Beyond Thymol and Carvacrol: Characterizing the Phenolic Profiles and Antioxidant Capacity of Portuguese Oregano and Thyme for Food Applications

Abstract

Featured Application

This research underscores the potential of Portuguese oregano and thyme accessions as rich sources of diverse phenolic compounds, particularly flavonoids and phenolic acids, which have been less studied compared to the dominant monoterpenes thymol and carvacrol. These compounds contribute significantly to the antioxidant properties of the plants, offering potential applications in the food industry. By incorporating these unique phenolic profiles into food formulations or active packaging, the antioxidant benefits can enhance the food shelf life, improve the nutritional value, and support health and well-being. This makes these accessions valuable for developing innovative, healthier food products.

Abstract

Oregano (Origanum vulgare L.) and thyme (Thymus mastichina L.) are popular aromatic plants used in the culinary sphere and are known for their antioxidant capacity. This study investigates the variability in the antioxidant capacity and phenolic profiles, focusing on flavonoids and phenolic acids, of oregano and thyme accessions from the Portuguese Bank of Plant Germplasm. The antioxidant capacity was assessed by measurements using the ferric-ion-reducing antioxidant power and DPPH radical scavenging activity assays, Total Phenolic Content, and individual phenolic compounds. Oregano extracts presented the highest total phenolic content (~2152 mg GAE/g DW), while thyme extracts showed the highest ferric-reducing antioxidant powers. However, both aromatic plants (~0.58 mmol TE/g DW) presented similar DPPH radical scavenging capacities. Rosmarinic acid was the major phenolic compound in methanolic extracts from oregano and thyme, but higher amounts were found in thyme (23.11 to 40.45 mg/g DW). The accessions of oregano and thyme showed statistical differences in the phenolic content and antioxidant capacity. This study provides valuable insights into the phenolic profile of Portuguese oregano and thyme, which could constitute potential sources of natural antioxidants and bioactive compounds, to be used by the food industry as natural food additives.

1. Introduction

Since ancient times, aromatic plants from the Lamiaceae family have been commonly used in the Mediterranean cuisine [1]. Oregano, thyme, rosemary, basil, peppermint, and sage are examples of these aromatic herbs used to impart flavor to food. The addition of these herbs to foods makes it possible to reduce salt consumption, making them even more essential for a healthy diet [2].

One of the most used aromatic plants is Origanum vulgare L. or oregano. The leaves are used fresh or dried for flavoring food such as meat, sausages, pizza, and salads [3]. This attribute is linked to the aromatic components of the essential oil, mainly composed of thymol and its isomer carvacrol [4].

Thyme (Thymus vulgaris) is a relative of the oregano genus, traditionally used in a fresh or dry form to season traditional dishes and aromatize olive oil [2]. Several species of thyme are known, but Thymus mastichina L. is endemic to the Iberian Peninsula and commonly known as “Bela-Luz” [3]. Thymol is also the principal active ingredient of oil extracted from Thymus species [4].

Besides their flavoring properties, aromatic plants provide health benefits, anti-inflammatory, antidiabetic, and anticancer effects, with cardio, hepatic, and neuroprotective actions [5,6]. These biological activities are mostly related to the presence of a high content of polyphenols with higher antioxidant activity in these aromatic herbs.

Furthermore, in recent years, the food industry has made efforts regarding the replacement of synthetic additives with natural alternatives, due to safety concerns about some food additives (such as butylated hydroxyanisole and butylated hydroxytoluene [7]). Additionally, consumers are concerned about food and health and prefer more natural foods [8]. Thus, oregano, thyme, and other aromatic herbs have a high potential to be used as a source of natural antioxidants. Moreover, several studies have used oregano and thyme to preserve food products, demonstrating their antioxidant and antimicrobial potential [9,10,11,12].

Although thymol and carvacrol are well-documented as the primary bioactive constituents of oregano and thyme, the roles of other phenolic compounds, such as flavonoids and phenolic acids, have been comparatively underexplored. These compounds also play critical roles in the overall antioxidant capacity and bioactive potential of these herbs. This study aims to address this gap by evaluating the antioxidant potential of methanolic extracts from Portuguese accessions of Origanum vulgare subsp. virens (Hoffmanns. and Link) Bonnier and Layens and Thymus mastichina L. conserved by the Portuguese Plant Germplasm Bank (BPVG). Moreover, to better understand the influence of different species, edaphoclimatic conditions, agronomic practices, and pests’ occurrences on biological activities, this study determines lesser-studied phenolic compounds, specifically flavonoids and phenolic acids, across different accessions. Furthermore, assessing their biological activity can provide valuable information for their incorporation into new food formulations or packaging solutions, with the aim of enhancing the nutritional value and/or extending the shelf life. They support well-being which makes them valuable ingredients for developing healthier food products.

2. Materials and Methods

2.1. Plant Material

A collection of 8 accessions of Origanum vulgare L. subsp. virens (Table 1) and 8 accessions of Thymus mastichina L. subsp. donyanae R. Morales (Table 2), progeny of seeds gathered in their natural habitats, were harvested at the beginning of the flowering period, during April and June of 2022. The initial stage of flowering is characterized by the appearance of 20 to 30% of flowers. During this phenological phase, the plants exhibit a dry matter content (DM) ranging from 20 to 25%, indicating a moisture content of approximately 80%.

These accessions were chosen due to the lack of data on their antioxidant capacity. The 16 accessions were conserved in the Portuguese Bank of Plant Germplasm—BPGV (Braga, Portugal)—and collected in different regions of Portugal (Figure 1).

2.2. Chemicals and Reagents

Methanol, ethanol, and acetonitrile were obtained from Merck (Darmstadt, Germany). Gallic acid, (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocâlteu reagent, and formic acid were purchased to Sigma (Sternheim, Germany). The phenolic compounds standards were purchased from Sigma and Extrasynthese (Lyon, France). 2,4,6-tripyridyl-S-triazine (TPTZ), ferric chloride, and sodium acetate were supplied by Fluka (Buchs, Germany). Iron sulfate and sodium carbonate were supplied by Panreac (Barcelona, Spain) and BDH (Poole, UK), respectively.

2.3. Preparation of Plant Extracts

Oregano and thyme extracts were prepared from freeze-dried material (Scanvac Cool Safe, Labogene Scandinavian by Design). Extraction was conducted using methanol at a liquid-to-solid ratio of 20 mL/g. The preparation of methanolic extracts was conducted following the method described by Serrano et al. [15]. The assays were performed in duplicate.

2.4. Determination of Antioxidant Capacity

The antioxidant capacity was assessed spectrophotometrically, measuring the absorbance using a UV–visible spectrophotometer (double-beam; Hitachi U-2010, Cincinatti, IL, USA). Before evaluation of antioxidant properties, plant extracts (dry residue of extraction—DW) were diluted in methanol at 0.01 g DW/mL for oregano and 0.001 g DW/mL for thyme. All determinations were performed in triplicate.

2.4.1. Total Phenolic Content (TPC)

The Folin–Ciocâlteu method was used for the determination of Total Phenolic Content (TPC), following the method described by Asami et al. [16]. Briefly, 1 mL of each sample was mixed with 0.5 mL Folin–Ciocâlteu reagent and distilled water. After 5 min, 1.5 mL of a 200 g/L sodium carbonate solution was added. The mixture was then diluted to 10 mL with distilled water and allowed to react at room temperature (20 °C) in the dark for 120 min [17]. A control using distilled water was also prepared. The absorbance was measured at 750 nm. The TPC of each sample was determined based on the standard curve (y = 122.62x − 0.0263, r² = 0.9955), using gallic acid as the standard. The results were expressed as g of gallic acid equivalents (GAE) per g of plant dry residue extract.

2.4.2. DPPH Radical Scavenging Activity Assay

The DPPH radical (2,2-diphenyl-1-picryl-hydrazyl) assay evaluates the sample’s capacity to inhibit lipid oxidation by determining its free radical-scavenging capacity. The DPPH radical assay was assessed using the modified method of Kondo et al. [18]. The assay is based on the reaction of the antioxidants in the sample with the radical DPPH, reducing it to diphenylpicrylhydrazine, leading to the discoloration of the solution [18].

For the assay, 100 µL of each sample was added at different concentrations to 2 mL of 0.07 mmol/L of ethanolic solution of DPPH radical. The mixture was shaken and left to stand for 60 min at room temperature (20 °C) in the dark. The absorbance of samples was measured at 517 nm. The absorption of the assay using ethanol was read as a negative control. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as standard. The results were expressed as micromoles of Trolox equivalents per g of plant dry residue extract (μmol TE/g DW), based on the standard curve (y = 0.001 x + 0.0139, r² = 0.9992).

2.4.3. Ferric-Ion-Reducing Antioxidant Power Assay (FRAP)

Ferric-ion-reducing antioxidant power (FRAP) measures the formation of a blue-colored Fe²⁺—2,4,6-tripyridyl-S-triazine (TPTZ) compound from the colorless oxidized Fe³⁺ form by the action of electron-donating antioxidants [19].

The FRAP assay was carried out using a modified colorimetric methodology of Serrano et al. [13]. Briefly, 200 μL of the diluted extracts (in 500 g/L methanol) were mixed with 1.8 mL FRAP reagent prepared according to Serrano et al. [13]. The absorbance of the blue complex was monitored at 593 nm and determined against water (control). The results were expressed as μmol Fe²⁺ per g of plant dry residue extract (μmol Fe²⁺/g DW), extrapolated from a standard curve (y = 0.002x − 0.025, r² = 0.9980) previously prepared using different concentrations of iron sulfate.

2.5. Determination of Phenolic Compounds by UHPLC-ToF-MS

The detection and quantification of phenolic compounds were performed using a High-Performance Liquid Chromatography (Nexera X2 Shimadzu) coupled to Time-of-Flight Mass Spectrometry (SCIEX, Foster City, CA, USA) equipped with a Turbo Ion Spray electrospray ionization, working in positive mode (ESI+), following the method reported by Teixeira et al. [20]. MultiQuant™ 3.0 and PeakView™ 2.2 software were used to obtain retention time and isotope mass to confirm the identity of each phenolic compound, compared to high pure analytical standards of phenolic compounds, with purity higher, between 95 and 99%.

For this determination, the methanol extracts of oregano (at 0.01 g DW/mL) and thyme (at 0.001 g DW/mL) were diluted to half their concentration with water acidified with 0.2% of formic acid to be finally solubilized in methanol: water (1:1, v:v) and 0.1% of formic acid. The determinations were performed in duplicate.

2.6. Statistical Analysis

The statistical analysis of the data was analyzed with IBM^® SPSS^® Statistics, version 28.0.1.1. (Chicago, IL, USA). Significance was defined at p < 0.05. The one-way analysis of variance (ANOVA) was used to compare the means between k samples when the normality and homogeneity of variances were demonstrated. The Tukey test was applied to examine the disparities among average values. For comparison between two samples, t-test was applied to compare means when the normality and homogeneity of variances were validated. The results that did not meet assumptions of normality (Shapiro–Wilk tests), were compared using the non-parametric Kruskal–Wallis H test. Results concerning the statistical evaluation were expressed as mean value plus the standard deviation (SD) of replicates. In addition, Pearson correlation coefficients (r) were determined in order to determine a statistically significant positive or negative relationship between antioxidant capacity, TPC, and the individual phenolic compounds of the methanolic extracts.

3. Results and Discussion

3.1. Antioxidant Capacity

3.1.1. Oregano

All methanolic extracts from O. vulgare showed an antioxidant capacity (Table 3). The samples presented a similar scavenging ability in both the DPPH radical assay and FRAP assay, ranging between 0.4476 and 0.6029 mmol TE/g DW and 1354.86 and 1932.33 µmol Fe²⁺/g DW, respectively. Likewise, the oregano extracts showed comparable phenolic contents, ranging between 1639.71 mg GAE/g DW for BPGV10423 and 2660.86 mg GAE/g DW for BPGV11408. This similarity between samples could indicate that, despite the different locations and consequently different climatic conditions, the high antioxidant potential of oregano is ensured. Only two samples of oregano were statistically different. Oregano BPGV11408, collected in Santarém, showed the highest TPC and the highest antioxidant capacity, with a significant difference (p < 0.05), while the oregano from Portalegre (BPGV10423) showed a significantly lower TPC and lower ferric reducing antioxidant power (p < 0.05). These results could be related to temperature, since Santarém was the location with the highest average temperature during the months of April to June, showing that the oregano may have increased the production of the phenolic content as a response to heat-induced stress, since these compounds are involved in the defense mechanism towards different abiotic stresses [21,22]. Also, Portalegre and Bragança were the districts with a lower rainfall volume during this period and correspond to the samples with a lower antioxidant capacity in FRAP and DPPH radical assays (BPVG10423 and BPVG11267). This may indicate that oregano under drought stress decreases the content of phenolic compounds. These findings are similar to those reported by Ancillotti et al. [22], which concluded that water stressed plants decrease the concentration of phenolics and antioxidant activity assessed by the DPPH radical assay.

Considering BPGV10442, the multiplication of the original BPGV10442 did not influence the antioxidant capacity or TPC of the oregano extract (p > 0.05).

Compared with other studies, our oregano samples showed the highest TPC. For example, Khorsand et al. [23] reported a lower amount of TPC (ranging from 20.3 to 35.5 mg GAE/g DW) in seven accessions of oregano belonging to three subspecies (virens, vulgare, and gracile) collected in Iran. A higher TPC was found by Yan et al. [24] (ranging from 79.5 mg to 147 mg GAE/g DW) in 42 accessions of O. vulgare from Germany National GeneBank, but below our TPC. More recently, on a Greek Island, the oregano collected by Michalaki et al. [25] showed a higher TPC (362.1 mg GAE/g DW) using Ultrasound-Assisted Extraction, but it was not comparable to our results due to a different extraction method. These unrelated results may suggest that several factors, such as extraction methods, environmental parameters, and post-harvesting conditions, may affect plant metabolism and, consequently, the expression of phenolic compounds [26,27].

Previously, Castilho et al. [28] reported a lower antioxidant capacity of O. virens from Madeira Island (0.0011 mmol TE/g DW by a DPPH radical assay). This might be related to the use of n-hexane as the extraction solvent and the use of activated carbon to remove chlorophylls, which might have co-extracted other compounds with antioxidant activity.

3.1.2. Thyme

All methanolic extracts from T. mastichina showed an antioxidant capacity by DPPH radical scavenging and Ferric-reducing antioxidant power assays (Table 4).

The samples presented a similar scavenging ability in the DPPH radical assay, ranging between 0.4812 and 0.7205 mmol TE/g DW. No statistical differences (p > 0.05) were found between the sub-samples, except for accessions BPGV10384 and BPGV12078. In BPGV10384, the chalk-green foliage (T2.2) subsample presented the highest DPPH radical scavenging ability (p < 0.05), while in BPGV12078, the subsample with larger leaves and revolute margin (T7.1) showed a lower scavenging ability when compared to the common morphotype.

In the FRAP, BPGV10381 presented a much lower ferric-reducing antioxidant power with a mean of 338.5 µmol Fe²⁺/g DW, while the other subsamples of seven types of thyme showed an antioxidant power ten times higher (mean ~3148 µmol Fe²⁺/g DW). One exception was found in BPGV10384, where the subsample with elliptical leaves and larger nodes (T2.1) showed a significantly (p < 0.05) lower antioxidant power when compared to the other subsamples, with 281.95 µmol Fe²⁺/g DW.

Comparing the subsamples with the common morphotype of T. mastichina, a significantly higher (p < 0.05) antioxidant capacity was found for BPGV11276, with 3990.88 µmol Fe²⁺/g DW, while the lower antioxidant capacity was found for BPGV11295 (2521.99 µmol Fe²⁺/g DW), though collected in Bragança.

The FRAP assay indicated intra-variability among subsamples. For example, in BPGV12078, the subsample with larger leaves and a revolute margin (T7.1) presented significantly (p < 0.05) lower antioxidant power when compared with the common morphotype.

No statistical differences were found among subsamples of BPGV12093; however, the FRAP assay indicated that the subsample with a light green foliage color (T8.4) had a similarly high antioxidant power of the common morphotype (T8.5) when compared to the other subsamples, possibly indicating that differences in the morphotype of thyme could influence the antioxidant capacity.

Other authors evaluated the antioxidant capacity of thyme extracts using different assays. For example, Taghouti et al. [3] used the ABTS radical, hydroxyl radicals, and nitric oxide radical scavenging assays, where the extract of T. mastichina showed a high antioxidant capacity with 0.20 mmol TE/g DW in the ABTS radical assay.

Regarding the TPC, the T. mastichina methanolic extracts showed very similar phenolic contents, varying between 365.64 and 529.46 mg GAE/g DW, and not being statistically different (p > 0.05) among subsamples. However, two exceptions to this intra-variability were observed in BPGV10384 and BPGV11288. In BPGV10384, the subsample with chalk-green foliage presented a significantly higher TPC (p < 0.05) than the common morphotype and the other subsample with a reddish fruiting branch with elliptical leaves and larger nodes. In BPGV11288, the subsample with dark green foliage presented a significantly lower TPC (p < 0.05) compared to the common morphotype of T. mastichina. No statistical differences (p > 0.05) were found among the common morphotypes of each thyme accession.

However, a previous study with methanolic extracts from T. mastichina growing wild in Spain reported a lower TPC when compared to our results (ranging between 2.90 and 9.15 mg GAE/g DW) [29]. This difference might be due to the application of a preliminary extraction with a petroleum ether, probably resulting in the co-extraction of antioxidant compounds. Other studies reported the TPC with different reference compounds (reference phenolic), which makes the comparison among extracts of thyme difficult. For example, Taghouti et al. [30] reported that the hydroethanolic extracts of T. mastichina collected in Portugal in 2020 showed 24.61 mg caffeic acid equivalents/g DW.

3.1.3. Comparison between Oregano and Thyme Extracts

Comparing our oregano and thyme extracts, oregano extracts presented the highest total phenolic content (~2152 mg GAE/g DW vs. ~436 mg GAE/g DW). Likewise, methanolic extracts of oregano (O. vulgare) showed a higher TPC than thyme (T. capitatus) extracts, with 55 mg GAE/g DW and 38.2 mg GAE/g DW, respectively. However, when compared to our results, the Greek plants seemed to have a lower TPC [31]. This could be related to the preparations of extracts since Greek plant extracts were prepared from dried samples and our extracts were prepared from freeze-dried samples. The freeze-dried process can preserve phenolic compounds and the other compounds responsible for the antioxidant capacity of plants [32].

However, our thyme extracts showed the highest ferric-reducing antioxidant powers (~3148 µmol Fe²⁺/g DW vs. ~1627 µmol Fe²⁺/g DW in oregano extracts), while similar DPPH radical scavenging abilities were found (~0.6259 mmol TE/g DW for thyme and ~ 0.5367 mmol TE/g DW for oregano). The same high antioxidant capacity of thyme extracts was found by Vallverdú-Queralt et al. [33], where ethanolic extracts (50%, v/v) of T. vulgaris from Spain showed a higher antioxidant capacity by the DPPH radical assay, with 1.15 mmol TE/g DW (compared to 0.78 mmol TE/g DW for O. vulgare from China). Contrary to these results, Senki et al. [31] study showed that oregano extracts presented a higher antioxidant capacity than thyme in the DPPH radical assay, with 139.8 mg TE/g DW and 56.2 mg TE/g DW, respectively; and the same tendency was found in the FRAP assay [31]. Different origins, edaphoclimatic conditions, harvest times, and methods of preparation of extracts could influence the antioxidant capacity and the phenolic composition of extracts. Also, the extraction solvent had an influence on the antioxidant capacity and phenolic composition of extracts, for instance, extracts from oregano and thyme with 50% ethanol showed the highest TPC when compared to ethanol and water [34].

3.2. Phenolic Compounds

To better understand the antioxidant capacity of oregano and thyme extracts, UHPLC-ToF-MS analysis was performed to analyze the composition and variation of the main phenolic compounds of extracts. The method was previously validated for the determination of 53 phenolic compounds, including phenolic acids and flavonoids. The method has a good analytical performance, with good linearity (determination coefficients (r² > 0.99)), with low limits of detection (LOD), and limits of quantification (LOQ). The LOQs for each compound are given in Table A1.

3.2.1. Oregano

Rosmarinic acid was the major phenolic compound found in all oregano extracts (Table 5), ranging between 1.67 mg/g DW for BPGV10442 and 3.44 mg/g DW for BPGV16272. Rosmarinic acid is an ester of caffeic acid with various biological activities demonstrated, such as anti-inflammatory, antioxidant, antiviral, antibacterial, and antitumoral activities [35,36]. Other phenolic acids were found in oregano, including gentisic acid (between 0.121 and 0.576 mg/g DW) and 4-hydroxybenzoic acid (between 0.0272 and 0.0747 mg/g DW), making this class of phenolic compound the major class in oregano extracts. The chromatograms of the major phenolic compounds found in oregano are shown in Figure 2.

Similar amounts of rosmarinic acid were reported by Khorsand et al. [23] (0.66–1.65 mg/g DW) in samples collected in Iran; however, a high concentration of rosmarinic acid was reported by Yan et al. [24] (18.69 mg/g DW), who evaluated 174 samples of O. vulgare from different origins (including Germany, Georgia, Italy, Albania, Spain, the Czech Republic, the USA, and Hungary). Contrary to our results, Oliveira et al. [37] reported that thymol was the major phenolic compound in oregano available in Portuguese markets, with 1.76 mg/g DW, followed by rosmarinic acid (0.207 mg/g DW) and carvacrol (0.117 mg/g DW). These differences in the individual phenolic content may also be related to the extraction method, for example, the rosmarinic acid content of the same oregano sample with microwave-assisted extraction (MAE) was 1.5 times higher when compared to conventional extraction [10]. Similarly, our analysis was limited by the lack of high purity standards for thymol and carvacrol, emphasizing the variability of phenolic profiles depending on the analytical methods and standards available.

No statistical difference was found in the content of kaempferol-3-O-B-rutinoside. The content of this flavonol ranged between 0.0088 and 0.0156 mg/g DW. This is contrary to Khorsand et al. [23] who found a large disparity in the kaempferol content, ranging between 0.005 and 0.898 mg/g DW.

Flavanones were one of the major groups of flavonoids present in oregano extracts, including naringenin, eriodyctiol, and sakuranetin, with higher levels in samples collected in Portalegre (p < 0.05), while the samples collected in Bragança showed a lower content of those compounds. This class is typical of citrus fruits, but was previously detected by Khorsand et al. [23] with higher levels (0.103 to 0.188 mg/g DW) in oregano collected in Iran.

Other minor compounds were found in oregano extracts, including genistin (4.88 μg/g DW), luteolin (3.60 μg/g DW), and apigenin (1.93 μg/g DW). Those flavones were also found in oregano from Iran [23], although in higher amounts; for example, 2.99 mg of luteolin and 0.022 mg of apigenin per gram of dried O. vulgare.

Furthermore, (-)-gallocatechin gallate was found in oregano extracts, between 0.0044 and 0.0062 mg/g DW, with higher levels for BPGV16286 and BPGV16427, with significant superiority (p > 0.05). This compound is a non-epimerized isomer of a catechin derivative (epigallocatechin gallate), characteristically of green tea, and is responsible for their antioxidant capacity and biological activities [38].

3.2.2. Thyme

In thyme methanolic extracts, rosmarinic acid was also the main phenolic compound quantified, with high concentrations (Table 6). The lowest content of rosmarinic acid was 23.11 mg/g DW in a subsample of BPVG11295 and the highest amount was 40.45 mg/g DW in a subsample of BPVG10384. Rosmarinic acid is the major phenolic compound reported in the literature [3,29,30,37,39,40], but the highest content of rosmarinic acid was reported by Taghouti et al. [3] (8 mg/g DW), followed by Oliveira et al. [37] (6.6 mg/g DW). The chromatograms of rosmarinic acid and other major phenolic compounds found in thyme are shown in Figure 3.

Quercitrin was the second major phenolic compound in thyme, ranging between 21.03 and 35.36 mg/g DW, followed by isorhamnetin-3-O-glucoside (3.82 to 14.59 mg/g DW) and luteolin (2.31 to 13.38 mg/g DW). Contrary to our results, several luteolin derivatives were found by Martins et al. [39] in a sample of T. vulgaris collected in Spain, including luteolin 7-O-glucuronide, luteolin 7-O-glucoside, luteolin O-hexoside, and luteolin O-diglucuronide, with luteolin 7-O-glucoside being the major compound with 26.43 mg/g. The presence of luteolin O-hexoside was also reported in T. mastichina samples by Taghouti et al. [3], but at lower levels (2.87 mg/g DW), more in agreement with our results. Other authors reported that salvianolic acids and their derivatives (K, I, A, and B) were another group of phenolic compounds present in high amounts in thyme extracts [3,41]; however, those compounds were not in the scope of our methodology.

Rutin and quercetin were other flavonols found in thyme extracts, although in lower concentrations. Different concentrations of quercetin were found between samples; for example, a significantly high amount of quercetin was present in a subsample with a common morphotype of BPVG12093 (T8.5) with 5.60 mg/g DW. Also, the common morphotype of BPVG11288 (T5.2) showed a significantly higher content of this flavonol when compared to the subsample with dark green foliage. Contrary, the common morphotype of BPVG11295 showed a more inferior (p < 0.05) content than the subsample with larger leaves and a revolute margin.

Naringenin and eriodyctiol, compounds from the flavanones class, were found in similar levels in all thyme extracts, between 1.1 and 4.2 mg/g DW, while sakuranetin, a 7-methoxy derivative of naringenin, was found in all thyme extracts but at levels twice as low as the other flavanones (between 0.7 and 2.4 mg/g DW). No flavanones were detected in samples of T. mastichina from Spain [3], but eriodyctiol was detected in lower levels in samples of T. vulgaris from Spain at 0.66 mg/g DW with 80% methanol [39] and at similar levels (0.60 mg/g DW) using 50% ethanol as the extraction solvent [34]. Interestingly, the subsample with larger leaves and a revolute margin from the BPVG12093 (T8.3) was the sample with a superior content of flavanones and flavones (p < 0.05). Also, the same tendency was found when comparing the subsamples of BPGV12078.

The isoflavone genistin was present in thyme, with the highest amount on a subsample of BPVG10381 (5.22 mg/g DW) and a lower level on BPVG11276 (1.40 mg/g DW). Lower quantities of 4-hydroxybenzoic acid were found in all thyme extracts, ranging from 0.34 to 0.65 mg/g DW, with no statistical differences (p > 0.05) between the common morphotypes for each Accession. Also, 4-O-caffeoylquinic acid was quantified in thyme, with the highest levels (p < 0.05) in subsamples with foliage of a citrus green color (T3.1—0.82 mg/g DW) and light green color (1.38 mg/g DW), when compared to other subsamples from the same Accession. However, to the best of our knowledge, no other study reports the presence of this phenolic compound in thyme.

Other vestigial compounds were detected in some thyme extracts, including syringic acid (T7.1, T8.5), kaempferol-3-O-B-rutinoside (T1.1), previously reported as minor compounds [29,34,39], and narirutin (T1.1), with no previous reports.

3.3. Correlations among Different Parameters

3.3.1. Oregano

Pearson’s correlation coefficients (r) among the main phenolic compounds and antioxidant capacity of oregano extracts are given in Figure 4. In the oregano extracts, the TPC and FRAP antioxidant assay presented a highly significant correlation of 0.802 (p < 0.01), thus indicating that the phenolic compounds present in Oregano vulgaris act as antioxidants. However, no correlation was observed between the TPC and DPPH radical assay (p = 0.494). The FRAP and DPPH radical assay showed a positive correlation with 0.778 (p < 0.01), indicating that the results of those assays corroborate the high capacity of these species to act as antioxidants.

Regarding the correlations among individual phenolics, none of the quantified 14 phenolic compounds in thyme were positively or negatively correlated with the TPC or antioxidant assays. However, there was not a significant correlation between the rosmarinic acid and TPC or between this main phenolic compound and antioxidant activity. Contrary to our results, Khorsand et al. [23] demonstrated a moderate correlation between the DPPH antioxidant activity and rosmarinic acid, but no significant correlation was observed between the FRAP activity and rosmarinic acid content. Also, Yan et al. [24] reported that there is no correlation between the antioxidant capacity measured by the oxygen radical absorbance capacity and the concentration of rosmarinic acid. Thus, although rosmarinic acid is the major phenolic compound detected in oregano extracts, other phenolics and constituents can also be present and, together, act synergistically, providing a strong antioxidant effect.

A correlation analysis showed some relations among the phenolic compounds. The results demonstrated a moderate negative correlation between rosmarinic acid and the presence of gentistic acid (r = −0.564, p < 0.05), rutin (r = −0.524, p < 0.05), and sakuranetin (r = −0.472, p < 0.05). Naringenin was strongly correlated with eriodyctiol (r = 0.877, p < 0.01) and sakuranetin (r = 0.924, p < 0.01), both flavanones, indicating that the presence of one compound from this class is related with the presence of another compound from the same class in similar amounts. Interestingly, the results showed a strong correlation between gentistic acid and sakuranetin (r = 0.828, p < 0.01).

3.3.2. Thyme

Pearson’s correlation coefficients (r) among the main phenolic compounds and the antioxidant capacity of the thyme extracts are given in Figure 5.

All of the samples of T. mastichina showed a high antioxidant capacity and a high TPC; however, not all of the samples with the highest rosmarinic acid content were amongst the samples that presented the highest values of the TPC or antioxidant capacity. The results showed no significant correlation between the Total Phenol Content and antioxidant assays, and no significant correlation between those assays and individual phenolic compounds. Contrary to our results, Delgado et al. [29] found that rosmarinic acid influenced the DPPH radical activity, as well as the FRAP assay, when analyzing methanolic extracts of T. mastichina from Spain, indicating that rosmarinic acid is responsible for the antioxidant capacity of thyme. Moreover, Méndez-Tovar et al. [42] also reported a highly significant correlation between the TPC and DPPH assay.

Nevertheless, some correlations were found among the phenolic compounds. For instance, the content of luteolin was strongly correlated with the apigenin (r = 0.780, p < 0.01), since they are compounds from the same flavonoid class. Also, a strong correlation between quercitrin and genistein (r = 0.708, p <0.01) was presented despite the fact that they belong to different flavonoid classes.

4. Conclusions

This study provides a comprehensive evaluation of the phenolic composition and the antioxidant capacity of different accessions of oregano and thyme grown in different locations in Portugal. By focusing on lesser-studied phenolic compounds, such as flavonoids and phenolic acids, this research highlights the complex phenolic profiles beyond the well-known monoterpenes thymol and carvacrol. The major phenolic compound present in both aromatic plants is rosmarinic acid, a common phenolic compound in aromatic plants from the Lamiaceae family and recognized for its inflammatory, antioxidant, antiviral, and antibacterial activities. However, no strong correlation was found between the individual phenolic compounds and the antioxidant capacity, suggesting a more complex and synergetic effect between the different compounds. The levels of individual phenolic compounds and antioxidant properties depend on many factors, such as edaphoclimatic conditions (including water and heat stress), the harvest time, drying conditions, and methods of the preparation of extracts. While statistical differences were found between different accessions, the results show a consistent pool/profile of the different phenolics amounts of the accessions and, in the case of thyme, even between morphotypes. Portuguese types of oregano and thyme represent a strong and consistent source of natural antioxidants and bioactive compounds that can be exploited for further food, pharmaceutical, or cosmetic applications, as well as to be used by the food industry as natural food additives. However, it is important to note that, like many aromatic plants, they can cause adverse effects if used improperly. Oregano and thyme may lead to gastrointestinal issues, allergic reactions, and interactions with certain medications, especially when used in high doses. These potential risks should be considered in their application and further studies are recommended to ensure their safe use.

Author Contributions

Conceptualization, C.S.; Data curation, A.R.S.M., A.S. and V.R.L.; Formal analysis, A.R.S.M., C.A., A.S. and V.R.L.; Funding acquisition, C.S., C.A. and A.S.-S.; Investigation, A.R.S.M., A.S. and V.R.L.; Methodology, A.R.S.M., A.S. and V.R.L.; Project administration, C.S.; Resources, A.R.S.M., A.S. and V.R.L.; Software, A.R.S.M. and A.S.; Supervision, C.S., C.A. and A.S.-S.; Validation, C.A., A.S. and V.R.L.; Visualization, A.S.-S.; Writing—original draft, A.R.S.M. and V.R.L.; Writing—review and editing, C.S., A.S., C.A. and A.S.-S. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be made available on request.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Tables

Figure 1. Geographical origins of the studied oregano (Origanum vulgare L. subsp. virens) and thyme (Thymus mastichina L. subsp. donyanae R. Morales) samples, their common morphotype, and data on average temperature and rainfall between April and June at the sampling points. Note: The data on average air temperature (°C) and total precipitation (mm) by geographical location were obtained from the National Institute of Statistics (INE) website and provided by the Portuguese Institute of the Sea and Atmosphere (IPMA) [13,14]. The bars indicate the average air temperature (°C) and the green line indicates total precipitation (mm) for each month.

Figure 2. Chromatograms (intensity vs. time, min) of rosmarinic acid and gentisic acid in oregano sample (BPGV10423).

Figure 3. Chromatograms (intensity vs time, min) of rosmarinic acid, quercitrin, isorhamnet-in-3-O-glucoside, and eriodyctiol in thyme sample (BPGV10381, sample 1.2).

Figure 4. Heatmap of the Pearson correlation coefficient (r) matrix between antioxidant capacity and individual phenolic compounds in oregano extracts. The abbreviations used are as follows: TPC—Total Phenol Content, DPPH—antioxidant activity based on 2,2-diphenyl-1-picrylhydrazyl assay, FRAP—antioxidant activity based on ferric reducing antioxidant power assay.

Figure 5. Heatmap of the Pearson correlation coefficient (r) matrix between antioxidant capacity and individual phenolic compounds in thyme extracts. The abbreviations used are as follows: TPC—Total Phenol Content, DPPH—antioxidant activity based on 2,2-diphenyl-1-picrylhydrazyl assay, FRAP—antioxidant activity based on ferric reducing antioxidant power assay.

Table 1

Identification of oregano (Origanum vulgare L. subsp. virens) accessions, including their origin, elevation, and field observations.

Accession *	Origin	Elevation (m)	Field Observations
BPGV10423	Ponte de SôrPortalegre	245	Elliptical oval bracts
BPGV10442	SouselPortalegre	1278	Common morphotype
BPGV10442 **	SouselPortalegre	1278	Common morphotype
BPGV11267	GimondeBragança	622	Dense foliage and sparse flowers
BPGV11280	ParâmioBragança	780	Common morphotype
BPGV11408	AlcanedeSantarém	297	Sparse foliage and smaller leaves
BPGV16272	Rio FrioViana do Castelo	333	Common morphotype
BPGV16286	SoajoViana do Castelo	127	Larger cordiform leaves with dentate margin, no petals
BPGV16427	AlpendrinhaCastelo Branco	650	Common morphotype

Common morphotype: ovate leaves with obtuse base, ciliated margin, and acute apex; pink and white petals and obovate bracts; * Accession according to the Portuguese Bank of Plant Germplasm (Braga, Portugal); ** This Accession corresponds to the multiplication from the original BPGV10442.

Table 2

Identification of thyme (Thymus mastichina L.) accessions, including their origin and field observations of each sub-sample.

Accession *	Origin	Sub-Sample	Field Observations
BPGV10381	Vila Velha de Rodão	1.1	Reddish fruiting branch with elliptical leaves and larger nodes
	Portalegre	1.2	Light green foliage color
BPGV10384	Ponte de SôrPortalegre	2.1	Reddish fruiting branch with elliptical leaves and larger nodes
		2.2	Chalk-green foliage
		2.3	Common morphotype
BPGV11264	Gimonde	3.1	More citrus-green foliage
	Bragança	3.2	Common morphotype
BPGV11276	DonaiBragança	4.1	Common morphotype
BPGV11288	Castanheira	5.1	Dark green foliage
	Bragança	5.2	Common morphotype
BPGV11295	Minas de Santo Adrião	6.1	Larger leaves and revolute margin
	Bragança	6.2	Common morphotype
BPGV12078	Vilarinho da Castanheira	7.1	Larger leaves and revolute margin
	Bragança	7.2	Common morphotype
BPGV12093	JunqueiraBragança	8.1	Reddish fruiting branch with elliptical leaves and larger nodes
		8.2	More brownish fruiting branches
		8.3	Larger leaves and revolute margin
		8.4	Light green foliage color
		8.5	Common morphotype

Common morphotype: Leaves and flowers distributed along the entire stem leaves with elliptical, flat marginal veins, and green foliage. * Accession according to the Portuguese Bank of Plant Germplasm (Braga, Portugal).

Table 3

Results of the Total Phenolic Content and Antioxidant Capacity in oregano extracts.

Accessions	TPCmg GAE/g DW	FRAPµmol Fe2+/g DW	DPPHmmol TE/g DW
BPGV10423	1639.71 ± 41.37 a	1354.86 ± 47.60 a	0.5011 ± 0.0310 ab
BPGV10442	2130.95 ± 62.48 abc	1626.06 ± 37.79 ab	0.5590 ± 0.0095 ab
BPGV10442	2171.24 ± 63.73 abc	1769.20 ± 27.09 ab	0.5739 ± 0.0263 ab
BPGV11267	1927.32 ± 272.69 ab	1548.89 ± 163.40 ab	0.4476 ± 0.0350 a
BPGV11280	2066.20 ± 239.83 abc	1564.58 ± 131.87 ab	0.5335 ± 0.0187 ab
BPGV11408	2660.86 ± 0.58 c	1932.33 ± 38.84 b	0.6029 ± 0.0192 b
BPGV16272	2486.45 ± 148.58 bc	1619.82 ± 78.03 ab	0.5440 ± 0.0510 ab
BPGV16286	2271.46 ± 28.90 abc	1667.45 ± 80.86 ab	0.5364 ± 0.0375 ab
BPGV16427	2014.73 ± 81.32 abc	1567.66 ± 45.15 ab	0.5320 ± 0.0064 ab

The results are expressed as mean ± standard deviation (SD), from three replicates. Different superscript letters indicate the significant difference (p < 0.05) in each assay. GAE—gallic acid equivalents; TE—Trolox equivalents; DW—dried weight.

Table 4

Results of the Total Phenolic Content and Antioxidant Capacity in thyme extracts.

Accession		TPCmg GAE/g DW	FRAPµmol Fe2+/g DW	DPPHmmol TE/g DW
BPGV10381	1.1	410.98 ± 10.87	342.60 ± 4.58	0.6143 ± 0.0001
	1.2	389.44 ± 2.71	334.38 ± 7.74	0.5879 ± 0.0220
BPGV10384	2.1	415.17 ± 21.24 a	281.95 ± 2.79 a	0.4812 ± 0.0371 a
	2.2	529.46 ± 9.72 b	3940.91 ± 34.40 c	0.6292 ± 0.0046 b
	2.3	448.94 ± 2.05 a	3362.87 ± 225.97 bABC	0.5738 ± 0.0363 ab
BPGV11264	3.1	460.59 ± 1.92	3431.37 ± 233.35	0.7205 ± 0.0137
	3.2	439.43 ± 19.19	3818.78 ± 16.06 BC	0.6922 ± 0.0091
BPGV11276	4.1	503.70 ± 15.97	3990.88 ± 2.80 C	0.6254 ± 0.0190
BPGV11288	5.1	371.96 ± 17.77 a	3395.88 ± 10.20	0.6205 ± 0.0525
	5.2	466.77 ± 14.71 b	3053.55 ± 427.24 AB	0.5780 ± 0.0471
BPGV11295	6.1	443.80 ± 23.58	2702.53 ± 56.21	0.6044 ± 0.0292
	6.2	418.92 ± 43.04	2521.99 ± 102.40 A	0.6051 ± 0.0384
BPGV12078	7.1	440.49 ± 12.17	2610.13 ± 2.27 a	0.5814 ± 0.0104 a
	7.2	467.09 ± 26.92	3011.71 ± 12.00 bAB	0.6791 ± 0.0089 b
BPGV12093	8.1	365.64 ± 19.16	2543.83 ± 3.42 a	0.6267 ± 0.0173
	8.2	408.59 ± 28.92	2928.82 ± 26.47 bc	0.6630 ± 0.0113
	8.3	444.87 ± 35.08	2861.80 ± 41.62 b	0.6332 ± 0.0211
	8.4	422.55 ± 43.92	3084.56 ± 9.95 c	0.7011 ± 0.0391
	8.5	428.57 ± 9.52	3109.83 ± 78.16 cABC	0.6758 ± 0.0197

The results are expressed as mean ± standard deviation (SD) from three replicates. Different letters indicate statistically significant differences (p < 0.05). Lowercase letters indicate statistically significant differences between the subsamples of each Accession of Thyme. Uppercase letters indicate statistically significant differences between the “common morphotype” of each Accession of thyme extracts. GAE—gallic acid equivalents; TE—Trolox equivalents; DW—dried weight.

Table 5

Phenolic compounds (mg/g DW) in oregano extracts.

Accession *	Rosmarinic Acid	Gentisic Acid	4-Hydroxybenzoic acid	Rutin	Kaempferol-3-O-B-rutinoside	Naringenin	Eriodictyol	Sakuranetin
BPGV10423	2.129 ± 0.128 ab	0.386 ± 0.007 cd	0.0537 ± 0.0014 bc	0.021 ± 0.0044 ab	0.0088 ± 0.0014 a	0.0307 ± 0.009 d	0.0368 ± 0.004 d	0.0042 ± 0.0002 c
BPGV10442	2.120 ± 0.166 ab	0.454 ± 0.022 d	0.0543 ± 0.0003 bc	0.0037 ± 0.0028 cd	0.0131 ± 0.0028 a	0.0305 ± 0.0005 d	0.0304 ± 0.005 d	0.0047 ± 0.0001 c
BPGV10442 **	1.667 ± 0.049 a	0.576 ± 0.007 e	0.0372 ± 0.0016 ab	0.094 ± 0.0043 e	0.0125 ± 0.0004 a	0.0332 ± 0.0007 d	0.0235 ± 0.008 c	0.0047 ± 0.0001 c
BPGV11267	2.252 ± 0.112 abc	0.232 ± 0.004 b	0.0321 ± 0.0010 a	0.035 ± 0.0036 bcd	0.0109 ± 0.0008 a	0.0077 ± 0.0001 a	0.0033 ± 0.002 a	0.0020 ± 0.0001 a
BPGV11280	2.706 ± 0.266 bcd	0.209 ± 0.006 b	0.0499 ± 0.0033 bc	0.039 ± 0.0022 cd	0.0123 ± 0.0021 a	0.0108 ± 0.0003 a	0.0083 ± 0.025 ab	0.0020 ± 0.0002 a
BPGV11408	3.109 ± 0.240 cd	0.240 ± 0.013 b	0.0561 ± 0.0048 c	0.045 ± 0.0029 d	0.0156 ± 0.0034 a	0.0113 ± 0.0005 a	0.0085 ± 0.025 ab	0.0021 ± 0.0001 a
BPGV16272	3.438 ± 0.343 d	0.236 ± 0.033 b	0.0747 ± 0.0086 d	0.027 ± 0.0034 abc	0.0129 ± 0.0014 a	0.0168 ± 0.0020 b	0.0137 ± 0.016 b	0.0031 ± 0.0001 b
BPGV16286	3.069 ± 0.109 cd	0.121 ± 0.023 a	0.0392 ± 0.0025 abc	0.016 ± 0.0005 a	0.0096 ± 0.0006 a	0.0229 ± 0.0007 c	0.0238 ± 0.007 c	0.0027 ± 0.0001 b
BPGV16427	3.274 ± 0.029 d	0.352 ± 0.007 c	0.0272 ± 0.0005 a	0.039 ± 0.0012 cd	0.0118 ± 0.0006 a	0.0193 ± 0.0004 b	0.0107 ± 0.002 b	0.0028 ± 0.0001 b

The results are expressed as mean ± standard deviation (SD) from four replicates. Different superscript letters indicate the significant difference (p < 0.05) between different Accessions for each phenolic compound. * Accession according to the Portuguese National Plant Germplasm Bank (Braga, Portugal). ** This Accession corresponds to the multiplication from the original BPGV10442.

Table 6

Phenolic compounds (mg/g DW) in thyme extracts.

Accession *		Rosmarinic Acid	Quercitrin	Isorhamnetin-3-O-glucoside	Eriodictyol	Luteolin	Genistin	Naringenin
BPGV10381	1.1	37.05 ± 0.662 b	31.07 ± 0.424	9.413 ± 0.134 a	1.422 ± 0.046 b	2.311 ± 0.046	3.782 ± 0.071 a	3.180 ± 0.074 b
	1.2	33.75 ± 0.299 a	35.36 ± 0.432	11.25 ± 0.433 b	1.068 ± 0.031 a	2.448 ± 0.082	5.219 ± 0.221 b	1.741 ± 0.210 a
BPGV10384	2.1	31.18 ± 1.13 a	21.03 ± 0.808 a	6.412 ± 0.334 b	3.099 ± 0.145 a	5.471 ±0.261 a	1.531 ± 0.073 a	2.232 ± 0.084 a
	2.2	40.45 ±0.749 b	23.45 ± 2.62 a	3.820 ± 0.083 a	4.160 ± 0.122 b	6.853 ± 0.517 b	2.189 ± 0.045 b	4.011 ± 0.334 b
	2.3	33.71 ± 0.899 aB	26.89 ± 0.869 aAB	7.254 ± 0.196 bA	2.508 ± 0.187 aBC	4.148 ± 0.070 aBC	2.453 ± 0.044 cBC	2.010 ± 0.231 aA
BPGV11264	3.1	35.63 ± 1.65	32.31 ± 2.01	12.99 ± 1.10	1.413 ± 0.085	4.859 ± 0.278 b	2.873 ± 0.334	2.362 ± 0.106
	3.2	32.34 ± 0.943 B	31.30 ± 2.25 B	11.50 ± 0.350 BC	1.570 ± 0.036 A	3.903 ± 0.117 aAB	2.548 ± 0.111 BC	2.115 ± 0.066 AB
BPGV11276	4.1	39.14 ± 1.06 C	22.44 ± 1.05 A	8.050 ± 0.539 A	3.142 ± 0.079 D	3.441 ± 0.123 AB	1.403 ± 0.096 A	3.129 ± 0.182 C
BPGV11288	5.1	36.38 ± 1.79	28.89 ± 0.928	7.831 ± 0.220 a	2.975 ± 0.227	5.308 ± 0.301	2.313 ± 0.146	2.384 ± 0.287
	5.2	39.59 ± 1.15 C	30.33 ± 1.31 B	14.59 ± 1.621 bC	2.805 ± 0.084 CD	5.120 ± 0.186 C	2.196 ± 0.242 ABC	2.638 ± 0.080 BC
BPGV11295	6.2	23.11 ± 0.701 a	28.64 ± 1.37	8.781 ± 0.658	1.519 ± 0.120 a	4.738 ±0.379 b	2.339 ± 0.368	1.774 ± 0.148
	6.3	27.76 ± 1.33 bA	26.94 ± 1.01 AB	7.023 ± 0.372 A	2.264 ± 0.096 bB	2.918 ± 0.156 aAB	2.019 ± 0.099 AB	1.797 ± 0.029 A
BPGV12078	7.1	31.60 ± 1.68	22.82 ± 0.721 a	8.000 ± 0.381 a	4.192 ± 0.343 b	11.37 ± 1.03 b	1.478 ± 0.132 a	2.997 ± 0.143
	7.2	34.66 ± 0.724 B	29.35 ± 2.31 bB	9.532 ± 0.218 bAB	2.702 ± 0.088 aBCD	4.531 ± 0.164 aBC	2.712 ± 0.107 bBC	3.009 ± 0.123 C
BPGV12093	8.1	31.31 ± 0.921 b	24.90 ± 0.268 a	4.944 ± 0.066 a	2.059 ± 0.060 b	6.253 ± 0.152 b	1.930 ± 0.221 a	1.116 ± 0.025 a
	8.2	32.37 ± 0.579 bc	32.51 ± 0.798 b	8.923 ± 0.222 bc	1.291 ± 0.036 a	2.950 ± 0.119 a	4.286 ± 0.136 c	1.170 ± 0.029 a
	8.3	29.34 ± 0.979 ab	23.82 ± 0.836 a	10.14 ± 0.343 c	3.937 ± 0.151 d	13.38 ± 0.806 c	2.674 ± 0.069 ab	2.635 ± 0.096 c
	8.4	26.82 ± 1.02 a	26.59 ± 0.965 a	13.41 ± 0.613 d	2.951 ± 0.217 c	6.469 ± 0.106 b	2.387 ± 0.130 ab	1.328 ± 0.045 a
	8.5	35.79 ± 0.299 cBC	26.99 ± 0.813 aAB	7.390 ± 0.268 bA	4.235 ± 0.069 dE	12.06 ± 0.539 cD	3.030 ± 0.382 bC	1.827 ± 0.050 bA
Accession *		Quercetin	Apigenin	4-Hydroxybenzoic acid	Rutin	Sakuranetin	4-O-Caffeoylquinic acid	Taxifolin
BPGV10381	1.1	nd	0.459 ± 0.020	0.584 ± 0.041	0.247 ± 0.125	1.418 ± 0.109	0.736 ± 0.037 b	0.204 ± 0.014 b
	1.2	nd	0.386 ± 0.064	0.629 ± 0.038	0.182 ± 0.082	1.521 ± 0.038	0.602 ± 0.049 a	0.137 ± 0.041 a
BPGV10384	2.2	nd	0.298 ± 0.193 a	0.411 ± 0.018 a	<LOQ	1.636 ± 0.038 b	0.436 ± 0.221	0.943 ± 0.088 b
	2.1	0.102 ± 0.001	0.447 ± 0.043 a	0.499 ± 0.019 b	0.107 ± 0.032	1.058 ± 0.083 a	0.568 ± 0.046	0.532 ± 0.017 a
	2.3	1.113 ± 0.583 aA	0.406 ± 0.022 aBC	0.423 ± 0.021 a	0.154 ± 0.039	1.452 ± 0.222 abB	0.628 ± 0.091 AB	0.999 ± 0.009 bD
BPGV11264	3.1	1.170 ± 0.417	0.514 ± 0.027 b	0.636 ± 0.029 b	0.163 ± 0.015	0.768 ± 0.250	0.816 ± 0.058 b	0.106 ± 0.022
	3.2	1.562 ± 0.070 AB	0.403 ± 0.013 aABC	0.468 ± 0.025 a	0.125 ± 0.046	1.351 ± 0.059 AB	0.470 ± 0.072 a	0.158 ± 0.013 A
BPGV11276	4.1	2.321 ± 0.114 AB	0.356 ± 0.045 AB	0.592 ± 0.048	<LOQ	1.372 ± 0.170 AB	0.747 ± 0.144 B	nd
BPGV11288	5.1	0.529 ± 0.003 a	0.530 ± 0.047	0.581 ± 0.031	<LOQ	1.834 ± 0.188b	0.656 ± 0.065	1.133 ± 0.024 b
	5.3	2.786 ± 0.067 bB	0.476 ± 0.015 C	0.393 ± 0.140	0.100 ± 0.018	1.279 ± 0.055 aAB	0.613 ± 0.021 AB	0.495 ± 0.037 aC
BPGV11295	6.2	1.663 ± 0.832 b	0.379 ± 0.078	0.522 ± 0.098	0.100 ± 0.021 b	0.985 ± 0.173	0.411 ± 0.096 b	nd
	6.3	0.585 ± 0.090 aA	0.325 ± 0.020 A	0.483 ± 0.133	0.070 ± 0.010 a	1.074 ± 0.073 AB	0.384 ± 0.121 a	nd
BPGV12078	7.1	nd	0.623 ± 0.070 b	0.451 ± 0.046 a	0.090 ± 0.072	2.348 ± 0.249 b	0.441 ± 0.055 a	0.301 ± 0.068
	7.2	nd	0.429 ± 0.020 aBC	0.651 ± 0.078 b	0.104 ± 0.006	1.364 ± 0.092 a	0.550 ± 0.031 bAB	nd
BPGV12093	8.1	0.866 ± 0.110 a	0.327 ± 0.018 a	0.377 ± 0.029 ab	0.097 ± 0.050 a	0.885 ± 0.075 ab	0.434 ± 0.037 a	0.086 ± 0.016 a
	8.2	nd	0.434 ± 0.031 a	0.463 ± 0.041 b	0.135 ± 0.064 ab	1.032 ± 0.055 b	0.455 ± 0.076 a	0.070 ± 0.003 a
	8.3	3.115 ± 0.100 b	1.244 ± 0.069 c	0.378 ± 0.029 ab	0.099 ± 0.007 a	1.272 ± 0.058 c	0.350 ± 0.021 a	0.055 ± 0.020 a
	8.4	1.548 ± 0.100 a	0.738 ± 0.029 b	0.339 ± 0.021 a	0.244 ± 0.046 b	0.780 ± 0.048 a	1.376 ± 0.075 b	0.035 ± 0.008 a
	8.5	5.597 ± 0.398 cC	0.785 ± 0.023 bD	0.448 ± 0.007 b	0.128 ± 0.008 ab	0.972 ± 0.026 bA	0.383 ± 0.010 aA	0.293 ± 0.030 bB

nd—not detected; LOQ—Limit of Quantification; The results are expressed as mean ± standard deviation (SD) from four replicates. Different letters indicate statistically significant differences (p < 0.05). Lowercase letters indicate statistically significant differences among the subsamples of each Accession of Thyme. Uppercase letters indicate statistically significant differences between the “common morphotype” of each Accession of thyme extracts. * Accession according to the Portuguese National Plant Germplasm Bank (Braga, Portugal).

Appendix A

Table A1

Limits of quantification (at µg/mL extract) for 53 phenolic compounds by UHPLC-ToF-MS.

Phenolic Acids
Hydroxycinnamic Acid Derivatives		Benzoic Acid Derivatives
Caffeic acid	0.050	Protocatechuic acid	0.100
p-coumaric acid	0.200	4-Hydroxybenzoic acid	0.100
o-coumaric acid	0.200	Gallic acid	0.050
trans-ferulic acid	0.100	Gentisic acid	0.200
Sinapic acid	0.200	Vanillic acid	0.100
1,5-Dicaffeoylquinic acid	0.050	Syringic acid	0.050
4-O-Caffeoylquinic acid	0.050
Chlorogenic acid	0.200
Neochlorogenic acid	0.100
Rosmarinic acid	0.500
Flavonoids
Flavan-3-ols		Flavanone
Epicatechin	0.020	Naringenin	0.010
Catechin	0.020	Eriodyctiol	0.020
(-)-Epigallocatechin	0.100	Sakuranetin	0.010
(-)-Epigallocatechin gallate	0.020	Eriocitrin	0.050
(-)-Gallocatechin	0.020	Hesperidin	0.050
(-)-Gallocatechin gallate	0.020	Narirutin	0.020
Epicatechin gallate	0.050	Pinocembrin	0.002
Flavonols		Flavone
Quercetin	0.020	Apigenin	0.010
Isoquercetin	0.020	Luteolin	0.020
Quercitrin	0.020	Sinensetin	0.010
Rutin	0.100	Tangeretin	0.020
Myricetin	0.050	Chrysin	0.010
Isorhamnetin	0.050	Luteolin-7-O-glucoside	0.010
Kaempferol	0.010	Anthocyanins
Isoflavone		5,7-Dimethoxyluteolinidin chloride	0.050
Genistein	0.020	Apigeninidin chloride	0.050
Genistin	0.020	Luteolinidin chloride	0.050
Flavanonol		Cyanidin-3-glucoside	0.100
Taxifolin	0.050	Non-Flavonoids
Chalcones		Ellagic acid	0.200
Phloridzin	0.200	Carnosic acid	0.500

References

1. Stefanaki, A.; van Andel, T. Mediterranean Aromatic Herbs and Their Culinary Use. Aromatic Herbs in Food: Bioactive Compounds, Processing, and Applications; Academic Press: Cambridge, MA, USA, 2021; pp. 93-121. [DOI: https://dx.doi.org/10.1016/B978-0-12-822716-9.00003-2]

2. Rodrigues, M.; Lopes, A.C.; Vaz, F.; Filipe, M.; Alves, G.; Ribeiro, M.P.; Coutinho, P.; Araujo, A.R.T.S. Thymus mastichina: Composition and Biological Properties with a Focus on Antimicrobial Activity. Pharmaceuticals; 2020; 13, 479. [DOI: https://dx.doi.org/10.3390/ph13120479] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33352776]

3. Taghouti, M.; Martins-Gomes, C.; Schäfer, J.; Santos, J.A.; Bunzel, M.; Nunes, F.M.; Silva, A.M. Chemical Characterization and Bioactivity of Extracts from Thymus mastichina: A Thymus with a Distinct Salvianolic Acid Composition. Antioxidants; 2019; 9, 34. [DOI: https://dx.doi.org/10.3390/antiox9010034] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31906063]

4. Escobar, A.; Pérez, M.; Romanelli, G.; Blustein, G. Thymol Bioactivity: A Review Focusing on Practical Applications. Arab. J. Chem.; 2020; 13, pp. 9243-9269. [DOI: https://dx.doi.org/10.1016/j.arabjc.2020.11.009]

5. Singh, N.; Yadav, S.S. A Review on Health Benefits of Phenolics Derived from Dietary Spices. Curr. Res. Food Sci.; 2022; 5, pp. 1508-1523. [DOI: https://dx.doi.org/10.1016/j.crfs.2022.09.009] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36132490]

6. Rathod, N.B.; Kulawik, P.; Ozogul, F.; Regenstein, J.M.; Ozogul, Y. Biological Activity of Plant-Based Carvacrol and Thymol and Their Impact on Human Health and Food Quality. Trends Food Sci. Technol.; 2021; 116, pp. 733-748. [DOI: https://dx.doi.org/10.1016/j.tifs.2021.08.023]

7. Galal, S.A.B.; Madhat Mousa, M.; Elzanfaly, E.S.; Hussien, E.M.; Amer, E.A.H.; Zaazaa, H.E. Quantitative Analysis of Residual Butylated Hydroxytoluene and Butylated Hydroxyanisole in Salmo Salar, Milk, and Butter by Liquid Chromatography–Tandem Mass Spectrometry. Food Chem.; 2024; 453, 139653. [DOI: https://dx.doi.org/10.1016/j.foodchem.2024.139653]

8. Fernandes, A.C.; Morais, C.; Franchini, B.; Pereira, B.; Pinho, O.; Cunha, L.M. Clean-Label Products: Factors Affecting Liking and Acceptability by Portuguese Older Adults. Appetite; 2024; 197, 107307. [DOI: https://dx.doi.org/10.1016/j.appet.2024.107307]

9. Mantzourani, I.; Daoutidou, M.; Nikolaou, A.; Kourkoutas, Y.; Alexopoulos, A.; Tzavellas, I.; Dasenaki, M.; Thomaidis, N.; Plessas, S. Microbiological Stability and Sensorial Valorization of Thyme and Oregano Essential Oils Alone or Combined with Ethanolic Pomegranate Extracts in Wine Marinated Pork Meat. Int. J. Food Microbiol.; 2023; 386, 110022. [DOI: https://dx.doi.org/10.1016/j.ijfoodmicro.2022.110022]

10. Lupuliasa, A.I.; Baroi, A.M.; Avramescu, S.M.; Vasile, B.S.; Prisada, R.M.; Fierascu, R.C.; Fierascu, I.; Sărdărescu, D.I.; Ripszky Totan, A.; Voicu-Bălășea, B. et al. Application of Common Culinary Herbs for the Development of Bioactive Materials. Plants; 2024; 13, 997. [DOI: https://dx.doi.org/10.3390/plants13070997]

11. Akbarbaglu, Z.; Mazloomi, N.; Sarabandi, K.; Ramezani, A.; Khaleghi, F.; Hamzehkollaei, A.R.; Jafari, S.M.; Hesarinejad, M.A. Stabilization of Antioxidant Thyme-Leaves Extract (Thymus vulgaris) within Biopolymers and Its Application in Functional Bread Formulation. Future Foods; 2024; 9, 100356. [DOI: https://dx.doi.org/10.1016/j.fufo.2024.100356]

12. Shen, Y.; Zhou, J.; Yang, C.; Chen, Y.; Yang, Y.; Zhou, C.; Wang, L.; Xia, G.; Yu, X.; Yang, H. Preparation and Characterization of Oregano Essential Oil-Loaded Dioscorea Zingiberensis Starch Film with Antioxidant and Antibacterial Activity and Its Application in Chicken Preservation. Int. J. Biol. Macromol.; 2022; 212, pp. 20-30. [DOI: https://dx.doi.org/10.1016/j.ijbiomac.2022.05.114] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35597375]

13. IPMA. Average Air Temperature (°C) by Geographical Location (Weather Station) and Month; Annual. 2022; Available online: https://www.ine.pt/xportal/xmain?xpid=INE&xpgid=ine_indicadores&indOcorrCod=0009895&contexto=bd&selTab=tab2&xlang=pt (accessed on 4 July 2024).

14. IPMA. Total Precipitation (Mm) by Geographical Location (Weather Station) and Month; Annual. 2022; Available online: https://www.ine.pt/xportal/xmain?xpid=INE&xpgid=ine_indicadores&indOcorrCod=0009911&contexto=bd&selTab=tab2&xlang=pt (accessed on 4 July 2024).

15. Serrano, C.; Oliveira, M.C.; Lopes, V.R.; Soares, A.; Molina, A.K.; Paschoalinotto, B.H.; Pires, T.C.S.P.; Serra, O.; Barata, A.M. Chemical Profile and Biological Activities of Brassica rapa and Brassica napus Ex Situ Collection from Portugal. Foods; 2024; 13, 1164. [DOI: https://dx.doi.org/10.3390/foods13081164] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38672837]

16. Asami, D.K.; Hong, Y.J.; Barrett, D.M.; Mitchell, A.E. Processing-Induced Changes in Total Phenolics and Procyanidins in Clingstone Peaches. J. Sci. Food Agric.; 2003; 83, pp. 56-63. [DOI: https://dx.doi.org/10.1002/jsfa.1275]

17. Singleton, V.L.; Rossi, J.A. Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am. J. Enol. Vitic.; 1965; 16, pp. 144-158. [DOI: https://dx.doi.org/10.5344/ajev.1965.16.3.144]

18. Kondo, S.; Yoshikawa, H.; Katayama, R. Antioxidant Activity in Astringent and Non-Astringent Persimmons. J. Hortic. Sci. Biotechnol.; 2004; 79, pp. 390-394. [DOI: https://dx.doi.org/10.1080/14620316.2004.11511778]

19. Benzie, I.F.F.; Strain, J.J. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay. Anal. Biochem.; 1996; 239, pp. 70-76. [DOI: https://dx.doi.org/10.1006/abio.1996.0292]

20. Teixeira, J.D.; Soares Mateus, A.R.; Sanchez, C.; Parpot, P.; Almeida, C.; Sanches Silva, A. Antioxidant Capacity and Phenolics Profile of Portuguese Traditional Cultivars of Apples and Pears and Their By-Products: On the Way to Newer Applications. Foods; 2023; 12, 1537. [DOI: https://dx.doi.org/10.3390/foods12071537]

21. Kumar, K.; Debnath, P.; Singh, S.; Kumar, N. An Overview of Plant Phenolics and Their Involvement in Abiotic Stress Tolerance. Stresses; 2023; 3, pp. 570-585. [DOI: https://dx.doi.org/10.3390/stresses3030040]

22. Ancillotti, C.; Bogani, P.; Biricolti, S.; Calistri, E.; Checchini, L.; Ciofi, L.; Gonnelli, C.; Del Bubba, M. Changes in Polyphenol and Sugar Concentrations in Wild Type and Genetically Modified Nicotiana Langsdorffii Weinmann in Response to Water and Heat Stress. Plant Physiol. Biochem.; 2015; 97, pp. 52-61. [DOI: https://dx.doi.org/10.1016/j.plaphy.2015.09.012]

23. Jafari Khorsand, G.; Morshedloo, M.R.; Mumivand, H.; Emami Bistgani, Z.; Maggi, F.; Khademi, A. Natural Diversity in Phenolic Components and Antioxidant Properties of Oregano (Origanum vulgare L.) Accessions, Grown under the Same Conditions. Sci. Rep.; 2022; 12, 5813. [DOI: https://dx.doi.org/10.1038/s41598-022-09742-4]

24. Yan, F.; Azizi, A.; Janke, S.; Schwarz, M.; Zeller, S.; Honermeier, B. Antioxidant Capacity Variation in the Oregano (Origanum vulgare L.) Collection of the German National Genebank. Ind. Crops Prod.; 2016; 92, pp. 19-25. [DOI: https://dx.doi.org/10.1016/j.indcrop.2016.07.038]

25. Michalaki, A.; Karantonis, H.C.; Kritikou, A.S.; Thomaidis, N.S.; Dasenaki, M.E. Ultrasound-Assisted Extraction of Total Phenolic Compounds and Antioxidant Activity Evaluation from Oregano (Origanum vulgare ssp. hirtum) Using Response Surface Methodology and Identification of Specific Phenolic Compounds with HPLC-PDA and Q-TOF-MS/MS. Molecules; 2023; 28, 2033. [DOI: https://dx.doi.org/10.3390/molecules28052033] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36903279]

26. Costa, D.C.; Costa, H.S.; Albuquerque, T.G.; Ramos, F.; Castilho, M.C.; Sanches-Silva, A. Advances in Phenolic Compounds Analysis of Aromatic Plants and Their Potential Applications. Trends Food Sci. Technol.; 2015; 45, pp. 336-354. [DOI: https://dx.doi.org/10.1016/j.tifs.2015.06.009]

27. Ribeiro-Santos, R.; Carvalho-Costa, D.; Cavaleiro, C.; Costa, H.S.; Albuquerque, T.G.; Castilho, M.C.; Ramos, F.; Melo, N.R.; Sanches-Silva, A. A Novel Insight on an Ancient Aromatic Plant: The Rosemary (Rosmarinus officinalis L.). Trends Food Sci. Technol.; 2015; 45, pp. 355-368. [DOI: https://dx.doi.org/10.1016/j.tifs.2015.07.015]

28. Castilho, P.C.; Savluchinske-Feio, S.; Weinhold, T.S.; Gouveia, S.C. Evaluation of the Antimicrobial and Antioxidant Activities of Essential Oils, Extracts and Their Main Components from Oregano from Madeira Island, Portugal. Food Control; 2012; 23, pp. 552-558. [DOI: https://dx.doi.org/10.1016/j.foodcont.2011.08.031]

29. Delgado, T.; Marinero, P.; Asensio-S.-Manzanera, M.C.; Asensio, C.; Herrero, B.; Pereira, J.A.; Ramalhosa, E. Antioxidant Activity of Twenty Wild Spanish Thymus mastichina L. Populations and Its Relation with Their Chemical Composition. LWT; 2014; 57, pp. 412-418. [DOI: https://dx.doi.org/10.1016/j.lwt.2013.12.041]

30. Taghouti, M.; Martins-Gomes, C.; Félix, L.M.; Schäfer, J.; Santos, J.A.; Bunzel, M.; Nunes, F.M.; Silva, A.M. Polyphenol Composition and Biological Activity of Thymus Citriodorus and Thymus vulgaris: Comparison with Endemic Iberian Thymus Species. Food Chem.; 2020; 331, 127362. [DOI: https://dx.doi.org/10.1016/j.foodchem.2020.127362]

31. Skendi, A.; Irakli, M.; Chatzopoulou, P. Analysis of Phenolic Compounds in Greek Plants of Lamiaceae Family by HPLC. J. Appl. Res. Med. Aromat. Plants; 2017; 6, pp. 62-69. [DOI: https://dx.doi.org/10.1016/j.jarmap.2017.02.001]

32. Vargas-Madriz, Á.F.; Kuri-García, A.; Luzardo-Ocampo, I.; Vargas-Madriz, H.; Pérez-Ramírez, I.F.; Anaya-Loyola, M.A.; Ferriz-Martínez, R.A.; Roldán-Padrón, O.; Hernández-Sandoval, L.; Guzmán-Maldonado, S.H. et al. Impact of Drying Process on the Phenolic Profile and Antioxidant Capacity of Raw and Boiled Leaves and Inflorescences of Chenopodium berlandieri ssp. berlandieri. Molecules; 2023; 28, 7235. [DOI: https://dx.doi.org/10.3390/molecules28207235]

33. Vallverdú-Queralt, A.; Regueiro, J.; Martínez-Huélamo, M.; Rinaldi Alvarenga, J.F.; Leal, L.N.; Lamuela-Raventos, R.M. A Comprehensive Study on the Phenolic Profile of Widely Used Culinary Herbs and Spices: Rosemary, Thyme, Oregano, Cinnamon, Cumin and Bay. Food Chem.; 2014; 154, pp. 299-307. [DOI: https://dx.doi.org/10.1016/j.foodchem.2013.12.106]

34. Muzolf-Panek, M.; Stuper-Szablewska, K. Comprehensive Study on the Antioxidant Capacity and Phenolic Profiles of Black Seed and Other Spices and Herbs: Effect of Solvent and Time of Extraction. J. Food Meas. Charact.; 2021; 15, pp. 4561-4574. [DOI: https://dx.doi.org/10.1007/s11694-021-01028-z]

35. Guan, H.; Luo, W.; Bao, B.; Cao, Y.; Cheng, F.; Yu, S.; Fan, Q.; Zhang, L.; Wu, Q.; Shan, M. A Comprehensive Review of Rosmarinic Acid: From Phytochemistry to Pharmacology and Its New Insight. Molecules; 2022; 27, 3292. [DOI: https://dx.doi.org/10.3390/molecules27103292] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35630768]

36. Zhao, J.; Xu, L.; Jin, D.; Xin, Y.; Tian, L.; Wang, T.; Zhao, D.; Wang, Z.; Wang, J. Rosmarinic Acid and Related Dietary Supplements: Potential Applications in the Prevention and Treatment of Cancer. Biomolecules; 2022; 12, 1410. [DOI: https://dx.doi.org/10.3390/biom12101410] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36291619]

37. Oliveira, A.S.; Ribeiro-Santos, R.; Ramos, F.; Castilho, M.C.; Sanches-Silva, A. UHPLC-DAD Multi-Method for Determination of Phenolics in Aromatic Plants. Food Anal. Methods; 2018; 11, pp. 440-450. [DOI: https://dx.doi.org/10.1007/s12161-017-1015-y]

38. Peng, H.; Shahidi, F. Oxidation and Degradation of (Epi)Gallocatechin Gallate (EGCG/GCG) and (Epi)Catechin Gallate (ECG/CG) in Alkali Solution. Food Chem.; 2023; 408, 134815. [DOI: https://dx.doi.org/10.1016/j.foodchem.2022.134815]

39. Martins, N.; Barros, L.; Santos-Buelga, C.; Silva, S.; Henriques, M.; Ferreira, I.C.F.R. Decoction, Infusion and Hydroalcoholic Extract of Cultivated Thyme: Antioxidant and Antibacterial Activities, and Phenolic Characterisation. Food Chem.; 2015; 167, pp. 131-137. [DOI: https://dx.doi.org/10.1016/j.foodchem.2014.06.094]

40. Martins-Gomes, C.; Steck, J.; Keller, J.; Bunzel, M.; Santos, J.A.; Nunes, F.M.; Silva, A.M. Phytochemical Composition and Antioxidant, Anti-Acetylcholinesterase, and Anti-α-Glucosidase Activity of Thymus carnosus Extracts: A Three-Year Study on the Impact of Annual Variation and Geographic Location. Antioxidants; 2023; 12, 668. [DOI: https://dx.doi.org/10.3390/antiox12030668]

41. Salehi, B.; Abu-Darwish, M.S.; Tarawneh, A.H.; Cabral, C.; Gadetskaya, A.V.; Salgueiro, L.; Hosseinabadi, T.; Rajabi, S.; Chanda, W.; Sharifi-Rad, M. et al. Thymus spp. Plants—Food Applications and Phytopharmacy Properties. Trends Food Sci. Technol.; 2019; 85, pp. 287-306. [DOI: https://dx.doi.org/10.1016/j.tifs.2019.01.020]

42. Méndez-Tovar, I.; Sponza, S.; Asensio-S-Manzanera, M.C.; Novak, J. Contribution of the Main Polyphenols of Thymus mastichina subsp. mastichina to Its Antioxidant Properties. Ind. Crops Prod.; 2015; 66, pp. 291-298. [DOI: https://dx.doi.org/10.1016/j.indcrop.2014.11.029]