonetii Boiss

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.


Introduction
ymus broussonetii Boiss ( ymus broussonetii) belongs to the Lamiaceae family and the genus of ymus. 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. is 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].
Due to the intensification of research on the pharmacological effects of T. 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.

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

Botanical Description and Geographical Distribution.
ymus 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 purplecolored, 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].
In comparison with wild-harvested and cultivated T. 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%), c-terpinene (8.9%), thymol (12.3%), and carvacrol (43.4%) for wildharvested 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]. e chemical analysis of polar fraction from T. 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].    Evidence-Based Complementary and Alternative Medicine summarizes all the studies which evaluated this activity in ymus broussonetii, including the plant part used, type of extract, the antibacterial test, the strains studied, and the key results. e literature screening indicated that scientists had investigated the effect of ymus 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). e 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.

Pharmacological
e results showed that the essential oil exhibited promising antibacterial power against the strains tested; Bacillus subtilis (V � 33 ± 0.4 mm), S. aureus Vibrio cholerae (V � 40 ± 0.4 mm) and P. aeruginosa (V � 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  Recently, Zerrifi and collaborators determined the in 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].

Antifungal Activity.
e antifungal activity of T. broussonetii EOs against many fungal strains was reported in several works [13,16,18,20,26,34]. e 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.
Saad 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.
e 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. erefore, these oils inhibited the growth of all fungal species with MIC values of 0.45 and 0.45 mg/mL for wild and cultivated ymus broussonetii, respectively. In another study, the essential oil of T. broussonetii was tested against two fungal strains (Candida albicans and Aspergillus brasiliensis). e results revealed a strong antifungal inhibition against Candida albicans with zones of inhibition of 35.67 ± 0.33 mm [26].

Antioxidant Activity.
Different studies have evaluated the antioxidant activity of extracts and EOs from different parts of T. 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 (IC 50 � 97.48 ± 2.24 μg/mL) and a high reducing power (EC 50 � 167.86 ± 1.46 μg/ml) compared with the standard antioxidants, quercetin, and BHT with IC 50 values of 1.07 ± 0.01 and4.21 ± 0.08 μg/mL, respectively, for DPPH and with EC 50 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. e results showed an interesting antioxidant effect of the wild and cultivated T. broussonetii EOs with IC 50 values of 132.23 ± 3.09 and 145.83 ± 3.47 μg/mL, respectively, for DPPH and with EC 50 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 (IC 50 � 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. e results revealed that both extracts exhibited a good antioxidant activity with FRAP capacity values

Anticancer
Activity. e anticancer properties of T. broussonetii have also been studied. Indeed, some investigations tested the efficiency of T. broussonetii essential oils  [21] on the P815 mastocytoma cell line using MTTassay. In this study, the essential oils exhibited an important dose-dependent cytotoxic effect against the P815 cell line (IC 50 � 0.016%).
In another study, the authors evaluated the cytotoxic activity of essential oils from two chemotypes of T. 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 IC 50 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. is 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].

Anti-Inflammatory Activity.
e antiedema effects of hexane, chloroform, and methanol extracts of T. broussonettii were evaluated on croton oil-induced ear edema in mice. e chloroform extract showed the highest activity, reducing the oedematous response by 47%, the ID 50 value of the indomethacin used as the reference drug (286 g/cm 2 ) is three times higher than that of the chloroform extract 93 g/cm 2 . e 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 ID 50 values of 56 and 132 g/cm 2 corresponding to 0.12 and 0.29 mol/cm 2 , respectively [28] (Table 6).

Anticorrosive Potential.
e essential oils of T. 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. e essential oil was found to be rich in bioactive substances, mainly carvacrol (39.51%) followed by benzene, 1methyl-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. e 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. e highest efficiency was 90% and reached 328 K. e 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).

Insecticidal Activities.
e T. broussonetti EOs were investigated for their insecticidal activity, using the larvae test sensibility technique. e 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%). e 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. e lethal concentration 50 (LC 50 ) during Evidence-Based Complementary and Alternative Medicine 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).

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 yeastinduced 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. e presence of these compounds can enhance this antipyretic activity [22] (Table 6).

Antinociceptive.
e immunostimulatory and neurotropic antistress effects of extracts (aqueous, ethyl acetate, and butanolic extracts) and EOs of T. broussonetii were evaluated at three doses. erefore, the aqueous and ethyl   [31] 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. e 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. e ethyl acetate extract increased both the number of traversed squares and the number of transitions without affecting the time spent in the dark compartment.
e aqueous extract exerted an anxiolytic effect on the animals, while it could rather enhance locomotor and exploratory activities. e 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).

Antiparasitic Activity.
In another work, the effect of T. broussonetii EOs was assessed on the experimental transmission of Toxoplasma gondii cysts in mice. ese 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).

Insecticidal Activity.
e insecticidal activity of T. broussonetii EO was screened using the contact toxicity assay. e oil proved insecticidal effectiveness against Tribolium castaneum Herbst. After 24 h of treatment, the LD 50 and LD 90 were 0.08 and 0.19 μl/cm 2 , respectively. ese results suggest that the contents of thyme EOs, in particular those obtained from the genus ymus, have a good botanical bioinsecticide potential against Tribolium castaneum Herbst [29]. e insecticidal activity of the EO of this plant was examined against Tribolium castanum by the contact toxicity assay. e essential oil exhibited the highest insecticidal activity with a median lethal time (TL 50 ) of 1.5 μL/cm 2 with LT 50 (lethal time required to kill 50% of the exposed insects) values of 30,36 (24,48) at a dose of 1 μl/cm 2 and 4,81 (3,99) at a dose of 1,5 μl/cm 2 , respectively and a LT 90 (lethal time required to kill 90% of the exposed insects) of 222,78(138,62-475,59) at a dose of 1 μl/cm 2 and 16,07 (11,08), respectively. e ymus 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).

Immunological and Behavioral Effects.
e antinociceptive effect of aqueous, butanol, and ethyl acetate extracts of T. 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]. e 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 ED 50 values of 177 (147-200) and 134 (95-170) mg/kg, respectively. e 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. ese obtained proved that T. broussonetii contains active compounds (polar and nonpolar) having antinociceptive activity with distinct mechanisms of action [31] (Table 6).

Toxicological
Investigations. An acute toxicity screening was carried out for T. 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. is was assessed using the Leitchfield and Wilcoxon method, and the effective lethal dose (LD 50 ) 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. e LD 90 s and LD 50 s were estimated to be 7.31 (5.64-13.54) and 4.47 (3.6-6.72) g/ kg, respectively [22].

Conclusion and Perspectives
Here, the phytochemistry, toxicology, and pharmacological properties of T. 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.
Evidence-Based Complementary and Alternative Medicine ese 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.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.