INTRODUCTION

Lamiaceae family of plants comprises of a variety of medicinal and aromatic plants. Among these, the members of Thymus genus are known to exhibit numerous biological activities, including antiviral, antifungal, and antioxidant activities.^1,2,3 Thymus genus mainly comprises of approximately 400 species of aromatic perennial plants and subshrubs, which grow mostly in Southern Europe, the Mediterranean region, North Africa, and Asia.⁴ Among these, 15 species, including Thymus munbyanus subsp. ciliatus (Desf.) Greuter & Burdet, are known to be distributed in Algeria. This subspecies is also known as Thymus ciliatus (Desf.) Benth., and it is addressed as “Zaatar” in Arabic. Several previous studies have reported the volatile chemical composition (essential oil) and antioxidant activities of T. munbyanus extracts; however, no information regarding the phytochemical nature of T. munbyanus extracts is available.^{5,6,7,8,9,10,11}

In this study, the secondary metabolites present in the aerial parts of T. munbyanus subsp. ciliatus were analyzed, isolated, and assessed for their in vitro antioxidant activities. To the best of our knowledge, this was the first study to identify and characterize compounds isolated from this plant.

MATERIALS AND METHODS

Chemicals and plant material

Most of the chemicals and reagents used in this study were purchased from Sigma (St. Louis, MO, USA). Some chemicals of analytical grade quality were procured from other commercial companies Roche (Darmstadt, Germany), Panreac Quimica (Spain) and MERCK (Germany). The aerial parts of T. munbyanus subsp. ciliatus were collected from Babor near Setif City, Algeria in May 2013. This plant was identified by Dr. W. Nouioua based on the information regarding Algerian flora.¹² A voucher specimen was deposited in the laboratory herbarium unit of the University of Constantine 1, VARENBIOMOL Research Unit (TC/123/05-13). The plant materials were stored at -20°C in a freezer until used for extraction.

Extraction and isolation of compounds

Air-dried parts (9.5 kg) of T. munbyanus subsp. ciliatus were powdered using a blender and extracted three times with MeOH/water (80/20, v/v). The hydromethanolic extracts were concentrated, dissolved in H₂O (1000 mL), and filtered. Further, the water-soluble part was extracted three times sequentially with chloroform, ethyl acetate, and n-butanol. Following extraction, chloroform (17.7 g), ethyl acetate (33 g), and n-butanol (59.2 g) extracts were collected.

Then, 32 g of ethyl acetate extract was fractionated using column chromatography, with a Sephadex LH-20 column. For elution, an isocratic system of CHCl₃/MeOH/hexane (7/2/1) was used and 26 fractions were collected. The precipitate from fraction 9 (155 mg) showed one spot contaminated with chlorophyll, which was washed with diethyl ether and acetone to give compound 5 (15 mg) and fraction 10 (2.5 g). The fractions were again fractionated using column chromatography, with a Sephadex LH-20 column, and elution was performed with an isocratic system of hexane/MeOH/CHCl₃ (1/2/7). This was subsequently followed by fractionation using a preparative column comprising silica gel, wherein elution with toluene/ethyl acetate/formic acid (10/6/1) yielded compounds 6 (3.1 mg), 11 (2.4 mg), 7 (5.5 mg), and 14 (2.7 mg). Moreover, fraction 11 (145 mg) and fraction 12 (124.18 mg) were purified on preparative plates of silica gel, and elution with toluene/ethyl acetate/formic acid (10/4/1) yielded compound 9 (1.7 mg) and compound 8 (7.3 mg), respectively. Further, fraction 14 (345 mg) was purified using a preparative column comprising silica gel, and elution with the same solvent mixture, at same ratio, resulted in the isolation of compounds 15 (5.5 mg), 12 (1.7 mg), and 1 (20 mg). Besides this, fraction 15 (3.12 g) was separated via column chromatography using a Sephadex LH-20 column, wherein samples were eluted isocratically with chloroform/methanol (7/3). This was followed by subsequent fractionation using a preparative silica gel, and elution with toluene/ethyl acetate/formic acid (10/2/1) yielded compounds 2 (3.2 mg), 3 (1.3 mg), 10 (3.4 mg), and 16 (9.1 mg).

The butanoic extract (10 g) was further subjected to fractionation on a polyamide column (SC6) with a gradient of toluene-MeOH, which further increased solvent polarity and yielded 21 fractions. Further, fraction 5 (0.37 g) was subjected to column chromatography on a Sephadex LH-20 column using isocratic CHCl₃/MeOH (6/4), which yielded 10 subfractions. Subfraction 7 (61.2 mg) was purified using thin-layer chromatography with EtOAc/MeOH/H₂O (18/1/1) to yield compound 13 (11 mg). Additionally, fraction 6 (76.3 mg) was separated using column chromatography, with Sephadex LH-20 as a stationary phase and methanol as a solvent phase, which resulted in the isolation of compound 4 (17 mg).

High-pressure liquid chromatography coupled with time-of-flight mass spectrometry (HPLC-TOF/MS) analysis

Phenolic contents present in organic solvent extracts, i.e., chloroform, ethyl acetate, and n-butanol extracts, were analyzed using HPLC-TOF/MS. HPLC analysis was performed using an Agilent 1260 Infinity Binary System (Agilent Technologies, Santa Clara, CA, USA) coupled with a 6210 TOF LC/MS detector on a ZORBAX SB-C18 (4.6x100 mm, 3.5 µm) column. Ultrapure water with 0.1% formic acid was used in mobile phase A, whereas mobile phase B contained 100% acetonitrile. The chromatographic separation was performed as per the following gradient: 0-1 min 10% B, 1-20 min 50% B, 20-23 min 80% B, and 23-30 min 10% B, with a flow rate of 0.6 mL. The column temperature was set at 35°C, and the injection volume was 10 µL. Retention times and m/z values were recorded for the phenolic compounds and in terms ofcompared with those of standard components. The crude extracts were dissolved in methanol at 25°C to obtain a concentration of 200 ppm and filtered using 0.45 µm PTFE filters.¹³

In vitro antioxidant assays

Total antioxidant capacity

To evaluate the antioxidant activities of the extracts and isolated compounds, ammonium phosphomolybdenum assay was performed, as previously described by Prieto et al.¹⁴ To prepare reaction solutions, sample solutions were prepared at different concentrations, 25, 50, and 100 µg/mL (0.3 mL each), mixed with 3 mL of reagent solution (ammonium molybdate-sodium phosphate-sulfuric acid), and vortexed to obtain homogeneous solutions. Further, the closed tubes containing the reaction solutions were incubated in a hot water bath for 90 min. Following this, the mixtures were cooled in an ice bath, and the absorbance of each solution was measured at 695 nm using a ultraviolet-visible spectrophotometer (UV-Vis) (Thermo Scientific Evaluation Array UV-Vis Spectrophotometer). For blank, 0.3 mL of solvent was used.

Free radical scavenging capacity

The scavenging activity of the samples was evaluated according to the method previously reported by Blois.¹⁵ In the present study, 1.5 mL of different concentrations of extracts and isolated compounds were mixed with 2,2-diphenyl-1-picrylhydrazyl (DPPH˙) solution (0.5 mL) in a test tube to get a homogeneous solution. Each mixture was incubated at 25°C for 30 min in a dark environment. The absorbance of resulting solutions was measured at 517 nm using a UV-Vis spectrophotometer (Thermo Scientific Evaluation Array UV-Vis spectrophotometer). The scavenging activity was calculated as per the following formula:

% activity = [(A_{1 (517 nm)} − A_{2 (517 nm)} ) / A_{1 (517 nm)}] × 100; where A₁ denotes control absorbance and A₂ denotes absorbance of the sample.

Statistical analysis

The antioxidant activity of the samples was evaluated in triplicates, and the results were presented as an average of the three experiments. Data were analyzed using SPSS 20.0 software, and p<0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION

Characterization of isolated compounds

The present study aimed to detect, isolate, and characterize the phenolic compounds present in the aerial extracts of T. munbyanus subsp. ciliatus. Two previously undescribed compounds, namely methyl 2,3,5,6-tetrahydroxybenzoate (1) and 4-hydroxy-5- methoxy-2-oxo-2H-pyran-3-carboxylic acid (16), and 14 known compounds, including 3 flavonoids; 3’,5,5’,7-tetrahydroxyflavanone (2), luteolin (3), and isorhamnetin-3-O-β-glucoside (4); a sterol glucoside named daucosterol (5); and 11 phenolic compounds, namely salicylic acid (6), pluchoic acid (7), methyl caffeate (8), ethyl caffeate (9), protocatechuic acid (10), ferulic acid (11), p-coumaric acid (12), rosmarinic acid (13), tyrosol (14), and protocatechuic aldehyde (15), were isolated from n-butanol and ethyl acetate extracts of T. munbyanus subsp. ciliatus. The structures of all compounds isolated from T. munbyanus are shown in Figure 1.

Compound (1) was obtained as colorless amorphous powder. The molecular formula of compound 1 was determined to be C₈H₈O₆ using EI-MS with negative ion m/z 198.08 (M-H)^-, which indicated that the molecule had five degrees of unsaturation. The ¹H- nuclear magnetic resonance (NMR) spectrum in acetone-d6 exhibited just two singlets: first at δ_H 7.33 (s, 1H, H-4), which indicated the presence of penta substituted benzene ring, and second at δ_H 3.88 (s, 3H), corresponding to the presence of methoxy groups.

Further, ¹³C NMR, HSQC, and DEPT spectra exhibited six carbon signals, including one methoxy group at δ_c 55.73 (C-8), one methine at δ_c 107.23 (C-4), and four quaternary carbons. The four quaternary carbons included three aromatic carbons at δ_c 120.59 (C-1), 147.39 (C-2/C-6), and 140.39 (C-3/C-5) and one carbon at δ_c 166.55 (C-7), which was characterized as a carbonyl carbon of an ester group.

A long-range C-H correlation was observed for C-4 aromatic proton in the HMBC spectrum (²J_CH and ³J_CH correlations) with C-3/C-5 and C-2/C-6. The structure was further confirmed by the HMBC spectrum. Thus, compound 1 was identified as methyl 2,3,5,6-tetrahydroxybenzoate.

Compounds 2-15 have been previously isolated and characterized, and data for these compounds are given in Supplementary Information.

Compound 16 was obtained as a yellowish-brown solid. The molecular formula for this compound was found to be C₇H₆O₆, which suggested the presence of five degrees of unsaturation. The ¹³C NMR and DEPT spectra further confirmed the presence of seven carbon atoms that included one methoxy group at δ_c 55.31; four vinyl carbons with one methine at δ_c 106.57 (C-6); quaternary carbon atoms at δ_c 128.11 (C-3), 137.54 (C-5), and 146.91 (C-4); and the carbonyl region with two peaks at δ_c 173.81 and 168.83, which were indicative of the presence of a carbonyl carbon of the carboxylic acid and carbonyl carbon of the α-pyrone ring, respectively. The ¹H NMR and HSQC spectra further confirmed the hypothesis. In particular, it displayed one singlet of one proton at δ_H 7.33 that was assigned to H-6. Besides this, three proton singlets were reported at δ_H 3.87 for OCH₃. The HMBC measurements showed long-range correlations between the protons at δ_H 7.33 and two quaternary carbons at δ_c 137.54 (C-5) and δ_c 146.91 (C-4). The HMBC experiments also showed connectivity between the methoxy protons at δ_H 3.87 and the quaternary carbon at δ_c 146.91 (C-4). These findings indicated that the carboxylic acid was connected to C-3, whereas a methoxy functional group was connected to C-5. Therefore, compound 16 was assigned the structure of 4-hydroxy-5-methoxy-2-oxo-2H-pyran-3-carboxylic acid.

Quantification of the polar constituents of T. munbyanus subsp. ciliatus extracts

The extracts of T. munbyanus were analyzed using HPLC-TOF/MS. The compounds isolated from the extracts were identified in terms of their retention times and m/z values, and these values were compared with those of standard samples. Altogether, the results of spectral analysis revealed the presence of 29 compounds, including 11 phenolic acids and 18 flavonoids and phenolics (Table 1). Interestingly, the concentration of these compounds was found to be very less in the chloroform extract. The highest concentrations of scutellarin, baicalin, and fumaric acid were reported in the butanoic extract; however, these compounds were present in very low amounts in the ethyl acetate extract. Whereas, quercetin-3-β-D-glucoside, caffeic, and 4-hydroxybenzoic acid were found to be the major consitutents of the ethyl acetate extract.

Thus, these results highlight that the analyzed extracts of T. munbyanus subsp. ciliatus, i.e., ethyl acetate and butanoic extracts, comprised of complex mixtures of plant secondary metabolites. These active ingredients, particularly flavonoids and phenolic acids, are previously known to exhibit antioxidant properties.^16,17

Total antioxidant capacity

The antioxidant activities of the isolated and extracted samples were measured and expressed as absorbance values. This assay involved the reduction of Mo⁺⁶ → Mo⁺⁵, and the antioxidant activity was confirmed if a green phosphate/Mo⁵⁺ complex was formed, which showed maximum absorption at 695 nm at acidic pH.¹⁴ This assay has been widely used to evaluate the total antioxidant capacity of various extracts and isolates. The results for total antioxidant activity of the isolates and extracts obtained from T. munbyanus subsp. ciliatus are shown in Figure 2. Here, higher absorbance of the antioxidant denoted the presence of antioxidant property. It was observed that ethyl acetate extract and compound 8 exhibited similar antioxidant activities at concentrations of 25, 50, and 100 µg/mL as compared to butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), p<0.05. Phenolic compounds have long been explored as a therapeutic option to prevent cancer, owing to its effective antioxidant potency.¹⁸ It has also been reported that compounds having antioxidant activity play an important role in the inhibition of lipid peroxidation.¹⁹ These results highlighted that the isolated compounds and different solvent extracts, obtained from T. munbyanus, acted as promising natural sources of nontoxic and natural antioxidants.

Free radical scavenging activity

DPPH˙ free radical scavenging assay is generally applied to determine the antioxidant levels of extracts in a relatively short period of time. In particular, the ability of compounds to deliver hydrogen is assessed. The utility of this assay has been established and compared with that of other experiments.²⁰ It has been shown that natural chemicals reduce DPPH˙ due to their ability to donate hydrogen atom.²¹ When an antioxidant and DPPH˙ (a synthetic radical) are mixed, the antioxidant gives an electron to DPPH₂ and the purple color is converted into yellow color.

Interestingly, the free radical scavenging activitiy of natural contents was found to increase in a dose-dependent manner, p<0.05. In particular, compounds 2, 7, 8, 10, and 15 displayed a higher scavenging activity at high doses than BHA and BHT (Figure 3). This might be attributed to two different -OH substitutions that favor DPPH˙ scavenging activity.²² The stabilization of radicals by two subsequent -OH substitutions of phenolic groups for compounds 3, 8, and 15 are shown in Figure 4. In particular, compounds 3, 8, and 15 showed their ability to remove radicals, which is attributed to their resonance stability and presence of hydroxyl groups. It can act as a radical inhibitor and stop the radical reaction. Plant phenolics generally include superoxide radicals, lipid alkoxyl radicals, lipid peroxyl radicals, and nitric oxide radical, which are known to exhibit cleansing, metal chelating, antiallergic, estrogenic, and antiviral effects.²³ In general, polymeric polyphenols are known to be more effective antioxidants than monomeric phenolics.²⁴ The presence of -OH group in the ortho or para position of phenol increases the antioxidant activity of a compound.

CONCLUSION

This is the first study to report the isolation and characterization of phenolic compounds from T. munbyanus subsp. ciliatus species grown in Algeria. In particular, the study reported the isolation of two new compounds, named methyl 2,3,5,6-tetrahydroxybenzoate and 4-hydroxy-5-methoxy-2-oxo-2H-pyran-3-carboxylic acid, and 14 previously known compounds that belonged to different chemical classes, namely flavonoids, sterol, and phenolic derivatives. The ethyl acetate extract exhibited excellent free radical scavenging activity in vitro, which correlated well with the presence of polyphenol derivatives in compounds 3, 8, and 15.

Thus, all these findings provide scientific basis for the use of T. munbyanus subsp. ciliatus derivatives as functional ingredients in Algerian traditional medicine.

ACKNOWLEDGMENTS

This work was supported by a grant from State Planning Organization of Turkey (DPT:2010K120720).

Conflict of interest: No conflict of interest was declared by the authors. The authors are solely responsible for the content and writing of this paper.