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J Food Sci Technol. 2016 April; 53(4): 1957–1965.
Published online 2015 December 23. doi:  10.1007/s13197-015-2144-1
PMCID: PMC4926906

Characterization of aroma-active and phenolic profiles of wild thyme (Thymus serpyllum) by GC-MS-Olfactometry and LC-ESI-MS/MS

Abstract

The present study was designed to characterize the volatile, aroma-active and phenolic compounds of wild thyme. Volatile components of T. serpyllum were extracted by use of the purge and trap technique with dichloromethane and analyzed by gas chromatography–mass spectrometry (GC-MS). The extraction method gave highly representative aromatic extract of the studied sample based on the sensory analysis. A total of 24 compounds were identified and quantified in Thymus serpyllum. Terpenes were qualitatively and quantitatively the most dominant volatiles in the sample. Aroma extract dilution analysis (AEDA) was used for the first time for the determination of aroma-active compounds of Thymus serpyllum. In total, 12 aroma-active compounds were detected in the aromatic extract by GC-MS-Olfactometry and terpenes were the most abundant compounds. High-performance liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was used for the phenolic compounds analysis. 18 phenolic compounds were identified and quantified in the T. serpyllum. Luteolin 7-O-glucoside, luteolin and rosmarinic acid were the most abundant phenolics in this herb.

Keywords: Wild thyme, Thymus serpyllum, Volatile, Aroma-active, LC-MS-MS, Phenolics

Introduction

Medicinal and aromatic plants (MAPs) have been used in folk medicine for as long as human being have existed in the world. 70.000 species are estimated to have been used in folk medicine in the world. World Health Organization (WHO) has listed about 20.000 species in the world that yield drugs and over 3.000 botanical raw materials in global commerce. Turkey is very rich in MAPs and one of the biggest exporters in the world. A total of 347 native taxa and 28.000 tons of MAPs that are exported annually in Turkey (Farnsworth and Soejarto 1991; Satil et al. 2008; Cindric et al. 2013).

Species belonging to the Lamiaceae family are perennial herbaceous MAPs obtained from Mediterranean region. Thymus, a member of Lamiaceae family, is polymorphic comprising 60 taxa of which 39 species grow in Turkey. The genus Thymus has been credited with a long list of pharmacological properties, such as antiseptic antitussive, and expectorant (Schulz et al. 2005). Herbal parts of Thymus species are aromatic plants known as “kekik” in Turkey. Also, Thymus serpyllum is a well-known MAP and wildly distributed in the mountainous region of Central Anatolia of Turkey.

Volatile compounds are one of the most important attributes of Thymus species, and can greatly influence the consumers’ acceptance of the thymus and related food products. Wild thyme (Thymus serpyllum) is regarded as a source of volatiles containing sesquiterpenes, alcohols, aldehydes, ketones, esters and acids (Raal et al. 2004; Lee et al. 2005). Many studies on the composition of volatiles from different Thymus species have been carried out (Venskutonis 1997; Condurso et al. 2013; Martins et al. 2015). The analytical studies performed on Thymus show the main volatiles are thymol, carvacrol, p-cymene, β-pinene, γ-terpinene, β-caryophyllene, 1-borneol, 1,8-cineole (Hudaib and Aburjai 2007; Safaei-Ghomi et al. 2009). Previously published data show remarkable differences in the volatile composition of Thymus serpyllum in different countries. Thymus serpyllum accumulates high amounts of thymol including 42.63 %, (in Pakistan), 64.6 % (in India), and 81.5 % (in Armenia) (Puri et al. 1985; Sattar et al. 1991; Loziene and Venskutonis 2002; 2006). Contrary to these countries, thymol and carvacrol were not detected in the Thymus serpyllum from Lithuania, Sweden, Finland and Poland (Loziene and Venskutonis 2002, 2006).

Up to now, more than 90 volatile compounds have been identified in different thyme samples (Condurso et al. 2013). But only a small fraction of this large number of volatiles in thyme actually contributes to the overall aroma. The technology of gas chromatography- olfactometry (GC–O) made it possible to divide identified volatiles into odour-active and non-odour-active compounds with regard to their existing concentration in the studied sample (Kesen et al. 2014). Additionally, the relative aroma intensity of each component can be determined by aroma extract dilution analysis (AEDA), which involves GC-O evaluation of a serial dilution series of an aromatic extract.

MAPs are known to be a rich source of phenolic compounds. Among the Lamiaceae species oregano, (Origanum vulgare L.), thyme (Thymus vulgaris L.), wild thyme (Thymus serpyllum L.) have been studied for their high content of phenolic compounds (Takacsova et al. 1995; Vichi et al. 2001). Nonetheless, LC-MS-MS analyses on these herbs were limited in the literature. Kulisic et al. (2006) reported the presence of eight phenolic compounds in the extract of wild thyme. Within the identified phenolics, rosmarinic acid was the main compound and followed by eriocitrin, luteolin-7-O-glucoside, apigenin-7-O-glucoside, quercetin, luteolin, apigenin, respectively.

No work has yet been published in this field to characterize the odour-active compounds of Thymus serpyllum. In the present paper, the aim was to investigate volatile and aroma-active compounds of Thymus serpyllum produced in the Central Anatolia region of Turkey. Sensory evaluation of the aromatic extract obtained by purge and trap was done to assess its representativeness, before the GC-MS olfactometric analysis. Furthermore, there is no information available about the phenolic composition using LC-MS-MS of the studied sample. This study was also undertaken to evaluate phenolic composition of Thymus serpyllum.

Materials and methods

Sample and chemicals

Sample

The whole-dries Thymus serpyllum was used as plant material and collected from the mountain region of the Kayseri province of Central Anatolia. The sample was gathered before full flowering stage in June of 2014. The plant sample was dried at room temperature (20-25 °C) and used for analysis.

Chemicals

Water used in this study was purified by a Millipore-Q system (Millipore Corp., Saint-Quentin, France). The reference volatile compounds were obtained from the following sources: α-pinene, camphene, 3-penten-2-ol, myrcene, delta-2-carene, limonene, delta-3-carene, γ-terpinene, p-cymene, acetic acid, linalool, terpinen-4-ol, isovaleric acid, 1-borneol, carvone, heptanoic acid, thymol, carvacrol, hexadecanoic acid, p-cymene-2,5-diol were purchased from Sigma-Aldrich (Steinheim, Germany). Standards of gallic acid, protocatechuic acid, caffeic acid, chlorogenic acid, naringin, rutin, rosmarinic acid and luteolin were purchased from Sigma-Aldrich (St Louis, MO, USA). Dichloromethane, sodium sulfate and 4-nonanol were obtained from Merck (Darmstad, Germany). Dichloromethane was freshly distilled prior to use.

Representativeness test for aromatic extract

Sample preparation and presentation

Wild thyme was evaluated using the descriptive and preference tests according to Poste et al. (1991). The panel was composed of ten assessors (seven females and three males between 20 and 45 years old) from Cukurova University, Food Engineering Department. The assessors were previously trained in sensory evaluation techniques. In the present study, we used a cardboard smelling strip (reference 7140 BPSI, Granger-Veyron, Lyas, France) for the checking representativeness of the wild thyme aromatic extract obtained purge and trap extraction technique. Three grams of fined samples were placed in 15 mL brown coded flask as a reference for representativeness tests. Aromatic extract obtained from dichloromethane was adsorbed onto a cardboard smelling strip. After 1 min (the time necessary for solvent evaporation) the extremities of the strips were cut off, then placed in dark coded flasks (15 mL) and presented to the panel after 15 min. All the samples were assessed at room temperature (20 °C) in neutral conditions.

Similarity and intensity tests

Similarity and intensity tests were performed to demonstrate the closeness between the odour of the extracts and the wild thyme. These test procedures were well documented in our previous studies (Selli et al. 2014).

Analysis of volatile compounds

Extraction of volatile compounds

The purge and trap system was used for the extraction process consists in a source of nitrogen controlled by a flow-meter. The needle of the source of N2 and the cartridge were installed through the septum to purge and trap the aroma compounds. As adsorbent, 200 mg of Lichrolut EN resins from Merck was chosen as the most suitable material for aroma compounds retention according to the literature (Rodriguez-Bencomo et al. 2015). The temperature of the vial sample was controlled by a thermostated. 3 g sample transferred into 20 mL vial than, the sample was pre-incubated at the extraction temperature for 10 min. The purge and trap process was carried out for 90 min with a nitrogen flow of 500 mL/min. After purging process, the compounds retained in the cartridge were eluted with dichloromethane. After dehydration by anhydrous sodium sulphate, the pooled organic extract was reduced to 5 mL in a Kuderna Danish concentrator fitted with a Snyder column at 40 °C (Supelco, St Quentin, France) and then to 0.5 mL under a gentle stream of nitrogen. Extracts were then stored at −20 °C in a glass vial equipped with a Teflon-lined cap before analysis. Each sample was extracted in triplicate and the concentration of volatiles was expressed as 4-nonanol equivalent.

GC-FID, GC-MS and GC-O analysis of volatile compounds

The gas chromatography (GC) system was consisted of Agilent 6890 chromatograph equipped with flame ionization detector (FID), Agilent 5973-Network-mass selective detector (MSD) (Wilmington, USA) and a Gerstel ODP-2 (Linthicum, MD, USA) sniffing port. Volatile compounds were separated on DB-Wax column (30 m length × 0.25 mm i.d. × 0.5 μm thickness, J&W Scientific Folsom, USA). 3 μL of extract was injected in pulsed splitless (40 psi; 0.5 min) mode. Injector and FID detectors were set at 270 °C and 280 °C, respectively. The flow rate of carrier gas (helium) was 1.5 mL min−1. The oven temperature of the DB-Wax column was increased from 50° to 250 °C at a rate of 4 °C/min with a final hold at 250 °C for 10 min.

The same oven temperature programs were used for the mass-selective detector. The MS (electronic impact ionization) conditions were as follows: ionization energy of 70 eV, mass range m/z of 30–300 a.m.u., scan rate of 2.0 scan s−1, interface temperature of 250 °C, and source temperature of 180 °C. The volatile compounds were identified by comparing their retention index and their mass spectra on the DB-Wax column with those of a commercial spectra database (Wiley 6, NBS 75 k) and of the instrument’s internal library created from the previous laboratory studies. Some of the identifications were confirmed by the injection of the chemical standards into the GC-MS system. Retention indices of the compounds were calculated by using an n-alkane series.

Aroma extract dilution analysis (AEDA)

The aroma extract was analyzed by GS-MS-O using two experienced sniffers. For the AEDA, the concentrated aromatic extract (200 μL) of Thymus serpyllum was stepwise diluted 1:1 using dichloromethane as the solvent to obtain dilutions of 1:1, 1:2, 1:4 and up to 1:1024 of the extract. The odour-active compounds perceived by the panelists were recorded when sniffing the effluent from the sniffing port. Sniffing of dilutions was continued until no odorant could be detected by GC-MS-O. Each odorant was thus assigned a flavour dilution factor (FD factor) representing the last dilution in which the odorants was still detectable. FD factor of aroma compounds increases, also the degree of odour activity increases (Selli et al. 2014).

Analysis of phenolic compounds

Extraction of phenolic

compounds The extraction of phenolic compounds was carried out according to Martins et al. (2015) after some modifications. Thymus serpyllum sample was grinded with laboratory blender and 5 g samples were prepared in duplicate. Sample was extracted with 20 ml of 75 % methanol using magnetic stir bar for 2 h. The extract was separated and the solid was washed with 5 mL of 75 % methanol. The two extracts were combined, filtered and evaporated at vacuum at 20°. Then, extract was filtered through a 0.45-μm pore size membrane filter before injection.

LC-DAD-ESI-MS/MS analysis

An Agilent 1100 HPLC system (Agilent Technologies, Palo Alto CA-USA) operated by Windows NT based ChemStation software was used. The HPLC equipment was used with a diode array detector (DAD). System consisted of a binary pump, degasser and auto sampler. The column used was a Phenomenex reversed-phase C-18 column (4.6 mm × 250 mm, 5 μm) (Torrance, CA, USA). The mobile phase consisted of two solvents: Solvent A, water/formic acid (99:1; v/v) and Solvent B, acetonitrile/solvent A (60:40; v/v). Phenolic compounds were eluted under the following conditions: 0.7 mL min−1 flow rate and the temperature was set at 25 °C, isocratic conditions from 0 to 10 min with 0 % B, gradient conditions from 0 to 5 % B in 30 min, from 5 to 15 % B in 18 min, from 15 to 25 % B in 14 min, from 25 to 50 % B in 31 min, from 50 to 100 % B in 3 min, followed by washing and reconditioning the column. The DAD was set at 280, 320, and 360 nm for real-time monitoring of the peak intensity, and full spectra (190–650 nm) were continuously recorded for thyme component identification. The identification and assignation of each compounds was performed by comparing their retention times and UV spectra to authentic standards and also confirmed by an Agilent 6430 LC-MS/MS spectrometer equipped with an electrospray ionization source. The electrospray ionization mass spectrometry detection was performed in negative mode with the following optimized parameters: capillary temperature 400 °C; drying gas N2 12 L/min; nebulizer pressure, 45 psi (Fecka and Turek 2008; Cui et al. 2015). Data gaining was performed using multiple reactions monitoring (MRM) method that only monitors specific mass transitions during preset retention times. Dynamic MRM also has the ability to simultaneously perform ESI in both positive and negative mode, allowing for the analysis of different phenolic classes within a single chromatographic run.

Results and discussion

Intensity and similarity evaluation

Intensity and similarity evaluation tests have great importance in evaluating the representativeness of the aromatic extracts (Selli et al. 2008). The similarity scores of the aromatic extract obtained by purge and trap was found to be 75.1 mm on a 100 mm unstructured scale. The similarity of the extract was found to be an acceptable level. When we compared it with the other studies, the similarity of the cherry tomato was found to be 70.4 by Selli et al. (2014), for olive oil extract 75.7 by Kesen et al. (2014). Regarding the intensity score of the aromatic extract, it was found to be 72.3 mm on a 100 mm unstructured scale. This score is also high and acceptable. Briefly, the similarity and intensity results indicated that the representative aromatic extract was achieved with the purge and trap method in order to determine the wild thyme volatile and aroma-active compounds.

Volatile composition of wild thyme

Table Table11 shows the volatile compounds, their retention indices and mean values (mg 100 g−1) with standard deviations in wild thyme. A total of 24 volatile compounds were identified in the sample (Fig. 1). Most volatiles detected in this study were consistent with those of previously published studies (Inouye et al. 2001; Paaver et al. 2008). The main chemical group of the volatile compounds in the wild thyme sample was terpenes. These compounds represented over 90 % of the total volatiles in wild thyme. Other chemical groups such as alcohols and acids were present in minor quantities. Safaei-Ghomi et al. (2009) reported that the leaf essential oil of wild thyme was largely made up of oxygenated monoterpenoids, sesquiterpene and monoterpen hydrocarbons. Thymol and carvacrol are the main components of essential oil of wild thyme. Thymol and carvacrol are of special commercial interest as the preservation of food because of their high potency as antibacterials and antifungal agents (Schulz et al. 2005). Thymol was found in essential oil of wild thyme from Japan 35 %, Pakistan 42.6 %, India 64.6 %, and 81.5 % Armenia (Puri et al. 1985; Sattar et al. 1991; Loziene and Venskutonis 2002, 2006). However, the content of carvacrol is usually lower than the thymol content (Paaver et al. 2008). The obtained results revealed that the most important compounds were thymol (1702 mg 100 g−1) and carvacrol (179 mg 100 g−1) in the studied sample. These volatiles were distinctive for T. serpyllum species and found as major compounds in other previous results, as well (Inouye et al. 2001; Saez 2001). Nonetheless, the content of the volatiles in T. serpyllum depends on different factors, cultivation conditions such as geographical origin, climatic conditions, soil, harvesting time etc. (Banaeva et al. 1998). Loziene and Venskutonis (2002, 2006) reported, T. serpyllum growing the northern climatic area do not accumulate the thymol and carvacrol in more remarkable amount.

Table 1
Volatile composition of wild thyme (Thymus serpyllum)
Fig. 1
The GC-MS chromatogram of identified volatile compounds of Thymus serpyllum (Peak numbers refer to Table Table11)

γ-Terpinene and p-cymene were the other main compounds which detected in wild thyme. p-Cymene was previously found as precursor of the biosynthesis of thymol and carvacrol in thyme oil. The amount of these compounds was quantified 90.4 mg 100 g−1 and 88.8 mg 100 g−1 in studied sample, respectively. In the literature, high concentrations of γ-terpinene and p-cymene were detected in essential oils of wild thyme herbs both from Latvia and Estonia (Raal et al. 2004). The other terpenes in our sample were previously identified in Thymus serpyllum and various thyme species (Saez 2001; Raal et al. 2004; Schulz et al. 2005; Safaei-Ghomi et al. 2009).

Aroma-active compounds of wild thyme

The results of the olfactometric analysis were given in Table Table2.2. The potent odorants were determined using AEDA for the determination of flavour dilution (FD) factor. Application of the ADEA on wild thyme extract revealed 12 aroma-active compounds by using purge and trap method. The FD factors of the compounds range from 16 to 1024 including, terpenes (9), acid (1) and unknown (2) compounds. Unknown odorants were detected by GC-MS-O, but could not be identified by GC-MS. The aroma-active compounds of wild thyme were dominated by terpenes. The compounds which have the highest FD factors were thymol (FD = 1024), sabinene hydrate (512), delta-3-carene (FD = 256), carvone (FD = 128), γ- terpinene (FD = 128), and myrcene (FD = 128).

Table 2
Aroma-active compounds of wild thyme (Thymus serpyllum)

Terpenes were the major aroma-active components of wild thyme. Nine terpenes were identified in the extract as aroma active-compounds (Table (Table2).2). These terpenes are extensive, as they already have been found in other varieties of Thymus species (Diaz-Maroto et al. 2005; Goodner et al. 2006). Among the terpenes, thymol (FD = 1024) was the most powerful aroma active compound to contribute to the aroma profile of the wild thyme providing the characteristic thyme odour, followed by sabinene hydrate (herbal) and delta-3-carene (mint). Thymol is part of a naturally occurring class of compounds known as biocides, and the threshold of it is 50 μg L−1 in aqueous solution (Diaz-Maroto et al. 2005). This study revealed that this compound is the main aroma active compound of Thymus serpyllum due to its highest concentration in the herb. Our present study supports earlier result on thyme that confirm thymol is most important contributor to thyme aroma profile (Diaz-Maroto et al. 2005; Goodner et al. 2006). The other aroma-active terpene in terms of the FD factor was sabinene hydrate. The FD factor of this compound was 512. The above mentioned compound has been detected as an important contributor to the thyme odour by Diaz-Maroto et al. (2005). Also, this compound was detected as one of the most abundant components of the Thymus species by many researchers (Rasooli and Mirmostafa 2002; Raal et al. 2004; Schulz et al. 2005; Safaei-Ghomi et al. 2009). Among the terpenes, α-pinene (green, woody), camphene (woody), myrcene (green), delta 3-carene (mint), γ- terpinene (citrus), terpinene-4-ol (vegetable), carvone (flowery) were the following aroma-active compounds which have the lowest FD factors detected by GS-MS-O. However, α-pinene, camphene, myrcene, delta-3-carene, γ-terpinene and terpinene-4-ol have been previously identified as aroma-active compounds in Thymus cultivars in previously data on the species (Diaz-Maroto et al. 2005; Goodner et al. 2006).

Two unknown compounds may contribute to general aroma of wild thyme. Unknown 1 (LRI = 1014), with herbal note and the FD factor of this compound was 32. The unknown 2 (LRI = 1702) was determined in wild thyme as providing high flowery not with a FD = 256.

Phenolic composition of wild thyme

The phenolic profile of T. serpyllum, obtained after hydro alcoholic extraction and mass spectral data for compounds identified in negative ionization mode were listed in Table Table3.3. A total of 18 phenolic compounds were identified, eight of which were phenolic acids, ten flavan-3-ols. As for the phenolic compounds, 10 of which (gallic acid, protocatechuic acid, protocatechuic acid-hexoside, apigenin 6,8-di-C-glucoside, naringin, luteolin-O-diglucuronide, kaempferol O-glucuronide, rosmarinic acid-glucoside, apigenin O-glucuronide and methyl kaempferol O-rutinoside) were identified for the first time in T. serpyllum. LC-ESI-MS/MS chromatogram of phenolic compounds extracted from T. serpyllum was given in Fig. 2.

Table 3
Retention time, mass spectral data and identification of phenolic compounds of wild thyme (Thymus serpyllum)
Fig. 2
LC-DAD and LC-ESI-MS/MS MRM chromatograms of the identified phenolic compounds of Thymus serpyllum

Among phenolic acids, gallic acid (peak 1), protocatechuic acid (peak2), caffeic acid (peak 5), chlorogenic acid (peak 6), and rosmarinic acid (peak 14) were identified according to their retention, mass and UV–vis characteristics by comparison with commercial standards and LC-ESI-MS/MS analysis. The presence of chlorogenic acid was previously reported in T. serpyllum by Fecka and Turek (2008) and Boros et al. (2010) also caffeic acid was identified in tea infusions and leaves in T. serpyllum (Kulisic et al. 2006; Fecka and Turek 2008; Boros et al. 2010; Miron et al. 2011). However, gallic acid and protocatechuic acid compounds were previously identified in T. vulgaris (Proestos et al. 2005; Miron et al. 2011; Roby et al. 2013; Martins et al. 2015). Peak 3 ([M-H] at m/z 315) presented characteristic UV and MS spectra consistent with a protocatechuic acid-hexoside. This compound was described in plants, such as those that are member of the Prunus genus, Salvia officinalis (Zimmermann et al. 2011) but never before in T. serpyllum. Peak 8 ([M-H] at m/z 193, and MS2 fragment ions at m/z 134, 117) could be assigned as ferulic acid. This compound was reported in wild thyme by Boros et al. (2010). Rosmarinic acid-glucoside (Peak 12; [M-H] at m/z 521) was another important phenolic acid in wild thyme. This compound is probably the most abundant soluble bound phenolic acid, and is a type of non-flavonoid catecholic compound, which is present in many plants (Nagy et al. 2011; Martins et al. 2015). In general, rosmarinic acid was the most abundant phenolic acid found in the present study; this compound is well known by its potent antioxidant activity, radical-scavenging and antiviral properties. Besides, it takes part in inhibit gonadotrophin release, xanthine oxidase and adenylate cyclase activities. Fecka and Turek (2008) and Berdowska et al. (2013) studied a sample of T. serpyllum from Poland, after hot aqueous extraction from the examined sample. Both identified and quantified rosmarinic acid as major compound detected.

The other phenolic compounds identified in the analyzed sample belong to the groups of flavones, flavonols and flavanones. Apigenin 6,8-di-C-glucoside (peak 4) was identified as a [M-H] deprotonated molecule (m/z 593) yielded ion fragments at m/z 503. Apigenin 6,8-di-C-glucoside was previously reported in T. vulgaris by Vergara-Salinas et al. (2012) Furthermore, luteolin-O-diglucuronide ([M-H] at m/z 637), methyl kaempferol O-rutinoside ([M-H] at m/z 607) and kaempferol-O-glucuronide ([M-H] at m/z 461) were tentatively identified in T. vulgaris by Martins et al. (2015). Peak 11 presented a pseudo molecular ion [M-H] at m/z 461, together with the characteristic fragment ions at m/z 285. This compound was assigned to luteolin 7-O-glucuronide owing to the identification of that compound in leaves from T. vulgaris by Dapkevicius et al. (2002) and Vergara-Salinas et al. (2012), wild thyme by Miron et al. (2011).

Luteolin 7-O-glucoside (peak 13) and luteolin (peak 18) were the most abundant phenolic compound detected in the sample. Luteolin 7-O-glucoside according to its mass spectrum, shows the pseudo molecular ion [M-H] at m/z 447, and characteristic fragment ions at m/z 285. Luteolin 7-O-glucoside was among the functional compounds in thyme that suppress oxidative stress. Moreover this compound was identified in thyme infusions (Kulisic et al. 2006); leaves from T. vulgaris (Dapkevicius et al. 2002; Vergara-Salinas et al. 2012); and T. serpyllum (Miron et al. 2011). Luteolin was identified as a [M-H] deprotonated molecule (m/z 285) yielded ion fragments at m/z 175 and 199. This compound has high radical scavenging ability. It was also largely identified in different thyme varieties and obtained by different extraction methodologies (Dapkevicius et al. 2002; Kulisic et al. 2006; Vergara-Salinas et al. 2012). Besides, Miron et al. (2011) reported that luteolin was the main identified compound in the wild thyme ethanolic extract.

In the literature, it is reported that variations in the phenolic profiles of Thymus species depend on cultivars, place of origin, agronomic techniques and harvesting processing. On the other hand, the use of wild thyme a dietary supplement, could support the health benefits, both in the treatment of diseases related to reactive species production and oxidative stress and against bacterial infections (Dapkevicius et al. 2002; Martins et al. 2015).

Conclusions

A total of 18 phenolic compounds were identified and quantified in the samples of which 10 compounds (peak 1, 2, 3, 4, 7, 9, 12, 15, 16 and 17) have not been reported previously in T. serpyllum. The phenolic profiling of T. serpyllum and specially the confirmation of the identity by LC-ESI-MS/MS of the three major phenolics in the herb (peak 13, 14 and 18) that could be used as chemical markers. With regard to the volatile profile, a total of 24 aroma compounds have been identified and quantified in the aromatic extract of T. serpyllum. Also, aroma extract dilution analysis (AEDA) was used for the first time for the determination of aroma-active compounds of Thymus serpyllum. In total 12 compounds were detected as aroma-active compounds. Within these thymol (FD = 1024), sabinene hydrate (FD = 512) and delta 3-carene (FD = 256) were the most powerful contributors to the aroma of the T. serpyllum.

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