Study of the antioxidant activity of Thymus sibthorpii Bentham (Lamiaceae)

Abstract

From the aerial parts of Thymus sibthorpii Bentham (Lamiaceae), five flavonoids apigenin (1), 7-methoxy-apigenin (2), naringenin (3), eriodictyol (4) and eriodictyol-7-glucoside (5), have been isolated together with caffeic acid methyl ester (6), rosmarinic acid (7) and rosmarinic acid methyl ester (8). The structures of the isolated compounds were established by spectroscopic methods. The extracts and the isolated compounds were tested for their free radical scavenging activity using the following in vitro assays: (i) interaction with the free stable radical of DPPH (1,1-diphenyl-2-picrylhydrazyl), (ii) inhibition of linoleic acid lipid peroxidation induced by the dihydrochloric acid of 2,2-azobis-2-amidinepropane (AAPH) and (iii) the scavenging activity of enzymatically produced superoxide anion. Their inhibitory activity toward soybean lipoxygenase was evaluated in vitro, using linoleic acid as a substrate. The antioxidant results of the extracts are discussed in terms of their constitution in phenolic compounds, which were determined following the Folin–Ciocalteu method.

Keywords:

• Antioxidant activity
• flavonoids
• Lamiaceae
• lipoxygenase
• Thymus sibthorpii

Introduction

The genus Thymus (family Lamiaceae) is one of the eight most important genera as regards number of species within the Lamiaceae family. It is a perennial herbaceous plant indigenous to central and southern Europe and it is now widely cultivated as a tea, spice and herbal medicine1. Thyme leaf tea is said to promote rest and sleep. It is widely used in folk medicine because of its anti-inflammatory, antirheumatic, digestive, antiulcer and antimicrobial activities in Europe. Its leaf is listed in the German and British Herbal Pharmacopeia and has been used as a stomachic, carminative, diuretic, urinary disinfectant and vermifuge. Additionally, based on its traditional medicinal use, thyme is proposed for the Monograph of EMA/HMPC to use in productive cough associated with cold2.

Many studies refer to a sufficient antioxidant activity of Thymus extracts, due to its high concentration of phenolic compounds. There is a correlation between the antioxidant activity and the phenolic compounds concentration of the extracts3. Thymus extracts behave as superoxide and peroxyl radical scavengers on the 2,2-diphenyl-1-picrylhydrazyl-hydrate (DPPH)4–6 and 2,2-azobis-2-amidinepropane (AAPH) systems3.

Ethanolic, hexane and methanolic Thymus subfraction can be considered with antioxidative properties on the above systems6. Furthermore, the Thymus methanolic extract is mentioned as a stronger radical scavenger than others and standards BHT and BHA.4 The polar subfraction of methanolic extract is a stronger radical scavenger than the nonpolar subfraction. An inhibitory efficiency of the polar subfraction on linoleic acid oxidation is also measured.7

Another research was conducted to isolate the major components from the aerial parts of Thymus sibthorpii Benth – a morphologically variable species of the sect Serpyllum (Miller) Benth8 and to evaluate their antioxidant and anti-inflammatory activities in vitro. Five flavonoids and three phenolic acids were isolated so far, by repeated chromatographic isolation of butanol soluble fraction. Their structures were elucidated as apigenin (1), 7-methoxy-apigenin (2), naringenin (3), eriodictyol (4) eriodictyol-7-glucoside (5), caffeic acid methyl ester (6), rosmarinic acid (7) and rosmarinic acid methyl ester (8), by the analysis of spectroscopic evidences. The group of flavonoids is long considered as antioxidant because it interferes in oxidative process with various mechanisms. Thus, flavonoids may inhibit enzymes either by suppressing free radical synthesis, by chelating element traces, by scavenging free radicals or by protecting the antioxidant defenses9,10.

Lipid peroxidation and biomolecular damage is a consequence of oxidative stress. Through flavonoids free radical scavenging activity, flavonoids may be considered as enzyme inhibitors of various enzymes like lipogygenase11, cyclooxygenase, xanthine oxidase, etc12. Apparently, there is a correlation between structure and the type of inhibition11.

Position, structure and the total number of glycosides in a flavonoid is crucial for making a flavonoid a strong and effective antioxidant. Therefore, α-glycones give stronger antioxidants than their corresponding glycosides. As for the flavonoid chemical structure, those with a 3′-4′ catechol structure in B ring are stronger lipid peroxidation inhibitor through peroxyl, superoxide and peroxynitrite radical-scavenging activity. An unsaturated 2–3 bond in conjugation with a 4-oxo function gives strong antioxidants. Moreover, the presence of a 3-OH bond between A and B rings with the presence of 3′-4′ catechol, enhance the antioxidant activity of flavan-3-ols and flavon-3-ols whereas an O-methylation of hydroxyl groups of flavonoids decreases their radical-scavenging ability9.

The extracts, as well as the isolated compounds were tested for their free radical-scavenging activity using the following in vitro assays: (i) interaction with the free stable radical of DPPH (1,1-diphenyl-2-picrylhydrazyl), (ii) inhibition of linoleic acid lipid peroxidation with the dihydrochloric acid of AAPH and (iii) the scavenging activity of superoxide anion using the xanthine – xanthine oxidase system. Finally, their inhibitory activity toward soybean lipoxygenase was evaluated in vitro, using linoleic acid as a substrate. The antioxidant behavior of the extracts are discussed taking under consideration their constitution in phenolic compounds, which were determined with the method of Folin-Ciocalteu.

Materials and methods

Collection of plant material

Aerial parts of Thymus sibthorpii Bentham were collected when in flower from Chortiatis mountain, on June 2006. Voucher specimens have been deposited at the Herbaria of the Balkan Botanical Garden of Kroussia and the Laboratory of Pharmacognosy, University of Thessaloniki (AUTH).

Extraction and isolation

Air-dried aerial parts of the plant (381.3 g) were exhaustively extracted with petroleum ether (40–60°) (PE), dichloromethane (DM) and methanol (M). The methanol extract was concentrated and the residue redissolved in boiling water. The water-soluble fraction was filtered and extracted successively with hexane (HE), diethylether (ET), ethyl acetate (EA) and n-butanol (BU). CC of the residue of ET (3.42 g) over silica gel using DM-M mixtures followed by further purification on CC (Sephadex LH-20 using methanol as eluent) were obtained compounds 1 (2.4 mg), 2 (3.7 mg), 3 (0.4 mg), 4 (10.0 mg), 6 (4.7 mg) and 7 (87.8 mg). CC of the residue of EA (1.38 g) over silica gel using DM-M mixtures followed by further purification on CC (Sephadex LH-20 using methanol as eluent) were obtained compounds 2 (1.5 mg), 5 (5.3 mg), 7 (167.9 mg) and 8 (3.9 mg).

Chromatography

Column chromatography (CC): silica gel 60 (Merck Art. 9385), gradient elution with the solvents mixtures indicated in each case; Sephadex LH-20 (Pharmacia), elution with MeOH. TLC: Cellulose (Merck, Art. 5552). Solvents: acetic acid–water 15:85. Spray reagent: Neu reagent13. Silica gel 60 F254s (Merck, Art. 5554); Solvents: mixture of CH₂Cl₂ with MeOH; mixture of CH₂Cl₂ with MeOH and water; mixture of cyclohexane with EtOAc; mixture of EtOAc with MeOH and water. Spray reagent: vanillin-H₂SO₄.

Spectroscopic data

UV spectra were recorded on a Hitachi U-2000 spectrophotometer, according to Mabry et al. (1970)14. NMR: The ¹H NMR spectra (300.0 MHz) were recorded in CD₃OD using Bruker Avance 300 spectrometer. Chemical shifts are reported in δ (ppm) values relative to TMS.

Antioxidant assays

Each in vitro experiment was performed at least in triplicate, and the standard deviation of absorbance was less than 10% of the mean. In all cases, significant difference from control: *p < 0.1 **p < 0.01 (Student’s t-test).

Determination of total phenolic content

The concentration of total phenolic compounds in the fractions was determined spectrophotometrically using Folin–Ciocalteu reagent following the previously described method15 with few modifications. A certain amount (100 μl) from the stock solution of the sample was mixed with 500 μl of Folin–Ciocalteu phenol reagent and 400 μl of 7.5% sodium carbonate solution. The mixture was shaken thoroughly and incubated for 1.5 h at 30 °C protected from light. The absorbance of the blue color produced was measured with a spectrophotometer at 765 nm. The concentration of total phenolic compounds for each extract was calculated on the basis of a standard curve obtained using gallic acid as a standard (12 serial 2-fold dilutions to give arrange of 0.01–0.001 mg/ml in triplicate). Results were expressed as milligrams of gallic acid equivalent (GAE) per 100 g of dried weight.

Interaction of the extracts and the tested compounds with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) stable free radical

DPPH assay test is very useful in the micromolar range, since it demands minutes to hours for both lipophilic and hydrophilic samples. Many studies have been previously reported concerning the antioxidant activity of natural substances tested with DPPH16. In the presence of an antioxidant, which can donate an electron to DPPH, the purple color is typical of the free DPPH radical decays and can be followed spectrophotometrically (517 nm). This interaction indicates its radical scavenging ability in an iron-free system.

A 10 and a 20 μl sample from the stock solution of the extracts (approximately 2.5 mg in 1 ml DMSO) or 10 μl of the isolated compounds (final concentration 50 μM) were dissolved in absolute ethanol to a final volume of 1 ml in which 1 ml DPPH (0.1 mM, in absolute ethanol) was added. The reaction mixture was kept at room temperature. The optical density (OD) of the solution was measured at 517 nm, after 20 and 60 min. The optical densities of the samples in the absence of DPPH were subtracted from the corresponding OD with DPPH. The % reduction values were determined and compared to appropriate standards.17

Inhibition of linoleic acid peroxidation induced by the dihydrochloric acid of 2,2-azobis-2-amidinepropane (AAPH)

This assay can be used to follow oxidative changes and to understand the contribution of each tested compound. The water soluble azo compound AAPH (2,2′-azobis-(2-amidinopropane)-dihydrochloride) is used as a free radical initiator for in vitro studies of free radical production. Production of conjugated dienehydroperoxide by the oxidation of linoleic acid sodium salt in an aqueous solution is monitored at 234 nm. This assay can be used to follow oxidative changes and to understand the contribution of each tested compound. An amount of 10 μl of the 16 mM sodium linoleate solution was added to the UV cuvette containing 0.93 ml of 0.05 M phosphate buffer, pH 7.4, prethermostated at 37 °C. The oxidation reaction was initiated at 37 °C under air by the addition of 50 μl of 40 mM AAPH solution. Oxidation was carried out in the presence of samples (extracts 10 μl or isolated compounds 10, 50, 100 μM) in the assay without antioxidant, and lipid peroxidation was measured in the presence of the same level of DMSO. The rate of oxidation at 37 °C was monitored by recording the increase in absorption at 234 nm caused by conjugated dienehydroperoxides18. Trolox has been tested as reference compound19.

Scavenging activity of superoxide anion using the xanthine–xanthine oxidase system

The superoxide anion was generated enzymatically by the xanthine–xanthine oxidase system and measured by the nitroblue tetrazolium (NBT) method20. To the reaction mixture in phosphate buffer pH 7.4 (0.1 M) containing xanthine, NBT, the tested isolated compounds (using two different concentrations: 10 μM and 100 μΜ) and xanthine oxidase (at final concentration 0.07 U/mL) were added. After incubating for 10 min at room temperature, the absorbance was recorded at 560 nm. Caffeic acid was evaluated as reference compound21.

Soybean lipoxygenase inhibition

The bioassay was performed according to a previously described procedure17. All samples (extracts 10 μl or isolated compounds 10 and 100 μΜ) were initially dissolved in DMSO (approximately 2.5 mg in 1 ml DMSO for the extracts). The incubation mixture (final volume 1 ml) consisted of 10 μl or 1 μl of the test sample, 100 μl of sodium linolate (0.1 mM) and 0.2 μl of the enzyme solution (1/9 × 10⁻⁴, w/v in saline) in buffer tris pH 9. After incubation at room temperature for 3 min, the conversion of the sodium linoleate to 13-hydroperoxylinoleic acid was recorded at 234 nm and compared with an appropriate standard inhibitor (Nordihydroguaeretic acid).

Results and discussion

Phytochemical analysis

The present study was carried out on the aerial parts of Thymus sibthorpii Bentham (Lamiaceae) a taxa native of Greece, Bulgaria and Turkey22. The crude extracts of the aerial parts of T. sibthorpii Bentham afforded five flavonoids apigenin (1),23 7-methoxy-apigenin (2),24 naringenin (3),25 eriodictyol (4)26 and eriodictyol-7-glucoside (5),27 together with caffeic acid methyl ester (6),28 rosmarinic acid (7) and rosmarinic acid methyl ester (8)29,30. The structures of the isolates were given in Figure 1. The spectroscopic data (UV-Vis and ¹H NMR) of all the compounds are given in the Supplementary Material.

Figure 1. Structures of the isolated compounds.

Rosmarinic acid methyl ester (8).

Total phenol and flavonoid content

The total polyphenol content was 5.4 mg of GAE/gr extract (Table 1) gives information of the extracts total phenolic content (1.5, 3.5, 0.4 mg of GAE/g extract, respectively). The total GAE phenolic content was 141.00 ± 0.90 μg/mg (14.1%, w/w)7. M (methanol) extract was the most rich in phenols followed by EA (ethyl acetate).

Table 1. Polyphenol content of different plant extracts.

Extracts	mg of GAE/1gr extract ± SD*
PE	1.5 ± 0.1*
DM	0.2 ± 0.005*
M	5.4 ± 0.25*
HE	2.5 ± 0.15*
ET	1.5 ± 0.008*
EA	3.5 ± 0.005*
BU	0.4 ± 0.0002**

*SD < 10% of the mean.

In all cases, significant difference from control: *p < 0.1 **p < 0.01 (Student’s t-test).

Bioactivity

The present study was an attempt to isolate compounds from Thymus sibthorpii Bentham and to test them as potent antioxidants, using the DPPH, AAPH and xanthine/xanthine oxidase assays and as LOX inhibitors. All the isolated compounds are known to be widely distributed in Lamiaceae family31.

Various extracts of the plant were tested on DPPH and LOX assay. DPPH assay provides a good oxidative reagent to describe the antioxidant profile of the Thymus extracts, as well as of the isolated compounds.

All the plant extracts were tested for their antioxidant activity using the DPPH assay. The reducing activity seems to be time and concentration dependent Table 2. Methanol (M), Et₂O (ET), EtOAc (EA), butanol (BU) and water (W) extracts, immediately interact with the free radical (20 min) on both concentrations. These results are correlated with the findings of Table 1, in which the total phenolics are presented. On a similar study referring to Thymus spathulifolius (Hausskn. and Velen.) the methanolic extract DPPH had an IC₅₀ of 16.15 ± 0.5 μg/ml, which was close to the synthetic antioxidant, BHT, (19.8 ± 0.5 μg/ml).

Table 2: % Interaction of different plant extracts with DPPH.

	±SD*
	(100 μl)		(10 μl)
Extracts	20 min	60 min	20 min	60 min
PE	38.4 ± 0.5*	62.2 ± 0.9**	17.9 ± 0.1*	34.2 ± 1.0*
DM	23.7 ± 0.2*	22.4 ± 0.7**	7.9 ± 0.3**	49.5 ± 0.4*
M	95.3 ± 1.9*	87.6 ± 1.0**	91.7 ± 1.3**	92.1 ± 0.6**
HE	89.8 ± 2.1*	91.1 ± 1.4**	39.8 ± 0.9**	60.2 ± 0.1**
ET	99.7 ± 1.8**	93.1 ± 1.9**	95.2 ± 2.1*	94.7 ± 2.1**
EA	92.8 ± 1.7*	92.1 ± 1.3*	92.4 ± 1.8*	95.9 ± 2.3**
BU	85.4 ± 1.2**	87.6 ± 1.6*	90.7 ± 0.9*	86.4 ± 1.8**
W	83.7 ± 1.0*	86.4 ± 1.1**	86.4 ± 1.3*	87.9 ± 1.2*
NDGA	94 ± 2.1**	97 ± 1.5**	88 ± 1.2*	96 ± 1.6*

*SD < 10% of the mean.

In all cases, significant difference from control: *p < 0.1 **p < 0.01 (Student’s t-test).

In Table 4, the % interaction values of the isolated compounds (final concentration 50 μM) are given with DPPH. Compound 1 (apigenin) exerted 50% antioxidant activity (Table 4) after 60 min comparable to the results published to another study of Zeghad and Merghem, 201332.

Table 3. Percentage soybean LOX inhibitory activity of extracts.

Extracts (10 μL)	% Inhibition ± SD*
PE	12.6 ± 1.0*
DM	48.3 ± 0.7*
M	14.1 ± 0.1*
HE	36.8 ± 0.9*
ET	27.1 ± 0.5*
EA	8 ± 0.3**
BU	13.5 ± 0.1*
W	9.7 ± 0.8*
NDGA	88 ± 2.1**

*SD < 10% of the mean.

In all cases, significant difference from control: *p < 0.1 **p < 0.01 (Student’s t-test).

Table 4. Percentage interaction of isolated compounds with DPPH.

Compounds (50 μΜ)	% interaction 20 min ± SD*	Interaction % 60 min ± SD*
1	31.3 ± 1.5*	54.4 ± 2.3*
2	5.8 ± 0.1*	16.4 ± 0.5*
3	17.3 ± 0.9*	21.1 ± 1.0**
4	54.3 ± 1.7**	53.7 ± 1.2**
5	61.9 ± 2.3*	58.9 ± 0.8**
6	78.8 ± 2.1*	79.9 ± 1.1**
7	81.6 ± 1.6**	81.2 ± 1.9*
8	65.0 ± 2.1*	70.2 ± 0.8**
NDGA	83 ± 1.1*	87 ± 1.5**

*SD < 10% of the mean.

In all cases, significant difference from control: *p < 0.1 **p < 0.01 (Student’s t-test).

Compounds 4 (eriodictyol), 5 (eriodictyol-7-glucoside), 6 (caffeic acid methyl ester), 7 (rosmarinic acid) and 8 (rosmarinic acid methyl ester) were found to highly interact with the free radical.

AAPH is a water-soluble azo compound, which is used as a free radical generator, on lipid peroxidation assays, therefore, in screening antioxidants’ efficiency21,33–35. All the isolated compounds were tested in three different concentrations (10 μM, 50 μM and 100 μM) for their ability to inhibit linoleic acid peroxidation. The results from compounds – antilipid peroxidation activity – are given in Table 5. Compound 4 (eriodictyol) was the strongest inhibitor with 100% inhibition at 50 μM. Compound 1 (apigenin) and 3 (naringenin) did not exhibit any inhibition, compounds 2 (7-methoxy apigenin) and 6 (caffeic acid methyl ester) exhibited a moderate inhibition at 100 μM, whereas compounds 5 (eriodictyol-7- glucoside), 7 (rosmarinic acid) and 8 (rosmarinic acid methyl ester) totally inhibited linoleic acid peroxidation at 100 μΜ.

Table 5. % Inhibition of linoleic acid peroxidation induced by the isolated compounds.

Compounds	10 μM	50 μM	100 μM
1	nt	nt	0.0
2	nt	Nt	46.6 ± 0.4*
3	nt	nt	0.0
4	79.2 ± 2.3*	100 ± 2.3**	100 ± 2.8**
5	22.7 ± 0.8*	nt	100 ± 3.3**
6	nt	0.0	50.2 ± 1.0*
7	45.8 ± 1.6**	nt	100 ± 1.8**
8	75.8 ± 3.1*	nt	100 ± 2.5**
Trolox	63 ± 1.3*	76 ± 2.5*	83 ± 1.2*

*SD < 10% of the mean; *SD < 10% of the mean; nt: not tested.

In all cases, significant difference from control: *p < 0.1 **p < 0.01 (Student’s t-test).

The ability of the tested compounds to scavenge the superoxide anion generated by the xanthine/xanthine oxidase system is showed in Table 7. Compounds 4 (eriodictyol) and 8 (rosmarinic acid methyl ester) presented a moderate inhibition at 10 μM concentration. Compounds 1 (apigenin), 5 (eriodictyol-7-glucoside), 6 (caffeic acid methyl ester) and 7 (rosmarinic acid) at concentration 100 μM, moderate inhibited the enzyme.

Table 6. Percentage soybean LOX inhibitory activity of the isolated compounds.

Compounds	10 μM	100 μM
1	0	29.2 ± 0.2**
2	0	34 ± 0.5**
3	19.8 ± 0.1*	72.4 ± 1.6**
4	nt	0
5	nt	39.3 ± 0.3*
6	22.9 ± 0.3**	87 ± 0.9*
7	25.9 ± 0.7**	92.4 ± 2.6*
8	10.6 ± 0.8**	79.4 ± 1.6**
NDGA	86 ± 1.9**	91 ± 2.1**

*SD < 10% of the mean; nt: not tested.

In all cases, significant difference from control: *p < 0.1 **p < 0.01 (Student’s t-test).

Table 7. Superoxide anion scavenging activity % of the isolated compounds.

Compounds	10 μM	100 μM
1	nt	15.2 ± 0.4
2	0	0
3	7.2 ± 0.5*	0
4	27.4 ± 1.1*	nt
5	nt	21.3 ± 1.1*
6	nt	13.7 ± 0.8**
7	nt	28.6 ± 1.3*
8	13.3 ± 0.25*	0
Caffeic acid	45% ± 0.3**IC50 = 600 μΜ ± 2.3**

*SD < 10% of the mean; nt: not tested.

In all cases, significant difference from control: *p < 0.1 **p < 0.01 (Student’s t-test).

All extracts and the isolated compounds were tested as possible LOX inhibitors. Lipoxygenase is a key enzyme in inflammation in the arachidonic acid metabolism to leukotrienes. In Table 3, the % inhibitions of the tested extracts (Chloroform extract 48.3%, Hexane extract 36.8% and Et₂O 27.1%) are given. In Table 6, the inhibitory activities of the isolated compounds at 10 μM and 100 μM concentration are given. Compounds 3 (naringenin), 6 (caffeic acid methyl ester), 7 (rosmarinic acid) and 8 (rosmarinic acid methyl ester) moderate inhibited LOX at 10 μM. Higher inhibition is observed at 100 μM. The inhibition values for compounds 1 (apigenin), 2 (7-methoxy-apigenin) and 5 (eriodictyol-7-glucoside) at 100 μM were found to be moderate inhibitors, whereas compound 4 (eriodictyol) did not present any inhibition at both concentrations.

Conclusions

Five flavonoids have been isolated from the aerial parts of Thymus sibthorpii Bentham (Lamiaceae). Both the extracts and the isolated compounds have been evaluated for their biological activity. Most of the extracts presented significant antioxidant activity in DPPH test. Among the isolated compounds, caffeic acid methyl ester (6) and rosmarinic acid (7) presented the highest antioxidant activity as well the strongest inhibitory activity against lipoxygenase. On the other hand, eriodictyol (4) was found to stronger inhibit the lipid peroxidation of linoleic acid. Further studies will be needed to clarify the antioxidant profile of Thymus sibthorpii Bentham and the isolated compounds.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Supplementary material available online

Funding

The authors are grateful to Dr. N. Krigas (Scientific collaborator of Department of Crop Production to Technological Education Institute of Thessaloniki for the identification of the plant material and to Dr. C. Litinas (Professor of Department of Chemistry, AUTH) for recording NMR spectra. This study was financially supported by the GSRT (015-ɛ 5838. 14–03-06).