Chemical Composition of Thymus Species from Bulgarian Flora

Abstract

The present study investigated the chemical composition of the main components of five commonly occurring Thymus species in Bulgaria: T. sibthorpii Benth., T. pulegioides L., T. glabrescens Willd. (syn. T. odoratissimus Mill.), T. callieri Borbas ex Velen. (syn. T. roegneri K. Koch), and T. zygioides Griseb. The phytochemical profiling of the Thymus species was performed using GC-MS for the analysis of essential oils and HPLC for the identification and quantification of phenolic compounds in the ethanolic extracts. Linalool was the dominant essential oil constituent in T. sibthorpii (48.17%), T. pulegioides (50.96%), and T. callieri (38.08%) while thymol prevailed in T. glabrescens (35.35%). A novel chemotype was observed in T. zygioides. The HPLC analysis confirmed rosmarinic acid as the major phenolic acid across all species. Rutin was the predominant flavonoid in four species whereas T. sibthorpii exhibited a remarkably high concentration of (+)-catechin. Overall, the high chemical diversity within the Thymus genus was confirmed. Due to the variability of compounds among plant species, the findings of the present study suggest that, along with essential oils, phenolic components may also contribute to the chemotaxonomic classification of the Thymus genus and influence the pharmacological activity of the species, which requires further study.

1. Introduction

Thyme is one of the most widespread aromatic plants from the Lamiaceae family. The Thymus genus comprises approximately 250 taxa, including 214 species and 36 subspecies, with a broad distribution across Europe, parts of the Mediterranean, the Middle East, and North Africa [1].

In general, the Thymus genus is characterized by various chemical constituents, classified into volatile and non-volatile fractions. The main part of the volatile fractions are the essential oils, whose composition serves as a basis for categorizing species into distinct chemotypes, representing the predominance of specific major constituents. These chemotypes reflect variations in the biosynthetic pathways of secondary metabolites, which are influenced by genetic, environmental, and ecological factors [2]. In Thymus species, common chemotypes are defined by the dominant terpenes, e.g., thymol, carvacrol, linalool, and geraniol [3,4,5]. The non-volatile fraction consists of phenolic compounds, such as rosmarinic, salvianolic, and caffeic acids; flavonoids, often with luteolin as the aglycone; and alkaloids, tannins, and saponins [6,7].

The herb has been well known since ancient times for its application in medicine, the food industry, and perfumery. In modern phytotherapy, thyme has been recognized as a potent remedy for respiratory diseases including bronchitis, asthma, and cough. Its efficacy is largely attributed to the essential oils, which possess bronchodilatory, antispasmodic, and antimicrobial properties [8,9]. These bioactive compounds help alleviate respiratory symptoms by reducing airway inflammation, relaxing bronchial muscles, and combating respiratory pathogens. The antioxidant activity is also extensively studied, at least in vitro, with significant attention to the polyphenols and volatile oil [10,11].

Over the years, the traditional use of thyme has been supported with pharmacologically proven evidence, leading the European Pharmacopoeia to issue monographs for thyme (Thymi herba); thyme oil, thymol-type (Thymi typo thymolo aetheroleum); and wild thyme (Serpylli herba). Different wild growing thyme species are native to Bulgaria, and the plant is widely used as a spice and medicinal herb—from simple tea to complex herbal medicinal products manufactured by the pharmaceutical industry [12]. For medicinal purposes, thyme should follow the standards of the European Pharmacopoeia, but in current practice, substitutions with other species are common due to botanical similarity [13].

Hence, there is a need to investigate indigenous Bulgaria species’ chemical composition and bioactivity, which presents a great challenge for scientists as the genus is characterized by high diversity and polymorphism. For this reason, some species are listed as synonyms in different taxonomic databases. According to the electronic database of “EuroPlusMed” [14], the Bulgarian flora features 18 Thymus species, whereas the fourth edition of the “Conspectus of the vascular flora of Bulgaria” lists two more [15]. In the abovementioned sources, it is visible that the Bulgarian flora lacks two of the most studied species worldwide—Thymus vulgaris and Thymus serpyllum.

Considering the widespread use of wild thyme as an herbal tea and source material for different types of phytopreparations, the present study aimed to investigate the chemotaxonomic differentiation among the species Thymus sibthorpii Benth., T. pulegioides L., T. glabrescens Willd. (T. odoratissimus Mill.), T. callieri Borbas ex Velen. (T. roegneri K. Koch), and T. zygioides Griseb., based on essential oil composition and phenolic profiles, in order to ensure accurate taxonomic classification.

2. Materials and Methods

2.1. Plant Material

Aerial parts (stems with leaves and flowers) of T. sibthorpii, T. pulegioides, T. glabrescens, T. callieri, and T. zygioides were collected during the flowering period from different localities of Northeastern Bulgaria’s Black Sea Floristic region. The following species, identified by Asst. Prof. Petya Boycheva from the Department of Biology, Faculty of Pharmacy, Medical University of Varna, were deposited as samples in the Herbarium of Sofia University St. Kliment Ohridski (

Table 1. Collection data for Thymus species.

Species	Voucher Number	Collection Date	Coordinates
T. sibthorpii Benth.	108368	May, 2024	43.2107° N, 27.5048° E
T. pulegioides L.	108369	May, 2024	43.2106° N, 27.5053° E
T. glabrescens Willd.	108370	May, 2024	43.1750° N, 27.5343° E
T. callieri Borbas ex Velen.	108366	May, 2024	43.1749° N, 27.5342° E
T. zygioides Griseb.	108367	May, 2024	43.2408° N, 27.2530° E

).

2.2. Isolation of Essential Oils

Air-dried at room temperature, plant materials were grounded to 0.5 mm, then subjected to hydrodistillation with distilled water for 3 h to extract the essential oil using a Clevenger-type apparatus. The oils obtained were collected and stored in a sealed container at 4 °C in a refrigerator until analysis.

2.3. Plant Extracts

The dry plant material was ground to 0.5 mm using a laboratory mill. For each extract, 2.00 g of the powdered sample was combined with 40 mL of 70% (v/v) ethanol in an Erlenmeyer flask, corresponding to a concentration of 50 mg/mL (5% w/v) of dry weight (DW) of plant material in the extraction solvent. The mixture was heated for 2 h under continuous stirring on a magnetic stirrer, with a reflux condenser attached to prevent solvent loss. After heating, the samples were subjected to ultrasonic extraction in a water bath sonicator for 30 min at room temperature without additional heating. The resulting mixtures were then cooled and stored at 4 °C for 48 h to ensure complete extraction of biologically active compounds. Finally, the extracts were filtered through standard paper filters and stored at 4 °C until HPLC analysis.

2.4. Chemicals and Reagents

Hydrocarbons used to determine retention indices (RIs) for the GC-MS analysis included octane (≥99%), nonane (≥99%), decane (≥99%), undecane (≥99%), dodecane (99%), tridecane (≥99%), tetradecane (≥99%), and hexadecane (≥99%), sourced from Merck KGaA (Darmstadt, Germany). Hexane, supplied by Sigma-Aldrich (Steinheim, Germany), was used for the dilution of EOs.

All analytical standards for the HPLC analysis were obtained from Sigma-Aldrich (Steinheim, Germany). All other solvents employed in the analysis were of analytical grade and purchased from local distributors.

2.5. GC-MS Analysis

The chemical composition of the essential oil samples was analyzed via Gas Chromatography coupled with Mass Spectrometry (GC-MS). The GC-MS analysis was carried out using a Bruker Scion 436-GC SQ MS (Bremen, Germany) instrument fitted with a ZB-5MSplus fused silica capillary column (30 m length, 0.25 mm internal diameter, and 0.25 µm film thickness). Helium was used as a carrier gas with a constant 1 mL/min flow rate. Before the analysis, samples were prepared by dilution of 20 µL of each EO sample with hexane to a concentration of 2%. The injection volume was 1 µL with an injector split ratio of 1:50.

The oven temperature was programmed as follows: starting at 60 °C for 1 min; increasing to 100 °C at 6 °C/min, then to 120 °C at 4 °C/min; followed by 140 °C at 6 °C/min and 180 °C at 10 °C/min; and finally to 230 °C at 13 °C/min, where it was held for 2 min. Mass spectra were obtained in full-scan mode over a 50–350 m/z mass range. RI values of the essential oil components were calculated based on the retention times of the C₈–C₁₆ n-alkane series injected under the same experimental conditions. Volatile constituents in the essential oils were identified by comparing their mass spectra and RI values with data from the Wiley NIST11 Mass Spectral Library (NIST11/2011/EPA/NIH) and published literature. The experiments were performed in triplicates, and the relative peak area percentage was calculated as a mean of the three independent measurements.

2.6. HPLC Analysis

The ethanolic extracts 50 mg/mL (5% w/v) DW were analyzed using High-Performance Liquid Chromatography (HPLC) according to Krasteva et al. 2022 [16]. The analysis was conducted on a Waters 1525 Binary Pump system (Waters, Milford, MA, USA), combined with a Waters 2484 Dual Absorbance Detector and analytical software Breeze 3.30, using a Supelco Discovery HS C18 column (5 µm, 250 mm × 4.6 mm). Before injecting into the HPLC system, the samples were prepared by dilution of each extract with ethanol to a concentration of 25 mg/mL (2.5% w/v) DW and filtered through a 0.25 µm membrane filter. Each sample injection was 20 µL, with a flow rate maintained at 1.0 mL/min. Gradient elution was performed using 1% acetic acid in water (Solvent A) and methanol (Solvent B). The gradient for Solvent A was adjusted as follows: from 0 to 36 min, it decreased from 90% to 78%; from 36 to 37 min, it dropped to 70%; from 37 to 47 min, further to 60%; from 47 to 58 min, to 54%; from 58 to 59 min, to 40%; from 59 to 71 min, down to 20%; from 71 to 72 min, it was increased back to 90%, and held at 90% until 75 min. Detection was carried out at 280 nm for gallic acid, protocatechuic acid, (+)-catechin, vanillic acid, syringic acid, (−)-epicatechin, p-coumaric acid, salicylic acid, and hesperidin and at 360 nm for chlorogenic acid, caffeic acid, ferulic acid, rutin, rosmarinic acid, quercetin, and kaempferol. The quantification of these compounds was based on calibration curves prepared from standard solutions at concentrations of 10, 15, 25, 50, 100, 200, and 500 µg/mL.

3. Results

3.1. Essential Oil Yield

The essential oil yields from the aerial parts of the five Thymus species were calculated as percentages of dried plant material. Thymus sibthorpii and Thymus glabrescens exhibited the highest yields (1.1% each), followed by T. pulegioides and T. callieri—1.0% and 0.9%, respectively. The lowest essential oil content was determined in T. zygioides—0.2%.

3.2. GC-MS Profiling of Thymus Species

The chemical composition of the essential oils obtained from the aerial parts of T. sibthorpii, T. pulegioides, T. glabrescens, T. callieri, and T. zygioides was determined using Gas Chromatography–Mass Spectrometry (GC-MS), identifying a total of 65 volatile compounds. Among the studied species, T. zygioides exhibited the highest number of detected components, with 42 compounds present in levels above 0.1%, while T. pulegioides had the lowest, with only 22 compounds exceeding this threshold. In total, 85.19% to 99.29% of the essential oil constituents were identified.

Table 2. Chemical composition (%) of T. sibthorpii (Ts), T. pulegioides (Tp), T. glabrescens (Tg), T. callieri (Tc), and T. zygioides (Tz) aerial parts’ essential oils; major constituents are highlighted in bold and tr indicates trace amounts—less than 0.05%.

№	Compound	RI	RI Lit. Data	Formula	Class of Compound	% Ts	% Tp	% Tg	% Tc	% Tz
1	3-Hexen-1-ol	870	874	C6H12O	O	–	–	tr	–	–
2	Tricyclene	922	922	C7H14O2	O	–	–	–	–	0.71
3	α-Thujene	924	924	C10H16	MH	–	–	1.39	tr	0.88
4	α-Pinene	930	930	C10H16	MH	0.11	0.1	0.65	0.18	8.07
5	Camphene	945	948	C10H16	MH	0.19	0.16	0.49	0.31	11.9
6	Sabinene	964	964	C10H16	MH	–	0.21	0.23	0.09	0.66
7	1-Octen-3-ol	968	967	C8H16O	O	0.65	0.66	0.91	0.81	–
8	3-Octanone	972	970	C8H16O	O	0.13	0.1	0.11	0.21	–
9	β-Pinene	977	977	C10H16	MH	0.31	0.9	1.08	0.24	1.24
10	3-Octanol	984	982	C8H18O	O	0.21	0.26	0.14	0.28	0.21
11	α-Phellandrene	995	995	C10H16	MH	–	–	0.21	–	–
12	δ-3-Carene	997	999	C10H16	MH	–	–	0.07	–	–
13	α-Terpinene	1006	1008	C10H16	MH	–	–	1.65	–	0.52
14	o-Cymene	1015	1018	C10H14	MH	0.15	–	6.93	0.17	0.94
15	Limonene	1021	1020	C10H16	MH	–	0.24	0.22	0.05	5.45
16	Eucalyptol	1026	1025	C10H18O	MO	0.27	0.28	1.05	0.48	3.05
17	cis-β-Ocimene	1040	1040	C10H16	MH	–	–	0.07	–	6.57
18	γ-Terpinene	1052	1050	C10H16	MH	0.17	0.09	8.42	0.08	1.75
19	cis-Sabinene hydrate	1064	1062	C10H18O	MO	–	0.13	0.28	–	–
20	cis-Linalool oxide	1065	1065	C10H18O2	MO	0.09	–	–	0.1	–
21	α-Terpinolene	1076	1077	C10H16	MH	–	–	0.11	–	0.47
22	Linalool	1093	1094	C10H18O	MO	48.17	50.96	22.84	38.08	0.12
23	Nonanal	1095	1096	C9H18O	O	–	–	–	–	0.17
24	cis-p-Menth-2-en-1-ol	1116	1116	C10H18O	MO	–	–	–	–	0.09
25	trans-p-Menth-2-en-1-ol	1136	1137	C10H18O	MO	–	–	–	–	0.1
26	cis-Verbenol	1139	1140	C10H16O	MO	–	–	–	–	0.13
27	Camphor	1142	1143	C10H16O	MO	–	–	–	–	0.52
28	Nerol oxide	1144	1144	C10H16O	MO	–	–	–	0.07	–
29	δ-Terpineol	1168	1169	C10H18O	MO	–	–	–	–	0.06
30	endo-Borneol	1172	1172	C10H18O	MO	0.67	0.56	1.62	1.09	1.26
31	Terpinen-4-ol	1181	1181	C10H18O	MO	0.07	0.25	0.74	0.18	2.77
32	α-Terpineol	1199	1198	C10H18O	MO	0.52	1.96	0.11	0.18	0.23
33	cis-Dihydrocarvone	1200	1200	C10H16O	MO	–	–	–	–	0.13
34	Decanal	1206	1205	C10H20O	O	–	–	–	–	0.2
35	trans-Piperitol	1210	1211	C10H18O	MO	–	–	–	–	0.05
36	Nerol	1226	1225	C10H18O	MO	0.86	–	–	1.78	0.2
37	Thymol methyl ether	1229	1231	C11H16O	MO	0.35	–	2.57	0.25	–
38	Carvacrol methyl ether	1238	1239	C11H16O	MO	–	–	4.17	–	–
39	β-Citral	1239	1239	C10H16O	MO	0.37	0.28	–	0.75	1.26
40	Geraniol	1262	1263	C10H18O	MO	18.67	13.48	–	27.66	–
41	α-Citral	1282	1279	C10H16O	MO	0.43	0.38	–	1.09	1.53
42	Bornyl acetate	1305	1302	C12H20O2	MO	–	–	–	–	0.21
43	Thymol	1312	1304	C10H14O	MO	0.22	–	35.35	–	0.5
44	Carvacrol	1323	1327	C10H14O	MO	–	–	0.17	–	–
45	α-Terpinyl acetate	1368	1365	C12H20O2	MO	–	3.01	–	–	–
46	Neryl acetate	1377	1376	C12H20O2	MO	0.06	–	–	0.06	0.45
47	Geranyl acetate	1406	1409	C12H20O2	MO	12.45	7.54	–	9.51	–
48	β-Bourbonene	1413	1417	C15H24	SH	0.6	0.37	0.13	0.9	0.87
49	β-Caryophyllene	1446	1442	C15H24	SH	3.09	2.12	1.54	2.42	5.42
50	β-Copaene	1454	1459	C15H24	SH	0.08	–	tr	0.14	–
51	cis-β-Farnesene	1471	1476	C15H24	SH	–	–	–	–	0.43
52	α-Caryophyllene	1475	1480	C15H24	SH	0.13	0.09	0.07	0.11	0.29
53	γ-Muurolene	1489	1484	C15H24	SH	–	–	–	–	0.23
54	Germacrene D	1495	1491	C15H24	SH	3.91	3.87	2.44	2.98	12.00
55	Elixene	1505	1492	C15H24	SH	–	–	–	0.12	7.13
56	α-Farnesene	1508	1508	C15H24	SH	–	–	–	–	0.35
57	β-Bisabolene	1511	1513	C15H24	SH	4.48	3	2.73	3.93	0.73
58	γ-Cadinene	1514	1515	C15H24	SH	–	–	–	–	0.22
59	δ-Cadinene	1518	1518	C15H24	SH	0.06	–	0.05	tr	1.25
60	β-Sesquiphellandrene	1521	1523	C15H24	SH	0.08	–	0.07	0.07	–
61	α-Bisabolene	1531	1531	C15H24	SH	0.07	–	0.54	0.22	–
62	Geranyl butyrate	1538	1540	C14H24O	MO	0.07	–	–	0.29	–
63	Spathulenol	1555	1557	C15H24O	SO	–	–	–	0.07	3.2
64	Caryophyllene oxide	1559	1561	C15H24O	SO	0.2	0.13	0.14	0.66	0.6
65	Neryl (S)-2-methylbutanoate	1565	1562	C15H26O2	MO	–	–	–	–	0.07
	Terpene classes
	Monoterpene hydrocarbons (MH)					0.93	1.7	21.52	1.12	38.45
	Oxygenated monoterpenes (MO)					83.27	78.83	68.9	81.57	12.73
	Sesquiterpene hydrocarbons (SH)					12.5	9.45	7.54	10.89	28.92
	Oxygenated sesquiterpenes (SO)					0.2	0.13	0.14	0.73	3.8
	Others (O)					0.99	1.02	1.16	1.3	1.29
	Total identified (%)					97.89	91.13	99.29	95.61	85.19

presents the percentage composition and retention indices of the individual components, listed in the order of their elution from the column.

The identified compounds were classified into five main groups: monoterpene hydrocarbons (MH), oxygenated monoterpenes (MO), sesquiterpene hydrocarbons (SH), oxygenated sesquiterpenes (SO), and other compounds (O). Oxygenated monoterpenes (MO) were the predominant class in most samples, contributing 83.27% in T. sibthorpii, 81.57% in T. callieri, 78.83% in T. pulegioides, and 68.9% in T. glabrescens. However, in T. zygioides, monoterpene hydrocarbons (38.45%) were the major components, followed by sesquiterpene hydrocarbons (28.92%). In contrast, oxygenated sesquiterpenes (SO) were found in smaller quantities, with a maximum of 3.8% in the T. zygioides sample.

The abundance of oxygenated monoterpenes in the essential oil of T. sibthorpii was indicated by the detection of linalool (48.17%), geraniol (18.67%), and geranyl acetate (12.45%) as the major components. This led to the classification of the species as a linalool chemotype.

The highest concentration of linalool was found in the sample of T. pulegioides—50.96%. Similar to T. sibthorpii, notable amounts of geraniol (13.48%) and geranyl acetate (7.54%) were detected. However, the presence of α-terpinyl acetate (3.01%) slightly differentiated it from the profile of T. sibthorpii.

T. glabrescens demonstrated association with the thymol chemotype as the principal constituent in the sample was thymol (35.35%). Additionally, linalool was detected in a significant amount—22.84%, followed by γ-terpinene—8.42%.

Another example of the linalool chemotype was T. callieri with linalool (38.08%), geraniol (27.66%), and geranyl acetate (9.51%) as the main compounds in the essential oil. The profile was similar to the one of T. sibthorpii, though with a higher geraniol content.

Unlike the other species, T. zygioides exhibited a much lower proportion of oxygenated monoterpenes (12.73%). The main compounds included germacrene D (12.0%), camphene (11.9%), α-pinene (8.07%), and elixene (7.13%).

3.3. Phenolic Composition of Thymus Ethanolic Extracts

The results of the High-Performance Liquid Chromatography (HPLC) analysis of ethanolic extracts from the aerial parts of five Thymus species—T. sibthorpii (Ts), T. pulegioides (Tp), T. glabrescens (Tg), T. callieri (Tc), and T. zygioides (Tz)—are presented in

Table 3. Content of the major phenolic compounds in ethanolic extracts of Thymus aerial parts, presented as μg/g DW of plant material; main compounds are highlighted in bold.

	Compounds	Content, μg/g DW of Plant Material
		Ts	Tp	Тg	Tc	Tz
Phenolic acids	Gallic acid	241.2	276.4	NF *	NF	175
	Protocatechuic acid	400	1001	602.6	318.6	360.2
	Chlorogenic acid	NF	278.2	NF	NF	NF
	Vanillic acid	545.6	758.6	438.2	461.4	489.8
	Caffeic acid	119.2	147.6	89.6	NF	164.2
	Syringic acid	134.6	189.8	242.8	154.2	125.2
	p-Coumaric acid	104.4	127.2	176.6	143.8	156.6
	Ferulic acid	229	7263.2	4280.6	6291	295
	Salicylic acid	1518.8	NF	NF	NF	NF
	Rosmarinic acid	11,483.8	15,783.8	11,667.4	12,444.8	70,77.6
Flavonoids	(+)-Catechin	34,720	299.6	303.6	202.6	286.6
	(−)-Epicatechin	1213.2	895.8	774	922.4	777.4
	Rutin	8281.8	11,652.4	7875.2	6510	10,681.6
	Hesperidin	774.2	NF	NF	NF	NF
	Quercetin	59.6	282.4	504.4	160.4	81
	Kaempherol	ULQ	31.8	57.8	ULQ **	ULQ

* NF—Not found; ** ULQ—Under the limit of quantification.

. The analysis confirmed the presence of sixteen compounds—ten phenolic acids and six flavonoids.

Among the phenolic acids, rosmarinic acid was the most abundant compound across all samples, with the highest concentration observed in Thymus pulegioides (15,783.8 μg/g), followed by Thymus callieri (12,444.8 μg/g), Thymus glabrescens (11,667.4 μg/g), and Thymus sibthorpii (11,483.8 μg/g). The lowest amount was found in Thymus zigioides (7077.6 μg/g), though still at substantial levels.

Ferulic acid was present in highly variable concentrations, with a peak in T. pulegioides (7263.2 μg/g) and T. callieri (6291 μg/g), and much lower amounts in the ethanolic extracts of T. sibthorpii and T. zigioides—229 and 295 μg/g, respectively. Protocatechuic acid was detected in notable amounts, with a high in T. pulegioides (1001 μg/g) and T. glabrescens (602.6 μg/g). Vanillic acid was consistent across all samples, ranging from 438.2 μg/g (T. glabrescens) to 758.6 μg/g (T. pulegioides). In contrast, a significant amount of salicylic acid was detected exclusively in Thymus sibthorpii—1518.8 μg/g. Some phenolic acids, including caffeic, syringic, and p-coumaric acids, were found in lower concentrations, generally below 200 μg/g across most samples.

Among flavonoids, (+)-catechin showed an exceptionally high concentration in Thymus sibthorpii (34,720 μg/g) while it was only present in 202.6–303.6 μg/g in the remaining extracts. Rutin was the major flavonoid found in the other four samples, with concentrations ranging from 6510 μg/g in Thymus callieri to 11,652.4 μg/g in Thymus pulegioides.

A moderate and consistent presence in all species was shown by (−)-epicatechin, highest in T. sibthorpii (1213.2 μg/g) and lowest in T. glabrescens (774 μg/g). Quercetin was detected at low levels, highest in T. glabrescens—504.4 μg/g. Hesperidin was found only in the ethanolic extract of Thymus sibthorpii (774.2 μg/g).

4. Discussion

4.1. Essential Oil Yield

The essential oil yield in the present study aligned with the values reported by other authors. Thymus pulegioides is the species with the most abundant data available. The highest concentration of the essential oil was reported in another Bulgarian study, where a yield of 1.18% was obtained by using micro hydrodistillation with diethyl ether as a solvent [17]. Results similar to ours (1.0%) are mentioned by Italian authors—1.11%, during the full flowering period in May, and Romanian researchers indicate 1% content for representatives of the species collected at a higher altitude (1800 m) [18,19]. The lowest essential oil yield has been reported for the species growing in Western Romania—only 0.49% [8].

The essential oil yield from T. glabrescens in our study (1.1%) fell between the previously reported values of 1.69% [17] and 0.73% [18]. For T. callieri, Bulgarian authors presented a maximum yield of 2.11% [17], whereas in our study, the value obtained was 0.9%. T. sibthorpii showed a prevailing yield of 1.1%, higher than the 0.83% reported earlier [17]. Among the species analyzed, T. zygioides exhibited the lowest essential oil yield (0.2%), while other Bulgarian authors reported a 0.87% concentration of the volatiles [20].

The comparative quantitative analyses highlighted the variability of essential oil content in Thymus species, which could be attributed to the ecological conditions, plant development stage, altitude, and extraction method employed.

4.2. Essential Oil Composition

Interestingly, our findings differed significantly from the other Bulgarian research on the essential oil of Thymus sibthorpii, which reported a novel chemotype: myrcen-8-yl acetate/linalool/myrcen-8-ol [17]. In contrast, myrcene and its derivatives were not detected in our samples. Greater similarities were observed with the species from Greece, which belonged to the linalool chemotype, with the notable exception of geraniol and geranyl acetate, which were also major constituents of our Thymus sibthorpii sample [21]. In that study, temporal variations in the chemical composition of the essential oil were discussed, confirming that linalool was the dominant compound during the flowering period, whereas in the pre-flowering stage, thymol was found to be in the highest concentration. Studies from Turkey have reported a distinctly different chemical profile of the essential oil, indicating that their population of the species belongs to the thymol/p-cymene and carvacrol/p-cymene chemotypes [22].

The analysis of the essential oil of Thymus pulegioides revealed linalool (50.96%) as the dominant component, followed by other oxygenated monoterpenes. This differed significantly from previous studies of the species collected from Southwestern Bulgaria (Vlahina Mts.), where α-terpinyl acetate (66.79%) was the major component [17]. However, our analysis detected α-terpinyl acetate only in T. pulegioides, and at a much lower concentration (3.01%). Reports from Croatia [23], Slovakia [15], Lithuania [3], and Germany [24] support the presence of a linalool chemotype within the species, where linalool concentrations ranged from 11.4% (Slovakia) to 80.29% (Lithuania). Carvacrol was frequently identified as the main component of the essential oil of this Thymus species from Croatia [24], Romania [25,26], Slovakia [15], and Germany [24], with concentrations varying between 23% and 63.2%. Additionally, populations from Bosnia and Herzegovina and Poland have been associated with a geraniol chemotype [27,28].

Despite the differences in the subsequent major constituents, our findings regarding the composition of the Thymus glabrescens essential oil align with previous studies from Bulgaria [17], Romania [29], Hungary [30], and Serbia [31,32]. These studies consistently reported thymol as the principal component, typically followed by γ-terpinene or p-cymene. In contrast, our sample was characterized by a notably high concentration of linalool (22.84%), which set it apart from the established chemotypic patterns of this species. To date, linalool has been reported only once in the volatile profile of T. glabrescens, and in that case, it was present in a much lower concentration (5.49%); the Serbian study in question classified the sample within the geraniol chemotype [33]. Other researchers have described a γ-terpinene chemotype of Thymus glabrescens [32,34]; in our sample, γ-terpinene was only the third most abundant compound, further highlighting the distinctiveness of our chemical profile.

Thymus callieri exhibited a markedly different essential oil composition compared to previously reported analyses. In contrast with our findings, which revealed the predominance of oxygenated monoterpenes—linalool, geraniol, and geranyl acetate—earlier studies on another Bulgarian population of the species described an essential oil dominated by aromatic compounds, with carvacrol (42.65%), thymol (13.38%), and γ-terpinene (12.04%) as the major constituents [17]. Furthermore, the essential oil composition reported for T. roegneri (a synonym of T. callieri) from Turkey also exhibited an aromatic profile; however, in this case, thymol (58.23%) and p-cymene (12.94%) were the dominant compounds while carvacrol was present only in a lower concentration (8.59%) [35].

Prior studies from Bulgaria presented a sharply contrasting chemical composition of the essential oil of Thymus zygioides, characterized by a high concentration of aromatic compounds (61.2%), with thymol comprising 51.2% of the total oil content. Further establishing the sample as a classic thymol chemotype was the presence of borneol, p-cymene, and γ-terpinene [36]. Similarly, essential oils of T. zygioides var. lycaonicus from Turkey consistently demonstrated high thymol levels (42–58%) and occasionally significant amounts of carvacrol (up to 62%) or geraniol (77%) [22]. The Greek variant of the species displayed a more balanced composition, with nearly equal amounts of thymol (19.5%), p-cymene (19.4%), and γ-terpinene (17.2%) [36]. In contrast to these reported chemotypes, our sample exhibited a strikingly lower percentage of oxygenated monoterpenes (12.73%) and was not dominated by thymol or other typical aromatic constituents. Instead, sesquiterpene hydrocarbons such as germacrene D and elixene, along with monoterpene hydrocarbons including camphene and α-pinene, emerged as the predominant components. Thymol was detected only in trace amounts (0.5%), suggesting a fundamentally different chemotypic profile.

4.3. Flavonoid and Phenolic Acid Content

Consistent with the broader literature on Thymus phytochemistry, rosmarinic acid was the most dominant phenolic acid across all samples, with the highest concentration in T. pulegioides (15,783.8 μg/g). This was in agreement with multiple previous studies reporting rosmarinic acid as the most abundant phenolic constituent in Thymus species, particularly during the budding and flowering stages [37,38]. The concentration observed in our study was even higher than the values reported for T. sibthorpii from Lithuania, where it reached 9323 μg/g [39].

Ferulic acid was detected in all five Thymus species, albeit at highly variable concentrations. Previous studies also present this phenolic acid as typical for the genus, reporting values between 3.58 and 8.43 μg/g for T. glabrescens and 3.56–12.5 μg/g for T. pulegioides [40]. Our results revealed significantly higher concentrations of ferulic acid in the ethanolic extracts of this species, namely 7263.2 μg/g for Thymus pulegioides and 4280.6 μg/g for Thymus glabrescens. However, the quantitative values of the constituents were not directly comparable due to differences in extraction methodologies, beginning with the choice of solvent. In our study, 70% ethanol was used as the extraction solvent, whereas the referenced study employed methanol.

Interestingly, the levels of caffeic acid, a precursor in the biosynthesis of rosmarinic acid, were relatively low across all species (89.6–164.2 μg/g) and did not correlate with rosmarinic acid concentrations. This lack of direct correlation supports observations from earlier studies indicating that caffeic acid accumulation does not necessarily correlate with the levels of its downstream product [39]. This uncoupled relationship may reflect differences in the regulation of the phenylpropanoid pathway enzymes across developmental stages or among different Thymus species.

Unlike previous reports on the phenolic composition of Thymus species, our analysis identified salicylic acid in the ethanolic extract of Thymus sibthorpii. Several studies have reported that the production of salicylic acid in plants is induced by elevated ozone levels or exposure to heat stress [41,42]. Salicylic acid pathways are activated to cope with environmental challenges. This response is thought to enhance plant tolerance to high temperatures by promoting basal thermotolerance and modulating key physiological processes [43].

Rutin emerged as the most abundant flavonoid in four of the Thymus extracts analyzed. Although the direct comparison of rutin concentrations across studies is limited by methodological differences, particularly the choice of extraction solvent, the flavonoid has been widely reported in Hungarian Thymus species, including T. pulegioides, T. glabrescens, and with the highest content observed in T. praecox [40]. Our findings were consistent with a Ukrainian study reporting high rutin levels in ethanolic extracts of T. serpyllum [44], as well as with a study from the Slovak Republic identifying rutin as a key flavonoid in three Thymus species, notably T. pulegioides [11]. The recurring detection of rutin across diverse Thymus taxa and geographic regions supports its potential use as a chemotaxonomic marker within the genus.

The exceptionally high concentration of (+)-catechin observed in the ethanolic extract of Thymus sibthorpii has not been previously reported in the literature. Existing studies indicated considerable variability in the accumulation of flavan-3-ols among Thymus species, with a general trend toward higher concentrations of (−)-epicatechin compared to its diastereomer [40,45]. This pattern was consistent with our findings in the other samples, where (−)-epicatechin levels ranged from 774 μg/g in T. glabrescens to 922.4 μg/g in T. callieri, while (+)-catechin was present at notably lower concentrations, ranging from 202.6 μg/g (T. callieri) to 303.6 μg/g (T. glabrescens).

4.4. Limitations of the Study

A limitation of the present study was the absence of dry extract preparation, which precluded the determination of extraction yields expressed as mg of dry extract per g of powdered plant material and the content of phenolic compounds per g of dry extract. Instead, all analyses were performed using crude liquid extracts prepared from plant material corresponding to a concentration of 50 mg/mL (5% w/v) of DW. Although this approach was deliberately chosen to obtain liquid extracts suitable for subsequent experimental applications, it inherently limited the possibility of evaluating and comparing extraction efficiency or yield across different solvents and extraction methodologies, which was beyond the scope of the current research. Another limitation of the study involved the chosen analytical techniques. A more comprehensive chemical evaluation of the studied species is needed for the confirmation of our findings, including metabolomic assessment through advanced analytical methods such as LC-MS and NMR-spectrometry. These considerations underline the need for future studies involving more diverse extraction methods and analytical techniques, as well as for pharmacological studies for the determination of the medicinal potential of these species and their extracts and essential oils.

5. Conclusions

A comparative phytochemical analysis of five widespread Thymus species from the North Black Sea floristic region was presented for the first time, integrating essential oil profiling via GC-MS and the phenolic composition analysis of ethanolic extracts via HPLC. Significant interspecific variability in both volatile and non-volatile constituents was observed, which may be attributed to environmental factors, genetic diversity, and ecological conditions, as suggested in previous works.

The results from the GC-MS analysis of the essential oils classified most of the species as belonging to the linalool chemotype—T. sibthorpii, T. callieri, and T. pulegioides were rich in oxygenated monoterpenes, especially linalool and geraniol. In contrast, T. glabrescens aligned with the thymol chemotype, while the composition of T. zygioides essential oil revealed a novel chemotypic profile, characterized by a high proportion of monoterpene and sesquiterpene hydrocarbons.

The HPLC analysis confirmed rosmarinic acid as the major phenolic acid in all samples, reaffirming its role as a key phytochemical marker within the Thymus genus. Among the flavonoids, rutin emerged as the principal one in four species, excluding T. sibthorpii, where an exceptionally high concentration of (+)-catechin was detected. This was an unprecedented finding among the Thymus species. In the remaining species, (−)-epicatechin was more evenly distributed and typically present in higher amounts. The detection of salicylic acid and hesperidin further distinguished the chemical profile of T. sibthorpii from the other species.

Overall, the high chemical diversity within the genus Thymus was confirmed. Along with the diversity of volatile compounds in these plant species, the results of the present study indicate that some non-volatile constituents, such as phenolic components, can also be used for the chemotaxonomic classification of the genus Thymus. In this regard, the standardization of the analytical methods used for analysis remains a key challenge.