Metabolite Profiles of Thymus longedentatus from Natural and Cultivated Areas

Abstract

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.

1. Introduction

Thymus species (Lamiaceae) rank among the most widely utilized aromatic and medicinal plants globally. Traditionally, they are consumed as herbal infusions, appreciated for their pleasant flavor and potential health benefits. Beyond their use in folk medicine, Thymus species serve as valuable raw materials in a broad range of industries, including perfumery, pharmaceuticals, food technology, and organic agriculture due to their complex secondary metabolite profiles and well-documented biological activities [1,2,3,4]. Their essential oils, in particular, are known for antimicrobial, antifungal, antioxidant, and insecticidal properties, which have been extensively studied and exploited in both traditional and modern applications. Although native primarily to the Mediterranean basin, North Africa, and parts of Asia, many Thymus taxa are cultivated widely to meet increasing industrial demand.

T. longedentatus (Degen & Urum.) Ronniger is a lesser known, yet highly promising, endemic species restricted to the Balkan Peninsula [5]. It is distinguished by its strong lemon aroma, attributable to a high content of citral isomers—neral and geranial—which are widely recognized for their sensory, antimicrobial, and phytotoxic properties. Despite its narrow natural range, T. longedentatus has attracted scientific interest due to its unique phytochemical composition and diverse biological activities, which suggest potential for commercial exploitation [6,7]. Moreover, essential oils from other plant species have also been reported to exhibit acetylcholinesterase inhibitory and repellent activities [6].

Recent studies have demonstrated a range of bioactivities associated with the essential oil (EO) and extracts of T. longedentatus. Notably, aqueous solutions of its EO have shown significant inhibitory effects on the germination of Trifolium pratense and Lolium perenne, suggesting allelopathic or herbicidal potential at relatively low concentrations (2 µL/mL). Furthermore, the EO of T. longedentatus has exhibited acetylcholinesterase (AChE) inhibitory activity, indicating potential for development as a bioinsecticidal agent. Comparable findings have been reported for essential oils of other plant species, which showed both repellent capacity and AChE inhibition [6]. These findings align with the broader trend of identifying natural alternatives to synthetic organophosphate pesticides [7]. In addition, antifungal assays have shown that the EO of T. longedentatus effectively inhibits the growth of major phytopathogens such as Botrytis cinerea, Phytophthora nicotianae, and Phytophthora cryptogea, further supporting its potential as a natural plant protection agent [7].

Despite these promising attributes, the ecological limitations of T. longedentatus—including its narrow geographic distribution and low population density—pose significant constraints on its sustainable use. The species is currently known to occur in specific regions of southeastern Bulgaria, particularly the Eastern Rhodopes and the Thracian Lowland. These habitat constraints underscore the urgent need to investigate the species’ cultivation potential and evaluate whether phytochemical fidelity can be maintained under ex situ conditions. The conservation and commercial utilization of T. longedentatus thus hinge on establishing effective propagation protocols and assessing the impact of cultivation on its metabolite composition.

The environmental conditions of the regions where T. longedentatus occurs—such as the Eastern Rhodopes, Thracian Lowland, and western Balkan area—have been the subject of several hydrogeological and geophysical investigations, which provide valuable context for understanding local edaphic and climatic influences on secondary metabolism. Previous studies have explored factors such as groundwater contamination risk and evapotranspiration dynamics in these regions [8,9], as well as geological determinants of radon potential [10]. Such background data support the hypothesis that environmental heterogeneity across these sites may contribute to observed phytochemical variation in plant populations.

The present study addresses this gap by performing a comparative analysis of the metabolic profiles of T. longedentatus populations from natural habitats and a cultivated provenance established near Sofia. Through the use of GC-MS and complementary chromatographic techniques, this work aims to assess whether key bioactive compounds—particularly citral isomers, phenolic acids, and triterpene constituents—are retained during cultivation. The findings are intended to inform both conservation strategies and the development of this endemic species as a viable resource for phytopharmaceutical and agrochemical applications.

2. Materials and Methods

2.1. Plant Material

Plant material of T. longedentatus was collected from three distinct locations in Bulgaria: natural populations in the Eastern Rhodopes (ER; 41°40′24.56″ N, 25°49′57.64″ E) and the Thracian Lowland (TL; 42°03′16.48″ N, 24°26′16.33″ E), representing habitats with contrasting climatic and edaphic conditions, as well as from cultivated plots near Sofia (CA) established with material originating from ER. This sampling design was intended to capture habitat-related variation in metabolite composition and to enable direct comparison between wild and cultivated populations. Wild-growing plants were taxonomically identified by Assoc. Prof. Ina Aneva (Institute of Biodiversity and Ecosystem Research, BAS). Voucher specimens (SOM 1379; SOM 1380) have been deposited in the Herbarium of the Institute of Biodiversity and Ecosystem Research (SOM), Sofia, Bulgaria. For essential oil extraction, approximately 300 g of air-dried aerial parts were harvested from each site, with collections made from multiple individuals pooled to obtain three biological replicates per location.

The Eastern Rhodopes (ER) are characterized by a continental-Mediterranean climate with mean annual temperatures around 13 °C and precipitation of 600–700 mm, on predominantly shallow, stony soils. The Thracian Lowland (TL) has a transitional continental climate, with warmer conditions (mean annual temperature 13–14 °C) and about 500–550 mm annual rainfall, on fertile alluvial soils. The cultivated plots near Sofia (CA) are located in a temperate continental zone with mean annual temperatures of 9–10 °C and precipitation of 550–600 mm, on brown forest soils. In these plots, irrigation was applied regularly, ensuring the absence of water deficit.

2.2. Extraction

2.2.1. Essential Oil

The aerial parts of the collected T. longedentatus samples were subjected to hydrodistillation using a Clevenger-type apparatus, following the standard procedure outlined in the European Pharmacopoeia [11]. Hydrodistillation of the aerial parts yielded 0.2% (v/w, based on dry weight) essential oil in samples from the natural populations and 0.3% in the cultivated population.

2.2.2. Methanolic Extracts

Powdered plant material (100 mg) was placed in Eppendorf tubes and extracted with 1 mL of methanol at room temperature for 24 h. Following extraction, the methanolic extracts were decanted into glass vials for GC/MS analysis or retained in Eppendorf tubes for HPTLC analysis. All extracts were subsequently evaporated to dryness under ambient conditions prior to further analysis.

2.2.3. Acetone Exudate

Aerial parts of the samples studied (3 g) were immersed in acetone for 5 min to extract surface compounds accumulated on the epidermis and within secretory structures. The resulting solution was filtered and evaporated to dryness under ambient conditions.

2.3. Derivatisation

Prior to GC/MS analysis, the dried methanolic extracts and acetone exudates were subjected to derivatization. Each sample was treated with 100 µL of pyridine and 100 µL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), followed by heating at 60 °C for 2 h to ensure complete silylation of functional groups.

2.4. Gas Chromatography/Mass Spectrometry (GC/MS) Analysis

GC–MS analysis was performed using a Thermo Scientific Focus gas chromatograph (Rodano, Milan, Italy) coupled with a Thermo Scientific DSQ mass selective detector (Austin, TX, USA), operating in electron ionization (EI) mode at 70 eV. Separation was achieved using an ADB-5MS capillary column (30 m × 0.25 mm × 0.25 µm). Analytical conditions followed protocols previously described by Berkov et al. [12] for derivatized extracts. Compound identification was carried out by comparing the acquired mass spectra with entries in the NIST 14 library and an in-house MS database, as well as by evaluating retention indices (RI) against published values [13]. Retention times (RT) were also recorded for each compound under the applied chromatographic conditions. Retention indices were calculated using a series of n-alkanes analyzed under identical chromatographic conditions. The relative abundance of each component was estimated based on its peak area in the total ion chromatogram.

2.5. High-Performance Thin Layer Chromatography Analysis

Preliminary quantification of rosmarinic acid content was performed using HPTLC, providing indicative estimates of compound abundance rather than definitive quantitative values. Standard solutions of rosmarinic acid (1.5, 2.5, and 5 µg per application spot on the HPTLC plate, prepared from a 1000 µg/mL stock solution) and 5 µL of each sample extract were applied to the plates. All plant extracts were spotted in triplicate. Chromatographic separation was carried out using a mobile phase consisting of chloroform:ethyl acetate:formic acid (50:40:10, v/v/v). After development, the plates were sprayed with ‘Naturstoffreagenz A’ reagent to visualize the compounds. Rosmarinic acid, with an Rf value of 0.64, was detected by its fluorescence emission under UV light at 336 nm. Images were captured using a digital camera, and densitometric analysis was performed with QuantiScan 2.1 software (Biosoft, San Francisco, CA, USA). Rosmarinic acid content in the samples was preliminarily estimated by comparing the peak areas of the unknowns to those of the standards applied on the same plate [14,15].

2.6. Total Phenol Content

The total phenolic content in the methanolic extracts of the studied samples was determined spectrophotometrically using the Folin–Ciocalteu reagent, following the procedure described by Dobreva et al. [16]. All determinations were performed in triplicate, and results are presented as mean ± standard deviation.

2.7. Methodological Considerations

GC-MS was used for compositional screening of essential oils and derivatized extracts, and HPTLC/Folin–Ciocalteu for indicative estimates of selected phenolics. These methods were chosen for their accessibility, reproducibility, and suitability for preliminary comparative assessments across multiple provenances. These methods provide indicative rather than definitive quantitative values. Future studies should employ higher-resolution and/or orthogonal techniques—such as UHPLC-HRMS/MS (Orbitrap/Q-TOF) for untargeted and targeted metabolomics, GC × GC-MS to improve separation of volatile isomers, HS-SPME-GC-MS for headspace volatiles, LC-MS/MS (MRM) for absolute quantification of key markers (e.g., rosmarinic, chlorogenic, and triterpene acids), GC-FID with internal standards for robust EO quantitation, and 1D/2D NMR for non-biased profiling—to refine identification and obtain absolute concentrations.

2.8. Statistical Analysis

Quantitative data for total phenolic content and rosmarinic acid content (mean ± SD; n = 3 per region) were analyzed using one-way ANOVA based on summary statistics, followed by Tukey’s HSD for pairwise comparisons. Statistical significance was set at p < 0.05. Essential-oil composition data are presented descriptively because replicate-level variance was not available in the current summary. Analyses were performed in standard statistical software.

3. Results

3.1. Essential Oils

The essential oil compositions of the studied T. longedentatus samples are presented in

Table 1. Main compounds in the essential oils of T. longedentatus from various origins [% area].

Compounds	RT	RI	CA	ER	TL
α-Pinene	7.61	932	0.30	0.64	2.87
Camphene	8.02	946	0.52	0.54	4.64
β-Pinene	8.75	974	0.13	0.81	0.22
1-Octen-3-ol	8.92	979	0.71	0.94	0.42
p-Cymene	10.04	1025	0.42	0.38	0.85
Eucalyptol	10.26	1026	2.69	5.48	2.20
Linalool	12.03	1095	2.52	0.27	4.70
Camphor	13.32	1141	0.66	2.14	7.31
Isoborneol	13.97	1155	0.53	0.57	4.13
Terpinen-4-ol	14.16	1174	0.44	0.60	0.89
α-Terpineol	15.03	1186	0.26	1.05	0.47
cis-Geraniol	15.43	1228	1.65	0.52	3.27
Neral	15.91	1235	30.36	25.63	12.14
Geranial	16.62	1264	38.45	33.88	15.63
Neryl acetate	18.40	1359	0.92	2.91	13.28
Geranyl acetate	18.91	1382	0.97	2.91	1.20
Caryophyllene oxide	23.29	1581	0.91	1.00	0.54

Legend: Eastern Rhodopes (ER), the Thracian Lowland (TL), and cultivated areas near Sofia (CA) Values are expressed as relative percentage of the total ion chromatogram (% of total GC–MS peak area). Abbreviations: RT—Retention Time; RI—Retention Index.

. While the oils exhibited similar qualitative profiles across all localities, notable quantitative differences in individual components were observed. Citral isomers—neral and geranial—were identified as the predominant constituents in all samples. In the cultivated area (CA) samples, these isomers accounted for more than 60% of the total oil content. In contrast, the essential oil from the Thracian Lowland (TL) population exhibited a significantly lower citral content (approximately 30%) but showed a markedly higher proportion of neryl acetate.

3.2. Methanolic Extracts

A range of primary and secondary metabolites were identified in the methanolic extracts of the studied T. longedentatus samples, as summarized in

Table 2. Compounds identified in the methanolic extracts and acetone exudates of T. longedentatus from various origins [% area].

Compounds	RT	RI	Methanolic Extracts			Acetone Exudates
			CA	ER	TL	CA	ER	TL
Lactic Acid	8.33	1066	0.92	0.46	0.02
Glycolic acid	8.78	1081	0.54	0.14	0.09
exo-Borneol	12.98	1195	0.35	0.06	0.25
Phosphoric acid	14.16	1296	0.31	0.04	0.04
Glycerol	14.28	1289	8.44	1.17	2.18	1.39	0.87	1.48
Thymol	15.23	1322	0.16	0.05	0.09	0.34	0.28	0.31
Succinic acid	15.38	1325	1.3	0.34	0.27	0.11	0.74	0.14
Fumaric acid	16.38	1353	Trace	0.02	0.11
Hydroquinone	17.67	1409	0.43	0.81	0.11	0.15	3.65	0.10
Geranic acid	17.93	1448	1.24	0.21	0.2
Malic acid	19.95	1473	1.91	0.38	0.84
Erytrithol	20.43	1503	0.09	0.01	0.18
Pyroglutamic acid	20.64	1515	0.44	0.04	0.09
Erythronic acid	21.75	1526	0.10	0.18	0.15
Hydroxybenzoic acid	23.32	1637	Trace	0.01	Trace	0.72	0.76	0.51
Vanillic acid	26.89	1776				0.22	0.14	0.16
Fructose 1	27.27	1800	6.45	7.97	4.78	0.47	1.77	0.28
Fructose 2	27.44	1815	8.96	2.01	11.62	1.02	2.90	0.48
Protocatechuic acid	27.58	1835	0.01	0.03	Trace	0.33	0.37	0.12
Quinic acid	28.29	1839	4.92	3.01	4.53	0.28	0.23	0.09
Hydroxycinnamic acid	29.57	1940	0.05	0.07	0.02
β-D-Glucopyranose	30.17	1945	5.7	6.81	14.07	1.40	1.23	1.99
Hexadecanoic acid	30.78	2040	0.63	0.34	0.25	2.45	2.46	0.89
Catechollactate	31.20	2080	0.26	1.72	2.25
myo Inositol	31.53	2090	1.18	0.88	5.21
Ferulic acid	31.57	2104	trace	0.02	Trace	0.26	0.1	trace
Caffeic acid	32.11	2141	0.1	0.31	0.27	0.29	0.49	0.38
Octadecadienoic acid	32.91	2204	0.15	0.3	0.39	0.24	1.60	0.11
Octadecatrienoic acid	32.98	2212	0.22	0.46	0.52
Octadecanoic acid	33.25	2238	0.06	0.16	0.18	0.76	1.16	0.13
Arbutin	36.24	2561	1.38	23.96	0.47
Sucrose	36.69	2628	15.3	24.78	15.55	1.23	0.77	0.28
Chlorogenic acid	42.37	3091	0.86	0.02	0.14
Oleanolic acid	49.25	3525				14.25	20.59	22.43
Ursolic acid	50.71	3530				15.84	10.08	5.04

Legend: Eastern Rhodopes (ER), the Thracian Lowland (TL), and cultivated areas near Sofia (CA). Values are expressed as relative percentage of the total ion chromatogram (% of total GC–MS peak area). Abbreviations: RT—Retention Time; RI—Retention Index.

. Key bioactive constituents included chlorogenic acid, exo-borneol, arbutin, hydroquinone, geranic acid, pinitol, and several phenolic acids such as caffeic, ferulic, and 4-hydroxybenzoic acid. Although the overall metabolic profiles were qualitatively similar across the different geographical origins, notable quantitative variations were observed. The cultivated area (CA) samples exhibited higher concentrations of chlorogenic acid, geranic acid, and several other metabolites. In contrast, the Eastern Rhodopes (ER) population showed markedly elevated levels of arbutin, hydroquinone, and related compounds. Additionally, monosaccharides were found in particularly high concentrations in samples from the Thracian Lowland (TL), indicating possible region-specific metabolic adaptations.

3.3. Acetone Exudates

The compounds identified in the acetone exudates of the studied T. longedentatus samples, as determined by GC/MS analysis, are summarized in Table 2. Triterpene acids—specifically oleanolic and ursolic acids—were the most abundant constituents across all samples. In addition to these dominant compounds, the exudates also contained various phenolic acids, terpenes, fatty acids, and saccharides, reflecting the chemically diverse composition of the external secretions associated with the aerial parts of the species.

3.4. Total Phenolic and Rosmarinic Content

The total phenolic content and rosmarinic acid concentration in the methanolic extracts of the studied T. longedentatus samples were determined using spectrophotometric and HPTLC methods, respectively (

Table 3. Total phenolic content (mg GAE/g DW) and rosmarinic acid content (mg/g DW) in aerial parts of T. longedentatus.

Samples	Total Phenolic Content [mg GAE/g DW]	Rosmarinic Acid Content [mg/g DW]
Cultivated areas (CA)	9.26 ± 0.39	1.96 ± 0.3
Eastern Rhodopes (ER)	14.66 ± 0.55	1.85 ± 0.2
Thracian Lowland (TL)	11.63 ± 0.10	1.82 ± 0.1

Values are expressed as mg/g dry weight (DW) of plant material. Total phenolic content determined as gallic acid equivalents (GAE).

). The highest total phenolic content was observed in the Eastern Rhodopes population, followed by the Thracian Lowland, while the cultivated material near Sofia showed the lowest value.

One-way ANOVA confirmed that these differences were statistically significant (F(2,6) = 194.27, p = 3.5 × 10⁻⁶). Tukey’s HSD indicated that all pairwise comparisons were significant (ER > TL > CA).

In contrast, rosmarinic acid content showed no significant differences among the three regions (F(2,6) = 2.17, p = 0.195).

4. Discussion

This study provides a preliminary comparative analysis of the metabolite composition of T. longedentatus from wild populations (Eastern Rhodopes and Thracian Lowland) and a cultivated population near Sofia. Although the species is geographically restricted to the Balkans, its phytochemical profile remained largely consistent across both natural and cultivated origins, with some quantitative variations. These results offer valuable insights into the ecological plasticity of the species and the feasibility of its ex situ cultivation for conservation and commercial purposes.

4.1. Preservation of Essential Oil Composition and Citral Dominance

The essential oil of T. longedentatus was dominated by citral isomers, neral and geranial, across all studied origins, in agreement with previous findings [5]. These compounds, known for their lemon-like aroma and broad biological activity spectrum, are of particular interest for applications in perfumery, food preservation, and natural plant protection. Their consistent presence and dominance—especially in the cultivated samples where citral content exceeded 60%—indicate the preservation of this key metabolic trait under cultivation conditions. This observation is noteworthy, given that EO composition in Thymus species can often be significantly influenced by environmental and edaphic factors.

The elevated citral levels in cultivated samples suggest that environmental conditions in the Sofia region not only support but may enhance the biosynthesis or accumulation of these compounds. The preservation of citral isomers during cultivation is critical, as these compounds are associated with antimicrobial, antifungal, and antioxidant activities, and even patented for herbicidal use in lemongrass-based formulations [17,18]. This underscores the suitability of cultivated T. longedentatus for the development of high-value EO-based products.

4.2. Secondary Metabolite Plasticity and Environment-Driven Variation

While qualitative composition remained largely consistent across samples, GC/MS analysis of methanolic extracts revealed significant quantitative differences in several individual metabolites. The quantitative differences in metabolite content may be explained by ecological contrasts among the sites: the Eastern Rhodopes are characterized by drier conditions and stony soils, while the Thracian Lowland features warmer temperatures and alluvial soils. In the cultivated plots, irrigation is applied regularly and no water deficit occurs, which may explain the markedly lower arbutin content compared to the natural populations. Cultivation conditions with regular irrigation, reduced competition, and stable soil management likely promoted the accumulation of certain metabolites such as citral isomers, chlorogenic acid, and geranic acid. By contrast, compounds associated with stress adaptation, such as arbutin and hydroquinone, were more abundant in wild plants exposed to natural abiotic stressors, particularly water deficit in the Eastern Rhodopes. This suggests that metabolite profiles of T. longedentatus reflect both genetic stability and plastic responses to environmental conditions. For instance, chlorogenic acid, a potent antioxidant phenolic acid, was markedly more abundant in the cultivated material. This is consistent with previous reports showing that elevated CO₂ levels, supplemental lighting, and mild abiotic stress can stimulate chlorogenic acid biosynthesis in other plant species [19,20].

In contrast, arbutin and hydroquinone—compounds with known antioxidant, skin-lightening, and antimicrobial properties—were significantly more concentrated in the Eastern Rhodopes samples. Arbutin biosynthesis is known to be enhanced under water deficit conditions [21], which may reflect the specific microclimatic or edaphic stressors present in this mountainous region. These findings suggest that different environmental pressures modulate secondary metabolism in T. longedentatus, leading to region-specific chemotypes without altering the core metabolite identity.

The TL population was distinguished by an elevated content of monosaccharides and sugar alcohols such as myo-inositol and pinitol. These osmoprotective molecules are known to accumulate under saline or osmotic stress and may be indicative of specific adaptations to the coastal environment. These results further support the hypothesis that secondary metabolite variation is influenced by abiotic factors, which can be leveraged to optimize phytochemical yields for specific industrial or pharmacological purposes.

4.3. Exudate Chemistry and Defensive Potential

The acetone-extracted exudates from aerial plant surfaces were found to be rich in triterpene acids, namely oleanolic and ursolic acids, compounds that have also been reported as major constituents in Thymus species with demonstrated antifungal and antioxidant activity [22]. These compounds are known for their multiple pharmacological effects, including anti-inflammatory, antimicrobial, hepatoprotective, and antitumor activities [23,24,25]. Their accumulation in surface tissues also suggests a protective ecological role, acting as barriers against herbivores, pathogens, and abiotic stress.

Interestingly, the content of these triterpenes was consistently high across all samples, confirming their role as constitutive defense compounds in T. longedentatus. However, a trend of higher ursolic acid concentration in wild populations, particularly from TL, may reflect additional adaptive functions in more ecologically dynamic environments. These results not only underscore the pharmacological significance of these exudates but also point to the possibility of exploiting T. longedentatus surface chemistry in the development of natural crop protection agents or functional cosmetics.

4.4. Rosmarinic Acid and Total Phenolics

The quantification of total phenolic content and rosmarinic acid—a hallmark compound of many Thymus species—further confirmed the influence of geographic origin on metabolite abundance. The highest total phenolic content was observed in the ER sample, corresponding to its elevated arbutin content. However, rosmarinic acid levels remained relatively uniform across all samples, suggesting that its biosynthesis is less sensitive to environmental variation, possibly due to its central role in basal oxidative defense mechanisms.

Rosmarinic acid has well-documented antiviral, anti-inflammatory, and neuroprotective effects, making it a key target for standardization in quality control protocols. The observed stability across provenances indicates that rosmarinic acid may serve as a reliable chemotaxonomic and quality marker in T. longedentatus-derived products.

4.5. Implications for Cultivation, Conservation, and Commercial Use

The high degree of chemical fidelity between the cultivated and Eastern Rhodopes populations—especially regarding essential oil composition and key phenolic and triterpene constituents—provides supportive evidence that T. longedentatus can be successfully domesticated without compromising its phytochemical integrity. The controlled cultivation conditions, characterized by regular irrigation and reduced abiotic stress, appear to favor the accumulation of citral isomers and chlorogenic acid, while compounds linked to stress adaptation, such as arbutin and hydroquinone, remain more abundant in wild habitats.

This provides supportive evidence with important implications for both conservation biology and the sustainable utilization of this species for commercial purposes. Given the increasing interest in plant-based bioactives and natural alternatives to synthetic agrochemicals and pharmaceuticals, T. longedentatus holds considerable promise. Its consistent production of citral-rich essential oil, coupled with a rich profile of pharmacologically active phenolics and triterpenes, positions it as a candidate for inclusion in functional food, cosmeceutical, and botanical pesticide development pipelines.

At the same time, the preservation of metabolite diversity across natural populations highlights the importance of in situ conservation and habitat protection to safeguard the species’ genetic and chemical diversity.

From a sustainability perspective, cultivation reduces the risk of overharvesting from the small and fragmented wild populations, while in situ conservation remains essential to preserve the species’ genetic and chemical diversity. Protection of natural habitats within the distribution range of T. longedentatus should be prioritized alongside the development of ex situ cultivation strategies.

4.6. Limitations and Future Directions

The present study represents a preliminary comparative analysis based on GC-MS screening and indicative assays (HPTLC, Folin–Ciocalteu). While these methods support between-group comparisons, they do not constitute comprehensive metabolomic profiling. Incorporating UHPLC-HRMS/MS metabolomics, GC × GC-MS, targeted LC-MS/MS quantification, HS-SPME-GC-MS, and NMR will improve metabolite coverage, confirm identifications, and provide absolute concentrations in subsequent studies.

5. Conclusions

This study provides the first preliminary comparative analysis of the metabolite composition of T. longedentatus across wild and cultivated populations, providing critical insights into the species’ phytochemical consistency, environmental responsiveness, and cultivation potential. Although some quantitative differences were observed, the overall qualitative profiles—particularly the dominance of citral isomers in the essential oil and the consistent presence of pharmacologically important phenolic and triterpene compounds—were largely preserved across all origins.

Cultivated plants retained the key traits of their wild progenitors while also showing an enhanced accumulation of citral and chlorogenic acid. Moreover, the enhanced accumulation of selected bioactive compounds such as chlorogenic and geranic acids under cultivation conditions suggests that targeted agronomic practices may further optimize the species’ phytochemical output.

The stable occurrence of rosmarinic acid and triterpene acids, together with region-specific enrichment of other valuable metabolites, underscores the dual importance of T. longedentatus as both a reservoir of therapeutic compounds and a candidate for sustainable industrial applications. Cultivation under controlled conditions increases the yield of commercially valuable metabolites such as citral and chlorogenic acid. In contrast, stress-related compounds like arbutin and hydroquinone remain typical of wild populations. This duality highlights the complementary roles of cultivation in optimizing phytochemical yields and of natural habitats in preserving stress-induced metabolic diversity.

These results demonstrate that cultivation can be used to increase the yield of commercially valuable metabolites, while natural habitats remain important reservoirs of stress-induced metabolic diversity. Together, ex situ cultivation and in situ conservation provide complementary strategies for sustainable use and long-term preservation of this Balkan endemic species.