Purification, chemical characterization and evaluation of the antioxidant potential of carvacrol from Thymus vulgaris

Abstract This work focuses on the purification, chemical characterization and evaluation of the antioxidant activity of carvacrol in order to determine its contribution in the high antioxidant potential of Thymus vulgaris essential oil (TEO). Firstly, 68% of carvacrol was purified from TEO using chromatography on silica gel column and then chemically characterized using spectroscopic techniques (IR, MS and 1H and 13C NMR). In vitro, the antioxidant activity has been determined using DPPH, ABTS•+ and iron chelating assays. All assays proved the strong radical scavenging and reducing power of carvacrol. In vivo, antioxidant capacity towards stressed Saccharomyces cerevisiae cells was investigated by evaluating cell viability, antioxidant enzymes’ activity, the level of lipid peroxidation (LPO) as well as the activity of succinate dehydrogenase (SDH). Using carvacrol in a dose dependent manner (6.25-25 μg/mL), cell viability was outstandingly improved by 34.5-55% compared to stressed cells. Antioxidant enzymes (CAT, SOD, GR) activities were also brought back to values comparable to control cells along with lower LPO (0.81±0.07 nmol/mg) and SDH (1.15± 0.07 μmol/min/mg of protein) at 25 μg/mL. These findings suggest that the powerful antioxidant properties of TEO found in our previous study were mainly associated to its main component (carvacrol) that showed higher antioxidant activity compared to the other components. Therefore, carvacrol can be of a great use as a pharmacological agent against damages related to oxidative stress.


Introduction
Oxidative stress is a complex biological process characterized by an overproduction of reactive oxygen species (ROS) leading to the destruction of REDOX balance and provoking damage to all cellular compounds including protein, lipid and DNA 1 .Eventually, several pathologies have been reported to be linked to oxidative stress in instance diabetes 2 , cancer 3 as well as neurological and cardiovascular diseases 4 .In order to counteract the damages of oxidative stress, various bioactive compounds obtained from natural resources including plants and algae have been used in recent years for their actual or presumed beneficial effect towards oxidative damage 2 .Hence, phenolic compounds have gained considerable interest in the scientific field as natural substances with antioxidant potential and have been of great use in pharmaceutical and other industries.They are known as strong natural antioxidants having a crucial role in a wide range of biological and pharmacological properties 5 .As a phenol, carvacrol is among the most important naturally occurring antioxidants that has been the object of many scientific studies and has highly pronounced antioxidant as well as other biological properties.It is well known to be present in Thymus, Satureja, and Origanum plants 6 .Thus, a study of fourteen Iranian Thymus species elucidated that the main components were carvacrol and thymol showing the quality of the Thymus species 7 .Moreover, T. vulgaris is exploited in various industries.It has, however, been widely used for medicinal purposes in pharmaceutical industry as anti-acne agent, fungicidal and antiviral drug and it also proved to possess hepatoprotective properties 8 .Furthermore, thyme is one of the most precious spices and food preservatives used in food industry in almost everywhere in the world.The extensive use of T. vulgaris is due to many of its pharmacological and biological features that are scientifically validated and which are mainly attributed to its composition on monoterpene derivatives including thymol and carvacrol 9 .
Accordingly, in our previous study, the phytochemical analysis of T. vulgaris EO as well as four other EOs has elucidated a difference qualitatively and quantitatively in their composition.
T. vulgaris contained a variety of aromatic bioactive compounds with carvacrol as main component (76.66%).This came along with high free radical neutralization potential and a considerable antioxidant activity against oxidative stress induced by H 2 O 2 in the yeast Saccharomyces cerevisiae 10 .Continuously to our work, our paper aims to expose in detail the purification, chemical characterization and evaluation of the antioxidant activity of carvacrol in comparison with the other components from TEO in order to determine its contribution as main component in the antioxidant potential featured by TEO.

Chemicals and reagents
In this study, all used chemicals were of high quality.Hexane, Ethyl acetate and dimethylsulfoxide (DMSO), Acetic acid,

Essential oil
In this study, TEO was received in pure concentration from Naturactive Laboratoires Pierre Fabre France.It was extracted from aerial parts of Thymus vulgaris plant in the European region and its batch number is 3665606000181.

Purification of main product
The main component was purified using column chromatography which is one of the most used techniques for the purification and separation of compounds.Thus, 10 g of TEO was placed above the column and absorbed on the upper part of the MERCK60 silica gel used as stationary phase.Subsequently, the eluent which was a mixture of hexane/ethyl acetate was added carefully.The mobile phase, as eluted, is collected in small fractions in tubes thus allowing the isolation and purification of the compounds.Then, the pure fractions were combined in a pre-weighed round-bottom flask.Finally, the solvent was removed using a rotary evaporator and then weighed the round bottom flask containing the dry compound.The majority product in fraction F1 (6.8 g). was purified using 3/97 of hexane/ ethyl acetate and the more polar fraction F2 (2.7 g) was eluted using ethyl acetate as eluent.

Physical and spectroscopic data of isolated constituent
Following the purification of the main product, a spectroscopic study was established allowing the identification of the spectral data of the product by comparison with those of reference compounds: MS mass spectrum, IR spectrum, 13 C NMR spectra.
The infrared spectra were recorded on an IRAffinity-1S SHIMADZU Fourier transformed device, it operates with a transmittance mode ranging from 400 cm-1 up to 4000 cm -1 , with a resolution of 4, scan number =80.

Gas chromatography analysis
The EO was subjected to gas chromatography/ mass spectral analysis (GC/ MS) on a Shimadzu GC-2010 chromatograph connected to Shimadzu QP-2010 mass spectrometer using an ionization voltage of 70 eV.The capillary column was BP-5 (30 m x 0.25 mm I.D.; 0.25 μm film thickness).The oven temperature was programmed from 60 to 200°C with an increasing rate of 3°C/min, followed by a 15°C/min increase to 280°C, then maintained for 10 min at this temperature.The injected volume was about 0.1 µL.The carrier gas was helium, set at a flow rate of 1 ml/min and adjusted at a linear speed of 36.5 cm/s.

Yeast strain and growth conditions
The in vivo antioxidant activity was evaluated using a wild type strain of Saccharomyces cerevisiae (YMES2).The strain was isolated from traditional Moroccan bread dough and was kindly provided by Professor Faouzi Errachidi from Faculty of Sciences and Technologies of Fes (FST).Yeast strain was grown in liquid YPG medium (1% yeast extract, 1% peptone, 2% glucose) with an orbital shaker at 160 rpm, for 24 hr.at 30°C with the ratio of flask volume/ medium of 5/1.

In vitro antioxidant activity DPPH free radical scavenging assay
The ability of the obtained fractions from TEO to reduce DPPH free radicals was evaluated according to Wu et al. 11 method in triplicate and with some modifications.Hence, 50 μL of each fraction at different concentrations (0.6-10 mg/mL) was added to 1,950 μL DPPH ethanol solution (60 μM).The mixture was left 30 min in the dark at room temperature then the absorbance was measured at 517 nm.To calculate the activity the equation below was used: A 0 is the control's absorbance after 30 min; A t is the absorbance of each EO fraction after 30 min.Ascorbic acid was used as positive control.
ABTS + free radical scavenging assay According to the method reported by Dorman et al. 12 , this test was performed in triplicate.It is based on the ability to neutralize the radical ABTS •+ generated by the reaction of K 2 S 2 O 8 (2.45 mM) with ABTS (7 mM) aqueous solution in the dark and at room temperature for 16 h.First of all, the absorbance was adjusted to 0.7 by adding ethanol to the prepared solution.Then, 20 μL of each sample at different concentrations were added to 1980 μL of ABTS •+ .Absorbance was measured at 734 nm at time 0 (A 0 ) and after 6 min (A 1 ).Ascorbic acid was used as positive control.

Reducing power
The capacity of both EO fractions to reduce iron (Fe 3+ ) in ferric chloride to ferrous (Fe 2+ ) was determined using the method described by Trabelsi 13 .Thus, samples at different concentrations (0.6-10 mg/mL) were added to the reactional mixture that contained 0.2 M phosphate buffer (pH 6.6), 1% (w/v) potassium ferricyanide, 10% TCA and 0.1% ferric chloride.The measurement of the absorbance was done at 700 nm and the ascorbic acid was used as positive control.The assay was carried out in triplicate.

Determination of cell viability
Cell viability was determined in normal conditions and in oxidative stress condition that was induced by hydrogen peroxide H 2 O 2 (2 mM) for an hour at 30°C/160 rpm.The assay was carried out in triplicate on yeast cells treated or not with TEO fractions at different concentrations that showed no cytotoxicity on cells after the test carried out according to Dani et al. 14 .Thus, after being diluted (10000 x), yeast cells were plated on solidified medium YPG that contains 1% yeast extract, 1% peptone, 2% glucose and 2% agar and incubated at 30°C for 72 h 15 .The colonies were then counted and the survival was expressed as percentage.

Biochemical analysis
Cell-free extract preparation procedure After the culture, yeast cells were harvested performing a 5-min centrifugation at 4°C/ 6000g.Cells contained in the pellet were washed three times with 20 mM Tris-HCl buffer (pH 7.5) and put back into suspension in the lysis buffer that contains 50 mM Tris-HCl buffer (pH 7.5), 1 mM ethylenediamine tetraacetic acid (EDTA), 10 mM 2-β-mercaptoethanol, 1 mM phenylmethylsulfony fluoride (PMSF), and 1% (v/v) glycerol at a ratio of 3 mL/g (wet weight).The suspension was placed in ice while using A Bandelin Sonopuls Sonifier (90%, 20 s, 12×).Later, a centrifugation (15,000g, 45 min at 4°C) was performed using a Sigma 2-16 K refrigerated centrifuge.The supernatant obtained was used for all enzyme activity assays.As for protein content, it was determined using bovine serum albumin (BSA) as standard according to the Bradford procedure 16 .

Catalase (CAT) activity
Catalase (CAT) is an antioxidant enzyme known for its role in catalyzing the decomposition of H 2 O 2 into O 2 and H 2 O. Therefore, the consumption of H 2 O 2 was monitored in order to determine the CAT activity.The reaction mixture was prepared using 7.5 mM H 2 O 2 in 50 mM sodium phosphate buffer (pH 7.0) added to 50 μl of the enzyme extract.At wavelength of 240 nm, the kinetics was measured 17 .The CAT activity was then calculated using the molar extinction coefficient of H 2 O 2 (0.0394 mM -1 cm -1 ).It is defined as μmol H 2 O 2 consumption/min/mg of protein.

Superoxide dismutase (SOD) activity
As described by Paoletti 18 , the reaction mixture used to determine the activity of superoxide dismutase enzyme was composed of 5 mM EDTA, 2.5 mM MgCl2, 3.9 mM 2-mercaptoethanol, 0.27 mM NADH in 50 mM potassium phosphate buffer (pH 7) in addition of 50 μL enzyme extract.The kinetics was measured at a wavelength of 340 nm.This activity measurement was based on the capacity of the enzyme extract to inhibit the oxidation of NADH by superoxide radicals.Therefore, The SOD activity unit is the amount of enzyme required to inhibit the oxidation of the initial rate of NADH by 50%, expressed in U/mg of protein.

Glutathione reductase (GR) activity
The measurement of the GR activity was done as mentioned by Di Ilio 19 .This evaluation is based on following the drop in the absorbance at a wavelength of 340 nm as a consequence of the oxidation of NADPH.Firstly, incubation at 37°C for 2 min of the mixture that contained: 1 mM EDTA, 0.5 mM GSSG, 50 mM potassium phosphate buffer (pH 7.4) and 50 μL of enzyme extract.After that, 100 μL of NADPH (0.1 mM) was added.At last, the kinetics was measured at 340nm and the unit was indicated as nmol NADPH oxidized per min per mg of protein.

Determination of lipid peroxidation
This test was carried out according to the method described by Samokyszyn & Marnett 20 , using thiobarbituric acid reactive substances (TBARS) in order to evaluate the capacity of the extract to inhibit the formation malondialdehyde which is a marker of lipid peroxidation.For this purpose, the mixture was prepared by adding 1 mL of the extract to 1 mL of the solution containing 0.375% thiobarbituric acid and 15% trichloracetic acid in 0.25 M hydrochloric acid.The mixture was then heated 15 min at a temperature of 100°C and quickly put into ice to cool it down and stop the reaction.Later on, centrifugation was done at 1,000 g for 10 min and the supernatant's absorbance was measured at a wavelength of 535 nm.Results were expressed as nmoles MDA equivalents per mg protein.

Succinate dehydrogenase (SDH) activity
As reported by King 21 , the activity of SDH was determined according to the reduction of Dichlorophenolindophenol (DCIP).It is as a chemical compound and is known to be used as a reagent and redox dye by the change of its color that goes from blue in the oxidized form to colorless when reduced.For this purpose, a mixture composed of 0.053 mM DCIP, 0.3 mM EDTA in 100 mM potassium phosphate buffer (pH 7.4) and 50 μL enzyme extract was incubated at 25°C for 10 min.Then 50 μL of KCN-Succinate (3.25 mg/mL of KCN in 0.5 M succinate) was added.At a wavelength of 625nm the activity of SDH was measured.The unit (μmol DCIP reduced/min/mg protein) was determined using molar extinction coefficient of DCIP (19,100 M -1 cm -1 ).

Statistical analysis
All graphics were done using GraphPad Prism 8.0.2 software for Windows (GraphPad Software Inc., San Diego, CA, USA).Differences between groups were tested for statistical significance using Two-way ANOVA followed by Sidak's multiple comparison test at a p < 0.05 significance level.All experiments were performed at least in triplicate and presented as mean values ± standard deviation (SD).

Results and Discussion
In continuation of our previous work on the phytochemical composition and antioxidant properties of TEO as well as others EOs which showed that TEO contains a variety of aromatic bioactive compounds, the majority of which was carvacrol (76.66%) 10 .Since carvacrol is well known for its antioxidant potential, it seemed interesting to us to carry out a comparative study of the antioxidant capacity between carvacrol purified from TEO and the rest of the compounds present in order to elucidate its crucial contribution in the high antioxidant activity of TEO.
First of all, the column chromatography over silica gel of TEO led to the purification of major constituent in first fraction F1 with 68%, together with a mixture of more polar constituents in second fraction F2.The purification was confirmed by GC analysis and the structure elucidation of the isolated constituent was identified by comparison of its spectroscopic properties 1 H and 13 C NMR data with those reported in the literature 22 .Its mass spectrum shows the molecular ion m/e 150 which corresponds to the molecular formula C 10 H 14 O.The IR spectrum shows a wide band around 3400 cm -1 characteristics of hydroxyl groups and the bands at 1610, 1480, 1430 cm -1 testify to the presence of an aromatic nucleus.As for the 1 H and 13 C NMR spectra of the purified product in (Supplementary files), they resemble to those of carvacrol.Its structure was confirmed by comparing these results with those of the same product reported in the literature as shown in Table 1.Thus, the structure of the isolated component in F1 was assigned to carvacrol.The chromatogram of carvacrol is shown in Fig. 1.Secondly, a preliminary analysis of the cytotoxicity of both fractions at range of concentrations (6.25-25 μg/mL) (Fig. 2A) has showed absolutely no toxicity on yeast cells.It is well known that hydrogen peroxide is a major contributor to oxidative stress and is extensively used in inducing oxidative stress in cellular models.As H 2 O 2 reacts Fe 2+ ions via the Fenton's reaction, the highly reactive hydroxyl radical (OH • ) is generated.Hence, it is considered as the main damaging and most dangerous of all ROS 23,24 .Thus, an investigation was carried out to determine their effect on cell viability using S. cerevisiae cells pretreated with Table 1. 13 2B showed in comparison with control cells that viability has significantly decreased to 42±2.12% (P < 0.001) when cells were only exposed to H 2 O 2 .Accordingly, ROS have been known for their implication in oxidative damage and eventually cell death 25 .However, the pretreatment with carvacrol has outstandingly improved the viability in a dose dependent manner (6.25-25 μg/ mL) by 34.5-55% compared to stressed cells.The pretreatment with the rest of the components has slightly improved the viability at a concentration of 25 μg/mL by 22% compared to stressed cells.This is clearly showing that cell viability has been restored after the use of carvacrol which is revealing its important protective effect against cell death caused by oxidative stress induced by H 2 O 2 .Moreover, the in vitro evaluation of antioxidant potential of both fractions has been assayed using three tests: DPPH, ABTS and reducing power RP.DPPH test is associated with the potential of an extract to scavenge DPPH • that are considered as stable free radicals 26 .In the present study, the DPPH • free radicals scavenging activity of the two fractions of TEO as well as ascorbic acid VC was investigated at different concentrations.Results in Fig. 3A showed that carvacrol clearly revealed a dosedependent DPPH • scavenging activity compared to F2.The percent of DPPH • scavenging at a range of concentrations 0.6-10 mg/mL was 22.61-83.58%,12.28-31.57%by carvacrol and other components respectively.Furthermore, purified 10 fig 3 carvacrol in F1 graphically displayed a lower IC 50 value compared to (F2) that showed a higher IC 50 value.On the other hand, ABTS scavenging test allows the measurement of the relative ability of natural extracts and pure compounds to scavenge ABTS •+ radicals generated in aqueous phase following the oxidation of ABTS with potassium persulfate 27 .As illustrated in Fig. 3B, carvacrol showed a high ABTS • + scavenging ability as the concentration increased.At a range of concentrations 0.6-10 mg/mL, the scavenging rates were 38.1-89.33 %.At the same range of concentrations, scavenging rates of other components of TEO were 10-33% showing low scavenging ability compared to those of carvacrol and VC.As for reducing power test, Fig. 3C illustrate the curve of reducing power for both fractions as well as ascorbic acid used as control.Carvacrol exhibited a high reducing power increasing in a concentration-dependent manner.As for the rest of the components, displayed a very low reducing power compared to ascorbic acid used as standard.The reducing power of both TEO fractions as well as VC at 10 mg/mL was in the following order: VC (2.162±0.23)> F1 (1.782±0.17)> F2 (0.172±0.011).These results confirmed the contribution of carvacrol in the reducing power activity 28 .The results of free radicals scavenging assays in the current study come in correlation with previous studies suggesting that the monoterpene (carvacrol) presents a high ability to scavenge DPPH • and ABTS •+ radicals through hydrogen-donating mechanism.This antiradical effect of carvacrol is due to its redox characteristics, which has a crucial role in quenching singlet and triplet oxygen, neutralizing radicals, and decomposing peroxides 29 .
Enzymatic antioxidants are defined as proteins that are implicated in the catalytic conversion of ROS as well as their by-products to nontoxic stable molecules.Thus, this defense system where all enzymes work cooperatively and represent a crucial defense mechanism against cell damage caused by oxidative stress 30 .As, SOD catalyzes the reaction in which superoxide anions are converted to hydrogen peroxide (H 2 O 2 ).CAT prevents the accumulation of hydrogen peroxide by catalyzing its decomposition to H 2 O and O 2 .Thus, GR acts by regularly providing intermediate 10 fig 4 metabolites to scavenge ROS 31 .In this study levels of CAT, SOD and GR were investigated.Results in Fig. 4 showed that the value was increased considerably after stressing yeast cells using H 2 O 2 compared to the control sample containing normal unstressed cells of S. cerevisiae.Hence, using H 2 O 2 the amounts of CAT, SOD and GR were higher by ~254%, ~218% and ~183% (P<0.001)respectively compared to the control.Accordingly, the presence of a strong positive correlation between CAT and SOD activities in yeast cells in a state of stress induced by different concentrations of H 2 O 2 has been reported 32,33 .This may be attributed to the response of yeast cells to peroxide stress.However, pretreatment with carvacrol at different concentrations (6.25-25 μg/mL) diminished impressively the antioxidant enzymes level to comparable levels of control sample in a dose-dependent manner.As for F2, a slight decrease was observed at 25 μg/mL.Moreover, it has been acknowledged that excessive levels of oxidants can also cause damage to lipids which is known as lipid peroxidation.Generally, this damage can be defined as an attack on lipids particularly polyunsaturated fatty acids by oxidants specifically hydroxyl radical (HO • ) 34 .The most mutagenic product of lipid peroxidation is malondialdehyde (MDA) which has been commonly used as biomarker for lipid peroxidation due to its reaction with thiobarbituric acid 35 .In our study, lipid peroxidation has been evaluated using the level of TBA-MDA adduct.Results in Fig. 5A showed a higher level (1.28±0.17nmol/mg of protein; P < 0.001) in cells exposed to H 2 O 2 compared to the control sample 0.76±0.07nmol/mg of protein which is 1.68-fold.This peak in the level of MDA is a consequence of an increase in free radicals.In contrast, we observed a significant decrease of the level of MDA in cells pretreated with pure carvacrol in a dose dependent manner compared to treatment with the other products.At 25 μg/mL, levels of MDA were 0.81±0.07and 0.94±0.03nmol/mg respectively for F1 and F2.In correlation with earlier studies, the observed results may be explained by the antioxidant effect of carvacrol and its important role in reducing lipid peroxidation by suppressing its reactions and so inhibiting the generation products like MDA 36,37 .
Found in the mitochondria, SDH is an enzyme of the respiratory chain that catalyzes the oxidation of succinate to fumarate as well as ubiquinone to ubiquinol.It is used as a metabolic marker for the oxidative stress evaluation 38 .In the present study, the evaluation of the effect of oxidative stress induced by H 2 O 2 on the SDH activity showed (Fig. 5B) a significant decrease to (0.61±0.05 μmol/ min/ mg of protein; P < 0.01) after the exposure to H 2 O 2 compared to the control sample (1.3 ± 0.17 μmol/ min/ mg of protein; P < 0.01).However, the treatment with carvacrol restored significantly in a dose dependent manner the activity of SDH to reach a value of (1.15±0.07μmol/ min/ mg of protein) close to the value in the control sample at 25 μg/mL.We also observed an increase in the activity at 25 μg/mL (0.90 ± 0.04 μmol/ min/ mg of protein) in cells pretreated with F2.As it has been reported, SDH has an important antioxidant role by controlling superoxides scavenging in the mitochondria 39 .Moreover, the restoration of the activity of SDH can be explained by the fact that it was protected against attack by free radicals using carvacrol which is a mono-terpene well known for its antioxidant properties and its important role in free radicals scavenging 40,41 .Whereof, TEO is a rich source of bioactive compounds mainly contains thymol, carvacrol, p-cymene and γ-terpinene 42 .Hence, high antioxidant activities have been disclosed for carvacrol and thymol in Thymus plants 43 .Thymol is an isomer of carvacrol and both are broadly used as flavourings in food industry and are considered safe for consumption by the European Commission 44 .In addition, carvacrol has been used as food additive to inhibit bacterial growth and contamination thanks to its antimicrobial activity and pleasant aroma.Furthermore, it has been reported that these compounds can act as free radical scavengers and play a key role in preventing chronic diseases by retarding the oxidative degradation caused by an excessive amount of ROS 7 .In general, the importance of this EO is attributed to its main constituents notably thymol and carvacrol which have been shown to exhibit a broad variety of properties and uses and significantly contribute to the antioxidant and antimicrobial 45 as well as anti-inflammatory properties of this oil 46 .These considerations warrant the introduction of carvacrol and species with high carvacrol content as natural antioxidants for beneficial applications in human health.

Conclusion
The present investigation confirms the main contribution of carvacrol which is a monoterpene phenol in the high antioxidant effect of the analyzed Thymus vulgaris essential oil.Hence, the treatment with carvacrol decreased lipid peroxidation, ameliorated cell viability and levels of antioxidant enzymes and metabolic enzyme were brought to levels comparable to control sample.In vitro, carvacrol has been found to act as an efficient DPPH • and ABTS •+ free radicals' scavenger and has also shown a high reducing power.Thus, the antioxidant potential of carvacrol is associated to the existence of the hydroxyl group (OH) linked to aromatic ring.These findings suggest that the antioxidant property exhibited by carvacrol makes it a great natural alternative to synthetic antioxidants and a pharmacological agent that may be efficient against oxidative damage caused by chronic stress.

Figure 3 .
Figure 3.The in vitro antioxidant activity of purified CAR and other components of TEO by (A) DPPH free radicals scavenging (B) ABTS free radicals scavenging and (C) Reducing power.Values are means ± SD of three independent experiments performed in triplicate