Targeted and Untargeted Metabolomics and Pharmacological Potential of Endemic Stachys sparsipilosa R. Bhattacharjee & Hub.-Mor.

Abstract

Species of the genus Stachys (Lamiaceae) are recognized for their ethnobotanical importance and chemical diversity. In this study, the essential oil (EOS) and solvent extracts of the endemic species Stachys sparsipilosa were investigated using integrated GC–MS and LC–ESI–QTOF/MS approaches. GC–MS analysis showed that identified constituents accounted for 94.62% of the total oil, with caryophyllene oxide, kauran-16-ol, and cubebol as major components. Targeted LC–MS analysis quantified prominent phenolic compounds, including chlorogenic acid, rutin, and hesperidin, while untargeted metabolomics tentatively annotated 168 metabolites belonging to phenolics, terpenoids, and other classes. Antioxidant capacity was evaluated using complementary in vitro assays, and enzyme inhibitory activities against α-amylase, α-glucosidase, tyrosinase, acetylcholinesterase, and butyrylcholinesterase were assessed in comparison with standard inhibitors. The extracts demonstrated measurable but generally moderate activities relative to the corresponding positive controls. The essential oil exhibited moderate, non-selective cytotoxic effects at relatively high concentrations, whereas solvent extracts showed limited activity within the tested range. Molecular docking analyses were performed as supportive tools to explore possible enzyme–ligand interactions. Overall, S. sparsipilosa displays a chemically diverse metabolite profile associated with composition-dependent bioactivities, providing a basis for further mechanistic and in vivo studies.

1. Introduction

The genus Stachys L. represents the most extensive lineage within the subfamily Lamioideae and is among the largest genera of the Lamiaceae family, encompassing approximately 370 taxa worldwide. Its primary distribution lies within the warm-temperate belt of the Mediterranean region and southwestern Asia. Additional centres of species diversification are reported from the Americas and southern Africa, whereas representatives of the genus are notably absent from Australia and New Zealand. Two major hotspots of diversity have been identified globally: one extending across southern and eastern Anatolia to the Caucasus and adjacent regions of northwestern Iran and northern Iraq, and another located in the Balkan Peninsula [1].

Members of Stachys have long been integrated into traditional medicinal systems across multiple continents, including Europe, Asia, Africa, and the Americas. They are commonly prepared as infusions, decoctions, or topical formulations. Ethnomedicinal records indicate their use for a wide range of ailments such as respiratory disorders, gastrointestinal complaints, inflammatory conditions, cardiovascular problems, and neurological disturbances. In East Asian practices, certain species are administered for infectious diseases and ischemic conditions, while Middle Eastern and Mediterranean traditions employ them for inflammatory, metabolic, and gynecological disorders. In Anatolia, Stachys species are frequently used in folk medicine for colds, cough, digestive complaints, fever, and metabolic diseases, including diabetes and neurodegenerative conditions [2,3,4,5].

The therapeutic relevance of Stachys species is attributed to their chemically diverse secondary metabolites. Essential oils are typically dominated by sesquiterpenes and monoterpenes such as (E)-caryophyllene, caryophyllene oxide, germacrene D, spathulenol, and α- and β-pinene. In addition, non-volatile fractions are rich in flavonoids, iridoid glycosides, phenylethanoid derivatives, diterpenes, fatty acids, and phenolic acids, many of which have been associated with antioxidant, anti-inflammatory, and enzyme-modulating activities [6].

Stachys sparsipilosa, an endemic species confined to Turkey, is primarily distributed in the Eastern Mediterranean region. It inhabits calcareous slopes and the understory of Pinus brutia forests at altitudes ranging from approximately 400 to 1700 m [1]. Despite its restricted distribution, the species has received limited scientific attention. To date, only a single investigation has explored its biological properties, focusing mainly on antioxidant and antimicrobial activities [7]. Comprehensive phytochemical profiling and evaluation of its broader bioactivity potential remain largely unexplored.

Therefore, this study aimed to (i) comprehensively characterize the volatile and non-volatile metabolome of the endemic species Stachys sparsipilosa by integrating GC–MS with targeted and untargeted LC–ESI–QTOF/MS, (ii) explore whether the observed chemical profile provides chemotaxonomically informative features within Stachys, and (iii) examine potential associations between major metabolite classes/abundant compounds and the measured bioactivities (antioxidant capacity and inhibition of α-amylase, α-glucosidase, tyrosinase, AChE, and BuChE), supported by exploratory molecular docking. The novelty of this work does not lie in the identification of entirely new metabolites or the proposal of a new chemotype, but rather in providing the first integrated metabolomic and multi-target bioactivity assessment of S. sparsipilosa, linking compositional patterns with functional screening data within a chemotaxonomic framework. The results are discussed in terms of identifying composition-dependent bioactivity patterns that may guide future fractionation/compound isolation studies and inform the rational evaluation of S. sparsipilosa as a candidate source of bioactive constituents for nutraceutical, cosmetic, and pharmaceutical research while acknowledging that further mechanistic and in vivo validation is required.

2. Materials and Methods

2.1. Chemicals

Acetylcholinesterase (AChE, from Electrophorus electricus), butyrylcholinesterase (BuChE, from equine serum), acetylthiocholine/butyrylthiocholine iodide, 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman’s reagent), galanthamine, tyrosinase (from mushroom), L-Dopa, kojic acid, α-glucosidase (Type I, E.C.3.2.1.20), p-nitrophenyl glucopyranoside, α-amylase (Type VI-B, E.C.3.2.1.1), hydrocarbon mixtures (C₇-C₃₀ n-alkanes), 1,1-Diphenyl-2-picrylhydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), potassium persulfate, trolox, 2,4,6-tris(2-pyridyl)-s-triazinen and ferric chloride were purchased from Sigma Aldrich (Saint Louis, MO, USA).

2.2. Plant Material

Stachys sparsipilosa (36°19′35″ N, 30°21′55″ E) was collected in May 2025 from Kumluca (Antalya, Türkiye). Botanical identification was performed by Prof. Dr. Hasan Yıldırım, and voucher sample (No. 11219) was deposited in the Herbarium of Ege University.

2.3. Isolation of the Essential Oil

The fresh flower parts of the plants (200 g) were subjected to hydrodistillation using a Clevenger-type apparatus with 2000 mL of distilled water. After a distillation period of 3 h, the essential oils were collected and dried over anhydrous sodium sulfate, measured to determine yield (%, w/w), and stored in amber vials at 4 °C until analysis.

2.4. Gas Chromatography-Mass Spectrometry Analysis

The analysis of essential oil was performed using an Agilent 7890B gas chromatograph equipped with a PAL RSI 85 liquid autosampler and an Agilent MSD 5977A mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Separation was achieved on an HP-5MS capillary column (30 m × 0.30 mm, 0.25 µm film thickness), with helium as the carrier gas at a flow rate of 0.7 mL/min. The oven temperature was programmed to start at 50 °C, increase to 210 °C at 4 °C/min and hold for 3 min, then rise to 280 °C at 20 °C/min and maintain for 5 min. Injections were carried out in split mode (1:50) with an injection volume of 1.0 μL. The interface, ion source, and quadrupole temperatures were set at 250 °C, 180 °C, and 150 °C, respectively. Electron impact ionisation was applied at 70 eV, and mass spectra were acquired over a range of 50–500 amu. Compound identification was performed by comparison with the WILEY mass spectral library.

2.5. Procedure for Sample Extraction

One gram of air-dried and powdered plant material was extracted with 15 mL of solvent (methanol, ethyl acetate, or diethyl ether) in 50 mL Falcon tubes. The mixtures were continuously agitated on a rotator (ISOLAB Laborgeräte GmbH, Eschau, Germany) at room temperature for 1 h. The extraction process was repeated three times using fresh solvent, and the supernatants were combined. The mixtures were centrifuged at 4000 rpm for 10 min to separate the liquid phase. Combined extracts were evaporated to dryness under reduced pressure using a rotary evaporator (Buchi, Flawil, Switzerland). The dried extracts were weighed to determine the extraction yield and stored at 4 °C until further analysis. Methanol, ethyl acetate, and diethyl ether were selected to represent solvents of increasing non-polarity, allowing for a broader coverage of metabolites with different polarity profiles. Methanol was used to extract polar phenolic and flavonoid-type compounds, ethyl acetate to recover semi-polar constituents, and diethyl ether to target relatively non-polar metabolites.

2.6. Quantitative Analysis of Phenolic Compounds

Chromatographic analysis of the extracts was carried out using an Agilent 6550 QTOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray ionisation (ESI) source to identify and characterise polyphenols. Separation was achieved on an Agilent Poroshell 120 SB-C18 column (100 × 3.0 mm, 2.7 μm particle size). The mobile phase consisted of water with 0.1% formic acid (solvent A) and acetonitrile (solvent B). The gradient program was as follows: 95–5% B (0–8 min), 75–25% B (8–16 min), 50–50% B (16–24 min), 25–75% B (24–30 min), 5–95% B (30–32 min), 25–75% B (32–34 min), 50–50% B (34–36 min), 75–25% B (38 min), and 95–5% B (38–42 min). The flow rate was maintained at 0.4 mL/min, with an injection volume of 10 μL for each sample or standard. The column temperature was set at 35 °C. ESI conditions included a nozzle voltage of 1000 V, a gas temperature of 290 °C, and a nebuliser pressure of 35 psi. Mass spectra were acquired over an m/z range of 50–1800, and data were processed using MassHunter Workstation Software (Qualitative Analysis, version B.06.01, Agilent Technologies, Santa Clara, CA, USA).

2.7. LC-QTOF-MS Screening

Chromatographic separation was performed using mobile phases with gradient elution as outlined in the corresponding table. Mobile phase A consisted of water with 0.1% formic acid, while mobile phase B was acetonitrile. The column was maintained at 35 °C, with an injection volume of 10 μL and a flow rate of 0.4 mL/min. The gradient program was as follows: 0 min, 5% B; 7.5 min, 25% B; 15 min, 50% B; 22.5 min, 75% B; 30 min, 95% B; 37.5 min, 5% B, followed by a 2.5 min reconditioning cycle. Mass spectrometric analysis was carried out on an Agilent 6550 iFunnel high-resolution accurate-mass QTOF-MS system equipped with an Agilent Dual Jet Stream electrospray ionisation (Dual AJS ESI, Agilent Technologies, Santa Clara, CA, USA) source, operating in both positive and negative ionisation modes. Instrument parameters were set as follows: desiccant gas flow, 14.0 L/min; nebuliser pressure, 35 psi; drying gas temperature, 290 °C; sheath gas temperature, 400 °C; and sheath gas flow, 12 L/min. MS/MS spectra were acquired using collision energies of 20 eV, and full-scan mass spectra were recorded over an m/z range of 50–1800. Data acquisition and processing were performed with MassHunter Workstation software (Agilent Technologies). Compound annotations were based on accurate mass measurements and database-matched MS/MS fragmentation patterns where available, using the METLIN metabolomics database and the METLIN_AM_PCDL library. Since MS/MS spectra were not available for all metabolites, identifications were considered putative.

2.8. Total Phenolic and Flavonoid Contents

The total phenolic content (TPC) and total flavonoid content (TFC) of the extracts were determined using colourimetric methods as previously described [8]. TPC values were expressed as milligrams of gallic acid equivalents (GAE) per gram of dried extract, while TFC values were expressed as milligrams of quercetin equivalents (QE) per gram of dried extract.

2.9. DPPH (1,1-Diphenyl-2-picrylhydrazyl) Assay

The DPPH radical scavenging activity was evaluated according to the method of Blois (1958) [9] with minor modifications. Briefly, 0.1 mM DPPH solution was mixed with the sample solution at appropriate concentrations and incubated for 30 min at room temperature in the dark. Absorbance was measured at 517 nm. Trolox was used as the reference antioxidant, and the results were expressed as milligrams of Trolox equivalents per gram of extract (mg TE/g extract).

2.10. ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonicacid)) Assay

The ABTS⁺ radical cation was generated by mixing equal volumes of 7.4 mM ABTS and 2.45 mM potassium persulfate solutions, followed by incubation at room temperature in the dark. The resulting solution was then diluted with methanol to obtain an absorbance of 0.700 ± 0.02 at 734 nm. Subsequently, 0.2 mL of the sample was mixed with 2.8 mL of the ABTS⁺ solution, and the absorbance was measured at 734 nm after incubation for two hours at room temperature [10]. Trolox was used as a positive control, and the results were expressed as milligrams of Trolox equivalents per gram of extract (mg TE/g extract).

2.11. CUPRAC (Cupric Ion Reducing) Assay

The cupric ion reducing antioxidant capacity (CUPRAC) assay was performed according to Apak et al. (2008) [11] with minor modifications. Briefly, the reaction mixture contained 10 mM CuCl₂, 7.5 mM neocuproine, ammonium acetate buffer (pH 7.0), and the sample solution. After incubation in the dark for 30 min at room temperature, absorbance was measured at 450 nm. Trolox was used as the reference antioxidant, and the results were expressed as milligrams of Trolox equivalents per gram of extract (mg TE/g extract).

2.12. FRAP (Ferric Reducing Antioxidant Power) Assay

The FRAP reagent was prepared by combining 25 mL of acetate buffer (0.3 M, pH 3.6), 2.5 mL of TPTZ (2,4,6-tris(2-pyridyl)-s-triazine) solution (10 mM in 40 mM HCl), and 2.5 mL of ferric chloride solution (20 mM). Subsequently, 0.1 mL of the sample was added to 2.0 mL of the FRAP reagent, and the mixture was incubated for 30 min at room temperature. The absorbance was recorded at 593 nm [12]. Trolox served as the positive control, and the results were expressed as milligrams of Trolox equivalents per gram of extract (mg TE/g extract).

2.13. Enzyme Inhibitory Activity

The enzyme inhibitory activities of the samples were assessed spectrophotometrically using a 96-well microplate reader. Tyrosinase inhibition was evaluated according to the dopachrome method, with kojic acid employed as the positive control. Sample solutions were prepared in potassium phosphate buffer (1.15 M, pH 6.8) containing 5% dimethyl sulfoxide, yielding seven concentrations ranging from 1000 to 1 μg/mL, together with a tyrosinase enzyme solution at 46 U/mL. The samples and enzyme were incubated in the microplate at 25 °C for 10 min, after which a 2.5 mM L-DOPA substrate solution was added and a second incubation was carried out for 20 min at the same temperature. Absorbance was subsequently measured at 475 nm [13].

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition assays were carried out using the Ellman method [14] with minor modifications [15]. Briefly, enzyme solutions and sample concentrations (0.001–1000 μg/mL) were incubated in microplates, and the reaction was initiated by adding the corresponding thiocholine substrates together with DTNB. Absorbance was monitored spectrophotometrically, and galanthamine was used as the positive control. The inhibitory activities against α-glucosidase [16] and α-amylase [17] were evaluated according to established spectrophotometric methods. Appropriate substrates were used for each enzyme, and absorbance changes were recorded using a microplate reader. Acarbose served as the positive control in both assays.

IC₅₀ values for the samples in all enzyme inhibition assays were calculated using GraphPad Prism 10 software, based on data from three independent experiments.

2.14. Molecular Docking Procedures

Molecular docking studies were performed to support the in vitro enzyme inhibition results and to explore the possible binding interactions of the major phytochemicals with the active sites of the target enzymes. All docking procedures, including ligand preparation, protein preparation, homology modelling, grid generation, and docking simulations, were conducted according to a previously established computational protocol described in detail by Emir et al. (2022) [18]. Docking calculations were carried out using a flexible docking approach, and the resulting binding poses were evaluated based on calculated binding energies and key interactions within the catalytic sites of the enzymes. The same docking parameters were consistently applied to all ligands to ensure comparability of the predicted binding affinities.

2.15. Cell Culture and Cytotoxicity (MTT) Assay

LNCaP (human prostate adenocarcinoma), PC-3 (human prostate adenocarcinoma), HeLa (human cervical carcinoma), HDF (human dermal fibroblast), and MRC-5 (human lung fibroblast) cell lines were procured from ATCC and maintained in our laboratory stocks under liquid nitrogen conditions. For cytotoxicity evaluations, cells were cultured in 100 mm culture dishes and sub-cultured until reaching a maximum confluency of approximately 70%, then incubated in a humidified atmosphere at 37 °C with 5% CO₂. To determine IC₅₀ values of the tested extracts at different concentrations, cells were seeded into 96-well plates, and viability assessments were initiated after a 24 h attachment period. Following 48 h exposure of the cell lines to the extracts, the MTT assay was applied by adding an MTT working solution to achieve a final concentration of 0.5 mg/mL and incubating for 4 h. Subsequently, the medium was discarded, and 200 µL of DMSO was introduced into each well to solubilise the formed formazan crystals. Absorbance measurements were carried out at 570–690 nm using a microplate reader (Varioskan, Thermo Fisher Scientific, Waltham, MA, USA), and the resulting data were subjected to statistical evaluation with GraphPad Prism (version 10.1.0).

2.16. Statistical Analysis

All antioxidant, enzyme inhibition, and cytotoxicity assays were performed in triplicate as independent technical replicates within each experimental run. Data were expressed as mean ± standard deviation (SD). Prior to one-way ANOVA, data were assessed for normal distribution and homogeneity of variances. Tukey’s post hoc test was subsequently applied to determine statistically significant differences (p < 0.05). Exploratory PCA was performed using SPSS (version 25, IBM Corp., Armonk, NY, USA) based on metabolite-class distribution data. Heatmap visualisation was generated to illustrate associations between metabolite classes and biological activities. These analyses are provided in the Supplementary Materials.

3. Results

3.1. Extraction and Essential Oil Composition

In the present investigation, methanol, ethyl acetate, and diethyl ether extracts, as well as the essential oil, were obtained from S. sparsipilosa. The extraction yields were 8.4% for methanol, 5.1% for ethyl acetate, and 3.9% for diethyl ether, calculated based on dry plant material. These samples were designated with the following codes: EOS (essential oil), MES (methanol extract), EAS (ethyl acetate extract), and DES (diethyl ether extract). The EOS was provided with a yield of 0.26% through hydrodistillation using a Clevenger apparatus. The chemical constituents identified are summarised in

Table 1. Chemical composition of essential oil.

No.	Compounds	RI a	RI b	Abundance
1	α-Pinene	939	939	0.21
2	Nonanal	1089	1089	0.14
3	L-Linalool	1106	1105	0.11
4	4-Terpineol	1177	1176	0.08
5	α-Terpineol	1190	1190	0.09
6	Decanal	1195	1195	0.11
7	Pulegone	1235	1234	0.15
8	α-Cubebene	1354	1353	0.47
9	α-Copaene	1367	1367	1.98
10	(-)-β-Bourbonene	1384	1384	0.88
11	β-Cubebene	1390	1390	0.57
12	β-Caryophyllene	1423	1423	1.89
13	α-Humulene	1456	1455	0.29
14	6,10-Dimethylundeca-5,9-dien-2-one	1460	1462	0.22
15	Germacrene-D	1485	1484	0.77
16	epi-Bicyclosesquiphellandrene	1490	1495	4.89
17	α-Muurolene	1517	1516	0.51
18	Cubebol	1522	1522	6.93
19	δ-Cadinene	1528	1528	2.49
20	(-)-Spathulenol	1582	1582	1.96
21	Caryophyllene oxide	1589	1589	11.96
22	Epicubebol	1607	1605	1.57
23	α-Humulene epoxide II	1608	1608	1.43
24	Di-epi-1,10-cubenol	1623	1621	1.37
25	α-Caryophylladienol	1635	1636	0.59
26	T-Muurolol	1648	1648	2.23
28	α-Cadinol	1653	1652	1.03
29	cis-10-Hydroxycalamene	1667	1668	0.81
30	Caryophyllenol-II	1675	1672	1.54
31	Cadalene	1684	1685	0.65
32	ent-Germacra-4(15),5,10(14)-trien-1β-ol	1694	1695	0.38
33	Hexahydrofarnesyl acetone	1835	1835	2.26
34	Nonadecane	1900	1900	1.26
35	Hexadecanoic acid, methyl ester	1928	1930	0.41
36	ent-Pimara-8(14),15-diene	1939	1938	1.38
37	Isopimara-7,15-diene	1950	1950	0.56
38	n-Hexadecanoic acid	1962	1961	0.67
39	Trachylobane	1965	1965	0.51
40	Manoyl oxide	1989	1989	4.06
41	Eicosane	2000	2000	0.31
42	Kaur-16-ene	2036	2036	5.72
43	Octadecanal	2037	2035	1.02
44	Hexadecanoic acid, 2-hydroxy-, methyl ester	2046	2044	0.82
45	Heneicosane	2100	2100	2.29
46	Phytol	2122	2122	3.05
47	4,4-Dimethyl-13.alpha.-androst-5-ene	-	2140	3.53
48	Docosane	2200	2200	3.21
49	1,19-Eicosadiene	-	2206	1.69
50	Kauran-16-ol	2210	2210	7.26
51	Tricosane	2300	2300	1.15
52	(-)-Kaur-16-en-19-al	2330	2332	1.67
53	4,8,12,16-Tetramethylheptadecan-4-olide	2364	2365	0.54
54	Tetracosane	2400	2400	0.21
55	Pentacosane	2500	2500	0.89
56	Heptacosane	2700	2700	1.85
	Sesquiterpene Hydrocarbons			15.39
	Oxygenated Sesquiterpene hydrocarbon			30.99
	Diterpene hydrocarbons			8.17
	Oxygenated diterpenes			16.04
	Alkanes			11.17
	Other			12.86
	Total			94.62

^a Retention index calculated on HP-5MS column using the homologous series; ^b Literature retention index found in Adams, NIST Chemical Web Book and/or NIST 14.

, together with their retention indices, relative percentages, and order of elution on the column. GC–MS analysis of EOS revealed a chemical composition accounting for 94.62% of the total oil. Oxygenated sesquiterpenes were the dominant class (30.99%), and the oil was mainly characterised by caryophyllene oxide (11.96%) and cubebol (6.93%). Sesquiterpene hydrocarbons accounted for 15.39%, represented primarily by β-caryophyllene, α-copaene, and δ-cadinene. Oxygenated diterpenes constituted 16.04% of the oil and were dominated by kauran-16-ol (7.26%) and manoyl oxide (4.06%), while diterpene hydrocarbons (8.17%) were mainly represented by kaur-16-ene (5.72%).

3.2. Targeted LC-ESI-QTOF/MS Analysis

The phytochemical composition of the extracts was comprehensively characterised through targeted metabolomic analyses. Twenty-two selected phenolic compounds were quantified using LC-ESI-QTOF/MS (

Table 2. Concentrations of phenolic compounds in different samples of S. sparsipilosa.

Compound Name	Formula	Exact Mass	MES	EAS	DES
Catechol	C6H6O2	109.0289	ND	ND	ND
4-Hydroxybenzoic acid	C7H6O3	137.0239	181.89 ± 0.73 a	ND	ND
Vanillic acid	C8H8O4	167.0344	48.24 ± 0.78 a	ND	ND
3-Hydroxybenzoic acid	C7H6O3	137.0239	260.12 ± 0.35 a	ND	ND
p-Coumaric acid	C9H8O3	163.0395	39.17 ± 0.88 c	140.67 ± 0.18 a	93.65 ± 0.22 b
Vitexin	C21H20O10	431.0978	483.36 ± 1.15 a	17.27 ± 0.44 b	4.41 ± 0.23 c
Hesperidin	C28H34O15	609.1819	601.67 ± 0.50 a	19.01 ± 1.15 c	35.39 ± 0.80 b
Chlorogenic acid	C16H18O9	354.0951	946.28 ± 0.66 a	233.28 ± 0.99 b	4.18 ± 0.77 c
Ferulic Acid	C10H10O4	193.0501	156.04 ± 1.00 b	183.41 ± 0.45 a	ND
Rutin	C27H30O16	609.1456	687.41 ± 0.56 a	270.48 ± 0.86 b	137.54 ± 0.28 c
Fisetin	C15H10O6	285.0399	176.80 ± 0.39 b	179.07 ± 1.04 a	ND
Morin	C15H10O7	301.0348	ND	104.92 ± 0.29 a	ND
Daidzein	C15H10O4	253.0501	2.37 ± 0.26 c	3.08 ± 0.28 a	2.40 ± 0.36 b
Galangin	C15H10O5	269.0450	5.82 ± 0.73 b	7.86 ± 1.14 a	1.59 ± 0.44 c
Quercetin	C15H10O7	301.0348	ND	241.68 ± 0.27 a	ND
Luteolin	C15H10O6	285.0399	30.05 ± 1.12 c	31.30 ± 0.66 b	63.16 ± 1.04 a
Isorhamnetin	C16H12O7	315.0505	33.08 ± 0.24 c	143.65 ± 0.43 a	52.13 ± 0.76 b
Naringenin	C15H12O5	271.0606	17.56 ± 0.79 c	69.86 ± 0.31 a	55.77 ± 0.91 b
Genistein	C15H10O5	269.0450	17.34 ± 0.23 c	38.23 ± 0.41 a	34.39 ± 0.30 b
Kaempherol	C15H10O6	285.0399	14.27 ± 0.40 c	108.42 ± 0.75 a	52.32 ± 0.93 b
Chrysin	C15H10O4	253.0501	1.63 ± 0.98 c	24.92 ± 0.40 a	19.41 ± 0.29 b
Apigenin	C15H10O5	269.0450	44.37 ± 0.23 a	34.36 ± 0.43 c	36.44 ± 0.81 b

Superscript letters indicate statistically significant differences between extracts within the same row (p < 0.05). Concentrations are given as µg/g of extract. ND: not detected.

). The phytochemical analysis revealed a diverse range of phenolic acids and flavonoids, including both simple and complex molecular structures. Among the simpler phenolic acids identified were 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, vanillic acid, and p-coumaric acid. More complex flavonoids such as vitexin, hesperidin, rutin, and chlorogenic acid were also present. Quantitative measurements indicated significant variation among the compounds. Chlorogenic acid had the highest concentration, recorded at 946.28 µg/g extract in MES, while rutin reached 270.48 µg/g extract in EAS. Quercetin demonstrated a concentration of 241.68 µg/g extract in EAS, whereas daidzein and chrysin were detected at comparatively low levels. Overall, the data underscore substantial chemical diversity among the identified metabolites and illustrate clear differences in their quantitative representation within the analysed samples.

3.3. Untargeted LC-ESI-QTOF/MS Analysis

As part of an untargeted metabolomic analysis, LC-ESI-QTOF-MS profiling resulted in the identification of a total of 168 metabolites belonging to phenolics, terpenes, iridoids, lactones, alkaloids, and primary metabolites. The results are shown in Table S1. Among the detected metabolites, phenolics constituted the most abundant class, encompassing 28 flavonoid glycosides, 4 phenylpropanoid glycosides, 4 polyphenols, 2 flavonoids, and 2 phenolic acids (Table S1).

3.4. Total Phenolic and Total Flavonoid Contents

The total phenolic and flavonoid contents of the extracts were quantitatively assessed using spectrophotometric methods. MES exhibited the highest total phenolic (76.32 mg GAE/g) and flavonoid contents (49.13 mg QE/g), followed by EAS (49.61 and 31.08 mg/g, respectively) and DES (19.43 and 10.42 mg/g, respectively).

3.5. Antioxidant Activity

The antioxidant activities of the extracts and essential oil were evaluated using DPPH, ABTS, FRAP, and CUPRAC assays (

Table 3. Antioxidant capacities of MES, EAS, DES and EOS.

Sample	DPPH	ABTS	FRAP	CUPRAC
MES	21.39 ± 0.17 b	57.45 ± 0.81 a	72.18 ± 0.29 a	72.49 ± 0.33 a
EAS	23.77 ± 0.26 a	49.16 ± 0.29 b	59.20 ± 0.63 b	63.28 ± 0.19 b
DES	12.96 ± 0.31 d	33.65 ± 0.21 c	39.84 ± 0.77 d	47.22 ± 0.96 c
EOS	14.46 ± 0.28 c	31.73 ± 0.62 d	40.09 ± 0.18 c	44.56 ± 0.53 d

Superscript letters indicate statistically significant differences between extracts within the same row (p < 0.05). Values are expressed as Trolox equivalents (mg TE/g sample).

). All samples demonstrated measurable radical scavenging and reducing capacities, with clear differences depending on the extraction solvent. Among the tested samples, MES exhibited the highest reducing power in the FRAP (72.18 ± 0.29 mg TE/g) and CUPRAC (72.49 ± 0.33 mg TE/g) assays, followed by EAS. In the DPPH and ABTS assays, EAS showed slightly higher activity than MES. DES and EOS displayed comparatively lower antioxidant capacities across all methods. Although the extracts showed consistent antioxidant responses, the obtained Trolox equivalent values indicate moderate activity when compared with the reference standard under equivalent assay conditions. The higher antioxidant capacity of MES and EAS is likely associated with their elevated total phenolic and flavonoid contents, suggesting that solvent polarity plays an important role in determining antioxidant performance. Overall, the results indicate solvent-dependent differences in redox activity rather than exceptionally strong radical scavenging effects.

3.6. Cytotoxic Activity

The cytotoxic effects of the extracts and essential oil were evaluated against LNCaP, PC-3, HeLa, HDF, and MRC-5 cell lines. Within the tested concentration range, MES, EAS, and DES did not exhibit measurable cytotoxic activity on any of the evaluated cell lines. In contrast, the essential oil (EOS) demonstrated dose-dependent inhibitory effects. The calculated IC₅₀ values were 84.01 µg/mL for MRC-5 and 152.1 µg/mL for HDF cells. For cancer cell lines, IC₅₀ values were 99.09 µg/mL for LNCaP, >200 µg/mL for PC-3, and 95.40 µg/mL for HeLa cells. To objectively assess cancer selectivity, selectivity indices (SI) were calculated by comparing IC₅₀ values obtained in normal and cancer cell lines. The resulting SI values were below 2 for all comparable comparisons (e.g., SI_MRC-5/LNCaP ≈ 0.85; SI_MRC-5/HeLa ≈ 0.88; SI_HDF/LNCaP ≈ 1.53; SI_HDF/HeLa ≈ 1.59), confirming the absence of selective antiproliferative activity. Overall, these IC₅₀ values indicate moderate cytotoxic effects occurring at relatively high concentrations, with comparable responses observed in both normal and cancer cell lines under the experimental conditions.

3.7. Enzyme Inhibitory Activity

The inhibitory activities of EOS, MES, EAS, and DES against selected enzymes were evaluated and compared with standard inhibitors (

Table 4. Enzyme inhibitory activities of MES, EAS, DES and EOS.

Sample	Amylase	Glucosidase	Tyrosinase	AChE	BuChE
MES	98.63 ± 0.28 c	298.33 ± 2.05 c	32.79 ± 0.28 d	49.23 ± 0.57 d	397.58 ± 1.77 b
EAS	46.19 ± 1.72 d	35.27 ± 1.92 d	79.33 ± 1.80 c	198.51 ± 1.72 b	225.81 ± 0.98 c
DES	188.69 ± 0.94 a	243.88 ± 1.08 d	128.53 ± 0.51 a	318.66 ± 1.08 a	409.42 ± 2.04 a
EOS	126.19 ± 1.31 b	547.63 ± 2.18 a	119.27 ± 1.22 b	76.19 ± 0.93 c	168.33 ± 1.01 d
Acarbose	8.75 ± 0.97 d	300.56 ± 6.65 b
Kojic Acid			7.90 ± 0.02 d
Galantamine				0.10 ± 0.01 d	1.04 ± 0.01 d

Superscript letters indicate statistically significant differences between extracts within the same row (p < 0.05). Values are shown as IC₅₀.

). Overall, the samples exhibited measurable but moderate inhibition relative to the corresponding positive controls. For AChE inhibition, MES showed the strongest activity among the extracts (IC₅₀ 49.23 µg/mL); however, this effect was markedly weaker than that of galantamine (IC₅₀ 0.10 µg/mL). EOS demonstrated comparatively lower AChE inhibition (IC₅₀ 76.19 µg/mL). In the case of BuChE, EOS exhibited the most pronounced effect among the samples (IC₅₀ 168.33 µg/mL), yet this activity remained substantially lower than galantamine (IC₅₀ 1.04 µg/mL). Regarding carbohydrate-hydrolysing enzymes, EAS showed the strongest α-amylase (IC₅₀ 46.19 µg/mL) and α-glucosidase inhibition (IC₅₀ 35.27 µg/mL) among the extracts. Nevertheless, these values were notably higher than those of acarbose for α-amylase (IC₅₀ 8.75 µg/mL), indicating moderate potency. For tyrosinase inhibition, MES exhibited the lowest IC₅₀ value (32.79 µg/mL) among the samples; however, this activity was considerably weaker than kojic acid (IC₅₀ 7.90 µg/mL). These findings indicate that the observed enzyme inhibition is moderate in magnitude and may reflect cumulative or synergistic effects of multiple phytochemical constituents rather than the action of a single highly potent inhibitor.

3.8. In Silico Docking Analysis

Eight major EOS compounds were docked into the active sites of AbTYR, TcAChE, PPA, and homology models of eqBuChE and Saccharomyces cerevisiae α-glucosidase using DOCK6. The docking scores are summarised in

Table 5. The calculated docking scores of the studied compounds inside the active sites of tyrosinase (PDB ID:2Y9X), α-amylase (PDB ID:1HX0), TcAChE (PDB ID:1DX6), and the active sites of homology models of α-glucosidase and eqBuChE, along with the absolute ranked positions.

Compounds	Tyrosinase	αAmylase	α-Glucosidase	TcAChE	eqBuChE
epi-Bicyclosesquiphellandrene	−19.95 (2)	−24.30 (1)	−29.33 (1)	−32.47 (1)	−28.22 (9)
Cubebol	−21.89 (1)	−27.64 (2)	−29.12 (1)	−32.81 (2)	−28.69 (24)
Caryophyllene oxide	−15.84 (3)	−23.99 (9)	−26.30 (1)	−27.20 (4)	−26.90 (1)
Manoyl oxide	−20.11 (1)	−27.99 (1)	−28.09 (1)	−25.41 (1)	−33.93 (1)
Kaur-16-ene	−18.91 (1)	−30.90 (1)	−20.83 (5)	−31.73 (1)	−31.42 (1)
Phytol	−43.96 (1)	−38.41 (7)	−47.63 (3)	−45.62 (5)	−46.35 (9)
4,4-Dimethyl-13.alpha.-androst-5-ene	−17.92 (2)	−28.32 (1)	−30.36 (1)	−30.85 (6)	−27.74 (62)
Kauran-16-ol	−21.41 (1)	−30.83 (1)	−32.79 (1)	−33.32 (1)	−32.72 (9)
Acarbose		−38.90 (18)	−71.46 (3)
Galantamine				−35.48 (18)	−33.76 (31)
Kojic acid	−21.06 (1)

.

AbTYR; Phytol, cubebol, and kauran-16-ol exhibited comparatively favourable grid scores among the essential-oil constituents, although kojic acid showed stronger predicted binding affinity (Table 5). Cubebol interacted with residues His85, His244, Val248, Asn260, His263, Phe264, Asn280, Gly281, Ser282, and Val283 (Figure 1). Kauran-16-ol displayed a similar interaction pattern (Figure 1). Phytol occupied the entrance of the copper-binding site and formed predominantly hydrophobic contacts (Figure 1).

α-Amylase (PPA): Phytol produced one of the lowest grid scores among the essential-oil constituents, although acarbose exhibited stronger predicted binding overall (Table 5). These ligands occupied a cavity formed by residues such as Trp58, Tyr62, His101, Arg195, Asp197, Lys200, and Glu233 (Figure 2). Hydrogen bonding was observed for phytol and kauran-16-ol, whereas kaur-16-ene interacted mainly through non-polar contacts (Figure 2).

α-Glucosidase: Within the homology model, phytol yielded the lowest grid score among EOS constituents, followed by kauran-16-ol and 4,4-dimethyl-13α-androst-5-ene (Table 1). Acarbose exhibited a lower grid score than the EOS compounds (Table 5). Phytol formed a hydrogen bond with Ser156 and additional hydrophobic contacts; kauran-16-ol and 4,4-dimethyl-13α-androst-5-ene interacted primarily via non-polar forces.

TcAChE and eqBuChE: Phytol achieved the lowest grid scores among the essential-oil constituents, while galantamine exhibited comparable or stronger predicted binding (Table 5). In TcAChE, phytol occupied both CAS and PAS, forming hydrophobic contacts and a hydrogen bond with Tyr70 (Figure 3). Kauran-16-ol formed two predicted hydrogen bonds with Tyr121, and cubebol formed two hydrogen bonds with Ser122 (Figure 3). In eqBuChE, phytol interacted with catalytic triad residues and formed a hydrogen bond with Asn83, whereas manoyl oxide and kauran-16-ol established primarily hydrophobic interactions, with kauran-16-ol additionally forming a hydrogen bond with Gly116 (Figure 3).

It should be emphasized that molecular docking provides a theoretical estimation of ligand–enzyme interactions and does not establish a definitive inhibition mechanism. The docking results are therefore interpreted as supportive and exploratory, complementing the experimentally observed moderate enzyme-inhibitory activities. Small differences in docking scores were not overinterpreted, as such variations may depend on algorithmic parameters and scoring functions.

4. Discussion

According to the literature, caryophyllene oxide has been identified as the predominant component in the essential oil of S. spectabilis Choisy ex DC. [19], S. cretica subsp. mersinaea (Boiss.) Rech.f. [19], S. palustris L. [20], and S. iva Griseb. [21]. The repeated occurrence of this oxygenated sesquiterpene as a dominant constituent across several Stachys taxa suggests that this compound may represent a conserved chemotaxonomic feature within the genus rather than a species-specific anomaly. The present chemical profile, which also includes diterpenoid derivatives such as kauran-16-ol and manoyl oxide, further indicates that the essential oil composition of S. sparsipilosa aligns with the broader terpenoid-rich metabolic tendencies previously described for the genus.

In the literature, the concentration of chlorogenic acid has been reported to be significantly elevated in phenolic compound analyses conducted on various Stachys species, including S. annua L. subsp. annua var. annua [22], S. cretica L. subsp. smyrnaea Rech Fil. [23], S. germanica subsp. heldreichii (Boiss.) Hayek [24], S. cretica L. subsp. kutahyensis Akcicek subsp. nov. [25], and S. cretica L. subsp. vacillans Rech. Fil. [26], suggesting that this compound may represent a chemotaxonomic marker of the genus. The consistent recurrence of chlorogenic acid as a quantitatively dominant metabolite across multiple taxa reinforces the hypothesis that this phenolic acid may play a taxonomic as well as a functional biochemical role within Stachys.

A comparative evaluation with previously reported Turkish Stachys species reveals that most taxa are characterised by sesquiterpene-hydrocarbon–dominant chemotypes, typically defined by germacrene-D and/or β-caryophyllene as major constituents [27]. In several Turkish representatives, these compounds occur at substantially higher relative abundances than observed in the present study. By contrast, the essential oil of S. sparsipilosa exhibited relatively low levels of these commonly dominant hydrocarbons and was instead characterised by a higher proportion of oxygenated sesquiterpenes and a notable presence of diterpenoid-type constituents, including kaurane-related compounds. When the comparison is restricted to Antalya-origin taxa, a similar sesquiterpene-hydrocarbon dominance has been reported for regional species such as S. sericantha as well as for the endemic S. aleurites [27], which was described as β-caryophyllene-dominant. In contrast, S. sparsipilosa, also collected from Antalya, departs from this regional tendency by exhibiting a compositional profile enriched in oxygenated sesquiterpenes and structurally diverse diterpenes. This divergence among taxa occurring within the same geographical region suggests that chemotype differentiation in Turkish Stachys is more strongly influenced by species-specific biosynthetic regulation than by geographic proximity alone. Such variation supports the chemotaxonomic relevance of quantitative compositional shifts within the genus.

Furthermore, the untargeted metabolomic screening, which revealed a wide range of phenolics, terpenes, iridoids, lactones, and alkaloids, illustrates that the phytochemical architecture of the species is not restricted to a limited compound group but instead reflects a metabolically diverse phytochemical system (Table S1). Such diversity is commonly associated with ecological adaptability and multifunctional biological activity in aromatic medicinal plants.

Comparable trends have been documented in previous studies on various Stachys species [22,24,25,26,28], in which methanolic extracts consistently exhibited elevated concentrations of total phenolics and flavonoids. This solvent-dependent enrichment pattern is widely recognised as a determinant of antioxidant capacity, particularly in electron-transfer-based assays. The predominance of phenolic compounds in polar extracts supports the view that extraction solvent polarity is a critical parameter influencing both qualitative and quantitative metabolite recovery. Rather than being attributable to a single antioxidant compound, the radical scavenging and reducing behaviours observed are more plausibly the result of cumulative or synergistic redox interactions among multiple phenolic acids and flavonoids. Such multi-compound antioxidant responses are frequently reported in botanical extracts where structurally diverse phenolics coexist within the same matrix. Among the identified phenolic constituents, chlorogenic acid, rutin, and hesperidin are well-documented for their antioxidant properties, which may contribute to the observed radical-scavenging and reducing activities of the extracts. Chlorogenic acid has demonstrated strong DPPH and NO radical-scavenging capacity, with reported SC₅₀ values of 18.4 µg/mL and 20.4 µg/mL, respectively [29], confirming its high electron-donating efficiency. In addition, chlorogenic acid exhibits ferrous ion-chelating activity, supporting its role as a secondary antioxidant by stabilising transition metals involved in ROS generation [30]. Similarly, phenolic acids, including chlorogenic acid, have been shown to display antioxidant effects in multiple systems such as DPPH scavenging, reducing power, β-carotene bleaching, and metal chelation assays [31]. Hesperidin has also been reported to exert significant DPPH and hydrogen peroxide scavenging activity in vitro [32], while rutin exhibits strong antioxidant potency associated with its redox-active flavonol structure and ability to inhibit radical-mediated oxidative processes [33]. Collectively, the presence of these phenolic compounds in the extracts may partially explain the observed DPPH, ABTS, FRAP, and CUPRAC activities, likely through combined radical-scavenging, electron-transfer, and metal-chelating mechanisms rather than a single dominant pathway.

Cholinesterase inhibitors, which are commonly employed in the management of Alzheimer’s disease, do not influence overall mortality rates; however, they are known to improve patients’ quality of life and contribute to disease stabilisation by enhancing cholinergic neurotransmission [34]. The detection of inhibitory activity against both AChE and BuChE, therefore, indicates potential neuromodulatory relevance and suggests that the phytochemical constituents present in the extracts may interact with multiple enzymatic targets within cholinergic pathways.

Diabetes mellitus represents a major global health challenge, and α-glucosidase and α-amylase inhibitors constitute an important class of therapeutic agents used in the management of postprandial hyperglycemia [35]. The concurrent inhibition of these carbohydrate-hydrolysing enzymes suggests that the observed bioactivity profile is not restricted to neurochemical modulation but extends to metabolic regulation as well.

Tyrosinase is a key enzyme responsible for the biosynthesis of melanin. However, the abnormal accumulation of melanin in localised regions of the skin can lead to the development of melanoma and various other pigment-related dermatological disorders [36]. Beyond its physiological role, tyrosinase also catalyses enzymatic browning reactions in fruits and vegetables, resulting in undesirable colour and quality changes [37]. Consequently, tyrosinase inhibitors have attracted considerable attention due to their potential applications in both the pharmaceutical and food industries. The simultaneous presence of antioxidant and tyrosinase inhibitory activities, therefore, reflects a multifunctional phytochemical behaviour that is consistent with the polyphenolic and terpenoid composition of the species.

The enzyme inhibition data indicate measurable activity across multiple targets; however, the observed IC₅₀ values were generally higher than those of the corresponding standard inhibitors. Therefore, the inhibitory effects should be interpreted as moderate in magnitude. The results likely reflect cumulative contributions of multiple phytochemical constituents rather than the presence of a highly potent single compound.

The major quantified phenolics-chlorogenic acid, rutin, and hesperidin-may collectively contribute to the multi-enzyme inhibition profile observed in the present study. Chlorogenic acid, the most abundant phenolic identified in the methanolic extract, may substantially contribute to the observed enzyme inhibition profile. Previous studies demonstrated that 5-O-caffeoylquinic acid inhibits α-amylase via mixed-type kinetics [38] and suppresses both α-amylase and α-glucosidase activities in vitro [39], with further kinetic evidence supporting mixed-type inhibition of α-glucosidase [40]. Chlorogenic acid has also been reported to inhibit acetylcholinesterase and butyrylcholinesterase activities [41]. In addition, extracts standardised in chlorogenic acid exhibited strong tyrosinase inhibition with low IC₅₀ values [42], supporting its potential interaction with copper-containing active sites. Therefore, the moderate inhibition of carbohydrate-hydrolysing enzymes, cholinesterases, and tyrosinase observed in the present study may be partially attributed to the high chlorogenic acid content of the extracts. Hesperidin may also contribute to the observed enzyme inhibition profile. It has been shown to inhibit α-glucosidase through an uncompetitive mechanism supported by kinetic and docking analyses [43]. Flavonoid–α-amylase interaction studies further indicate that structurally related compounds can interact near the catalytic site and exert mixed-type inhibition [44]. In addition, hesperidin has been reported to inhibit tyrosinase activity via a noncompetitive mechanism [45]. Beyond carbohydrate-hydrolysing enzymes, hesperidin has also demonstrated acetylcholinesterase inhibitory activity in vitro [41]. Collectively, these findings suggest that hesperidin, as one of the major constituents, may partially contribute to the multi-enzyme inhibition pattern detected in the present study. Rutin, one of the major flavonoids identified in the extract, may contribute to the observed enzyme inhibition profile. It has been reported to inhibit α-amylase and α-glucosidase in vitro, showing approximately 53% inhibition for both enzymes, supported by docking analyses indicating stable interactions within the catalytic pocket [46]. In addition, computational and kinetic studies have demonstrated that rutin acts as a competitive tyrosinase inhibitor by interacting with active-site histidine residues and copper ions [47]. Furthermore, rutin has been shown to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in vitro, supporting its contribution to cholinesterase modulation [48]. Collectively, these findings indicate that rutin may contribute to the multi-enzyme inhibition pattern observed in the present extracts.

However, due to the limited number of extract types evaluated in the present study, formal statistical correlation analyses between individual metabolite concentrations and bioactivity parameters were not considered methodologically robust. Therefore, the relationships discussed here should be interpreted as exploratory and literature-supported rather than statistically validated correlations.

The essential oil was characterised by a high proportion of oxygenated sesquiterpenes, with caryophyllene oxide identified as one of the major constituents. Caryophyllene oxide has been reported to exhibit notable cholinesterase inhibitory activity, including both AChE and BChE inhibition, as well as antioxidant effects, which may partially account for the enzyme-inhibitory properties observed in the present study [49]. Moreover, previous investigations have demonstrated that individual terpenoids present in Salvia essential oils, including caryophyllene oxide, contribute to anticholinesterase activity, although their isolated effects do not fully explain the total activity of the whole oil [50]. In addition, experimental studies on combinations of terpenoids have revealed that synergistic and additive interactions may occur among essential oil constituents, leading to enhanced enzyme inhibition compared to individual compounds [51]. Similarly, caryophyllene oxide and other major terpenes have shown significant α-glucosidase inhibitory activity, and synergistic interactions between selected components have been reported [52]. Therefore, the enzyme-inhibitory effects of the essential oil in the present study are likely attributable to both the intrinsic activity of caryophyllene oxide and the complex interactive behaviour of the terpenoid matrix.

Research demonstrates that essential oils and extracts derived from various Stachys species exhibit inhibitory activity against AChE, BuChE, α-glucosidase, α-amylase, and tyrosinase. However, the majority of findings are presented in terms of equivalent standard compounds, and there exists a limited number of studies offering IC₅₀ values.

Tundis [53] reported the inhibitory effects of S. lavandulifolia Vahl extracts against AChE, BuChE, and tyrosinase. The n-hexane extract exhibited the highest AChE inhibition, while polar extracts demonstrated greater tyrosinase inhibitory potential compared to non-polar fractions. These findings suggest that solvent polarity exerts a decisive influence on enzyme inhibition profiles, a pattern that aligns with the solvent-dependent trends observed in the present investigation.

Additionally, the inhibitory activities of S. guyoniana and S. chasmosericea extracts [54] indicate that moderate to strong enzyme inhibition is not an isolated phenomenon within the genus but rather a recurring phytochemical-pharmacological characteristic influenced by extraction methodology and metabolite distribution.

The eight essential-oil constituents included in the docking analysis were selected based on their relative abundance in the GC–MS profile (Table 1), representing the major components of the oil, namely caryophyllene oxide (11.96%), cubebol (6.93%), kauran-16-ol (7.3%), kaur-16-ene (5.71%), epi-bicyclosesquiphellandrene (4.89%), manoyl oxide (4.06%), phytol (3.8%), and 4,4-dimethyl-13α-androst-5-ene (3.6%). This selection was intended to prioritise structurally diverse and quantitatively relevant constituents with a higher likelihood of contributing to the experimentally observed enzyme inhibition. Although docosane was also detected among the oil constituents, it was not included in the docking analysis due to its saturated hydrocarbon structure lacking functional groups capable of forming specific interactions (e.g., hydrogen bonding) with enzyme active sites, making meaningful docking predictions unlikely. The docking simulations provide a structural and mechanistic framework that complements the experimental enzyme inhibition assays. The interaction patterns observed for diterpenoid and sesquiterpenoid constituents such as phytol, cubebol, and kauran-16-ol indicate that hydrogen bonding and hydrophobic interactions may contribute significantly to enzyme–ligand stabilisation.

Nevertheless, the relative abundance of individual compounds suggests that no single constituent is likely to be solely responsible for the overall inhibitory behaviour. Instead, the collective presence of multiple bioactive molecules with overlapping but distinct binding capacities supports the hypothesis of multi-component synergy. Such synergistic interactions are frequently reported in essential oil and phenolic-rich extract research and provide a plausible explanation for why whole-extract activity profiles often exceed the predicted effects of isolated compounds.

Overall, the docking affinity trends were generally consistent with the moderate enzyme inhibition observed experimentally. Compounds showing favourable binding interactions in silico were among the relatively abundant constituents of the essential oil, supporting their potential contribution to the measured bioactivity. However, no direct linear relationship between docking scores and experimental IC₅₀ values is implied, as whole-extract activity reflects the combined and potentially synergistic effects of multiple constituents rather than single-compound binding energies. Accordingly, the docking results are interpreted as mechanistic support for the experimental findings rather than quantitative predictors of inhibitory potency.

In light of the cytotoxicity findings obtained in the present study, EOS exhibited only a moderate and cell line–dependent antiproliferative effect, while MES, DES, and EAS remained essentially inactive within the tested concentration range. The relatively higher IC₅₀ values observed for both normal fibroblast models (MRC-5 and HDF) and several cancer cell lines. With respect to cytotoxicity, the essential oil exhibited moderate antiproliferative effects at relatively high concentrations, without clear selectivity between normal and cancer cell lines. These findings suggest limited potency under the tested experimental conditions and do not support strong anticancer implications without further investigation. Observations suggest that the cytotoxic potential of the investigated samples is strongly influenced by their chemical composition and may differ substantially from other members of the genus. Therefore, positioning the present results within the broader literature on Stachys species—where both markedly stronger and more selective antiproliferative activities have been reported—provides an essential framework for interpreting the comparatively moderate activity profile observed here and for discussing species- and extract-dependent variability in cytotoxic behaviour. Previous studies indicate that Stachys species display variable but often remarkable cytotoxic potential depending on the plant taxon, extract type, and target cell line [55,56,57,58,59,60,61,62,63]. The essential oil of Stachys cataonica (EOC) demonstrated antiproliferative activity on both malignant and normal cell models, with reported IC₅₀ values of 7.71 µg/mL on MRC-5 fibroblasts and 16.37 µg/mL on LNCaP prostate carcinoma cells. In comparison, the essential oil of Stachys sivasica (EOS) showed weaker or non-calculable activity on LNCaP but an IC₅₀ of 11.69 µg/mL on MRC-5 [55].

In prostate cancer models, Stachys parviflora root extracts exhibited high potency against PC-3 cells with an IC₅₀ of 17.2 µg/mL, approaching the activity of the positive control doxorubicin. The isolated diterpenoid 1-hydroxy-tanshinone IIA from this species was identified as a key bioactive component (IC₅₀: 22.64 µg/mL), inducing apoptosis by increasing Bax and decreasing Bcl-2 protein levels [56]. Solvent extracts of Stachys duriaei showed more moderate, dose-dependent effects against PC-3 cells, where the n-butanolic extract (BESD) and its fraction (BF1SD) produced IC₅₀ values of 196.40 µg/mL and 281.10 µg/mL, respectively. Mechanistically, BESD induced cell cycle arrest at the G2/M phase, while BF1SD caused a dual blockade at G0/G1 and G2/M [57]. A markedly stronger cytotoxic profile has been reported on HeLa cervical carcinoma cells for several species. Stachys annua extracts showed very low IC₅₀ values of 0.099 µg/mL for natural material and 0.211 µg/mL for micropropagated samples [58]. Additionally, Stachys koelzii essential oil inhibited HeLa cells with an IC₅₀ of 0.06 mg/mL [59]. Further reports emphasise this variability: the essential oil of S. viticina achieved approximately 95% inhibition at 1.25 mg/mL [60], while S. palaestina volatile oil inhibited HeLa proliferation by 99.27% at 1 mg/mL [61]. Ethanolic extracts of S. cretica ssp. mersinaea reduced HeLa cell viability to 34% at 100 µg/mL [62]. Stem extracts of S. recta and S. palustris also inhibited HeLa growth by more than 25% at 10 µg/mL [63].

Collectively, these findings suggest that numerous Stachys taxa exhibit promising antiproliferative activity, particularly against HeLa and PC-3 cell lines. However, the concurrent activity observed on normal fibroblasts such as MRC-5 indicates that biological selectivity remains highly species- and extract-dependent.

Overall, the present findings expand the currently limited knowledge on Stachys sparsipilosa and place this endemic species within a broader phytochemical and pharmacological context. The integrated evaluation of its chemical composition and multi-target bioactivity provides a structured basis for interpreting composition–activity relationships in this taxon. These results may support future comparative and mechanism-oriented studies within the genus Stachys.

5. Conclusions

In conclusion, the present study provides a comprehensive phytochemical characterization of Stachys sparsipilosa through integrated GC–MS and LC–ESI–QTOF/MS analyses combined with in vitro bioactivity assays and exploratory molecular docking. The essential oil and solvent extracts exhibited a chemically diverse composition dominated by terpenoids and phenolic compounds. The investigated samples demonstrated measurable antioxidant and enzyme-inhibitory activities; however, these effects were generally moderate when compared with standard reference compounds. The essential oil showed moderate, non-selective cytotoxic effects at relatively high concentrations, while solvent extracts displayed limited activity under the tested conditions. Molecular docking analyses supported the experimental findings by suggesting plausible enzyme–ligand interactions, but should be considered exploratory rather than definitive evidence of the mechanism. Overall, the results highlight composition-dependent bioactivity patterns and contribute to the phytochemical knowledge of this endemic species. Further fractionation, mechanistic studies, and in vivo investigations are required to clarify the biological relevance and potential applications of the identified constituents.