Exploring the metabolite composition and biological activities of Thymus canoviridis via volatile and non-volatile fraction analysis

Thymus canoviridis, an endemic species in Türkiye, was studied for its chemical composition and biological activities, with emphasis on food-related applications. GC-MS analysis of the essential oil from aerial parts revealed an exceptionally high carvacrol content (99.9%), highlighting its potential as a natural preservative. LC-MS/MS profiling of ethanol extracts (aerial and root) identified rosmarinic acid as the dominant phenolic (885.53 ± 7.25 μg/g in aerial part; 721.08 ± 6.14 μg/g in root), along with notable levels of apigenin and quinic acid. The aerial extract showed higher total phenolic (114.39 ± 1.86 mg PEs/g) and flavonoid contents (47.80 ± 0.94 mg QE/g) than the root extract. In vitro antioxidant assays revealed strong activity for both extracts: the root was more active in DPPH (IC₅₀: 24.32 ± 0.84 μg/mL) and CUPRAC (A_0.5: 9.99 ± 0.02 μg/mL), while the aerial part extract was superior in ABTS (IC₅₀: 8.36 ± 0.05 μg/mL). The essential oil exhibited outstanding ABTS (IC₅₀: 0.43 ± 0.02 μg/mL) and CUPRAC (A_0.5: 3.36 ± 0.07 μg/mL) activity. Enzyme inhibition assays showed strong α-glucosidase inhibition (IC₅₀: 683.35 ± 3.75 μg/mL) by the oil and selective butyrylcholinesterase inhibition (69.61 ± 1.84% at 200 μg/mL). Antimicrobial tests demonstrated significant activity against Candida tropicalis (MIC: 19.53 μg/mL) and Staphylococcus aureus (MIC: 39.06 μg/mL). Taken together, T. canoviridis represents a promising source of bioactive compounds with antioxidant, antimicrobial, and enzyme-inhibitory properties suitable for functional food and clean-label preservation applications.

1 Introduction

The growing demand for natural and health-promoting ingredients in the food and nutraceutical industries has fueled the exploration of underutilized aromatic and medicinal plants. Members of the genus Thymus (Lamiaceae), comprising over 200 species primarily distributed across the Mediterranean region, have long been recognized for their culinary, aromatic, and therapeutic properties (1, 2). Many species of the genus Thymus (Lamiaceae) have long been consumed across Mediterranean cuisines—particularly in Türkiye and Greece—as herbal teas, aromatic spices in meat and dairy dishes, and natural preservatives in local food preparations (3, 4). However, despite extensive research on commonly used Thymus taxa such as T. vulgaris and T. serpyllum, certain endemic species from Anatolia remain chemically and biologically underexplored, representing untapped resources for the development of novel bioactive ingredients.

T. canoviridis Jalas is an endemic species distributed in limited areas of Türkiye (5). Although relatively less studied, this species is known to be used by local populations in infusions and as a seasoning herb in meat-based dishes, similar to other culinary Thymus taxa (3). Despite its traditional culinary use, comprehensive scientific studies investigating its phytochemical composition and biological functionality remain limited—particularly regarding (i) the comparative chemical composition of its aerial and root parts, (ii) the integration of volatile and non-volatile metabolite profiles, and (iii) the evaluation of its enzyme inhibitory mechanisms relevant to metabolic and neuroprotective health.

One of the distinguishing features of the Thymus genus is its chemotypic diversity, often characterized by essential oil profiles dominated by either thymol or carvacrol, two structurally related monoterpenic phenols with distinct bioactivities and sensory profiles (6). In the present study, T. canoviridis essential oil was found to contain an exceptionally high proportion of carvacrol, exceeding 99% of its volatile content. This unusually high carvacrol concentration suggests that T. canoviridis could serve as a valuable natural source of carvacrol for potential applications in phytotherapy, food preservation, and the development of functional ingredients. To our knowledge, such a high-purity carvacrol has not been previously reported for T. canoviridis, making it an important candidate for both phytochemical standardization and functional food innovation.

Carvacrol has gained significant attention in recent years due to its multifaceted biological properties, including antioxidant, antimicrobial, anti-inflammatory, and enzyme-inhibitory effects. These characteristics make it a valuable candidate for applications in functional foods, bioactive packaging, and natural preservatives (7, 8). Moreover, the interest in carvacrol has surged since the COVID-19 pandemic, owing to its supportive role in respiratory health and immune regulation, further promoting its incorporation into dietary supplements and phytotherapeutic formulations (9).

Phenolic compounds and essential oil components from Thymus species have been increasingly studied for their enzyme inhibitory activities, particularly against α-glucosidase, urease, acetylcholinesterase, and tyrosinase—enzymes linked to diabetes, neurodegenerative diseases, and skin disorders (10–13). The integration of such natural enzyme inhibitors into functional food systems has emerged as a promising strategy for metabolic health regulation and anti-aging nutrition.

In this first comprehensive study of T. canoviridis, we present an integrated metabolite and bioactivity profiling covering both volatile and non-volatile fractions. The essential oil composition was analyzed by GC-MS, while phenolic constituents of ethanol extracts from aerial and root parts were identified and quantified using LC-MS/MS. The antioxidant, antimicrobial, and enzyme inhibitory activities of these extracts were also evaluated. By combining chemical characterization with functional assays, this study expands the current phytochemical understanding of T. canoviridis and provides a scientific basis for its valorization as a bioactive and functional ingredient within the food and nutraceutical sectors.

2 Materials and methods

2.1 Chemicals and instruments

Phytochemical analysis of the aerial and root extracts, along with the essential oil of T. canoviridis, was conducted using LC-MS/MS (Shimadzu, Kyoto, Japan) and GC-MS (Agilent Technologies Inc., Santa Clara, CA, USA). Antioxidant, antimicrobial, and enzyme inhibitory activities were measured using a BioTek PowerWave XS microplate reader and a UV–Vis spectrophotometer. All analytical-grade reagents and standards used in the assays were obtained from Merck (Germany), Sigma-Aldrich (Germany), Applichem (Germany), and Fluka (Germany).

2.2 Plant material

The aerial parts and roots of T. canoviridis were collected during the flowering stage in June 2014 from Kemaliye, Erzincan province (Türkiye), located in Eastern Anatolia. The plant material was harvested by Assoc. Prof. Dr. Yeter Yeşil and Prof. Dr. Mehmet Boga. Taxonomic identification was confirmed by Assoc. Prof. Dr. Yeter Yeşil. A voucher specimen (ISTE: 104454) has been deposited at the Herbarium of Istanbul University, Faculty of Pharmacy. The collection site is characterized by a continental climate, with hot and dry summers and cold winters. The plant material was harvested from calcareous, rocky, and well-drained soil typical of the region's mountainous terrain.

2.3 Preparation of essential oil and extracts

2.3.1 Essential oil extraction

Aerial parts of T. canoviridis (100 g) were subjected to hydrodistillation with 500 mL of distilled water for 3 h using a Clevenger-type apparatus. The essential oil obtained was dried over anhydrous sodium sulfate and diluted with dichloromethane (1:3, v/v) prior to GC-MS analysis. A 1 μL aliquot was injected into the GC-MS system.

2.3.2 Preparation of ethanolic extracts

The plant was separated into aerial (11.24 g) and root (12.77 g) parts, ground with a high-speed blender, and macerated with ethanol (3 × volume) for 3 × 24 h (72 h). The pooled filtrates were evaporated to dryness under vacuum at 40 °C. Dried extracts were stored at −20 °C. For LC-MS/MS analysis, extracts were dissolved in methanol to a final concentration of 0.25 μg/mL and filtered through a 0.2 μm filter.

2.4 GC-MS analysis of essential oil

The essential oil was analyzed using an Agilent 7890A GC system coupled with a 5975 MSD. An HP-Innowax FSC column (60 m × 0.25 mm, 0.25 μm film thickness) was used for separation. The injector and detector were set at 250 and 280 °C, respectively. The oven temperature was programmed from 60 °C (1 min) to 190 °C at 20 °C/min (hold 60 min), then to 220 °C at 1 °C/min (hold 10 min). Helium was used as carrier gas (1 mL/min). Compound identification was based on retention indices calculated using co-injected n-alkanes (C8–C30) and mass spectral matching against NIST 05, Wiley 8, and in-house libraries. Quantification was expressed as relative peak areas. Methodology was adapted from Zengin et al. (14).

2.5 LC-MS/MS analysis of phenolic compounds

Phenolics in ethanol extracts were analyzed using a Shimadzu Nexera UHPLC system coupled with tandem MS. Separation was achieved using a C18 Inertsil ODS-4 column (150 × 4.6 mm, 3 μm) at 40 °C. Mobile phases consisted of water (5 mM ammonium formate + 0.1% formic acid) (A) and methanol with same additives (B). The gradient was: 0 min−40% B; 20 min−90% B; 24 min−40% B; 29 min—end. Flow rate: 0.5 mL/min; injection volume: 4 μL. Conditions were adapted from Ertaş et al. (15).

2.6 Determination of total phenolic and flavonoid contents

Total phenolic and flavonoid contents were determined using Folin–Ciocalteu and aluminum chloride colorimetric methods, respectively (16, 17). Results were expressed as μg pyrocatechol equivalents (PEs) and μg quercetin equivalents (QEs):

• Abs = 0.0409 × pyrocatechol (μg) + 0.0581 (R² = 0.9924).

• Abs = 0.0325 × quercetin (μg) – 0.0601 (R² = 0.9984).

2.7 Antioxidant activity assays

Antioxidant capacity was evaluated using DPPH, ABTS, and CUPRAC assays. Experimental procedures followed Ersoy et al. (18). Results for radical scavenging activities (DPPH and ABTS) are expressed as IC₅₀ values (μg/mL), while reducing power in the CUPRAC assay is expressed as A_0.5 (μg/mL), defined as the concentration of sample giving an absorbance of 0.5. The extracts were tested at concentrations of 10, 25, 50, and 100 μg/mL. Total phenolic and flavonoid contents were also determined and expressed as μg pyrocatechol equivalents (PEs) and μg quercetin equivalents (QEs) per mg extract, respectively.

2.8 Enzyme inhibitory activity assays

Enzyme inhibition activities were determined against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) (18), tyrosinase, urease, and α-glucosidase using previously described methods Ersoy et al. (19). Extracts were tested at four concentrations (25, 50, 100, and 200 μg/mL) to establish dose–response relationships. Results were expressed as % inhibition at a fixed concentration (200 μg/mL) for AChE, BChE, tyrosinase, and urease (20), while α-glucosidase inhibition (21) was presented as IC₅₀ values (μg/mL).

2.9 Antimicrobial activity assays

MICs of extracts were determined using the broth microdilution method against standard bacterial and yeast strains (22–24). Reference strains included: Staphylococcus aureus ATCC 29213, S. epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 4352, Proteus mirabilis ATCC 14153, Candida albicans ATCC 10231, C. parapsilosis ATCC 22019, and C. tropicalis ATCC 750. Standard antibiotics (e.g., cefuroxime-Na, ceftazidime, amikacin, clotrimazole) were used as controls. Extract concentrations ranging from 5000 to 1.25 μg/mL were tested.

2.10 Statistical analysis

All data were expressed as mean ± standard deviation (SD) of three independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey's Honestly Significant Difference (HSD) post-hoc test to determine differences among extract groups (TCA, TCR, TCEO). A p-value less than 0.05 was considered statistically significant. Analyses and visualizations were conducted using Python (v3.10) and the statsmodels and scipy libraries.

3 Results

3.1 Volatile compound profile of T. canoviridis essential oil (TCEO)

GC-MS analysis of the essential oil from the aerial parts of T. canoviridis led to the identification of 99 volatile compounds (Table 1). Among them, carvacrol was identified as the dominant constituent, accounting for 99.9% of the total volatile composition. The total ion chromatogram (TIC) is presented in Figure 1, illustrating the prominent peak corresponding to carvacrol. Other typical Thymus volatiles such as thymol, p-cymene, or γ-terpinene were either absent or detected in trace amounts.

Table 1

Table 1. Chemical composition of the essential oil from the aerial parts of Thymus canoviridis identified by GC-MS.

Figure 1

Figure 1. TIC Chromatogram of Thymus canoviridis essential oil.

3.2 Phenolic composition of ethanol extracts from T. canoviridis (TCA and TCR)

The LC-MS/MS analysis of the ethanol extracts from the aerial (TCA) and root (TCR) parts of T. canoviridis led to the detection of 21 and 20 phenolic compounds, respectively (Table 2). In total, 25 distinct phenolic compounds were identified across both plant parts, with notable quantitative and qualitative differences between them.

Table 2

Table 2. Quantitative content (μg/g dry extract) of phenolic compounds in ethanol extracts from aerial and root parts of T. canoviridis as determined by LC-MS/MS.

Among the compounds detected, nicotiflorin (8975.52 μg/g), quinic acid (6404.85 μg/g), and rosmarinic acid (2371.36 μg/g) were the most abundant in the aerial part. The TCA extract also contained high levels of naringenin (1028.79 μg/g), cosmosiin (760.90 μg/g), and apigenin (234.16 μg/g), along with caffeic acid (722.29 μg/g) and fumaric acid (645.65 μg/g).

In comparison, the TCR extract was characterized by rosmarinic acid (6,448.00 μg/g), hesperidin (99.85 μg/g), and caffeic acid (581.05 μg/g) as dominant components. Notably, several phenolic acids such as tr-ferulic acid (211.71 μg/g) and protocatechuic acid (218.35 μg/g) were also present in appreciable amounts. Flavonoids, including naringenin, cosmosiin, and nicotiflorin, were significantly less concentrated in the roots compared to the aerial parts.

These findings reflect a distinct partitioning of phenolic compounds between the aerial and root tissues of T. canoviridis, underscoring tissue-specific metabolic pathways and functional specialization.

3.3 Total phenolic, flavonoid content and antioxidant activity of T. canoviridis

The ethanol extracts of the aerial (TCA) and root (TCR) parts of T. canoviridis, along with the essential oil (TCEO), were evaluated for their total phenolic and flavonoid contents, as well as antioxidant activity using DPPH, ABTS, and CUPRAC assays (Table 3).

Table 3

Table 3. Total phenolic and flavonoid contents, and antioxidant activity (DPPH, ABTS, and CUPRAC assays) of ethanol extracts from aerial and root parts of T. canoviridis^*.

The TCR extract exhibited the highest total phenolic content with 144.12 ± 2.60 μg PEs/mg extract, followed by TCEO (64.19 ± 0.83 μg PEs/mg) and TCA (59.56 ± 0.87 μg PEs/mg). In contrast, the TCA extract showed the highest flavonoid content (35.35 ± 1.05 μg QEs/mg), which was roughly double that of the root extract (17.35 ± 0.52 μg QEs/mg). The essential oil had only minor flavonoid presence (4.31 ± 0.38 μg QEs/mg).

Antioxidant assays revealed notable differences in radical scavenging and reducing capacities between the samples. In the DPPH assay, the root extract (TCR) exhibited stronger activity (IC₅₀ = 24.32 ± 0.84 μg/mL) compared to the aerial extract (TCA, IC₅₀ = 33.08 ± 0.54 μg/mL). However, the essential oil (TCEO) demonstrated the weakest DPPH activity (IC₅₀ = 273.42 ± 1.65 μg/mL).

Interestingly, in the ABTS assay, TCEO was the most potent (IC₅₀ = 0.43 ± 0.02 μg/mL), outperforming both extracts (TCA: 8.36 ± 0.05 μg/mL, TCR: 9.72 ± 0.08 μg/mL), and even showing comparable results with standard compounds such as BHT and BHA. This strong ABTS activity may be attributed to the hydrophobic antioxidant potential of carvacrol-rich oil.

In the CUPRAC assay, TCR again showed superior reducing ability (A_0.5 = 9.99 ± 0.02 μg/mL), followed by TCA (10.59 ± 0.04 μg/mL) and TCEO (3.36 ± 0.07 μg/mL). Among standard antioxidants, BHA showed the strongest activity in all assays.

According to the one-way ANOVA followed by Tukey HSD test, the phenolic content of TCR was significantly higher than TCA and TCEO (p < 0.001), while the flavonoid content of TCA was significantly higher than TCR and TCEO (p < 0.001). TCEO exhibited significantly lower IC₅₀ in the ABTS assay compared to TCA and TCR (p < 0.001), despite having the lowest total phenolic and flavonoid content. These findings suggest that the ABTS scavenging effect of TCEO may be attributed to non-phenolic compounds present in the essential oil.

These findings highlight a tissue-specific antioxidant profile, with the root extract (TCR) being particularly rich in phenolic acids contributing to DPPH and CUPRAC activity, while the essential oil's hydrophobic nature and carvacrol content make it more effective in the ABTS system.

3.4 Enzyme Inhibition activity of T. canoviridis

The ethanol extracts from the aerial (TCA) and root (TCR) parts, along with the essential oil (TCEO) of T. canoviridis, were evaluated for their inhibitory effects on several clinically relevant enzymes, including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, urease, and α-glucosidase (Table 4).

Table 4

Table 4. Enzyme inhibition activity of ethanol extracts from aerial and root parts of T. canoviridis^a.

Among the tested samples, the essential oil (TCEO) demonstrated the highest inhibition against both AChE (69.75 ± 1.14%) and BChE (69.61 ± 1.84%), showing comparable effectiveness to the standard drug galanthamine (AChE: 78.92 ± 1.04%; BChE: 78.22 ± 0.58%). The aerial extract (TCA) exhibited moderate BChE inhibition (33.93 ± 0.67%), while the root extract (TCR) showed weaker inhibition (13.89 ± 0.62%). None of the ethanol extracts showed measurable AChE activity at 200 μg/mL.

The α-glucosidase inhibition assay revealed that TCEO exhibited moderate activity with an IC₅₀ value of 683.35 ± 3.75 μg/mL, comparable to the standard acarbose (IC₅₀ = 667.40 ± 2.20 μg/mL). TCA displayed weaker inhibition (IC₅₀ = 954.55 ± 1.48 μg/mL), and TCR showed no significant activity (IC₅₀ > 1800 μg/mL).

None of the samples exhibited noteworthy activity against tyrosinase or urease at the tested concentration (200 μg/mL). In contrast, the standard inhibitors kojic acid and thiourea showed 95.05 ± 0.37% and 98.37 ± 0.40% inhibition against tyrosinase and urease, respectively.

The essential oil (TCEO) exhibited significantly higher BChE inhibition compared to both ethanol extracts (TCA and TCR) (p < 0.001). This difference was confirmed by one-way ANOVA and Tukey HSD post-hoc tests. The potent BChE inhibition by TCEO, despite its low phenolic/flavonoid content, suggests the presence of active volatile compounds selectively targeting BChE. Further phytochemical and in silico analyses are warranted to elucidate the underlying mechanism.

These results underline the potential of carvacrol-rich essential oil of T. canoviridis as a natural cholinesterase and α-glucosidase inhibitor, suggesting possible applications in managing neurodegenerative and metabolic disorders.

3.5 Antimicrobial activity of T. canoviridis

The ethanol extracts from the aerial (TCA) and root (TCR) parts, as well as the essential oil (TCEO) of T. canoviridis, were assessed for their antimicrobial potential against a panel of Gram-positive and Gram-negative bacteria and Candida species, using the microbroth dilution method (Table 5). Among the tested samples, the root extract (TCR) showed the strongest antibacterial activity, particularly against Staphylococcus aureus ATCC 29213 (MIC: 39.06 μg/mL) and Candida tropicalis ATCC 750 (MIC: 39.06 μg/mL). The aerial extract (TCA) also exhibited activity against S. aureus (MIC: 156.25 μg/mL) and C. tropicalis (MIC: 19.53 μg/mL), indicating notable anti-staphylococcal and antifungal properties.

Table 5

Table 5. Minimum inhibitory concentrations (MIC, μg/mL) of ethanol extracts from aerial and root parts of T. canoviridis against tested microorganisms.

The essential oil (TCEO), although rich in carvacrol, showed limited antimicrobial efficacy with relatively high MIC values (≥2500 μg/mL) for Gram-negative bacteria including E. coli, K. pneumoniae, and P. mirabilis. Against Gram-positive bacteria, TCEO was moderately active, with MICs of 5000 μg/mL against S. aureus and S. epidermidis, and 10,000 μg/mL against E. faecalis. Interestingly, TCEO exhibited the highest antifungal activity against C. albicans (MIC: 312.5 μg/mL), followed by C. parapsilosis (625 μg/mL) and C. tropicalis (1,250 μg/mL). This is consistent with the known antifungal properties of carvacrol-rich essential oils.

As positive controls, the following standard antimicrobials were used in parallel assays: Cefuroxime-Na (1.2 μg/mL for S. aureus ATCC 29213), Cefuroxime (9.8 μg/mL for S. epidermidis ATCC 12228), Amikacin (128 μg/mL for E. faecalis ATCC 29212), Ceftazidime (2.4 μg/mL for P. aeruginosa ATCC 27853), Cefuroxime-Na (4.9 μg/mL for E. coli ATCC 25922 and K. pneumoniae 4352), Cefuroxime-Na (2.4 μg/mL for P. mirabilis ATCC 14153), Clotrimazole (4.9 μg/mL for C. albicans ATCC 10231), Amphotericin B (0.5 μg/mL for C. parapsilosis ATCC 22019 and 1 μg/mL for C. tropicalis ATCC 750).

The observed stronger activity of the polar ethanol extracts, particularly TCR, against Gram-positive bacteria and Candida strains compared to the essential oil may be attributed to the high content of phenolic compounds such as rosmarinic acid, caffeic acid, and flavonoids, which possess synergistic antimicrobial mechanisms.

These findings suggest that both polar and non-polar fractions of T. canoviridis exhibit differential antimicrobial profiles, with ethanol extracts being more effective against Gram-positive pathogens and Candida spp., while the essential oil retains moderate antifungal activity. Such profiles support the potential use of T. canoviridis in developing food preservatives or natural antimicrobial agents for health-related applications.

4 Discussion

The present study aimed to comprehensively evaluate the chemical composition and biological activities of the essential oil and ethanol extracts obtained from the aerial and root parts of T. canoviridis, an endemic species to Türkiye. Essential oil profiling via GC-MS, phenolic compound quantification using LC-MS/MS, and in vitro biological activity assessments including antioxidant, enzyme inhibitory, and antimicrobial assays were conducted. The findings provide new insights into the phytochemical richness and bioactivity spectrum of this relatively understudied Thymus species. The exceptionally high carvacrol content observed in the essential oil, along with distinct phenolic profiles and notable bioactivities, underscore the potential of T. canoviridis as a valuable source of bioactive compounds for pharmaceutical, nutraceutical, and food industry applications. These results also contribute to the growing body of knowledge regarding the chemotaxonomic diversity within the Thymus genus and highlight the importance of regional phytochemical investigations.

In the essential oil composition of T. canoviridis analyzed in the present study, carvacrol was identified as the predominant compound, remarkably constituting 99.9% of the total content. Existing studies indicate that although a few species may exhibit carvacrol concentrations exceeding 90%, such instances are exceedingly rare. For example, Nooshkam et al. (25) reported that carvacrol was the major component of the essential oils of Satureja khuzistanica and S. rechingeri from Iran, ranging from 95.9% to 96.7%. Their investigation across multiple localities consistently revealed extraordinarily high carvacrol concentrations. Similarly, Figuérédo et al. (26) identified Origanum dubium (82.7%) and O. minutiflorum (86.1%) as the most carvacrol-rich oregano species among Mediterranean taxa, including those from Türkiye, Greece, Italy, and Morocco. Other species such as O. compactum, O. dictamnus, O. onites, and O. vulgare ssp. hirtum also contained high carvacrol levels, ranging from 55.9% to 76.4%. The first report on the essential oil composition of T. canoviridis was published in 1998, based on samples collected from Bayburt. In that study, carvacrol was identified as the major component, but at a relatively modest concentration of 29.51%, alongside significant amounts of geraniol (13.25%) and thymol (9.49%) (27). More recent investigations have demonstrated much higher carvacrol levels in this species: 52.87% in the study by Güven et al. (28) and 72.88% in that of Yigitkan and Firat (29). The striking variation between earlier and current results may be attributed to chemical polymorphism. For instance, Aboukhalid et al. (30) analyzed essential oils from 527 individuals of O. compactum collected from 88 different locations. Some samples contained no carvacrol at all, while others reached up to 96.3%. Pirbalouti et al. (31) also explored this phenomenon by examining ten populations of S. khuzistanica from various Iranian localities. Although all samples were carvacrol-dominant, concentrations varied significantly (42.5–94.8%), with higher levels generally observed in plants growing at elevated altitudes. In another study, Emrahi et al. (32) demonstrated that moderate water stress significantly increased carvacrol content (by up to 23%) in O. vulgare subspecies.

Türkiye is a leading exporter of oregano oil, derived from multiple species—including Origanum, Thymus, Coridothymus, Thymbra, Satureja, and Lippia—that are widely cultivated and processed for essential oil production. Carvacrol is considered the primary bioactive constituent in these species. Among them, Origanum taxa are often highlighted for their high carvacrol content, with concentrations reported to reach up to 84% in Turkish species. However, certain Thymus species such as T. migricus and T. kotschyanus var. glabrescens have also been found to be rich in carvacrol, with levels up to 78% (33). To the best of our knowledge, the present study is the first to report a Thymus species with carvacrol content as high as 99.9%, possibly making T. canoviridis the first known Thymus taxon composed almost entirely of a single compound. Such monocomponent essential oils are exceptionally uncommon. As a notable exception, Tsuruoka et al. (34) identified 4aα,7α,7aα-nepetalactone as the sole compound in the essential oil of Nepeta sibirica. Extensive studies have characterized the essential oil compositions of various Thymus species from Türkiye, revealing considerable chemotypic diversity. For instance, Boga et al. (11) reported that camphor was predominant in T. convolutus (12.7%) and T. sipyleus (13.1%), while T. fallax was rich in bicyclogermacrene (21.5%). In T. kotschyanus var. kotschyanus, carvacrol (48.5%) and thymol (22.5%) were the main constituents. In contrast, T. haussknechtii was found to contain 28.2% carvacrol. Meanwhile, Küçükbay et al. (35) reported that T. kotschyanus var. kotschyanus primarily consisted of geraniol (55.0–59.1%) and geranyl acetate (27.1–28.8%). Other varieties, such as T. kotschyanus var. eriophorus and T. kotschyanus var. glabrascens, were characterized by carvacrol (57.2%) and its biosynthetic precursor p-cymene (11.0%). Additionally, T. serpyllum was shown to contain thymol (1702 mg/100 g) and carvacrol (179 mg/100 g) as major components (36). In another study, the endemic T. argaeus was reported to be rich in linalool, α-terpineol, and linalyl acetate (37). These findings collectively underscore the remarkable variability in essential oil profiles among Thymus species, which is influenced by a multitude of genetic, geographic, and environmental factors.

In the present study, LC-MS/MS analysis revealed that rosmarinic acid was the major phenolic constituent in both aerial and root ethanol extracts of T. canoviridis, accompanied by notable amounts of apigenin, quinic acid, and other hydroxycinnamic acid derivatives. A previous UPLC-MS/MS study investigated the water and methanol extracts of T. canoviridis leafy flowers, revealing that the methanol extract was notably richer in phenolic compounds. In comparison with the current study, there are considerable consistencies in the phytochemical profiles. Specifically, secondary metabolites such as apigenin, rosmarinic acid, and quinic acid were consistently identified across all tested extracts. Furthermore, compounds like naringenin, caffeic acid, fumaric acid, and ferulic acid were present in three out of the four extracts analyzed (28). Among the Thymus species naturally growing in Türkiye, rosmarinic acid stands out as a frequently occurring and dominant phenolic constituent. For instance, T. nummularius was reported to contain a particularly high amount of rosmarinic acid, reaching 131.899 ± 6.463 mg/g dry extract (15). Similarly, T. pectinatus and T. convolutus were also identified as rosmarinic acid-rich species, with this compound being the major phenolic in both (38). In another study, T. cariensis (2501.3 ± 178.34 μg/g), T. praecox subsp. grossheimii (2166.60 ± 154.48 μg/g), and T. pubescens (2499.32 ± 178.20 μg/g) were also shown to accumulate considerable levels of rosmarinic acid in their aerial parts (13). Additionally, other species such as T. leucostomus (34.8%), T. brachychilus (15.801 μg/g), T. argaeus (6.574 μg/g), T. fallax (9631.71 ± 686.74 μg/g), T. haussknechtii (10579.5 ± 754.32 μg/g), T. kotschyanus var. kotschyanus (1800.18 ± 128.35 μg/g), and T. sipyleus (2924.30 ± 208.50 μg/g) were also characterized as rosmarinic acid-rich taxa (11, 37–40).

In our study, the total phenolic content of T. canoviridis extracts was notably higher than the total flavonoid content, with the aerial part extract exhibiting slightly greater phenolic accumulation compared to the root extract. In the study by Köksal et al. (41), the ethanol extract of T. vulgaris was found to contain 158 μg gallic acid/mg extract of total phenolics and 36.6 μg quercetin/mg extract of total flavonoids. Similarly, Ertaş et al. (15) reported that in T. nummularius, the total phenolic content was higher than the total flavonoid content, consistent with the results of the present study. Niculae et al. (42) also demonstrated high total phenolic content in a 70% ethanol extract of T. marschallianus, further supporting the trend seen across different Thymus species.

4.1 Antioxidant activity

In the present study, both ethanol extracts and the essential oil of T. canoviridis exhibited substantial antioxidant activity, as evidenced by their performance in DPPH, ABTS, and CUPRAC assays, with the aerial and root extracts showing differential strengths across methods, and the essential oil demonstrating remarkable ABTS scavenging capacity despite its limited DPPH activity. In terms of antioxidant activity, the root extract (DPPH IC₅₀: 24.32 ± 0.84 μg/mL) exhibited stronger scavenging potential than the aerial part extract (DPPH IC₅₀: 33.08 ± 0.54 μg/mL). For the ABTS assay, the aerial part extract (ABTS IC₅₀: 8.36 ± 0.05 μg/mL) outperformed the root extract (ABTS IC₅₀: 9.72 ± 0.08 μg/mL). The root extract also demonstrated superior reducing power in the CUPRAC assay (A_0.5: 9.99 ± 0.02 μg/mL) compared to the aerial parts (A_0.5: 10.59 ± 0.04 μg/mL). However, both extracts showed weaker activity than synthetic standards such as BHA, α-tocopherol (α-TOC), and BHT. Overall, the root extract showed better performance in both DPPH scavenging and cupric ion reduction, while the aerial extract showed better ABTS scavenging activity. Despite being less potent than synthetic standards, both extracts exhibited considerable antioxidant potential, particularly in the ABTS assay. The essential oil of T. canoviridis (TCEO) had a significantly higher DPPH IC₅₀ (273.42 ± 1.65 μg/mL), indicating relatively weak activity compared to BHA (7.88 ± 0.20 μg/mL), α-TOC (16.30 ± 0.79 μg/mL), and BHT (58.86 ± 0.50 μg/mL). In contrast, TCEO exhibited a remarkably strong ABTS IC₅₀ value (0.43 ± 0.02 μg/mL), outperforming all tested synthetic standards. CUPRAC activity was also high (A_0.5: 3.36 ± 0.07 μg/mL), though not superior to BHA and BHT. These results emphasize that while TCEO has limited efficacy in DPPH scavenging, it is highly effective in ABTS scavenging and cupric ion reduction. The discrepancies across assays highlight the importance of using multiple antioxidant evaluation methods to assess different modes of action. In line with the current findings, Üstüner et al. (43) reported that the ethanol extract of T. sipyleus subsp. rosulans inhibited 50% of DPPH radicals at 104.91 μg/mL. Akin and Saki (44) showed that ethanol extract from T. vulgaris had 77% inhibition at 400 μg/mL, while Köksal et al. (41) noted a DPPH IC₅₀ of 12.1 μg/mL. The methanol extract of T. nummularius exhibited exceptional DPPH activity (IC₅₀: 5.73 μg/mL) exceeding that of BHT and α-TOC (15). These results align well with the current study, where both T. canoviridis extracts displayed notable DPPH scavenging potential. Eroglu Özkan et al. (13) demonstrated DPPH IC₅₀ values of 34.97, 51.44, and 41.80 μg/mL for T. cariensis and the aerial/root extracts of T. pubescens, respectively, aligning with the present values for T. canoviridis. Regarding ABTS activity, the IC₅₀ values of 8.36 and 9.72 μg/mL for aerial and root extracts were better than that of α-TOC. Küçükaydin et al. (12) reported comparable results for T. cariensis and T. cilicicus. Köksal et al. (41) reported a higher ABTS IC₅₀ of 54.08 μg/mL for T. vulgaris, whereas Bendjabeur et al. (45) found 7.44 μg/mL for T. algeriensis. Likewise, Boga et al. (11) showed values between 6.48 and 18.75 μg/mL for five Thymus species, with the most active being the root extract of T. haussknechtii. In the CUPRAC assay, both extracts of T. canoviridis were more effective than α-TOC. The ethanol extract of T. algeriensis showed an A_0.5 of 19.40 μg/mL (45), while T. nummularius also surpassed α-TOC in efficacy (15). Boga et al. (11) and Eroglu Özkan et al. (13) further supported this trend, reporting high CUPRAC activities in various Thymus taxa. In the present study, the essential oil of T. canoviridis demonstrated notable inhibitory activity against the α-glucosidase enzyme, with an IC₅₀ value of 683.35 ± 3.75 μg/mL—nearly comparable to that of the reference compound acarbose (IC₅₀ = 667.40 ± 2.20 μg/mL). This finding highlights the potential of T. canoviridis essential oil, which comprises 99.9% carvacrol, as a promising candidate for further antidiabetic investigations.

4.2 Enzyme inhibition activity

In the present study, the essential oil of T. canoviridis, characterized by an exceptionally high carvacrol content (99.9%), exhibited a strong α-glucosidase inhibitory effect, with an IC₅₀ value comparable to that of the reference drug acarbose. For instance, Salazar et al. (46) reported that Origanum vulgare essential oil, containing high levels of thymol and carvacrol, effectively inhibited α-glucosidase, and synthetic derivatives of these compounds exhibited potent activity through a mixed-type inhibition mechanism. Similarly, Singh et al. (47) emphasized the relevance of oxygenated monoterpenes like thymol and carvacrol in α-glucosidase inhibition. Ali (48) investigated the essential oil of T. vulgaris, composed primarily of thymol (55.88%), linalool (13.71%), carvacrol (8.36%), and p-cymene (6.00%). The oil showed potent α-glucosidase inhibitory activity, with an IC₅₀ value of 125.1 ± 4.25 μg/mL, further supporting the bioactivity of carvacrol-rich compositions.

In our study, T. canoviridis essential oil displayed selective cholinesterase inhibition, showing no significant activity against acetylcholinesterase (AChE) but exhibiting a strong inhibitory effect on butyrylcholinesterase (BChE), with 69.61 ± 1.84% inhibition at 200 μg/mL. The notable BChE inhibition shown by TCEO can be ascribed to its rich content of oxygenated monoterpenes such as carvacrol and borneol, which may interact favorably with the enzyme's broader active site, consistent with earlier findings on Thymus species. Regarding cholinesterase inhibition, our findings align with earlier literature reporting selective inhibition patterns. While T. canoviridis essential oil exhibited no notable activity against acetylcholinesterase (AChE), it significantly inhibited butyrylcholinesterase (BChE) with 69.61 ± 1.84% inhibition at 200 μg/mL. This pattern has also been observed in other Thymus taxa. For example, Boga et al. (11) reported that the ethanol extract from the aerial parts of T. fallax displayed notable BChE inhibitory activity (34.48 ± 0.60%) but was inactive against AChE and tyrosinase. Kindl et al. (49) investigated the AChE inhibition profile of various Thymus species, including T. longicaulis, T. praecox subsp. polytrichus, T. pulegioides, T. serpyllum subsp. serpyllum, T. striatus, and T. vulgaris, using the Ellman colorimetric method. The extracts exhibited dose-dependent AChE inhibition, ranging from 10 to 28%, 23 to 39%, and 64 to 86% at concentrations of 0.25, 0.5, and 1 mg/mL, respectively. Similarly, Ekin et al. (50) found that the ethanol extract of T. zygioides var. lycaonicus showed no activity against AChE at 2000 μg/mL but displayed significant BChE inhibition (30.92 ± 1.44%). Orhan et al. (51) also reported that the ethanol extract of T. praecox subsp. caucasicus var. caucasicus was inactive against AChE, even at 1000 μg/mL, corroborating the selectivity trend seen in our results. Collectively, the findings support the growing body of evidence that Thymus species, particularly their essential oils or ethanol extracts, may preferentially target BChE or α-glucosidase over AChE, suggesting selective enzyme modulation that could be valuable in managing neurodegenerative or metabolic disorders.

4.3 Antimicrobial activity

In the present study, the antimicrobial potential of T. canoviridis was assessed through the broth microdilution method, following the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The Minimum Inhibitory Concentration (MIC) values were determined against a panel of ten pathogenic strains, including six bacterial (Gram-positive and Gram-negative) and four fungal species. These included Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, Candida albicans, Candida parapsilosis, and Candida tropicalis. Among the tested extracts, the ethanol extract of the root part (TCR) showed the most pronounced antibacterial activity against S. aureus, with a MIC value of 39.06 μg/mL, while the aerial part (TCA) demonstrated moderate inhibition at 156.25 μg/mL. Notably, both aerial and root ethanol extracts exhibited strong antifungal activity against C. tropicalis, with MIC values of 19.53 μg/mL and 39.06 μg/mL, respectively, indicating remarkable sensitivity of this fungal strain to T. canoviridis phytochemicals. These findings are consistent with previous literature that underscores the antimicrobial potential of various Thymus species. For example, in a study by Oztürk et al. (52), the methanol extract of T. fallax demonstrated broad-spectrum antibacterial activity with MICs ranging from 31.25 to 500 μg/mL against strains such as Arthrobacter atrocyaneus, Bacillus sphaericus, Enterobacter hormaechei, Staphylococcus cohni cohni, Pseudomonas syringae, and Kocuria rosea. Afonso et al. (53) reported strong activity of T. herba-barona, T. pseudolanuginosus, and T. caespititius against S. aureus, with remarkably low MIC values of 0.6, 1.6, and 3.5 μg/mL, respectively, suggesting that certain Thymus species possess compounds with potent Gram-positive antibacterial activity. Similarly, Naz et al. (54) demonstrated that T. linearis extracts showed inhibitory zones ranging from 12 to 23 mm against Salmonella typhi, S. aureus, and Citrobacter freundii. Boga et al. (11) emphasized the antifungal efficacy of T. convolutus and T. haussknechtii, particularly against C. tropicalis, with MIC values similar to those observed in the current study (19.53 μg/mL). Furthermore, Eroglu Özkan et al. (13) reported comparable antifungal activity of T. cariensis, T. praecox subsp. grossheimii, and T. pubescens against C. tropicalis, with MICs ranging from 19.53 to 78.12 μg/mL. Collectively, these results support the relevance of T. canoviridis as a promising natural antimicrobial agent, particularly in the context of fungal infections and Gram-positive bacterial pathogens such as S. aureus.

Given the essential oil of T. canoviridis contains carvacrol at a remarkably high level (99.9%), it can be considered as a promising natural bioactive agent. Due to carvacrol's well-documented antimicrobial, antifungal, antioxidant, and anti-inflammatory properties, the essential oil may find practical applications in food preservation, dermatological preparations, natural pesticides, and veterinary products as a natural and eco-friendly alternative to synthetic agents.

5 Conclusion

The present study provides a detailed phytochemical and biological evaluation of T. canoviridis, emphasizing its potential as a valuable ingredient for food and nutraceutical applications. The essential oil was found to be remarkably rich in carvacrol (99.9%), a compound widely recognized for its antimicrobial, antioxidant, and preservative properties. Ethanol extracts of both aerial and root parts demonstrated notable phenolic richness, particularly in rosmarinic acid and apigenin, contributing to their strong antioxidant capacities. The extracts and essential oil exhibited selective enzyme inhibitory activities, especially against butyrylcholinesterase and α-glucosidase, which may offer added value in the context of functional food formulations aimed at metabolic and cognitive health support. Moreover, the pronounced antifungal activity against Candida tropicalis and antibacterial effect against Staphylococcus aureus underline the potential of T. canoviridis-derived preparations as natural preservatives in food systems.

Overall, this work represents the first integrated investigation of both volatile and non-volatile fractions of T. canoviridis, highlighting its distinctive phytochemical and bioactivity profile. These findings contribute significantly to the limited scientific knowledge on endemic Anatolian Thymus species and establish a foundation for the future valorization of T. canoviridis as a promising functional ingredient in food and health-promoting applications.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.

Author contributions

EE: Investigation, Methodology, Writing – original draft. SE: Investigation, Methodology, Writing – original draft. EÇ: Investigation, Methodology, Writing – original draft. SI: Methodology, Writing – original draft. GT: Investigation, Methodology, Writing – original draft. GZ: Investigation, Methodology, Writing – original draft. EM: Investigation, Methodology, Writing – original draft. YY: Investigation, Methodology, Writing – original draft. HS: Investigation, Methodology, Writing – original draft. MB: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing. EE: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This research was supported by the Scientific Research Projects Unit of Batman University under project number BTÜBAP-2018-SYO-1. The authors acknowledge the financial support of DÜBAP (Dicle University Scientific Research Projects Coordinator) for the publication fees.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnut.2025.1675586/full#supplementary-material

References

1. Stahl-Biskup E, Saez F. Thyme: The Genus Thymus. Medicinal and Aromatic Plants—Industrial Profiles: Essential Oil Chemistry of Thymus. Boca Raton, FL: CRC Press (2002). p. 75–124.

Google Scholar

2. Khoury M, Stien D, Eparvier V, Ouaini N, El Beyrouthy M, Arnold N, et al. Report on the medicinal use of eleven Lamiaceae species in Lebanon and rationalization of their antimicrobial potential by examination of the chemical composition and antimicrobial activity of their essential oils. Evid-Based Complement Altern Med. (2016) 2016:2547169. doi: 10.1155/2016/2547169

PubMed Abstract | Crossref Full Text | Google Scholar

3. Hammoudi Halat D, Krayem M, Khaled S, Younes S. A focused insight into thyme: biological, chemical, and therapeutic properties of an indigenous Mediterranean herb. Nutrients. (2022) 14:2104. doi: 10.3390/nu14102104

PubMed Abstract | Crossref Full Text | Google Scholar

4. Jalil B, Pischel I, Feistel B, Suarez C, Blainski A, Spreemann R, et al. Wild thyme (Thymus serpyllum L.): a review of the current evidence of nutritional and preventive health benefits. Front Nutr. (2024) 11:1380962. doi: 10.3389/fnut.2024.1380962

Crossref Full Text | Google Scholar

5. POWO. Thymus canoviridis Jalas. Plants of the World Online. Royal Botanic Gardens, Kew. (2024). Available online at: https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:460998-1 (Accessed May 24, 2025).

Google Scholar

6. Etri K, Pluhár Z. Exploring chemical variability in the essential oils of the thymus genus. Plants. (2024) 13:1375. doi: 10.3390/plants13101375

PubMed Abstract | Crossref Full Text | Google Scholar

7. Sharifi-Rad M, Varoni EM, Iriti M, Martorell M, Setzer WN, Contreras MM, et al. Carvacrol and human health: a comprehensive review. Phytother Res. (2018) 32:1675–87. doi: 10.1002/ptr.6103

PubMed Abstract | Crossref Full Text | Google Scholar

8. Bomfim Nda S, Nakassugi LP, Oliveira JFP, Kohiyama CY, Mossini SAG, Grespan R, et al. Antifungal activity and inhibition of fumonisin production by Rosmarinus officinalis L. essential oil in Fusarium verticillioides (Sacc) Nirenberg. Food Chem. (2015) 166:330–6. doi: 10.1016/j.foodchem.2014.06.019

PubMed Abstract | Crossref Full Text | Google Scholar

9. Baser KHC, Haskologlu IC, Erdag E. An updated review of research into carvacrol and its biological activities. Rec Nat Prod. (2025) 19:308–49. doi: 10.25135/rnp.504.2502.3428

Crossref Full Text | Google Scholar

10. Afonso AF, Pereira OR, Cardoso SM. Health-promoting effects of thymus phenolic-rich extracts: antioxidant, anti-inflammatory and antitumoral properties. Antioxidants. (2020) 9:814. doi: 10.3390/antiox9090814

PubMed Abstract | Crossref Full Text | Google Scholar

11. Boga M, Eroglu Özkan E, Ersoy E, Tuncay E, Yeşil Çantürk Y, Çinar E. Identification and quantification of phenolic and volatile constituents in five different Anatolian thyme species using LC–MS/MS and GC-MS, with biological activities. Food Biosci. (2021) 43:101141. doi: 10.1016/j.fbio.2021.101141

Crossref Full Text | Google Scholar

12. Küçükaydin S, Çayan F, Tel-Çayan G, Duru ME. HPLC-DAD phytochemical profiles of Thymus cariensis and T. cilicicus with antioxidant, cytotoxic, anticholinesterase, anti-urease, anti-tyrosinase, and antidiabetic activities. S Afr J Botany. (2021) 143:155–63. doi: 10.1016/j.sajb.2021.07.018

Crossref Full Text | Google Scholar

13. Eroglu Özkan E, Ersoy E, Yeşil Çantürk Y, Mataraci Kara E, Çinar E, Sahin H, et al. The therapeutic potential of ethnomedicinally important Anatolian thyme species: a phytochemical and biological assessment. Front Pharmacol. (2022) 13:923063. doi: 10.3389/fphar.2022.923063

PubMed Abstract | Crossref Full Text | Google Scholar

14. Zengin G, Sarikürkçü C, Aktümsek A, Ceylan R. Antioxidant potential and inhibition of key enzymes linked to Alzheimer's diseases and diabetes mellitus by monoterpene-rich essential oil from Sideritis galatica Bornm. endemic to Turkey. Rec Nat Prod. (2016) 10:195–206.

Google Scholar

15. Ertaş A, Boga M, Yilmaz MA, Yeşil Y, Tel G, Temel H, et al. A detailed study on the chemical and biological profiles of essential oil and methanol extract of Thymus nummularius (Anzer tea): Rosmarinic acid. Ind Crops Prod. (2015) 67:336–45. doi: 10.1016/j.indcrop.2015.01.064

Crossref Full Text | Google Scholar

16. Moreno MIN, Isla MI, Sampietro AR, Vattuone MA. Comparison of the free radical-scavenging activity of propolis from several regions of Argentina. J Ethnopharmacol. (2000) 71:109–14. doi: 10.1016/S0378-8741(99)00189-0

PubMed Abstract | Crossref Full Text | Google Scholar

17. Boga M, Ertaş A, Eroglu-Özkan E, Kizil M, Çeken B, Topçu G. Phytochemical analysis, antioxidant, antimicrobial, anticholinesterase and DNA protective effects of Hypericum capitatum var. capitatum extracts. S Afr J Botany. (2016) 104:249–57. doi: 10.1016/j.sajb.2016.02.204

Crossref Full Text | Google Scholar

18. Ersoy E, Eroglu-Özkan E, Boga M, Mat A. Evaluation of in vitro biological activities of three Hypericum species (H. calycinum, H confertum, and H perforatum). S Afr J Botany. (2020) 130:141–7. doi: 10.1016/j.sajb.2019.12.017

Crossref Full Text | Google Scholar

19. Ersoy E, Eroglu-Özkan E, Boga M, Yilmaz MA, Mat A. Anti-aging potential and anti-tyrosinase activity of three Hypericum species with focus on phytochemical composition by LC–MS/MS. Ind Crops Prod. (2019) 141:111735. doi: 10.1016/j.indcrop.2019.111735

Crossref Full Text | Google Scholar

20. Zahid H, Rizwani GH, Kamil A, Shareef H, Tasleem S, Khan A, et al. Anti-urease activity of Mimusops elengi Linn (Sapotaceae). European J Med Plants. (2015) 6:223–30. doi: 10.9734/EJMP/2015/12240

Crossref Full Text | Google Scholar

21. Demirci Kayiran S, Tavli ÖF, Mataraci Kara E, Kaplan A, Sahin H, Boga M, et al. Hypericum empetrifolium subsp. empetrifolium: An assessment of its antifungal, antidiabetic, anti-aging, and neuroprotective potential. Front Pharmacol. (2025) 16:1618761. doi: 10.3389/fphar.2025.1618761

PubMed Abstract | Crossref Full Text | Google Scholar

22. Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard–Second Edition; M27-A2. Wayne, PA: Clinical and Laboratory Standards Institute (CLSI) (1997).

Google Scholar

23. Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 7th ed.; Approved Standard M7-A7 (2006). Wayne, PA, USA: Clinical and Laboratory Standards Institute (CLSI).

Google Scholar

24. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing: 7th Informational Supplement; M100-S20 (2010). Wayne, PA: Clinical and Laboratory Standards Institute (CLSI).

Google Scholar

25. Nooshkam A, Mumivand H, Hadian J, Alemardan A, Morshedloo MR. Drug yield and essential oil and carvacrol contents of two species of Satureja (S. khuzistanica Jamzad and S rechingeri Jamzad) cultivated in two different locations. J Appl Res Med Aromat Plants. (2017) 6:126–30. doi: 10.1016/j.jarmap.2017.04.002

Crossref Full Text | Google Scholar

26. Figuérédo G, Cabassu P, Chalchat JC, Pasquier B. Studies of Mediterranean oregano populations. VI: Chemical composition of essential oils of Origanum elongatum Emberger et Maire from Morocco. J Essential Oil Res. (2006) 18:278–80. doi: 10.1080/10412905.2006.9699087

Crossref Full Text | Google Scholar

27. Başer KHC, Kirimer N, Tümen G, Duman H. Composition of the essential oil of Thymus canoviridis Jalas. J Essential Oil Res. (1998) 10:199–200. doi: 10.1080/10412905.1998.9700879

Crossref Full Text | Google Scholar

28. Güven L, Can H, Ertürk A, Demirkaya Miloglu F, Koca M, Ince F, et al. Comprehensive metabolic profiling of Thymus canoviridis (endemic) and Thymus pubescens var. pubescens using UPLC-MS/MS and evaluation of their antioxidant activities, enzyme inhibition abilities, and molecular docking studies. S Afr J Botany. (2024) 165:478–93. doi: 10.1016/j.sajb.2023.12.015

Crossref Full Text | Google Scholar

29. Yigitkan S, Firat M. Essential oil contents and biological activities of Thymus canoviridis Jalas and Thymus sipyleus Boiss. Ankara Üniversitesi Eczacilik Fakültesi Dergisi. (2024) 48:1068–77. doi: 10.33483/jfpau.1484485

Crossref Full Text | Google Scholar

30. Aboukhalid K, Lamiri A, Agacka-Mołdoch M, Doroszewska T, Douaik A, Bakha M, et al. Chemical polymorphism of Origanum compactum grown in all natural habitats in Morocco. Chem Biodiv. (2016) 13:907–26. doi: 10.1002/cbdv.201500511

PubMed Abstract | Crossref Full Text | Google Scholar

31. Pirbalouti AG, Moalem E, Yousefi M, Malekpoor F, Yousef-Naanaie S. Influence of ecological factors on carvacrol content of Satureja khuzestanica Jamzad. J Essential Oil Bear Plants. (2011) 14:630–8. doi: 10.1080/0972060X.2011.10643982

Crossref Full Text | Google Scholar

32. Emrahi R, Morshedloo MR, Ahmadi H, Javanmard A, Maggi F. Intraspecific divergence in phytochemical characteristics and drought tolerance of two carvacrol-rich Origanum vulgare subspecies: subsp. hirtum and subsp gracile. Ind Crops Prod. (2021) 168:113557. doi: 10.1016/j.indcrop.2021.113557

Crossref Full Text | Google Scholar

33. Baser KHC. Biological and pharmacological activities of carvacrol and carvacrol bearing essential oils. Curr Pharm Des. (2008) 14:3106–19. doi: 10.2174/138161208786404227

PubMed Abstract | Crossref Full Text | Google Scholar

34. Tsuruoka T, Bekh-Ochir D, Kato F, Sanduin S, Shataryn A, Ayurzana A, et al. The essential oil of Mongolian Nepeta sibirica: A single component and its biological activities. J Essential Oil Res. (2012) 24:555–9. doi: 10.1080/10412905.2012.729925

Crossref Full Text | Google Scholar

35. Küçükbay F, Kuyumcu E, Celen S, Azaz AD, Arabaci T, Yildiz B. Chemical composition and antimicrobial and antioxidant activities of three Turkish thyme essential oils. J Essential Oil Bear Plants. (2013) 16:661–71. doi: 10.1080/0972060X.2013.854498

Crossref Full Text | Google Scholar

36. Sonmezdag AS, Kelebek H, Selli S. Characterization of aroma-active and phenolic profiles of wild thyme (Thymus serpyllum) by GC-MS-Olfactometry and LC-ESI-MS/MS. J Food Sci Technol. (2016) 53:1957–65. doi: 10.1007/s13197-015-2144-1

PubMed Abstract | Crossref Full Text | Google Scholar

37. Sagdic O, Ozkan G, Aksoy A, Yetim H. Bioactivities of essential oil and extract of Thymus argaeus, Turkish endemic wild thyme. J Sci Food Agric. (2009) 89:1537–41. doi: 10.1002/jsfa.3513

Crossref Full Text | Google Scholar

38. Akman TÇ Simşek S Akşit Z Akşit H Aydin A Tüfekçi AR . Liquid chromatography–tandem mass spectrometry profile and antioxidant, antimicrobial, antiproliferative, and enzyme activities of Thymus pectinatus and Thymus convolutus: In vitro and in silico approach. J Sci Food Agric. (2024) 104:5313–27. doi: 10.1002/jsfa.13286

Crossref Full Text | Google Scholar

39. Zengin G, Atasagun B, Aumeeruddy MZ, Saleem H, Mollica A, Bahadori MB, et al. Phenolic profiling and in vitro biological properties of two Lamiaceae species (Salvia modesta and Thymus argaeus): A comprehensive evaluation. Ind Crops Prod. (2019) 128:308–14. doi: 10.1016/j.indcrop.2018.11.027

Crossref Full Text | Google Scholar

40. Fayez S, Fahmy NM, Zengin G, Yagi S, Uba AI, Eldahshan OA, et al. LC-MS/MS and GC-MS profiling, antioxidant, enzyme inhibition, and antiproliferative activities of Thymus leucostomus Hausskn. and Velen extracts. Arch Pharm. (2023) 356:e2300444. doi: 10.1002/ardp.202300444

Crossref Full Text | Google Scholar

41. Köksal E, Bursal E, Gülçin I, Korkmaz M, Çaglayan C, Gören AC, et al. Antioxidant activity and polyphenol content of Turkish thyme (Thymus vulgaris) monitored by liquid chromatography and tandem mass spectrometry. Int J Food Prop. (2017) 20:514–25. doi: 10.1080/10942912.2016.1168438

Crossref Full Text | Google Scholar

42. Niculae M, Hanganu D, Oniga I, Benedec D, Ielciu I, Giupana R, et al. Phytochemical profile and antimicrobial potential of extracts obtained from Thymus marschallianus Willd. Molecules. (2019) 24:3101. doi: 10.3390/molecules24173101

PubMed Abstract | Crossref Full Text | Google Scholar

43. Üstüner O, Anlas C, Bakirel T, Üstün Alkan F, Diren Sigirci B, Ak S, et al. In vitro evaluation of antioxidant, anti-inflammatory, antimicrobial and wound healing potential of Thymus sipyleus Boiss. subsp rosulans (Borbas) Jalas. Molecules. (2019) 24:3353. doi: 10.3390/molecules24183353

Crossref Full Text | Google Scholar

44. Akin M, Saki N. Antimicrobial, DPPH scavenging and tyrosinase inhibitory activities of Thymus vulgaris, Helichrysum arenarium and Rosa damascena Mill. ethanol extracts by using TLC bioautography and chemical screening methods. J Liquid Chromatogr Related Technol. (2019) 42:204–16. doi: 10.1080/10826076.2019.1591977

Crossref Full Text | Google Scholar

45. Bendjabeur S, Benchabane O, Bensouici C, Hazzit M, Baaliouamer A, Bitam A, et al. Antioxidant and anticholinesterase activity of essential oils and ethanol extracts of Thymus algeriensis and Teucrium polium from Algeria. J Food Measure Characterization. (2018) 12:2278–88. doi: 10.1007/s11694-018-9845-x

Crossref Full Text | Google Scholar

46. Salazar MO, Osella MI, Arcusin DEJ, Lescano LE, Furlan RLE. New α-glucosidase inhibitors from a chemically engineered essential oil of Origanum vulgare L. Ind Crops Prod. (2020) 156:112855. doi: 10.1016/j.indcrop.2020.112855

Crossref Full Text | Google Scholar

47. Singh A, Singh K, Kaur K, Sharma A, Mohana P, Prajapati J, et al. Discovery of triazole tethered thymol/carvacrol-coumarin hybrids as new class of α-glucosidase inhibitors with potent in vivo antihyperglycemic activities. Eur J Med Chem. (2024) 263:115948. doi: 10.1016/j.ejmech.2023.115948

PubMed Abstract | Crossref Full Text | Google Scholar

48. Ali A. Chemical composition, α-glucosidase inhibitory and anticancer activity of essential oil of Thymus vulgaris leaves. J Essential Oil Bear Plants. (2021) 24:1–9. doi: 10.1080/0972060X.2021.1973575

Crossref Full Text | Google Scholar

49. Kindl M, BlaŽeković B, Bucar F, Vladimir-KneŽević S. Antioxidant and anticholinesterase potential of six Thymus species. Evid-Based Complement Altern Med. (2015) 2015:403950. doi: 10.1155/2015/403950

PubMed Abstract | Crossref Full Text | Google Scholar

50. Ekin HN, Deliorman Orhan D, Erdogan Orhan I, Orhan N, Aslan M. Evaluation of enzyme inhibitory and antioxidant activity of some Lamiaceae plants. J Res Pharm. (2019) 23:749–58. doi: 10.12991/jrp.2019.184

Crossref Full Text | Google Scholar

51. Orhan I, Senol FS, Gülpinar AR, Kartal M, Sekeroglu N, Deveci M, et al. Acetylcholinesterase inhibitory and antioxidant properties of Cyclotrichium niveum, Thymus praecox subsp. caucasicus var caucasicus, Echinacea purpurea and E pallida. Food Chem Toxicol. (2009) 47:1304–10. doi: 10.1016/j.fct.2009.03.004

Crossref Full Text | Google Scholar

52. Oztürk S, Ercişli S. Broad-spectrum antibacterial properties of Thymus fallax methanol extract. Pharm Biol. (2008) 43:609–13. doi: 10.1080/13880200500301928

Crossref Full Text | Google Scholar

53. Afonso AF, Pereira OR, Neto RT, Silva AMS, Cardoso SM. Health-promoting effects of Thymus herba-barona, Thymus pseudolanuginosus, and Thymus caespititius decoctions. Int J Mol Sci. (2017) 18:1879. doi: 10.3390/ijms18091879

PubMed Abstract | Crossref Full Text | Google Scholar

54. Naz SAM, Wani TA, Hussain A, Alqahtani SS, Alothman ZA. In vitro phytochemical and antimicrobial screening of Thymus linearis. Bang J Pharmacol. (2015) 10:21–6. doi: 10.3329/bjp.v10i1.20639

Crossref Full Text | Google Scholar