We present a study on the taxonomy of eleven Thymus species, belonging to two sections and occurring naturally in Bulgaria. Four DNA barcoding markers—matK, rbcL, trnH-psbA and ITS—were applied to discriminate the species and to reveal their phylogenetic relationships. The results showed that rbcL has the lowest discriminating power regarding the studied species, while the other markers yielded results fitting better to the existing taxonomic schemes based on morphological traits. However, even in the case of better performing markers, the results were not straightforward—morphologically distinct species belonging to different sections were grouped together, and closely related species appeared genetically distinct. The results are typical for taxonomically complex groups, such as the genus Thymus, characterized in Bulgaria with great diversity, high percentage of endemism and still requiring a full and comprehensive taxonomic study. The results are discussed in the light of unresolved taxonomic problems and application of DNA barcoding methods.

The genus Thymus, a member of the Lamiaceae family, encompasses species renowned for their medicinal properties. This study aimed to determine the metabolite composition and free radical scavenging potential of three endangered species with restricted distribution in Bulgaria: T. perinicus (Velen.) Jalas, T. jalasaianus Stoyanov & Marinov, and one endemic to the Balkans – T. aznavourii Velen., all fall under the section Hyphodromi. Non-polar (chloroform and diethyl ether) and polar (methanol) extracts were prepared from the studied species. GC/MS and HPTLC analyses were applied to identify the metabolites. Antioxidant capacity was assessed using the DPPH test, while the Folin-Ciocalteu colourimetric assay measured total phenolic content (TPC). Chromatographic analyses revealed a rich array of primary and secondary metabolites, some known for their health-promoting properties. HPTLC identified compounds such as rosmarinic acid, lupeol, uvaol, ursolic, and oleanolic acids, while GC/MS detected terpenes like thymol, caryophyllene, cis–β-farnesene, and germacrene D, as well as phenolic acids such as 4-hydroxybenzoic, 4-hydroxycinnamic, vanillic, and caffeic acids, alongside fatty acids, fatty alcohols, and saccharides. Methanol extracts exhibited significant antiradical activity (IC50 < 50 µg/mL), with TPC values ranging from 7.44 to 17.97 mg GAE/g extract, aligning with DPPH assay findings. This study represents the first report on the chemical composition of extracts with varying polarity from T. jalasaianus and T. aznavourii. It offers valuable insights into the phytochemistry of these Thymus species, highlighting their potential applications across diverse industries and the need for their conservation.

Thymus longedentatus (Degen & Urum.) Ronniger is a Balkan endemic species valued for its essential oils and phenolic compounds, yet little is known about its phytochemistry under cultivation. This study compared the metabolite profiles of wild populations from the Eastern Rhodopes (ER) and Thracian Lowland (TL) with a cultivated population near Sofia (CA). Hydrodistillation yielded 0.2% essential oil (EO) in wild plants and 0.3% in cultivated plants. GC–MS analysis revealed citral isomers (neral and geranial) as dominant constituents, exceeding 60% in cultivated samples. Methanolic extracts and acetone exudates contained rosmarinic acid and triterpene acids consistently across all populations. Quantitative differences were observed in stress-related metabolites: arbutin and hydroquinone were enriched in wild plants, while chlorogenic and geranic acids were higher in cultivated plants. These findings demonstrate that cultivation preserves the main phytochemical profile of T. longedentatus while modulating the abundance of specific compounds, offering potential for sustainable utilization and conservation.

This study investigates the phytochemical composition and biological activity of eight Thymus species distributed in Bulgaria, with a focus on taxa from section Hyphodromi. High-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC–MS) were used to characterize the methanolic (MeOH) and diethyl ether (Et₂O) extracts, respectively. MeOH extracts revealed high concentrations of phenolic acids, particularly rosmarinic acid, salicylic acid, and flavonoid glycosides, with T. atticus, T. jalasianus, and T. leucotrichus showing the most diverse profiles. Et₂O extracts were dominated by triterpenic acids (ursolic and oleanolic) and monoterpenes such as thymol and carvacrol, especially in T. zygioides and T. leucotrichus. All MeOH extracts exhibited significant antioxidant activity in the DPPH assay (IC₅₀ < 50 µg/mL), with T. jalasianus and T. atticus demonstrating the strongest effects. Radical scavenging potential generally followed the trend of total phenolic content. Moderate acetylcholinesterase-inhibitory activity was observed only in T. zygioides and T. leucotrichus. The study reports for the first time data on the biological activity and metabolic composition of extracts from endemic and rare species and from the species of Bulgarian origin. The results provide new data on the phytochemical composition and in vitro antioxidant and acetylcholinesterase-inhibitory activities of selected Thymus species, contributing to the characterization of their overall in vitro biochemical profiles.