Next Article in Journal
Next Article in Special Issue
The Profile and Content of Polyphenols and Carotenoids in Local and Commercial Sweet Cherry Fruits (Prunus avium L.) and Their Antioxidant Activity In Vitro
Previous Article in Journal
Comparison of Mitochondrial Superoxide Detection Ex Vivo/In Vivo by mitoSOX HPLC Method with Classical Assays in Three Different Animal Models of Oxidative Stress
Previous Article in Special Issue
Impact of Cold versus Hot Brewing on the Phenolic Profile and Antioxidant Capacity of Rooibos (Aspalathus linearis) Herbal Tea

Antioxidants 2019, 8(11), 515;

Differentiation of Phenolic Composition Among Tunisian Thymus algeriensis Boiss. et Reut. (Lamiaceae) Populations: Correlation to Bioactive Activities
Laboratory of Plant Biotechnology, Department of Biology, National Institute of Applied Science and Technology, B.P. 676, Tunis CEDEX 1080, Tunisia
QOPNA & LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
Authors to whom correspondence should be addressed.
Received: 1 October 2019 / Accepted: 24 October 2019 / Published: 28 October 2019


Twelve Tunisian Thymus algeriensis populations growing wild in different bioclimatic zones, extending from the subhumid to the upper-arid bioclimates, were compared regarding their phenolic composition and their ability to serve as antioxidant, anti-acetylcholinesterase, and antibacterial agents. A significant variation of phenol profile was observed between the analyzed populations, as assessed by ultra-high-performance liquid chromatography coupled with a diode array detector and an electrospray mass spectrometer (UHPLC-DAD-ESI/MSn) technique. Rosmarinic acid was the main phenolic compound in most populations (383.8–1157.8 µg/mL extract), but still, those from the upper-arid bioclimatic zone were distinguished by the presence of carvacrol (1374.7 and 2221.6 µg/mL extract), which was absent in the remaining ones. T. algeriensis methanolic extracts were found to possess a substantial antioxidant and anti-acetylcholinesterase activities, with significant variation observed between populations, which were correlated to their phenolic contents. The antibacterial activity of the extracts tested against seven bacteria was revealed only by populations collected from upper-arid bioclimate and mainly associated with the presence of carvacrol. Extracts revealed a bacteriostatic effect against all bacteria (MIC = 1.4 mg/mL). Yet, the bactericidal activity (MBC = 1.4mg/mL) was restricted to the gram-negative bacteria Escherchia coli.
thyme; UHPLC-DAD-ESI/MSn; phenolic compounds; antioxidant activity; anti-acetylcholinesterase activity; antibacterial activity

1. Introduction

Nowadays, there is great interest in medicinal plants and their bioactive compounds, namely antioxidant compounds, to be used as health-promoting agents in distinct industrial fields. Thymus species are aromatic plants known for their richness in bioactive phytochemicals [1], including phenolic acids and flavonoids [2].
In most parts of the world, thyme is considered one of the most valuable spices/food preservatives in the food industry [3]. In addition, it is commonly used in folk medicine because of its medicinal properties, including antispasmodic, antioxidant [4,5,6], antifungal [7,8], antibacterial [9,10,11], antitumor [12] and anti-aflatoxigenic [13] properties.
In Tunisia, the genus Thymus L. includes Thymus capitatus (L.) Hoffm. et Link., (= Thymbra capitata (L.) Cav = Coridothymus capitatus (L.) Rchb. F. = Satureja capitata L. = Thymus cephalotus L), Thymus numidicus Poir., Thymus algeriensis Boiss. and Reut., (= Thymus hirtus subsp. algeriensis (Boiss. et Reut.)), and Thymus vulgaris L. [14]. Thymus algeriensis is an endemic species of Morocco, Algeria, Tunisia and Libya [14,15] and described to be a diploid (2n = 2x = 30) and gynodioecious species [16]. In Tunisia, this species has a wide geographical distribution and grows spontaneously in diverse bioclimatic zones, extending from the subhumid to the lower arid bioclimates. It is largely used, fresh or dried, in food, as a culinary herb and in folk medicine, mainly due to its protection against abortion and respiratory and digestive tube disorders [12,17].
The chemical composition and biological properties of essential oils of T. algeriensis from different geographical areas have been reported [18,19,20,21]. In turn, to the best of our knowledge, there are only a few studies focusing on the phenolic composition of this species, although previous bibliography highlighted its richness in flavonoids [22,23] and phenolic acids [24], thus suggesting its suitability to serve as a source of bioactive compounds. This work is complementary to that performed by members of our group [16,25] that focused on the genetic variability of T. algeriensis with regard to its essential oils. In the present study, variability of phenolic compounds is assessed in twelve Tunisian populations, collected from four different bioclimatic zones, while the potency of their respective methanolic extracts to serve as antioxidant and antibacterial agents, or to inhibit acetylcholinesterase activity, are correlated with their phenolic profile. Please note that the study of the chemical polymorphism in the natural populations of T. algeriensis species can provide new insights that may result in the selection of populations with high phenolic/bioactive components to be used by food and pharmaceutical industries [3].

2. Materials and Methods

2.1. Chemicals

Rosmarinic acid, carvacrol, luteolin-7-O-glucoside, kaempferol, apigenin-7-O-glucoside, and eriodictyol-7-O-glucoside were purchased from Extrasynthese (Genay, France). Methanol, n-hexane, and acetonitrile with HPLC purity were obtained from Lab-Scan (Lisbon, Portugal). Water was treated in a Direct-Q® water purification system (Merck Life Science, Darmstadt, Germany). Folin-Ciocalteu reagent was obtained from Panreac (Barcelona, Spain), sodium carbonate (Na2CO3), aluminum chloride (AlCl3), gallic acid, rutin, 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), butylated hydroxytoluene (BHT), lyophilized acetylcholinesterase (AChE, from electric eel, type VI-S), Iodure acetylthiocholine, DTNB 5,5′-dithio-bis-[2-nitrobenzoic acid] (DTNB), and Donepezil were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 2,4,6-tripyridyls-triazine (TPTZ) and β-carotene were obtained from Fluka (Munich, Germany). Tryptic soy broth was purchased from Scharlau Microbiology (Barcelona, Spain).

2.2. Plant Material

Leaves from twelve populations of T. algeriensis belonging to different geographic and bioclimatic zones in Tunisia, namely sub humid (Sh), upper semi-arid (Usa), mean semi-arid (Msa), lower semi-arid (Lsa), and upper arid (Ua) (Table 1), were collected during the flowering stage, to assure maximal phenolic amounts and homogeneity among samples (differences between individuals within and between populations are known to be more evident at the vegetative stage). The collected plants were identified by Pr. M. Boussaid from the INSAT (Department of Biology), and voucher specimens were deposited in the Herbarium of the National Institute of Applied Science and Technology of Tunis (T.a. INSAT, 15). From each population, ten individuals at the flowering phase were sampled randomly in an area exceeding 2 ha. Plant materials were dried for 7 days, at room temperature, in the absence of direct sunlight, reaching a final moisture level close to 10%, and then they were powdered by grinding.

2.3. Preparation of Extracts

The extracts were prepared following the general procedure previously described [26]. In detail, 1 g of dried leaves of T. algeriensis were macerated in 10 mL of methanol for 24 h, at room temperature. Extraction with methanol was reported as the most effective for the recovery of phenolic compounds [27]. The samples were filtered and stored +4 °C for general analysis.

2.4. Analysis of Phenolic Compounds

2.4.1. Determination of Total Phenolic and Flavonoid Contents

Total phenolic content was determined using the Folin-Ciocalteu method [28], with some modifications. In more detail, 0.5 mL of diluted sample extract was added to 2 mL of Folin-Ciocalteu reagent, followed by the adding of 2.5 mL of sodium carbonate solution (7.5%) and reading of absorbance at 760 nm, after incubation for 90 min in the dark. Results were expressed as gallic acid equivalents per g of plant dried weight (mg GAE/g leaves DW).
Estimation of total flavonoid content in each extract was performed using the AlCl3 method, as reported previously by [26], with minor modifications. One milliliter of each diluted sample was mixed with 1 mL of AlCl3 solution (2%), followed by the reading of absorbance at 430 nm after 15 min of incubation. The percentage contents of flavonoids were expressed as mg rutin equivalents per g of plant dried weight (mg RE/g leaves DW).

2.4.2. Identification and Quantification of Phenolic Compounds by UHPLC-DAD-ESI/MSn

The individual phenolic compounds of the twelve populations of Tunisian T. algeriensis were identified by UHPLC-DAD-ESI/MSn analysis, following the procedure previously described [29], performed on a Ultimate 3000 (Dionex Co., San Jose, CA, USA) apparatus equipped with an ultimate 3000 Diode Array Detector (Dionex Co., San Jose, CA, USA) and coupled with a Thermo LTQ XL (Thermo Scientific, San Jose, CA, USA) ion trap mass spectrometer equipped with an ESI source. The column used was a 100 mm length, 2.1 mm i.d., 1.9 μm particle diameter, end-capped Hypersil Gold C18 column (Thermo Scientific, San Jose, CA, USA), and its temperature was maintained at 30 °C. Quantification of phenolic compounds was performed by peak integration, using the external standard method, with the closest reference compound available [30].

2.5. Antioxidant Assays

2.5.1. DPPH• Scavenging Test

The free radical scavenging activity of extracts was measured using the 1,1-diphenyl-2-picryl-hydrazil radical (DPPH•), following the procedure previously described by Zaouali et al. [31]. One milliliter of diluted extract was added to 3 mL of the methanol-DPPH• solution (4 × 10−5 M) and stored in the dark. After 30 min, the decrease in absorbance was measured at 517 nm against a blank (methanol solution). Trolox was used as a positive control. Results were expressed as EC50 (the efficient concentration to scavenge 50% of DPPH•).

2.5.2. β-Carotene Bleaching Test

The β-carotene method was carried out according to [31]. The absorbance was measured at 470 nm. The same procedure was repeated with the synthetic antioxidant butylated hydroxytoluene (BHT) as a positive control. Results were expressed as EC50. An extract concentration providing 50% inhibition (EC50) was obtained, plotting inhibition percentage versus extract solution concentrations.

2.5.3. Ferric-Reducing Antioxidant Power (FRAP) Assay

The ferric-reducing ability of extracts was determined as described by Zaouali et al. [31]. The FRAP reagent was freshly prepared by mixing acetate buffer (300 mM, pH 3.6), TPTZ solution (10 mM TPTZ in 40 mM HCl), and FeCl3-6H2O (20 mM) in a ratio of 10:1:1. To perform the assay, 900 μL of FRAP reagent was mixed with 90 μL distilled water and 30 μL of the diluted samples. The absorbance was measured at 593 nm, using FRAP working solution as a blank. A standard curve was prepared using different concentrations of FeSO4.6H2O. Results were expressed in mmol Fe2+/L of extract.

2.6. Acetylcholinesterase Inhibition Assay

The anti-acetylcholinesterase activity was assayed by the spectrophotometric method of Elden et al. [32], with some modifications. Then, 20 μL of methanolic extracts (at different concentrations) was mixed with 25 μL of the enzyme solution (0.28 U/mL). After incubation during 15 min at 37 °C, the reaction was then initiated with the addition of 100 μL of acetylcholine solution (0.15 mM), and 500 μL of 0.3 mM 5,5-dithiobis-2-nitrobenzoic acid was added to 355 μL of Tris-HCl buffer (50 mM, pH 8.0, containing 0.1% bovine serum albumin. Results were expressed as EC50 (concentration providing 50% of AChE inhibition). Donepezil was used as a positive control.

2.7. Antibacterial Activity

2.7.1. Bacterial Strains

The antibacterial activity of methanolic extracts was evaluated against 7 standards bacteria, namely Staphylococcus aureus, Streptococcus feacalis, Bacillus cereus, Staphylococcus epidermis (gram-positive), and Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumonia (gram-negative). Bacterial strains were cultured overnight at 37 °C in nutrient broth (Scharlau Microbiology, Barcelona, Spain).

2.7.2. Well-Diffusion Method

Antibacterial tests were carried out via the well-diffusion method [33], using 100 µL of suspension of the tested bacteria, containing 105 CFU/mL of bacterial strains spread on Tryptic soy agar. Then, 70 µL of methanolic extracts were introduced into the well. The inoculated plates were incubated for 24 h at 37 °C. After incubation, the diameters of inhibition zones were used as a measure of antibacterial activity. Gentamicin (30 µg/disc) and dimethyl sulfoxide (DMSO) were used as a positive and negative control, respectively.

2.7.3. Determination of Minimum Inhibitory (MIC) and Bactericidal (MBC) Concentrations

The MIC was defined as the lowest concentration of the total extracts that induces no visible growth of bacteria [34]. Referring to results of the MIC assay, the minimum bactericidal concentration (MBC) was determined. Then, 50 µL from each methanol extract, showing growth inhibition zone, was added to 5 mL of Triptic soy agar (TSA) broth tubes and incubated for 24 h at 37 °C. From tubes which showed no growth, 0.1 mL of cells was spread on TSA agar plates. MBCs were determined as the highest dilution at which no growth occurred on the plates.

2.8. Statistical Analysis

The analysis of variance (ANOVA procedure), followed by Duncan’s multiple range tests (SAS 9.1.3 program, SAS Institute Inc, Cary, NC, USA), was used to assess the variation of phenol contents and biological activities among populations. The relationship between populations and biological activities was investigated by the Principal Component Analysis (PCA), using the MVSP 3.1 program (Kovach Computing Services, Pentraeth, Wales). The classification of populations according to their phenolic compounds was evaluated using cluster analysis (MVSP program). Correlations between phenolic compounds and their biological activities were carried out with PROC CORR procedure using SAS version 9 (SAS Institute Inc, Cary, NC, USA).

3. Results and Discussion

3.1. Total Phenol and Flavonoid Contents

The contents and composition of total phenols and flavonoids differ according to genotype, geographical, and ecological factors [35]. In our work, the contents of phenolic compounds of T. algeriensis populations were variable (Table 2), with maximum levels found in plants grown in the upper arid bioclimatic zone (32–34 mg GAE/g leaves DW), intermediate values in population Ta 10 from the lower semi-arid bioclimatic zone (17.1 mg GAE/g leaves DW), and lower levels, not exceeding 14.8 mg GAE/g leaves DW, being found in the remaining samples. In a similar trend, total flavonoids assumed maximum amounts in upper arid samples (approximately 10–11 mg ER/g leaves DW), while variable amounts ranging from 3 to 9 mg ER/g leaves DW were found in the lower semi-arid, mean semi-arid, upper semi-arid populations, and sub humid. As compared to previous literature data, the total amount of phenolic compounds herein found were in general superior to those revealed for T. algeriensis plants grown in Gafsa, Tamerza, and Kairouan in Tunisia (7.08–8.81 mg GAE/g leaves DW) [23], and, in particular, those of upper arid zone also overcome the ones reported for T. algeriensis from Algeria (18.7 mg GAE/g leaves DW) [36].

3.2. Characterization of Phenolic Compounds in T. algeriensis Populations

The individual phenolic components of the distinct Thymus populations were elucidated through UHPLC-DAD-ESI-MSn analysis of the respective methanolic extracts. Distinct phenolic compounds were identified among the populations (Table 3, Figure 1). With the exception of Ta 11 and Ta 12 from upper arid rosmarinic acid (peak 8, UVmax at 289 sh, and 328 nm, [M − H] at m/z 359) was the main phenolic component identified in the extracts. This is consistent with the general abundance of Thymus plants in caffeic acid derivatives, in particular rosmarinic acid [30,37,38,39,40], and also agrees with the recent work of Ziani et al. [24], who showed that, in opposition to aqueous extracts (dominated by flavones), the hydroalcoholic (in that specific case hydroethanolic) extract of a T. algeriensis specimen from Algeria was mainly rich in rosmarinic acid. Besides, all the methanolic extracts contained other caffeic derivatives, namely a caffeoyl derivative of rosmarinic acid (eluted in peak 11, [M − H] at m/z 537→493, 359), salvianolic acid K and E ([M − H] at m/z 555 and m/z 717 in peaks 10 and 12, respectively), and monomethyl lithospermate (peak 14, [M − H] at m/z 551→519, 359), which are common compounds in Thymus plants [2,24,41,42,43,44].
Flavonols were also detected as major phenolic components of T. algeriensis methanolic extracts, mostly represented by kaempferol glycosides, particularly kaempferol-O-hexoside and kaempferol-O-hexuronide (peaks 4 and 6, [M − H] at m/z 447 and m/z 461, respectively, and both with UVmax at 268 and 339 nm). Interestingly, Ziani et al. [24] also pointed that kaempferol-O-hexuronide was a major compound in aqueous or hydroethanolic extracts of Algerian T. algeriensis, but, to our knowledge, kaempferol-O-hexoside was not previously reported in this species. In regard to the flavone pool, this was represented by luteolin-O-hexuronide (([M − H] at m/z 461 and UVmax at 255, 266, and 345 nm eluted in peak 5), apigenin-di-C-hexoside (peak 2, [M − H] at m/z 593→473, 503, and 575), and apigenin-O-hexuronide (peak 6, [M − H] at m/z 445→269), which were all detected before, in the work of Ziani et al. [24]. Moreover, albeit not previously reported, scutellarein (i.e., 4,5,6,7,4’-tetrahydroxyflavone) derivatives were also found (peaks 13, 16, and 17). The first exhibited a pseudo-molecular ion [M − H] at m/z 623 and two fragments at m/z 461 and 285, which suggest the loss of a hexose moiety (−162 Da) and the simultaneous loss of hexose and hexuronic units (−162–176 Da), respectively, hence being assigned to a scutellarein-O-hexoside-hexuronide. In turn, the compound eluted in peak 16 corresponded to a dimethoxyscutellarein ([M − H] at m/z 313→298), probably cirsimaritin, since this was previously detected in Thymus plants [45,46], and the compound eluted in peak 17 ([M − H] at m/z 343→329) was herein tentatively identified as a tetramethoxyscutellarein derivative, based on its MS2 fragmentation pattern, which indicated the loss of one to four methyl units (ions at m/z 329, 313, 299, and 285).
Like the other described Thymus species, the methanolic extracts of T. algeriensis also contained flavanones, mainly represented by eriodictyol (peak 7, UVmax at 288 and 330sh nm, [M − H] at m/z 287→151, 269) and an O-hexoside derivative eluted at RT 2.8 ([M − H] at m/z 449). In addition, samples from upper arid revealed the presence of naringenin (peak 15, UVmax at 289, [M − H] at m/z 271→151).
Regardless of the presence of characteristic phenolic compounds from Thymus plants in the methanolic extracts of the distinct T. algerienses populations (Ta 1–12), specific features were found in some populations. The most striking one was related to the phenolic monoterpene carvacrol, which assumed high levels in samples from the upper arid bioclimatic zone (2222–1375 µg/mL extract for Ta 11 and Ta 12, respectively), standing in contrast to its absence in the extracts from the remaining populations (Table 4). This discrepancy is probably due to environmental and/or genetic factors [47] and also partially justifies the absence of this compound in the work of Ziani et al. [24].
Moreover, high levels of rosmarinic acid were found in population Ta 4 (1157 µg/mL extract) from upper semi-arid bioclimate, followed by populations Ta 10 (1083 µg/mL extract) from lower semi-arid bioclimate and those of the upper arid zones (957 and 807.2 µg/mL extract for Ta 11 and Ta12, respectively), while minimum contents were detected in population Ta 2 (383.8 µg/mL extract) from upper semi-arid bioclimatic zone. As for caffeoyl rosmarinic acid, contents varied between 39.2 µg/mL extract (Ta 1) and 232.2 µg/mL extract (Ta 7), while a moderate average was observed in populations Ta 10 (186.1 µg/mL extract), Ta 11 (206.6 µg/mL extract), and Ta 12 (183 µg/mL extract).
Regarding flavonols, minimum and maximum amounts of the most relevant compound (kaempferol-O-hexuronide) were found in populations Ta 3 and Ta 11 (202.9 and 862.80 µg/mL extract, respectively), while kaempferol-O-hexoside assumed relevant values in Ta 9 and Ta 5. For the flavone pool, apigenin-O-hexuronide was mainly present in population Ta 1 (112.8 µg/mL extract) from the sub humid bioclimate, and it was not detected in populations Ta 11 and Ta 12 from the upper arid zone. Apigenin-C-di-hexoside was found in all samples, with values ranging between 10.4 µg/mL extract (Ta 6) and 62.6 µg/mL extract (Ta 9).
According to their phenolic compounds, the cluster analysis divided the T. algeriensis populations in two major groups (Figure 2). The first one (I) was represented by the populations Ta 12 and Ta 11, which were distinguished by their richness in kaempferol-O-hexuronide and the presence of carvacrol. The second group (II) was subdivided into two subgroups, one formed by populations Ta 4, Ta 10, and Ta 7, overall characterized by rosmarinic acid abundance, and the other included seven populations (Ta 1, 2, 3, 5, 6, 8, and 9) that were characterized by lower amounts of rosmarinic acid.

3.3. Antioxidant Activity

Polyphenols were reported to display several biological effects, including antioxidant activity [30]. The screening of the antioxidant capacity of the methanolic extracts was evaluated by three methods, namely the DPPH• (1,1-diphenyl-2-picryl-hydrazil radical) scavenging assay, β-carotene bleaching test, and the ferric reducing antioxidant power (FRAP).
A significant variation was observed between populations (Table 5), regarding their ability to scavenge DPPH•. In more detail, populations Ta 11 and Ta 12, from the upper arid bioclimatic zones, exhibited the best antiradical capacity (EC50 = 8.9 and 10.3 µg/mL, respectively), which was even higher than that of the synthetic compound Trolox.
From the remaining populations, a considerable activity was also observed for populations Ta 10 from lower semi-arid, and Ta 4 and Ta 7 from the upper semi-arid (EC50 values of 19.9, 22.7, and 26.6 µg/mL, respectively), while Ta 2 and Ta 3 from the same bioclimatic zone had low scavenging activity (54.5 and 52.3 µg/mL, respectively). Less-promising results were revealed by Ziani et al. [20] for infusion, decoction and hydroethanolic T. algeriensis extracts (EC50 48–131 µg/mL), and even reported by Nickavar and Esbati. [48] for other Thymus species (31.47–48.68 µg/mL), which exhibited DPPH• EC50 values lower than those of the reference commercial compounds.
Notably, Thymus populations from the upper arid zone were also the most efficient regarding the potential to protect β-carotene from bleaching. The same trend was found in FRAP assay, with values of 16.7 and 20.6 mmolFe2+/L found for populations Ta 11 and Ta12, contrasting with those of the remaining samples (lower than 7 mmolFe2+/L).

3.4. Anti-Acetylcholinesterase Activity

Phenolic compounds are also claimed to modulate intracellular events involved in distinct neurodegenerative diseases, including the inhibition of AChE, i.e., a central therapeutic target in Alzheimer’s disease [49]. To our knowledge, to the present, only a few studies investigated the ability of Thymus extracts in modulating the activity of AChE [50,51,52]. T. algeriensis methanolic extracts showed moderate ability to inhibit AChE, with significant variations among populations (Table 5). The weakest activity was observed for the extracts of population Ta 3 (EC50 = 3 mg/mL) from upper semi-arid bioclimatic zone. An intermediate activity was revealed for the populations Ta 1, 8, and 10, with EC50 values ranging from 1 to 1.2 mg/mL, while populations Ta 11 and Ta 12 from the upper arid zone showed the best activity (EC50 of 0.2 and 0.1 mg/mL, respectively). Nevertheless, the inhibitory activity was lower than that of Donepezil (EC50 = 18 ± 0.1 μg/mL), a specific inhibitor of acetylcholinesterase, used as a positive control. Curiously, Kindl et al. [52] revealed that ethanolic extracts from T. longicaulis, T. pulegioides, and T. vulgaris exhibited had a lower inhibitory activity against AChE when compared to the reference galantamine (EC50 values of 0.66–0.67 mg/mL vs EC50 = 0.12 μg/mL).

3.5. Antibacterial Activity

The in vitro antibacterial activity of the methanolic extracts estimated by the diameter of inhibition also varied significantly among populations. In fact, only samples collected from upper arid bioclimatic zone, characterized by the presence of carvacrol, showed considerable antibacterial activity (Table 6). The highest activity was observed against E. coli, with inhibition zones of 14.5 and 13 mm being recorded for populations Ta 11 and Ta 12, respectively. In turn, inhibition zones for gram-positive strains varied between 10 and 14 mm, with the best activities observed for S. feacalis.
The bacteriostatic and bactericidal effectiveness of extracts estimated by MIC and MBC are shown in Table 6. Extracts from upper arid revealed a bacteriostatic effect against all bacteria strains (MIC = 1.4 mg/mL). Although, the bactericidal activity (MBC = 1.4mg/mL) was restricted to the gram-negative bacteria E. coli.

3.6. Correlations between Bioactivity and Phenolic Components

According to axes 1 and 2 (93.27% of the total inertia), the plot of the principal component analysis (PCA) based on the antioxidant, anti-acetylcholinesterase, and antibacterial activities showed two major groups (Figure 3). The first group, at the positive side of axis 1, enclosed ten populations that were less bioactive. The second group, situated at the negative side of axis 1, formed by the two populations Ta 11 and Ta 12 from upper arid bioclimate, characterised by the best antioxidant, anti-acetylcholinesterase, and antibacterial activities.
As expected, significant correlations were observed between total phenol and flavonoid contents and the three antioxidant assays determined by the antiradical activity (r = −0.81; r = −0.72, p < 0.01, respectively), β-carotene bleaching inhibitory activity (r = −0.94, p < 0.01; r = −0.69, p < 0.05, respectively), and FRAP assay (r = 0.96, p < 0.01; r = 0.70, p < 0.05, respectively) (Table 7). These results are also in line with previous studies that underlined good correlations between polyphenolics and antioxidant activity, due to the effectiveness of these compounds as free-radical scavengers and antioxidants [55].
Regarding specific compositions, DPPH and β-carotene bleaching activities were also correlated to carvacrol (r = −0.63; p < 0.05, r = −0.79; p < 0.01), rosmarinic acid (r = −0.83, r = −0.76; p < 0.01), caffeoyl rosmarinic acid (r = −0.72, r = −0.77; p < 0.01, respectively), kaempferol-O-hexuronide (r = −0.77, r = −0.76; p < 0.01), and Eriodictyol (r = −0.58, r = −0.68; p < 0.05). Several studies were in accordance with our results revealing free-radical scavenging activity attributed to carvacrol [56,57], rosmarinic acid, and luteolin derivates [38,52,58].
Regarding the anti-acetylcholinesterase activity, a significant correlation was observed between the carvacrol and the EC50 values of the enzyme inhibitory (r = −0.60, p < 0.05) (Table 7). The capacity of the carvacrol as possessing such property was reported by Aazza et al. [59] and Jukic et al. [60]. Besides, our results suggest that other phenolic compounds contribute to the inhibition of the acetylcholinesterase activity. Indeed, a significant correlation was observed between the anti-acetylcholinesterase activity and total phenols (r = −0.64, p < 0.05) and kaempferol-O-hexuronide (r = −0.58, p < 0.05).

4. Conclusions

This study was carried out in order to describe the phenolic composition of twelve Tunisian T. algeriensis populations harvested in different geographical and bioclimatic zones, and to evaluate their biological activities. Rosmarinic acid seems to characterize T. algeriensis species, although significant variations in its levels, as well as in other major phenolic compounds, were found. Moreover, populations collected from the most arid zones were characterized by the highest phenolic and flavonoid contents and distinguished by the presence of carvacrol, which was absent in the remaining populations.
All extracts revealed substantial antioxidant activity, as well as anti-acetylcholinesterase and antibacterial activities. The variation of chemical and biological activities among the populations should lead to the selection of plants collected from the most arid zone, with a high potential of antioxidant, anti-acetylcholinesterase, and antibacterial activities, in order to use them in health-care and food industries.

Author Contributions

R.J. contributed to investigation, data curation, and writing the original draft; I.B.H.Y. contributed to sample collection; M.B. contributed to sample collection and writing—review; A.M.S.S. contributed to resources and writing—review; S.M.C. contributed to data curation, supervision, resources, and writing—review; Y.Z. contributed to sample collection, supervision, and writing—review.


The authors thank the Tunisian Ministry of Scientific Research and Technology, the National Institute of Applied Science and Technology, and the University of Aveiro for financial support. Thanks are also due to the University of Aveiro, FCT/MEC for the financial support to the QOPNA research Unit (FCT UID/QUI/00062/2019), through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement. Susana M. Cardoso thanks the research contract under the project AgroForWealth (CENTRO–01–0145–FEDER000001), funded by Centro2020, through FEDER and PT2020.

Conflicts of Interest

The authors declare that there are no conflict of interest.


  1. Kasrati, A.; Jamali, C.A.; Fadli, M.; Bekkouche, K.; Hassani, L.; Wohlmuth, H.; Leach, D.; Abbad, A. Antioxidative activity and synergistic effect of Thymus saturejoides Coss. essential oils with cefixime against selected food–borne bacteria. Ind. Crops. Prod. 2014, 61, 338–344. [Google Scholar] [CrossRef]
  2. Afonso, A.; Pereira, O.; Neto, R.; Silva, A.; Cardoso, S. Health–Promoting Effects of Thymus herba barona, Thymus pseudolanuginosus, and Thymus caespititius Decoctions. Int. J. Mol. Sci. 2017, 18, 1879. [Google Scholar] [CrossRef] [PubMed]
  3. Tohidi, B.; Rahimmalek, M.; Arzani, A. Essential oil composition, total phenolic, flavonoid contents, and antioxidant activity of Thymus species collected from different regions of Iran. Food Chem. 2017, 220, 153–161. [Google Scholar] [CrossRef] [PubMed]
  4. Nickavar, B. Analysis of the essential oils of two Thymus species from Iran. Food Chem. 2005, 90, 609–611. [Google Scholar] [CrossRef]
  5. Safaei-Ghomi, J.; Ebrahimabadi, A.; Djafari-Bidgoli, Z.; Batooli, H. GC/MS analysis and in vitro antioxidant activity of essential oil and methanol extracts of Thymus caramanicus Jalas and its main constituent carvacrol. Food Chem. 2009, 115, 1524–1528. [Google Scholar] [CrossRef]
  6. Parveen, A.; Kyunn, W.W. Antioxidant and anti–cholinergic activities of phenolic compounds isolated from Thymus linearis collected from dir, Pakistan. Vegetos 2016, 29, 41–46. [Google Scholar]
  7. Pinto, E.; Pina-Vaz, C.; Salgueiro, L.; Goncalves, M.J.; Costa-de-Oliveira, C.; Cavaleiro, C.; Ana Palmeira, A.; Aca’cio Rodrigues, A.; Martinez-de-Oliveira, J. Antifungal activity of the essential oil of Thymus pulegioides on Candida, Aspergillus and dermatophyte species. J. Med. Microbio. 2006, 55, 1367–1373. [Google Scholar] [CrossRef]
  8. Giordani, R.; Hadef, Y.; Kaloustian, J. Compositions and antifungal activities of essential oils of some Algerian aromatic plants. Fitoterapia 2008, 79, 199–203. [Google Scholar] [CrossRef]
  9. Loziene, K.; Venskutonis, P.R.; Sipailiene, A.; Labokas, J. Radical scavenging and antibacterial properties of the extracts from different Thymus pulegioides L. chemotypes. Food Chem. 2007, 103, 546–559. [Google Scholar] [CrossRef]
  10. De Martino, L.; Bruno, M.; Formisano, C.; De Feo, V.; Napolitano, F.; Rosselli, S.; Senatore, F. Chemical Composition and Antimicrobial Activity of the Essential Oils from Two Species of Thymus Growing Wild in Southern Italy. Molecules 2009, 14, 4614–4624. [Google Scholar] [CrossRef]
  11. Hazzit, M.; Baaliouamer, A.; Veríssimo, A.R.; Faleiro, M.L.; Miguel, M.G. Chemical composition and biological activities of Algerian Thymus oils. Food Chem. 2009, 116, 714–721. [Google Scholar] [CrossRef]
  12. Nikolić, M.; Glamočlija, J.; Ferreira, I.C.F.R.; Calhelha, R.C.; Fernandes, Â.; Marković, T.; Marković, D.; Giweli, A.; Soković, M. Chemical composition, antimicrobial, antioxidant and antitumor activity of Thymus serpyllum L., Thymus algeriensis Boiss. and Reut and Thymus vulgaris L. essential oils. Ind. Crops Prod. 2014, 52, 183–190. [Google Scholar] [CrossRef]
  13. Razzaghi-Abyaneh, M.; Shams-Ghahfarokhi, M.; Rezaee, M.-B.; Jaimand, K.; Alinezhad, S.; Saberi, R.; Yoshinari, T. Chemical composition and antiaflatoxigenic activity of Carum carvi L., Thymus vulgaris and Citrus aurantifolia essential oils. Food Control 2009, 20, 1018–1024. [Google Scholar]
  14. Pottier-Alapetite, G. Flore de la Tunisie Angiospermes Dicotylédones Gamopétales; Ministère de l’Enseignement Supérieur et de la Recherche Scientifique et le Ministère de l’Agriculture: Tunis, Tunisie, 1981; pp. 809–811.
  15. Le Floc’h, E.; Boulos, L. Flore de la Tunisie, Catalogue Synonymique Commente; Le Floc’h: Montepellier, France, 2008; p. 461. [Google Scholar]
  16. Ben El Hadj Ali, I.; Zaouali, Y.; Bejaoui, A.; Boussaid, M. Variation of the Chemical Composition of Essential Oils in Tunisian Populations of Thymus algeriensis Reut. (Lamiaceae) and Implication for Conservation. Chem. Biodivers 2010, 7, 1279–1289. [Google Scholar]
  17. Zouari, N.; Ayadi, I.; Fakhfakh, N.; Rebai, A.; Zouari, S. Variation of chemical composition of essential oils in wild populations of Thymus algeriensis Boiss. et Reut., a North African endemic Species. Lipids Health Dis. 2012, 11, 28. [Google Scholar] [CrossRef]
  18. Amarti, F.; Satrani, B.; Ghanmi, M.; Farah, A.; Abderrahman Aafi, A.; Aarab, L.; El Ajjouri, M.; Chaouch, A. Composition chimique et activité antimicrobienne des huiles essentielles de Thymus algeriensis Boiss. & Reut. et Thymus ciliatus (Desf.) Benth. du Maroc. Biotechnol. Agron. Soc. Environ. 2010, 14, 141–148. [Google Scholar]
  19. El Ajjouri, M.; Ghanmi, M.; Satrani, B.; Amarti, F.; Rahouti, M.; Aafi, A.; Ismaili, R.; Farah, A. Composition chimique et activité antifongique des huiles essentielles de Thymus algeriensis Boiss. & Reut. et Thymus ciliatus (Desf.) Benth. contre les champignons de pourriture du bois. Acta Bot. Gall. 2010, 157, 285–294. [Google Scholar]
  20. Kouache, B.; Brada, M.; Saadi, A.; Fauconnier, M.L.; Lognay, G.; Heuskin, S. Chemical Composition and Acaricidal Activity of Thymus algeriensis Essential Oil against Varroa destructor. Nat. Prod. Commun. 2017, 12, 135–138. [Google Scholar] [CrossRef]
  21. Dob, T.; Dahmane, D.; Benabdelkader, T.; Chelghoum, D. Studies on the essential oil composition and antimicrobial activity of Thymus algeriensis Boiss. et Reut. Int. J. Aromather. 2006, 16, 95–100. [Google Scholar] [CrossRef]
  22. Benkiniouar, R.; Rhouati, S.; Touil, A.; Seguin, E.; Chosson, E. Flavonoids from Thymus algeriensis. Chem. Nat. Compd. 2007, 43, 3. [Google Scholar] [CrossRef]
  23. Guesmi, F.; Ben Farhat, M.; Mejri, M.; Landoulsi, A. In–vitro assessment of antioxidant and antimicrobial activities of methanol extracts and essential oil of Thymus hirtus sp. algeriensis. Lipids Health Dis. 2014, 13, 138. [Google Scholar] [CrossRef] [PubMed]
  24. Ziani, B.E.C.; Heleno, S.A.; Bachari, K.; Dias, M.I.; Alves, M.J.; Barros, L.; Ferreira, I.C.F.R. Phenolic compounds characterization by LC–DAD– ESI/MSn and bioactive properties of Thymus algeriensis Boiss. & Reut. and Ephedra alata Decne. Food Res. Int. 2018, 116, 312–319. [Google Scholar] [PubMed]
  25. Ben El Hadj Ali, I.; Guetat, A.; Boussaid, M. Chemical and genetic variability of Thymus algeriensis Boiss. et Reut. (Lamiaceae), a North African endemic species. Ind. Crops. Prod. 2012, 40, 277–284. [Google Scholar] [CrossRef]
  26. Chetoui, I.; Messaoud, C.; Boussaid, M.; Zaouali, Y. Antioxidant activity, total phenolic and flavonoid content variation among Tunisian natural populations of Rhus tripartita (Ucria) Grande and Rhus pentaphylla Desf. Ind. Crops. Prod. 2013, 51, 171–177. [Google Scholar]
  27. Rocchetti, G.; Blasi, F.; Domenico Montesano, D.; Ghisoni, S.; Marcotullio, M.C.; Sabatini, S.; Cossignani, L. Luigi Lucinia Impact of conventional/non–conventional extraction methods on the untargeted phenolic profile of Moringa oleifera leaves. Food Res. Int. 2019, 115, 319–327. [Google Scholar] [CrossRef] [PubMed]
  28. Singleton, V.L.; Orthofer, R.; Lamuela–Raventos, R.M. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent. Methods Enzymol. 1999, 299, 152–178. [Google Scholar]
  29. Pereira, O.R.; Catarino, M.D.; Afonso, A.F.; Silva, A.M.S.; Cardoso, S.M. Salvia elegans, Salvia greggii and Salvia officinalis Decoctions: Antioxidant Activities and Inhibition of Carbohydrate and Lipid Metabolic Enzymes. Molecules 2018, 23, 3169. [Google Scholar] [CrossRef]
  30. Pereira, O.R.; Peres, A.M.; Silva, A.M.S.; Domingues, M.R.M.; Cardoso, S.M. Simultaneous characterization and quantification of phenolic compounds in Thymus x citriodorus using a validated HPLC–UV and ESI–MS combined method. Food Res. Int. 2013, 54, 1773–1780. [Google Scholar] [CrossRef]
  31. Zaouali, Y.; Bouzaine, T.; Boussaid, M. Essential oils composition in two Rosmarinus officinalis L. varieties and incidence for antimicrobial and antioxidant activities. Food Chem. Toxicol. 2010, 48, 3144–3152. [Google Scholar] [CrossRef]
  32. Eldeen, I.M.S.; Elgorashi, E.E.; Van Staden, J. Antibacterial, anti–inflammatory, anti cholinesterase and mutagenic effects of extracts obtained from some trees used in South African traditional medicine. J. Ethnopharmacol. 2005, 102, 457–464. [Google Scholar] [CrossRef]
  33. Murray, P.R.; Baron, E.J.; Pfaller, M.A.; Tenover, F.C.; Yolken, H.R. Manual of Clinical Microbiology, 6th ed.; ASM Press: Washington, DC, USA, 1995; pp. 15–18. [Google Scholar]
  34. Okeke, M.I.; Iroegbu, C.U.; Eze, E.N.; Okoli, A.S.; Esimone, C.O. Evaluation of the root of Landolphia owerrience for antibacterial activity. J. Ethnopharmacol. 2001, 78, 119–127. [Google Scholar] [CrossRef]
  35. Liu, Q.; Tang, G.Y.; Zhao, C.N.; Feng, X.L.; Xu, X.Y.; Cao, S.Y.; Meng, X.; Li, S.; Gan, R.Y.; Li, H.B. Comparison of antioxidant activities of different grape varieties. Molecules 2018, 23, 2440. [Google Scholar] [CrossRef] [PubMed]
  36. Boulanouar, B.; Abdelaziz, G.; Aazza, S.; Gago, C.; Miguel, M.G. Antioxidant activities of eight Algerian plant extracts and two essential oils. Ind. Crops. Prod. 2013, 46, 85–96. [Google Scholar] [CrossRef]
  37. Özgen, U.; Mavi, A.; Terzi, Z.; Kazaz, C.; Asçı, A.; Kaya, Y.; Seçen, H. Relationship between chemical structure and antioxidant activity of luteolin and its glycosides isolated from Thymus sipyleus subsp. sipyleus var. sipyleus. Rec. Nat. Prod. 2011, 5, 12–21. [Google Scholar] [CrossRef]
  38. Costa, P.; Gonçalves, S.; Valentão, P.; Andrade, P.B.; Coelho, N.; Romano, A. Thymus lotocephalus wild plants and in vitro cultures produce different profiles of phenolic compounds with antioxidant activity. Food Chem. 2012, 135, 1253–1260. [Google Scholar] [CrossRef]
  39. Delgado, T.; Marinero, P.; Manzanera, M.C.A.S.; Asensio, C.; Herrero, B.; Pereira, J.A.; Ramalhosa, E. Antioxidant activity of twenty wild Spanish Thymus mastichina L. populations and its relation with their chemical composition. LWT–Food Sci. Technol. 2014, 57, 412–441. [Google Scholar] [CrossRef]
  40. Saija, A.; Speciale, A.; Trombetta, D.; Leto, C.; Tuttolomondo, T.; La Bella, S.; Licata, M.; Virga, G.; Bonsangue, G.; Gennaro, M.C.; et al. Phytochemical, Ecological and Antioxidant Evaluation of Wild Sicilian Thyme: Thymbra capitata (L.) Cav. Chem. Biodivers 2016, 13, 1641–1655. [Google Scholar] [CrossRef]
  41. Nagy, T.O.; Solar, S.; Sontag, G.; Koenig, J. Identification of phenolic components in dried spices and influence of irradiation. Food Chem. 2011, 128, 530–534. [Google Scholar] [CrossRef]
  42. Pereira, O.R.; Cardoso, S.M. Overview on Mentha and Thymus Polyphenols. Current Analytical Chemistry. Curr. Anal. Chem. 2013, 9, 382–396. [Google Scholar] [CrossRef]
  43. Afonso, A.F.; Pereira, O.R.; Válega, M.; Silva, A.M.S.; Cardoso, S.M. Metabolites and Biological Activities of Thymus zygis, Thymus pulegioides, and Thymus fragrantissimus Grown under Organic Cultivation. Molecules 2018, 23, 1514. [Google Scholar] [CrossRef]
  44. Schött, G.; Liesegang, S.; Gaunitz, F.; Gleß, A.; Basche, S.; Hannig, C.; Speer, K. The chemical composition of the pharmacologically active Thymus species, its antibacterial activity against Streptococcus mutans and the antiadherent effects of T. vulgaris on the bacterial colonization of the in situ pellicle. Fitoterapia 2017, 121, 118–128. [Google Scholar] [CrossRef] [PubMed]
  45. Adzet, T.; Vila, R.; Caaigural, S. Chromatographic analysis of polyphenols of some Iberian thymes. J. Ethnopharmacol. 1988, 24, 147–154. [Google Scholar] [CrossRef]
  46. Horwath, A.B.; Grayer, R.J.; Keith–Lucas, D.M.; Simmonds, M.S.J. Chemical characterisation of wild populations of Thymus from different climatic regions in southeast Spain. Biochem. Syst. Ecol. 2008, 36, 117–133. [Google Scholar] [CrossRef]
  47. Jaouadi, R.; Cardoso, S.M.; Silva, A.M.S.; Ben Hadj Yahia, I.; Boussaid, M.; Zaouali, Y. Variation of phenolic constituents of Tunisian Thymus capitatus (L.) Hoff. et Link. populations. Biochem. Syst. Ecol. 2018, 77, 10–15. [Google Scholar] [CrossRef]
  48. Nickavar, B.; Esbati, N. Evaluation of the Antioxidant Capacity and Phenolic Content of Three Thymus Species. J. Acupunct. Meridian Stud. 2012, 5, 119–125. [Google Scholar] [CrossRef]
  49. Costa, P.; Gonçalves, S.; Valentão, P.; Andrade, P.B.; Romano, A. Accumulation of phenolic compounds in in vitro cultures and wild plants of Lavandula viridis L’Hér and their antioxidant and anti–cholinesterase potential. Food Chem. Toxicol. 2013, 57, 69–74. [Google Scholar] [CrossRef]
  50. Orhan, I.; Kartal, M.; Tosun, F.; Şener, B. Screening of Various Phenolic Acids and Flavonoid Derivatives for their Anticholinesterase Potential. Z. Nat. C. 2007, 62, 829–832. [Google Scholar] [CrossRef]
  51. Orhan, I.; Senol, F.S.; Gülpinar, A.R.; Kartal, M.; Sekeroglu, N.; Deveci, M.; Kan, K.; Sener, B. Acetylcholinesterase inhibitory and antioxidant properties of Cyclotrichium niveum, Thymus praecox subsp. caucasicus var. caucasicus, Echinacea purpurea and E. pallida. Food Chem. Toxicol. 2009, 47, 1304–1310. [Google Scholar] [CrossRef]
  52. Kindl, M.; Blažeković, B.; Bucar, F.; Vladimir-Knežević, S. Antioxidant and Anticholinesterase Potential of Six Thymus Species. Evid. Based Complement. Alternat. Med. 2015, 2015, 403950. [Google Scholar] [CrossRef]
  53. Petrovic’, N.V.; Petrovic’, S.S.; Dzˇamic’, A.M.; C’iric’, A.D.; Risti’c, M.S.; Milovanovi’c, S.L.; Petrovi’c, S.D. Chemical composition, antioxidant and antimicrobial activity of Thymus praecox supercritical extracts. J. Supercrit Fluids. 2016, 110, 117–125. [Google Scholar] [CrossRef]
  54. Ebrahimi, S.N.; Hadian, J.; Mirjalili, M.H.; Sonboli, A.; Yousefzadi, M. Essential oil composition and antibacterial activity of Thymus caramanicus at different phenological stages. Food Chem. 2008, 110, 927–931. [Google Scholar] [CrossRef] [PubMed]
  55. Chizzola, R.; Michitsch, H.; Franz, C. Antioxidative Properties of Thymus vulgaris Leaves: Comparison of Different Extracts and Essential Oil Chemotypes. J. Agric. Food Chem. 2008, 56, 6897–6904. [Google Scholar] [CrossRef] [PubMed]
  56. Faleiro, L.; Miguel, G.; Gomes, S.; Costa, L.; Venâncio, F.; Teixeira, A.; Figueiredo, A.C.; Barroso, J.G.; Pedro, L.G. Antibacterial and Antioxidant Activities of Essential Oils Isolated from Thymbra capitata L. (Cav.) and Origanum vulgare L. J. Agric. Food Chem. 2005, 53, 8162–8168. [Google Scholar] [CrossRef] [PubMed]
  57. Fachini-Queiroz, F.C.; Kummer, R.; Estevão-Silva, C.F.; de Barros Carvalho, M.D.; Cunha, J.M.; Grespan, R.; Bersani-Amado, C.A.; Cuman, R.K.N. Effects of Thymol and Carvacrol, Constituents of Thymus vulgaris L. Essential Oil, on the Inflammatory Response. Evid. Based Complement. Alternat. Med. 2012, 2012, 657026. [Google Scholar] [CrossRef]
  58. Zhu, F.; Asada, T.; Sato, A.; Koi, Y.; Nishiwaki, H.; Tamura, H. Rosmarinic Acid Extract for Antioxidant, Antiallergic, and α–Glucosidase Inhibitory Activities, Isolated by Supramolecular Technique and Solvent Extraction from Perilla Leaves. J. Agric. Food Chem. 2014, 62, 885–892. [Google Scholar] [CrossRef]
  59. Aazza, S.; Lyoussi, B.; Miguel, M.G. Antioxidant and Antiacetylcholinesterase Activities of Some Commercial Essential Oils and Their Major Compounds. Molecules 2011, 16, 7672–7690. [Google Scholar] [CrossRef]
  60. Jukic, M.; Politeo, O.; Maksimovic, M.; Milos, M.; Milos, M. In Vitro acetylcholinesterase inhibitory properties of thymol, carvacrol and their derivatives thymoquinone and thymohydroquinone. Phytother. Res. 2007, 21, 259–261. [Google Scholar] [CrossRef]
Figure 1. Chromatographic representation of Ta 12 population at 280 nm. Numbers in the figure correspond to the UHPLC-DAD-ESI-MSn peaks described in Table 3.
Figure 1. Chromatographic representation of Ta 12 population at 280 nm. Numbers in the figure correspond to the UHPLC-DAD-ESI-MSn peaks described in Table 3.
Antioxidants 08 00515 g001
Figure 2. Cluster analysis performed on phenolic compounds of T. algeriensis extracts. Ta 1–12 correspond to the code number as detailed in Table 1. Sh, Usa, Msa, Lsa, and Ua correspond to sub humid, upper semi-arid, mean semi-arid, lower semi-arid, and upper arid bioclimatic zones, respectively (See Table 1).
Figure 2. Cluster analysis performed on phenolic compounds of T. algeriensis extracts. Ta 1–12 correspond to the code number as detailed in Table 1. Sh, Usa, Msa, Lsa, and Ua correspond to sub humid, upper semi-arid, mean semi-arid, lower semi-arid, and upper arid bioclimatic zones, respectively (See Table 1).
Antioxidants 08 00515 g002
Figure 3. Plot of the principal component analysis performed on values of antioxidant, anti-acetylcholinesterase, and antibacterial activities. Ta 1–Ta 12 correspond to the code number as detailed in Table 1. DPPH•, β-carotene, FRAP: antioxidant assays. S1: E.coli, S2: P. aeruginosa, S3: K. pneumoniae, S4: S. aureus, S5: B. cereus, S6: S. epidermis, S7: S. feacalis.
Figure 3. Plot of the principal component analysis performed on values of antioxidant, anti-acetylcholinesterase, and antibacterial activities. Ta 1–Ta 12 correspond to the code number as detailed in Table 1. DPPH•, β-carotene, FRAP: antioxidant assays. S1: E.coli, S2: P. aeruginosa, S3: K. pneumoniae, S4: S. aureus, S5: B. cereus, S6: S. epidermis, S7: S. feacalis.
Antioxidants 08 00515 g003
Table 1. Main ecological traits of the investigated T. algeriensis populations.
Table 1. Main ecological traits of the investigated T. algeriensis populations.
PopulationsCodeBioclimatic Zone aRainfall
KorbousTa 1Sub humid (Sh)55036°50′10°35′
EssabahiaTa 2upper semi-arid (Usa)45036°36′10°10′
Dj MansourTa 345036°17′9°36′
JendoubaTa 466036°25′8°44′
Dj chahidTa 552036°22′9°18′
MaktherTa 652035°51′9°12′
KesraTa 752035°48′9°21′
SilianaTa 8mean semi-arid (Msa)52035°51′9°12′
SersTa 924536°6′9°40′
SousseTa 10lower semi-arid (Lsa)16735°30′10°50′
ToujeneTa 11upper arid (Ua)10033°27′10°08′
MatmataTa 1210033°32′9°58′
a Bioclimatic zones were defined according to Emberger’s (1966) pluviothermic coefficient.
Table 2. Total phenolic compounds (mg GAE/g leaves DW) and total flavonoid (mg ER/g leaves DW) contents of Thymus algeriensis.
Table 2. Total phenolic compounds (mg GAE/g leaves DW) and total flavonoid (mg ER/g leaves DW) contents of Thymus algeriensis.
Sh Usa Msa Lsa Ua
Ta 1 Ta 2Ta 3Ta 4Ta 5Ta 6Ta 7 Ta 8Ta 9 Ta 10 Ta 11Ta 12
TPC9.1 hi ± 0.1 9 hi ± 0.28.0 i ± 0.114.8 d ± 0.812.0 ef ± 0.39.8 gh ± 0.412.9 e ± 0.3 8.2 hi ± 0.211.1 gf ± 0.2 17.1 c ± 0.3 34.4 a ± 0.831.6 b ± 1
TF2.8 h ± 0.1 4.8 f ± 0.27.2 e ± 0.29.3 b ± 0.17.5 e ± 0.17.0 e ± 0.24.0 g ± 0.2 4.0 g ± 0.17.8 cd ± 0.1 8.2 c ± 0.2 10.6 a ± 0.210.3 a ± 0.3
Ta 1–12 correspond to the code number as detailed in Table 1. Sh, Usa, Msa, Lsa, and Ua: sub humid, upper semi-arid, mean semi-arid, lower semi-arid, and upper arid bioclimatic zones, respectively (See Table 1). TPC: total phenolic compounds, TF: total flavonoids. Numbers in lines followed by the same letter are not significant at p > 0.05 (Duncan’s multiple range test).
Table 3. Phenolic compounds identified by UHPLC-DAD-ESI-MSn in the methanolic extracts of Thymus algeriensis.
Table 3. Phenolic compounds identified by UHPLC-DAD-ESI-MSn in the methanolic extracts of Thymus algeriensis.
PeaktR (min)λmax (nm)(m/z)MS2ions (m/z)Probable Compound
11.4ND341179Caffeoyl hexoside b
21.6271, 330593473, 503, 575Apigenin-di-C-hexoside b
32.8284, 325sh449259,287Eriodictyol-O-hexoside a
43.2268, 339447285Kaempferol-O-hexoside b
55.9255, 266, 345461285Luteolin-O-hexuronide b
68.8267, 343445269Apigenin-O-hexuronide b
79.2288, 330sh287151, 269Eriodictyol a
89.5287sh, 328359161,179, 224Rosmarinic acid a
99.7268, 339461285Kaempferol-O-hexuronide b
1010.1288sh, 324555493, 357, 393, 313Salvianolic acid K b
1110.7291sh, 324537493, 359Caffeoyl rosmarinic acid b
1212.2289sh, 325717519Salvianolic acid E b
1313.0273, 330623461, 285Scutellarein-O-hexoside-hexuronide b
1414.5298sh, 325551519, 359Monomethyl lithospermate b
1514.9289271151Naringenin a
1616.3277, 333313298, 271Cirsimaritin b
1716.6283, 331343329, 313, 299 285Tetramethyl-scutellarein b
1817.3276ND-Carvacrol a
ND: not detected. a Compound identification was based on comparison to standard. b Compound identification was based on interpretation of UV spectral and MS data, plus comparison to literature.
Table 4. Contents (µg/mL extract) of major phenolic compounds in Thymus algeriensis for the selected populations.
Table 4. Contents (µg/mL extract) of major phenolic compounds in Thymus algeriensis for the selected populations.
Compounds Sh Usa Msa Lsa Ua
Ta 1 Ta 2Ta 3Ta 4Ta 5Ta 6Ta 7 Ta 8Ta 9 Ta 10 Ta 11Ta 12
Phenolic acids
Rosmarinic acid 531.3 g ± 0.5 383.8 j ± 0.5410.4 h ±0.71157.8 a ±2.7593.6 f ±2.1410.9 h ±0.7756.3 e ± 0.7 391.3 i ± 0.5596.4 f ± 0.3 1083.2 b ± 3.5 957.0 c ± 1.0807.2 d ± 3.0
Caffeoyl rosmarinic acid 39.2 j ± 0.1 64.9 h ± 0.178.9 f ± 0.185.5 e ± 0.173.9 fg ± 0.245.8 i ± 0.0232.2 a ± 0.2 74.3 g ± 0.1101.6 d ± 0.1 186.1 c ± 0.3 206.6 b ± 1.1183.0 c ± 0.5
Eriodictyol hexoside -h 6.3 f ± 0.131.9 c ± 0.140.0 b ± 0.139.1 b ± 0.13.5 g ± 0.25.7 f ± 0.1 28.7 d ± 0.152.8 a ± 0.1 -h 9.1 e ± 0.26.0 f ± 1.1
Eriodictyol 4.1 h ± 0.1 16.9 b ± 0.14.4 hg ± 0.312.4 d ± 0.74.4 hg ± 0.88.2 f ± 0.111.4 d ± 0.1 1.1 i ± 0.15.5 g ± 0.2 9.9 e ± 0.4 14.7 c ± 0.142 a ± 0.1
Kaempferol-O-hexoside -j 83.9 h ± 0.3228.1 d ± 0.1326.3 c ± 0.2360.4 b ± 0.5-j118.4 e ± 0.2 95.3 g ± 0.1439.6 a ± 0.3 10.0 i ± 0.2 -j108.4 ± 0.1 f
Kaempferol-O-hexuronide 256.3 h ± 0.3 363.2 f ± 1.9202.9 j ± 1.7552.0 c ± 0.6213.2 ij ± 12.7216.5 ij ± 0.7526.4 d ± 0.5 225.2 i ± 0.4446.6 e ± 9.4 297.1 g ± 0.3 862.8 a ± 1.2655.7 b ± 2.6
Luteolin-O-hexuronide -g -g-g25.3 b ± 0.120.6 c ± 0.1-g12.7 d ± 0.1 3.0 f ± 0.127.9 a ± 0.1 5.7 e ± 0.1 -g-g
Apigenin-C-di-hexoside 18.4 f ± 2.7 10.7 g ± 0.121.7 e ± 0.154.1 b ± 0.153.3 b ± 0.110.4 g ± 0.155.3 b ± 0.3 53.4 b ± 0.162.6 a ± 0.1 37.7 d ± 0.2 40.2 c ± 0.154.2 b ± 0.1
Apigenin-O-hexuronide 112.8 a ± 0.1 6.8 c ± 0.13.8 e ± 0.16.1 d ± 0.13.5 ef ± 0.19.8 b ± 0.13.8 e ± 0.1 6.1 d ± 0.13.1 f ± 0.1 1.4 g ± 0.1 -h-h
Phenolic terpene
Carvacrol -c -c-c-c-c-c-c -c-c -c 2221.6 a ± 2.51374.7 b ± 5.0
Ta 1–12 correspond to the code number as detailed in Table 1. Sh, Usa, Msa, Lsa, and Ua: sub humid, upper semi-arid, mean semi-arid, lower semi-arid, and upper arid bioclimatic zones, respectively (See Table 1). Numbers in lines followed by the same letter are not significant at p > 0.05 (Duncan’s multiple range test).
Table 5. Antioxidant and anti-acetylcholinesterase activities of T. algeriensis methanolic extracts.
Table 5. Antioxidant and anti-acetylcholinesterase activities of T. algeriensis methanolic extracts.
Bioactivities Sh Usa Msa Lsa Ua
Ta 1 Ta 2Ta 3Ta 4Ta 5Ta 6Ta 7 Ta 8Ta 9 Ta 10 Ta 11Ta 12
Antioxidant activity
DPPH• (µg/mL) 42.7 c ± 2.5 54.5 b ± 2.152.3 b ± 1.422.7 fg ± 0.937.8 d ± 0.640.7 cd± 1.026.6 f ± 1.4 68.8 a ± 1.032.4 e ± 1.0 19.9 g ± 1.1 8.9 h ± 0.110.3 h ± 0.4
β-carotene (mg/mL) 1.43 e ± 0.0 1.50 d ± 0.11.81 a ± 0.01.04 h ± 0.01.35 f ± 0.31.60 b ± 0.01.13 g ± 0.0 1.60 b ± 0.01.53 c ± 0.1 0.40 i ± 0.0 0.03 k ± 0.00.06 j ± 0.0
FRAP (mmolFe2+/L) 2 f ± 0.0 1.2 gh ± 0.00.3 i ± 0.014.8 d ± 0.06.8 c ± 0.01.8 fg ± 0.05.1 d ± 0.0 1.0 hi ± 0.04.0 e ± 0.0 6.5 c ± 0.05 16.7 b ± 0.120.6 a ± 0.2
Anti-acetylcholinesterase (mg/mL) 1.0 ef ± 0.0 1.3 e ± 0.13.0 a ± 0.10.7 g ± 0.02.3 b ± 0.10.9 f ± 0.12.1 c ± 0.1 1.0 ef ± 0.01.6 d ± 0.1 1.2 e ± 0.0 0.2 hi ± 0.00.1 i ± 0.0
With the exception of FRAP, values are expressed in EC50 values, i.e., sample concentration providing 50% of inhibition. DPPH• scavenging activity: EC50 values for the positive control Trolox: EC50 = 13 µg/mL; β-carotene bleaching inhibition: EC50 values for the positive control BHT: EC50 = 29.4 ± 0.02 μg/mL; anti-acetylcholinesterase activity: EC50 values for the positive control Donepezil: 18 ± 0.1 μg/mL. Ta 1–12 correspond to the code number, as detailed in Table 1. Sh, Usa, Msa, Lsa, and Ua correspond to sub humid, upper semi-arid, mean semi-arid, lower semi-arid, and upper arid bioclimatic zones, respectively (See Table 1). Numbers in the same line followed by the same letter are not significant at p > 0.05 (Duncan’s multiple range test).
Table 6. Antibacterial activity estimated by diameter of inhibition (mm), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) (mg/mL) of T. algeriensis extracts.
Table 6. Antibacterial activity estimated by diameter of inhibition (mm), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) (mg/mL) of T. algeriensis extracts.
Bacteria Ta 11 Ta 12 Gentamicin
E. coli10,53614.5 a ± 13.0 b ± 0.01.4-18.0 ± 0.0
P. aeruginosa902710.0 a ± 0.51.4- 9.0 a ± 0.01.4-12.0 ± 0.0
K. pneumoniae10,03110.5 a ± 0.51.4- 10.0 a ± 0.01.4-17.0 ± 0.0
S. aureus653811.5 a ± 0.51.4- 10.0 a ± 0.01.4-21.0 ± 0.0
B. cereus11,77813.5 a ± 0.51.4- 11.0 a ± 0.51.4-17.0 ± 0.0
S. epidermis12,22812.0 a ± 0.01.4- 10.5 b ± 0.01.4-27.0 ± 0.5
S. feacalis10,54114.0 a ± 0.01.4- 13.0 a ± 0.51.4-12.0 ± 0.0
Ta 11 and Ta 12 correspond to samples collected from Toujene and Matmata respectively (Table 1). ATCC: American Type Culture Collection; Inh Zone: Inhibition zone expressed in mm. Numbers in the same line followed by the same letter are not significant at p > 0.05 (Duncan’s multiple range test).Results demonstrated that only samples containing carvacrol exhibited inhibition zones. This is in accordance with the work of Petrović et al. [53], who suggested that most of the antimicrobial activity of Thymus genus appeared to be associated with high amounts of monoterpenic phenols (e.g., carvacrol). In fact, carvacrol is considered to be a biocidal, resulting in bacterial membrane perturbations that lead to leakage of intracellular ATP and potassium ions and ultimately cell death [54].
Table 7. Correlations between phenolic content, antioxidant, and anti-acetylcholinesterase activities.
Table 7. Correlations between phenolic content, antioxidant, and anti-acetylcholinesterase activities.
Compounds Antioxidant Activity Anti-AChE
DPPHβ-CaroteneFRAP Anti-AChE
Total phenols −0.81 **−0,94 **0.96 ** −0,64 *
Flavonoids −0.72 **−0.69 *0.70 * −0.37 ns
Phenolic acids
Rosmarinic acid −0.83 **−0.76 **0.54 ns −0.42 ns
Caffeoyl rosmarinic acid −0.72 **−0.77 **0.67 * −0.20 ns
Eriodictyol hexoside 0.20 ns0.40 ns−0.22 ns 0.37 ns
Eriodictyol −0.58 *−0.68 *0.78 ** −0.54 ns
Apigenin-C-di-hexoside −0.38 ns−0.31 ns0.39 ns −0.07 ns
Apigenin-O-hexuronide 0.19 ns0.22 ns−0.26 ns −0.08 ns
Luteolin-O-hexuronide −0.18 ns0.15 ns−0.11 ns 0.23 ns
Kaempferol-O-hexuronide −0.77 **−0.76 **0.78 ** −0.58 *
Kaempferol-O-hexoside 0.04 ns0.33 ns−0.14 ns 0.44 ns
Phenolic terpene
Carvacrol −0.63 *−0.79 **0.87 ** −0.60 *
*, ** = significant at p < 0.05 and p < 0.01, respectively; ns = not significant.
Back to TopTop