A Comparative Study on the Phenolic Compositions and Biological Activities of Stachys annua, Stachys cretica, and Endemic Stachys tmolea from Türkiye

Abstract

In this study, the phenolic compound profiles and bioactivity properties of aerial part extracts obtained from Stachys annua, Stachys cretica, and the endemic species Stachys tmolea, all of which are recognized as medicinal plants in Türkiye, were investigated. In this context, phenolic components in the flavonoid-subgroup extracts like flavanones, flavonols, flavan-3-ols, flavan 3-ols acid hydrolysis, flavones, and in the phenolic acids and their liquid-solid phase acid-base hydrolysis extracts were determined by reversed-phase high-performance liquid chromatography coupled with a diode array detector (RP-HPLC-DAD). Also, the total-phenolic, total-flavonoid, and total-tannin contents of the Stachys species were estimated. Among the evaluated antioxidant capacities of the flavonoid and phenolic acid extracts, S. annua exhibited the strongest scavenging capacities against 2,2-diphenyl-1-picrylhydrazyl and hydroxyl-radicals with half-maximal inhibitory concentration (IC₅₀) values of 5.53 ± 0.32 and 1.09 ± 0.02 µg mL^-1, respectively. S. tmolea indicated the highest nitric-oxide radical scavenging and metal-chelating capacities. The highest enzyme inhibitory activities were determined in S. annua for acetylcholinesterase and in S. cretica for tyrosinase. The phenolic acid extracts displayed moderate cytotoxic activity against ACC-201, OE-33, HeLa, and HepG2 cells with IC₅₀ values ranging from 20.1 ± 1.25 to 45.3 ± 1.35 µg mL^-1. The results show that the phenolic composition and bioactivity properties of investigated Stachys species are potentially promising for applicability in the food and pharmaceutical industries.

Keywords:
antioxidant; cytotoxicity; enzyme inhibition; phenolics; Stachys spp.

Introduction

Phenolics with bioactive properties produced by plants are secondary metabolites, and their applications in fields such as food and medicine are of great importance due to their antioxidant, anti-inflammatory, antibacterial, antitumor, antimutagenic, and anticancer properties.¹-³ They act as antioxidants by scavenging a variety of free radicals, inhibiting primary-chain radical initiation reactions, binding metal ion catalysts, and decomposing primary oxidation products into non-radical species.²,⁴,⁵ In conditions where the balance between intracellular free radical levels and antioxidant systems is disrupted, various oxidations in biomolecules such as proteins, lipids, and DNA, neurodegenerative and cardiovascular diseases, increased apoptosis, aging, cancer, and other disorders play a triggering role.¹,³,⁵ Therefore, the consumption of natural plant sources containing phenolic compounds is of great importance for a healthy life.¹-⁴

In this context, Stachys L. is one of the largest genera of the family Lamiaceae, comprising approximately 300 species.⁶-⁸ It is widely distributed throughout the temperate regions of the Mediterranean, and Southwest Asia, North and South America, and South Africa. In Türkiye, Stachys L. is represented by 81 taxa, 51 of which are endemic.⁷ Previous studies⁸-¹¹ have shown that Stachys species contain bioactive components such as phenolics, volatile oils, iridoids, and diterpenoids, which are responsible for many of their biological activities. Plants of this genus are widely used in folk medicine, as food additives, and herbal teas. Historically, they have been used to treat various diseases, including influenza, ulcers, infections, asthma, diabetes, rheumatism, and neuropathy.⁹,¹² Previous studies⁸,¹⁰,¹³ reported chlorogenic acid, verbascoside, and apigenin 7-glucoside as the major phenolic compounds in Stachys tmolea and Stachys cretica subsp. anatolica, whereas hesperidin, kaempferol, and apigenin were identified in S. cretica subsp. smyrnaea. In addition, the essential oil profiles of aerial part extracts of Stachys officinalis collected in Serbia have been investigated.¹⁴ Another study indicated significant antioxidant, antimicrobial, and cytotoxic activities in extracts of Stachys acerosa Boiss., Stachys nenthamiana Boiss., Stachys byzantina K. Koch, Stachys lavandulifolia Vahl., Stachys obtusicrena Boiss., Stachys persica S. G. Gmel. ex C. A. Mey., Stachys pilifera Benth., Stachys pubescens Ten., and Stachys spectabilis Choisy ex DC. collected from Iran.¹⁵

In the present study, the phenolic compound profiles and biological activities of aerial part extracts of Stachys annua, S. cretica, and the endemic species S. tmolea, which are known as medicinal plants in Türkiye were investigated. Phenolic profiling included flavanones, flavonols, flavan 3 ols, acid hydrolysed flavan-3-ols (AH), flavones, phenolic acids, and their liquidand solidphase acid-base hydrolysis extracts. In addition, the total-phenolic content (TPC), total flavonoid content (TFC), and total-tannin content (TTC) of the methanolic extracts were determined. The antioxidant properties of the extracts were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH^•), hydroxyl (HO^•), and nitric oxide (NO^•) radical scavenging assays, as well as metal chelating activity, total reducing power (TRP), and ferric reducing antioxidant power (FRAP) assays. The inhibitory effects of the extracts on acetylcholinesterase (AChE) and tyrosinase were also investigated. Furthermore, the cytotoxic activities of the Stachys extracts against human cervical carcinoma (HeLa), lung carcinoma (ACC 201), oesophageal adenocarcinoma (OE-33), hepatocellular carcinoma (HepG2), and breast adenocarcinoma (MCF-7) cell lines were determined.

Experimental

Materials and reagents

In this study, all chemicals were of analytical or high performance liquid chromatography (HPLC) grade and purchased from Sigma-Aldrich. Ultrapure water obtained from a Milli-Q purification system was used for the preparation of all solutions. The ACC-201 (23132/87) and OE-33 (ACC-706) cancer cell lines were obtained from the German collection of microorganisms and cell cultures (DSMZ), whereas HeLa (CCL-2), MCF 7 (HTB 22), and HepG2 (HB-8065) cell lines were obtained from the American type culture collection (ATCC). Roswell Park Memorial Institute (RPMI) 1640 and Dulbecco Modified Eagle Medium (DMEM) culture media, as well as fetal bovine serum, were purchased from GIBCO-BRL.

Plant materials

S. annua (L.), S. cretica subsp. smyrnaea Rech. Fil., and S. tmolea Boiss. were collected from Acıpayam village (37°22’31”N, 29°13’14”E) (FAMER 1418) in Denizli Province, and Bozdağlar village (38°14’31”N, 28°10’27”E) (FAMER 1466) and Bozdağlar-Ödemiş (38°18’20”N, 28°07’44”E) (FAMER 1459) in Izmir Province, Türkiye, respectively. Taxonomic identification of the collected specimens was carried out by Dr. M. Nakiboglu. Voucher specimens were deposited in the Herbarium of the Fauna and Flora Research and Application Centre (FAMER), Dokuz Eylül University, Türkiye. All Stachys samples were collected during the flowering period. The flowers and leaves of each species were separated, air-dried at room temperature in the dark, and subsequently lyophilized. Equal amounts (15 g each) of the dried flowers and leaves were mixed, ground using a mechanical grinder, and sieved to obtain a particle size of 0.30-1.0 mm. A 20 g portion of the resulting powder mixture from each Stachys species was subjected to Soxhlet extraction with n-hexane for 5 h to remove lipophilic constituents. After extraction, the n-hexane phase was discarded, and the defatted plant material was dried using a rotary evaporator to remove residual solvent. The resulting samples were lyophilized and stored at -20 °C until preparation of phenolic extracts. Flavonoid subgroup and phenolic acid extractions were subsequently performed with 3 and 7 g portions of the defatted plant material, respectively.¹⁶-²¹

Preparation of flavonoid subgroup extractions

For the flavanone extraction, 3 g of defatted dry plant material was extracted with 120 mL of ethanol/water (4:1, v/v) medium in a water bath at 90 °C for 2 h. After cooling, the extract was centrifuged at 5,000 rpm for 10 min, and the supernatant was concentrated under reduced pressure using a rotary evaporator to obtain the dried extract.¹⁶

For flavonol extraction, 3 g of defatted dry plant material was extracted under reflux in a Soxhlet apparatus using 150 mL of 85% aqueous methanol and 45 mL of 25% HCl for 1 h.¹⁷ After cooling the extract was concentrated using a rotary evaporator and dried under reduced pressure.

For flavan-3-ols extraction, 3 g defatted dry plant material was extracted with 60 mL of methanol in an ultrasonic bath at 65 °C for 2 h. This extraction procedure was repeated twice, after which the combined extracts were centrifuged at 5,000 rpm for 10 min. The solvent was removed under reduced pressure using a rotary evaporator.¹⁸

Flavan-3-ol-acid hydrolysis (AH) extracts were prepared by sequential liquid-liquid extraction of flavan 3 ol extracts with 45 mL of diethyl ether followed by an equal volume of ethyl acetate. The combined organic extracts were then subjected to acid hydrolysis in 100 mL of 2.5 M HCl/methanol (4:1, v/v) at 100 °C for 2 h in a water bath.¹⁹

For flavone extraction, 3 g defatted dry plant material was stirred in 150 mL of diethyl ether at 25 °C for 20 min.²⁰ The mixture was filtered, and the solid residue was extracted twice in 75 mL portions of diethyl ether for 10 min each. The filtrates were combined and concentrated under reduced pressure using a rotary evaporator.

For each Stachys species, five flavonoid subgroup extracts were obtained. The extracts were subsequently dried by rotary evaporation followed by lyophilization to obtain powdered samples for subsequent analyses.

Preparation of phenolic acid extractions

Initially, 7 g of powdered defatted Stachys material was extracted by stirring with 120 mL of methanol/water (4:1, v/v) at 25 °C for 1 h. After solvent evaporation and lyophilization, half of the resulting extract was dissolved in 14 mL of water adjusted to pH 2.0. Extraction with 14 mL of diethyl ether liquid-liquid was then performed three consecutive times. The ether phase, containing the free phenolic acids (FPAs) was collected and concentrated under reduced pressure.²¹ The remaining aqueous phase was adjusted to pH 7.0 with 2 M NaOH and concentrated under reduced pressure using a rotary evaporator. The resulting residue was redissolved in 14 mL of 2 M NaOH and stirred at 25 °C for 4 h. The solution was then adjusted to pH 2.0 with 6 M HCl and extracted three times with diethyl ether as mentioned above. The ether phase obtained at this stage contained the base-hydrolysed phenolic acids (BHPAs). Subsequently the remaining aqueous phase was treated with 14 mL of 6 M HCl at 95 °C for 20 min and extracted with ethyl ether. The resulting ether fraction was designated as the acid-hydrolysed phenolic acids (AHPA) extract. The FPA, BHPA, and AHPA extracts were concentrated under reduced pressure and subsequently lyophilized to obtain a dry powder for further analyses.

The remaining half of the original extract was used for solid phase hydrolysis. The solid residue remaining after the FPA extraction was hydrolysed with 65 mL of 2 M NaOH at 25 °C for 4 h. After separation of the liquid phase, the remaining solid residue was further hydrolysed with 6 M HCl at 95 °C for 1 h. Both liquid phases were separately extracted with diethyl ether. The resulting extracts were designated as phenolic acid solid, base hydrolysis 1 (PASB₁) and as phenolic acid-solid, acid hydrolysis 2 (PASA₂), respectively. Finally, the remaining solid residue was subjected first to acid hydrolysis and subsequently to base hydrolysis under the same experimental conditions. The corresponding extracts were designated as phenolic acid: solid-acid hydrolysis 1 (PASA₁) and phenolic acid: solid-base hydrolysis 2 (PASB₂), respectively.

In total, 12 extracts were prepared from for each Stachys species, comprising five flavonoid subgroup extracts and seven phenolic acid extracts.

RP-HPLC-DAD analysis of phenolic compounds

Analyses of phenolic compounds in the Stachys extracts were performed using a high-performance liquid chromatography system (HPLC, Agilent 1200 Series, USA) equipped with a ultraviolet diode array detector (UV-DAD) and a fluorescence detector. HPLC was carried out on a C18 reverse-phase column (250 mm × 4.6 mm, 5 µm particles). Dried extracts and phenolic standards were dissolved in methanol (99.7%), filtered through 0.45 µm membrane filters, and analysed in triplicate using the reversed-phase high-performance liquid chromatography coupled with a diode array detector (RP-HPLC-DAD) system. Phenolic standards solutions were prepared in methanol at a concentration of 1 mg mL^-1. Determination of phenolic compounds was achieved considering the peak areas of calibration curves generated according to the retention times (t_R) of the standard chromatograms. The standards used, their corresponding t_R values, and the calibration curve equations (Supplementary Information (SI) section, Table S1) are provided as supplementary data, together with the chromatograms of the extracts (Figures S1-S26, SI section) and the flavonoid and phenolic acid profiles (Tables S2-S27, SI section). The injection volume was 20 µL, and the column temperature was 30 °C. The limit of detection (LOD) and limit of quantification (LOQ) were determined using the standard deviation of the response and the slope of the calibration curve or estimated based on signal-to-noise ratios of 3 and 10 for LOD and LOQ, respectively. Method sensitivity was evaluated by calculating the relative standard deviation (RSD %) from three replicate measurements at the LOD and LOQ levels using the following equation:

(1) $$\text{RSD}=\text{(standard deviation / mean)}\times 100$$

Analysis of flavonol extracts was carried out according to the method described by Olszewska.¹⁷ The mobile phase consisted (A) 0.5% orthophosphoric acid in water and (B) methanol, using the following gradient program at room temperature: 0-10 min, 40-60% B; 10-21 min, 60% B; 21-23 min, 60-40% B; 23-26 min, 40% B; 26 30 min, 40% B. The flow rate was 1 mL min^-1, and detection was performed at 370 and 254 nm. Analysis of flavan-3-ols and flavan-3-ol-(AH) compounds were performed according to the method describing by de Villiers et al.¹⁸ A mobile phase consisted (A) 2% acetic acid in water and (B) acetonitrile/water (70:30, v/v), using the following gradient program: 0-3 min, 5% B; 3-8 min, 5-15% B; 8-10 min, 15-20% B; 10-12 min, 20-25% B; 12-20 min, 25 40% B; 20-30 min, 40-80% B. UV detection was carried out at 280 nm with a flow rate of 1.2 mL min^-1. For the analysis of flavanones, a mobile phase consisted (A) 0.6% acetic acid in water and (B) methanol, using the following gradient program: 0 5 min, 20-40% B; 5 8 min, 40% B; 8-12 min, 40 60% B; 12-25 min, 60% B; 25-30 min, 60 20% B.¹⁶ The flow rate was 0.4 mL min^-1, and UV detection was performed at 285 nm. For flavone analysis, the mobile phase consisted (A) water/formic acid (19:1, v/v) and (B) methanol, using the following gradient program: 0-5 min, 50% B; 5 30 min, 80% B; 30 min, 80% B. The flow rate was 1 mL min^-1, and detection was performed at 350 nm.²⁰ For the analysis of phenolic acids, the mobile phase consisted (A) acetonitrile and (B) 2% acetic acid in water, using the following gradient program: 0-30 min, 85-100% B; 30-55 min, 50 85% B; 55-60 min, 0-50% B; 60-65 min, 0-100% B.²¹ The flow rate was 1 mL min ¹. Hydroxybenzoic acid and hydroxycinnamic acid derivatives were estimated at 280 and 320 nm, respectively.

Total phenolic, flavonoid, and tannin contents

TPC of the Stachys extracts was determined using the Folin-Ciocalteu method. The reaction mixture consisted Folin-Ciocalteu reagent (2 M), sodium carbonate (7%, m/v), and Stachys extracts at concentrations ranging from 10 to 100 µg mL^-1. After incubation at room temperature for 2 h, the absorbance was measured at 765 nm using gallic acid as the calibration standard. TPC was expressed as mg gallic acid equivalents per g dry weight extract (mg_gae g_dwe ¹).²² TFC was determined using the aluminium chloride method.²³ Extract samples were mixed with AlCl₃.6H₂O (10%, m/v) and potassium acetate (1 M), followed by vortex mixing. The reaction mixture was incubated at room temperature in the dark for 30 min, after which the absorbance was measured at 415 nm. TFC was expressed as mg quercetin equivalents per g dry weight extract (mg_qe g_dwe ¹). TTC was determined using the vanillin-HCl assay.²⁴ The reaction mixture consisted of the extract, vanillin solution (4%, m/v), and concentrated HCl was used. After vortex mixing, the mixture was incubated at room temperature in the dark for 20 min, and the absorbance was measured at 500 nm. TTC was expressed as mg tannic acid equivalents per g dry weight extract (mg_tae g_dwe^-1).

Antioxidant properties

The antioxidant capacities of the Stachys extracts were evaluated by determining their scavenging activities against reactive oxygen species, reactive nitrogen species, and synthetic free radicals using spectrophotometric methods. The radical scavenging activities of the phenolic extracts were expressed as half-maximal inhibitory concentration (IC₅₀) values, representing the concentration required to inhibit 50% of the radicals. The DPPH^• assay based on the reduction of the stable nitrogen centred free radical DPPH^• by electronor hydrogen-donating antioxidant compounds. For the determination of DPPH^• radical scavenging activity, extract solutions prepared in methanol of concentrations ranging 1 to 300 µg mL^-1 were mixed with DPPH^• solution (1 mM) and incubated in the dark at room temperature for 30 min. The absorbance was measured at 517 nm.²⁵ Vitamin-C (0-35 µg mL^-1) and butylated hydroxytoluene (BHT) (0-10 µg mL^-1) were used as positive controls.

HO^• scavenging activity was determined using the 2-deoxyribose assay, which evaluate the ability of antioxidant compounds to compete with 2-deoxyribose for hydroxyl radicals generated by the Fenton reaction. This competition inhibits the degradation of 2-deoxyribose to malondialdehyde, which is subsequently quantified spectrophotometrically.²⁶ The reaction mixture was (final volume, 1.0 mL) contained FeCl₃ (100 µM), ethylenediaminetetraacetic acid (EDTA, 104 µM), H₂O₂ (100 µM), vitamin-C (100 µM), deoxyribose (2.8 mM), phosphate buffer (20 mM, pH 7.4), and Stachys extracts at concentrations ranging from 1 to 100 µg mL^-1.²⁶ The mixtures were incubated at 37 °C for 60 min, followed by the addition of 1 mL thiobarbituric acid (1%, m/v) and 1 mL trichloroacetic acid (TCA, 2% m/v). The reaction mixtures were then incubated in a boiling water bath for 15 min, and the absorbance was measured at 535 nm. BHT (0-10 µg mL^-1) was used as the positive control.

NO^• scavenging activity was determined using the Griess assay, which evaluates the ability of antioxidant compounds, to inhibit the formation of nitrite ions generated from sodium nitroprusside. The resulting nitrite ions were quantified colorimetrically.²⁷ The reaction mixture consisted of sodium nitroprusside (10 mM), phosphate-buffered saline (20 mM, pH 7.4), and the Stachys extracts (10-300 µg mL^-1). After incubation at 25 °C for 150 min, 0.5 mL of the reaction mixture was mixed with 0.5 mL of Griess reagent, prepared as a 1:1 (v/v) mixture of 2% (m/v) sulphanilamide and 0.2% (m/v) naphthyl ethylenediamine dihydrochloride. The resulting mixtures were incubated at 25 °C for 30 min.²⁷ The control A_o was prepared by replacing the extract with phosphate buffer. Absorbance was measured at 546 nm. Vitamin-C (0-65 µg mL^-1) was used as the positive control.

Metal chelating activity was determined using the ferrozine assay, which is based on the formation of a stable, water-soluble coloured complex between ferrozine and Fe^II ions.²⁸ The chelating capacity of the extracts was evaluated by measuring the inhibition of Fe^II-ferrozine complex formation. The reaction mixture consisted of FeCl₂ (1 mM), extracts (10-300 µg mL^-1), and acetate buffer (100 mM, pH 4.0). After incubation at room temperature for 30 min, ferrozine (5 mM) was added, and the absorbance was measured at 562 nm.²⁸ EDTA (0-10 µg mL^-1) was used as the positive control.

The TRP and FRAP of the Stachys extracts were evaluated to determine their electron-donating capacities. Both assays are based on the reduction of Fe^III to a Fe^II by antioxidant compounds, resulting in a measurable colour change.²⁹,³⁰ TRP was determined according to the method of Oyaizu.²⁹ The reaction mixture consisted of K₃Fe(CN)₆ (1%, m/v), phosphate buffer (0.2 M, pH 6.6), and extracts at varying concentrations (10-100 µg mL^-1). After incubation at 50 °C for 30 min, TCA (10%, m/v) was added, and the mixture was centrifuged. An aliquot of the supernatant was then mixed with FeCl₃ (0.1%, m/v), and the absorbance was measured at 700 nm. Increased absorbance indicated greater TRP. The TRP values were expressed as mg vitamin-C equivalents per g dry weight extract (mg_vce g_dwe^-1). For the FRAP assay, 75 µL of Stachys extract (10-100 µg mL^-1) was mixed with 1425 µL of FRAP reagent consisting of 25 mL (300 mM, pH 3.6) acetate buffer, 2.5 mL of 10 mM 2,4,6-tris(2 pyridyl)-s triazine, and 2.5 mL of 20 mM FeCl₃.³⁰ The reaction mixtures were incubated at 37 °C in the dark for 30 min, and the absorbance was measured at 593 nm. FRAP values were expressed as mg vitamin-C equivalents per g dry weight extract (mg_vce g_dwe^-1).³¹

Inhibition of AChE and tyrosinase activities

The inhibitory activities of the Stachys extracts against AChE and tyrosinase activities were determined according to the methods described by Uysal et al.³² Galantamine (0-400 µg mL^-1) and kojic acid (0-500 µg mL^-1) were used as positive controls for AChE and tyrosinase assays, respectively. For the AChE inhibition assay, the reaction mixture consisted of 50 μL of extract, 125 μL of 3 mM 5,5-dithio-bis(2-nitrobenzoic acid) and 25 μL of 15 mM acetylthiocholine iodide. Subsequently, 25 μL of AChE solution (0.28 U mL^-1) was added, and the mixture was incubated for 15 min. Absorbance was then measured at 405 nm. For the tyrosinase inhibition assay, the reaction mixture consisted of 25 µL of extract, 40 µL of tyrosinase solution (200 U mL ¹), 100 µL of phosphate buffer (40 mM, pH 6.8), and 40 µL of L-DOPA (10 mM). After incubation at 25 °C for 5 min, the absorbance was measured at 492 nm. Enzyme inhibitory activities were expressed as IC₅₀ value (µg mL^-1).

MTT cell proliferation assay

The cytotoxic effects of Stachys extracts against HeLa (ATCC^® CCL-2), ACC-201 (DSMZ 23132/87), OE 33 (DSMZ ACC706), HepG2 (ATCC HB-8065), and MCF-7 (ATCC HTB-22) cancer cell lines were evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide (MTT) assay according to the method described by Mosmann.³³ Cells were seeded to 96-well plates at 1 × 10⁵ cells per well and cultured at 37 °C in a humidified atmosphere of 5% CO₂. The cells were treated with Stachys extracts at concentrations ranging from 10 to 150 µg mL^-1 prepared in 0.4% dimethyl sulfoxide (DMSO) for 24 and 48 h. Cells treated with culture medium containing 0.4% DMSO alone served as the negative control, while mitomycin-C was used as the positive control. Following incubation, 20 μL MTT solution (5 mg mL^-1) was added to each well, and the plates were incubated for 4 h at 37 °C. The resulting formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm using a microplate reader. Cell viability was expressed as the percentage of absorbance relative to the untreated control. IC₅₀ values, defined as the extract concentrations causing 50% inhibition of cell viability, were calculated from dose-response curves generated for each cancer all line. The negative control (0.4% DMSO) showed no significant effect on cell viability, whereas cancer cells, the positive control mitomycin-C (50 µg mL^-1) inhibited the proliferation of HeLa, ACC 201, OE-33, and MCF-7 cells by 90.84, 87.64, 82.35, and 76.86%, respectively, after 48 h treatment.

Statistical analyses

All experiments were performed in triplicate and the results are represented as the mean ± standard deviations (SD). Statistical significance was analysed using the Student’s t-test, with p < 0.05 considered statistically significant.

Results and Discussion

Phenolic compounds of Stachys extracts

Flavonoids and phenolic acids, which represent major classes of natural phenolic antioxidants, play essential roles in healthy nutrition by maintaining intracellular redox homeostasis and contributing to the prevention of various disorders, including aging-related diseases and cancer.¹,³ The major flavonoid and phenolic acid compounds were estimated in total 12 subgroup extracts obtained from the aerial parts of S. annua, S. cretica, and S. tmolea (Tables 1 and 2). Estimation was performed based on the peak areas from calibration curves generated according to the t_R of the corresponding standard chromatograms.

Table 1
Flavonoid compounds in the subgroup extracts of the S. annua, S. cretica, and S. tmolea samples determined by RP-HPLC-DAD analysis. Results were expressed as mean ± standard deviation of three independent replicates

Subgroup extracts of flavonoids / nm	S. annua / (mg gdwe-1)	S. cretica / (mg gdwe-1)	S. tmolea / (mg gdwe-1)
Flavanone / 285 nm
Hesperidin	41.44 ± 1.26	1.78 ± 0.07	10.99 ± 0.85
Naringenin	1.11 ± 0.05	3.33 ± 0.14	0.98 ± 0.04
Hesperetin	0.62 ± 0.01	9.53 ± 0.26	5.31 ± 0.12
Flavonol / 254, 370 nm
Rutin	1.92 ± 0.07	nd	nd
Myricetin	1.15 ± 0.03	nd	0.34 ± 0.03
Quercetin	1.34 ± 0.05	0.25 ± 0.02	nd
Kaempferol	0.75 ± 0.07	2.31 ± 0.07	9.88 ± 0.23
Isorhamnetin	3.05 ± 0.10	nd	1.99 ± 0.06
Isoquercitrin	9.75 ± 0.22	nd	40.89 ± 1.03
Quercitrin	1.72 ± 0.08	nd	0.57 ± 0.02
Flavan-3-ol / 280 nm
Catechin	nd	nd	nd
Epicatechin	3.02 ± 0.15	1.26 ± 0.05	1.33 ± 0.01
Epigallocatechin	nd	8.94 ± 0.14	6.04 ± 0.09
Flavan-3-ol-(AH) / 280 nm
Catechin	11.37 ± 0.77	nd	nd
Epicatechin	116.63 ± 1.52	10.42 ± 1.01	312.02 ± 4.65
Epigallocatechin	52.72 ± 1.10	88.19 ± 1.33	66.66 ± 1.17
Flavone / 350 nm
Apigenin	3.27 ± 0.10	0.12 ± 0.01	0.45 ± 0.03
Luteolin	0.83 ± 0.07	0.34 ± 0.05	0.55 ± 0.04
Eupatorin	3.99 ± 0.11	0.23 ± 0.02	0.24 ± 0.01
Diosmin	7.70 ± 0.15	nd	nd
Chrysoeriol	nd	nd	nd
Total amount	262.38 ± 0.33	126.70 ± 0.25	468.72 ± 0.56

Table 2
Hydroxybenzoic acid components in the extracts of the S. annua, S. cretica, and S. tmolea samples determined by RP-HPLC-DAD analysis. Results were expressed as mean ± standard deviation of three independent replicates

Hydroxybenzoic acid derivatives
Phenolic acid extracts / 280, 320 nm	Gallic / (mg gdwe-1)	Benzoic / (mg gdwe-1)	Vanillic / (mg gdwe-1)	4-Hydroxy-benzoic / (mg gdwe-1)	Syringic / (mg gdwe-1)	Proto-catechuic / (mg gdwe-1)
Free phenolic acids (FPA)
S. annua	0.24 ± 0.01	3.71 ± 0.19	2.16 ± 0.05	0.74 ± 0.03	1.35 ± 0.06	0.41 ± 0.01
S. cretica	2.99 ± 0.16	3.28 ± 0.07	5.31 ± 0.18	4.02 ± 0.28	1.26 ± 0.04	1.63 ± 0.06
S. tmolea	6.96 ± 0.39	nd	7.06 ± 0.29	3.84 ± 0.16	7.06 ± 0.09	2.26 ± 0.17
Base-hydrolysed phenolic acids (BHPA)
S. annua	nd	55.15 ± 1.12	1.42 ± 0.04	28.97 ± 0.90	14.15 ± 0.31	0.62 ± 0.01
S. cretica	1.29 ± 0.04	172.80 ± 2.65	57.76 ± 1.52	31.35 ± 1.02	nd	27.88 ± 0.87
S. tmolea	0.11 ± 0.01	53.60 ± 1.13	7.39 ± 0.68	10.58 ± 0.14	27.40 ± 0.85	15.01 ± 0.31
Acid-hydrolysed phenolic acids (AHPA)
S. annua	1.42 ± 0.04	7.35 ± 0.32	58.49 ± 0.72	26.05 ± 0.85	11.89 ± 0.14	2.83 ± 0.05
S. cretica	1.79 ± 0.04	nd	32.52 ± 0.35	15.89 ± 0.33	15.89 ± 0.31	nd
S. tmolea	10.91 ± 0.12	nd	58.34 ± 0.86	47.08 ± 1.11	18.67 ± 0.38	4.41 ± 0.07
Phenolic acids of solid hydrolysis solid phase hydrolysis: first acid (A1) then base (B2)
PASA1
S. annua	nd	nd	2.08 ± 0.05	nd	5.84 ± 0.08	148.32 ± 2.37
S. cretica	5.13 ± 0.18	nd	3.18 ± 0.07	1.03 ± 0.04	3.04 ± 0.07	93.53 ± 1.51
S. tmolea	13.92 ± 0.54	nd	10.35 ± 0.62	3.95 ± 0.07	6.11 ± 0.09	73.01 ± 1.21
PASB2
S. annua	nd	nd	10.84 ± 0.74	7.37 ± 0.13	9.01 ± 0.31	15.28 ± 0.87
S. cretica	4.43 ± 0.12	5.44 ± 0.18	20.64 ± 0.81	4.72 ± 0.07	11.86 ± 0.45	32.69 ± 0.93
S. tmolea	3.56 ± 0.07	nd	0.86 ± 0.02	8.48 ± 0.24	7.87 ± 0.10	2.07 ± 0.04
PASB1
S. annua	nd	24.44 ± 0.84	1.53 ± 0.06	1.26 ± 0.07	3.86 ± 0.05	0.73 ± 0.03
S. cretica	nd	nd	nd	nd	nd	nd
S. tmolea	nd	95.50 ± 1.45	5.37 ± 0.18	1.40 ± 0.04	nd	9.99 ± 0.11
PASA2
S. annua	nd	nd	5.04 ± 0.17	nd	9.22 ± 0.12	2.44 ± 0.10
S. cretica	nd	nd	nd	nd	nd	nd
S. tmolea	nd	nd	2.19 ± 0.04	3.57 ± 0.07	11.24 ± 0.34	2.25 ± 0.05

• mg g_dwe-1: mg g dry weight extract
• ^-1
  ; nd: not detected.

As shown in Table 1, epicatechin and epigallocatechin were the predominant among the 5 flavonoid subgroup extracts, and detected in the flavan-3-ol-(AH) fractions of all three Stachys species. The third most abundant flavonoid differed among the species, being hesperidin in the flavanone fraction of S. annua, hesperetin in the flavanone fraction of S. cretica, and isoquercitrin in the flavonol fraction of S. tmolea. Comparison of flavonoid contents revealed the following order: S. tmolea > S. annua > S. cretica as 468.72 ± 0.56; 262.38 ± 0.33; 126.70 ± 0.25 mg g_dwe^-1, respectively.

In the study by Bursal et al.¹² epigallocatechin and catechin were not detected in the leaf extracts of S. annua collected from Türkiye whereas epicatechin was estimated at 4.5 ± 0.5 and 62.7 ± 6.4 µg g_ext^-1 in the methanolic and aqueous extracts, respectively. Similarly, catechin and epicatechin were not detected in the methanolic and aqueous extracts of the aerial parts of S. cretica subsp. anatolica collected from Türkiye.⁸ Bahadori et al.⁹ reported that kaempferol, hesperidin, quercetin, and apigenin were the major flavonoids in the methanolic extract of aerial parts of S. cretica subsp. mersinaea from Türkiye with concentrations of 1910, 25770, 1900, and 2360 µg g_ext ¹, respectively. In the present study, hesperetin was identified for the three investigated Stachys species. In addition, epigallocatechin was detected in S. cretica and S. tmolea, while diosmin was detected in S. annua. Likewise, myricetin, quercitrin, and isoquercitrin were identified in S. annua and S. tmolea.⁸-¹⁰,¹²,¹³,³⁴ Furthermore, the flavan 3 ol-(AH) fractions, which were investigated in the present study exhibited the highest catechin levels following acid hydrolysis.

The hydroxybenzoic and hydroxycinnamic acid derivatives identified in the aerial part extracts of S. annua, S. cretica, and S. tmolea are presented in Tables 2 and 3. Benzoic acid, protocatechuic acid, and vanillic acid were the three predominant phenolic acids in both S. cretica and S. tmolea. No significant difference was observed between the protocatechuic acid content of S. tmolea PASA₁ and trans-cinnamic acid content of S. tmolea-PASB₂ (p > 0.05). In contrast, the major phenolic acids identified in S. annua were rosmarinic acid, trans-cinnamic acid, and protocatechuic acid.

Table 3
Hydroxycinnamic acid components in the extracts of the S. annua, S. cretica, and S. tmolea samples determined by RP-HPLC-DAD analysis. Results were expressed as mean ± standard deviation of three independent replicates

Hydroxycinnamic acid derivatives
Phenolic acid extracts / 280, 320 nm	Rosmarinic / (mg gdwe-1)	o-Coumaric / (mg gdwe-1)	p-Coumaric / (mg gdwe-1)	Caffeic / (mg gdwe-1)	Ferulic / (mg gdwe-1)	Sinapic / (mg gdwe-1)	t-Cinnamic / (mg gdwe-1)
Free phenolic acids (FPA)
S. annua	244.62 ± 7.42	0.99 ± 0.07	0.47 ± 0.02	2.46 ± 0.12	0.75 ± 0.06	201.23 ± 3.95	nd
S. cretica	5.42 ± 0.18	0.22 ± 0.01	0.81 ± 0.05	0.32 ± 0.01	0.45 ± 0.02	16.41 ± 0.65	nd
S. tmolea	9.05 ± 0.21	0.10 ± 0.01	1.31 ± 0.07	0.82 ± 0.05	0.67 ± 0.04	8.23 ± 0.17	nd
Base-hydrolysed phenolic acids (BHPA)
S. annua	7.58 ± 0.19	nd	7.05 ± 0.20	2.05 ± 0.05	0.78 ± 0.03	15.13 ± 0.92	1.35 ± 0.03
S. cretica	4.69 ± 0.13	1.29 ± 0.04	0.71 ± 0.03	nd	2.11 ± 0.06	10.14 ± 0.27	35.62 ± 1.07
S. tmolea	0.83 ± 0.02	0.58 ± 0.01	6.46 ± 0.23	5.02 ± 0.21	35.35 ± 1.03	3.36 ± 0.06	2.66 ± 0.05
Acid-hydrolysed phenolic acids (AHPA)
S. annua	2.61 ± 0.05	0.21 ± 0.01	5.07 ± 0.31	5.53 ± 0.28	3.64 ± 0.19	5.19 ± 0.28	0.49 ± 0.02
S. cretica	5.13 ± 0.28	3.68 ± 0.09	5.59 ± 0.27	16.13 ± 0.95	3.86 ± 0.07	44.37 ± 1.34	0.41 ± 0.01
S. tmolea	5.18 ± 0.19	0.83 ± 0.06	2.90 ± 0.05	5.27 ± 0.24	1.73 ± 0.03	9.11 ± 0.13	0.68 ± 0.04
Phenolic acids of solid hydrolysis solid phase hydrolysis: first acid (A1) then base (B2)
PASA1
S. annua	2.08 ± 0.07	0.59 ± 0.04	nd	1.40 ± 0.07	0.31 ± 0.01	12.63 ± 0.23	nd
S. cretica	5.67 ± 0.22	1.13 ± 0.09	1.34 ± 0.08	2.09 ± 0.11	1.54 ± 0.07	23.46 ± 0.85	nd
S. tmolea	3.95 ± 0.13	2.24 ± 0.08	0.91 ± 0.03	4.79 ± 0.17	0.49 ± 0.03	23.27 ± 1.15	nd
PASB2
S. annua	1.25 ± 0.03	nd	5.31 ± 0.29	3.46 ± 0.16	1.08 ± 0.03	22.46 ± 0.90	nd
S. cretica	2.30 ± 0.11	0.79 ± 0.05	0.52 ± 0.03	0.68 ± 0.03	2.14 ± 0.12	21.01 ± 1.12	1.19 ± 0.04
S. tmolea	2.11 ± 0.08	0.61 ± 0.02	0.26 ± 0.01	nd	3.01 ± 0.06	70.30 ± 1.47	nd
Phenolic acids of solid phase hydrolysis: first base (B1), then acid (A2)
PASB1
S. annua	0.43 ± 0.02	nd	1.35 ± 0.04	1.20 ± 0.05	2.87 ± 0.12	6.06 ± 0.22	nd
S. cretica	nd	nd	nd	nd	nd	nd	nd
S. tmolea	1.21 ± 0.07	0.33 ± 0.01	1.11 ± 0.06	nd	nd	7.03 ± 0.16	19.63 ± 0.88
PASA2
S. annua	0.58 ± 0.03	0.76 ± 0.04	2.34 ± 0.15	nd	nd	34.34 ± 1.03	nd
S. cretica	nd	nd	nd	0.13 ± 0.01	tr	nd	nd
S. tmolea	0.77 ± 0.01	0.37 ± 0.01	1.36 ± 0.07	nd	nd	6.16 ± 0.23	nd

Kocak et al.³⁴ previously quantified phenolic acids in the methanolic extracts of aerial parts of S. annua subsp. annua var. annua collected from Türkiye and reported chlorogenic acid (6.28 ± 0.153 mg g_ext^-1), benzoic acid (6.12 ± 0.128 mg g_ext^-1), and rosmarinic acid (1.58 ± 0.077 mg g_ext^-1) as the predominant compounds. Chlorogenic acid was not detected in any of the Stachys species analyzed in the present study; however, rosmarinic acid and benzoic acid were present at relatively high concentrations in the FPA and BHPA extracts, respectively. Previous studies⁹ have also shown that extracts of S. cretica subsp. mersinaea contain high levels of benzoic acid (3080 µg g^-1) and gallic acid (520 µg g^-1) in the ethyl acetate extract, as well as chlorogenic acid (1800 µg g^-1) in the methanolic extract. Likewise, aerial part extracts of S. tmolea collected from Afyonkarahisar, Türkiye, were reported to contain chlorogenic acid (1120.14 ± 41.24 µg g^-1 dry plant in the methanolic extract), together with 4-hydroxybenzoic acid and protocatechuic acid (29.66 ± 0.12 and 11.82 ± 0.05 µg g^-1 dry plant, respectively) in the aqueous extract.¹⁰ Overall, the concentrations of phenolic compounds determined in the present study (Tables 1-3) were generally higher than those reported in previous studies.⁹,¹⁰,³⁴ These discrepancies are likely attributable to differences in plant material, extraction procedures, solvent systems, geographical origin, and climatic conditions affecting the investigated Stachys species.⁸-¹⁰,¹²,¹³,³⁵ Consistent with these findings, Khanavi et al.³⁶ reported that although Stachys species are generally rich in phenolic compounds, their composition and abundance are strongly influenced by the extraction method and solvent employed.

Total-phenolic, total-flavonoid, and total-tannin contents of Stachys extracts

Phenolic compounds are among the most abundant secondary metabolites in plants and comprise a wide range of structurally diverse compounds, from simple to complex, such as tannins. The TPC, TFC, and TTC of the methanolic extracts of S. annua, S. cretica, and S. tmolea are presented in Table 4.

Table 4
Total-phenolic, -flavonoid and -tannin contents of Stachys species. Results were expressed as mean ± standard deviation of three independent replicates

Stachys species	Total phenolics / (mggae gdwe-1)	Total flavonoids / (mgqe gdwe-1)	Total tannins / (mgtae gdwe-1)
S. annua	228.41 ± 3.92	65.38 ± 1.52	173.00 ± 4.17
S. cretica	147.75 ± 6.35	68.88 ± 3.45	157.00 ± 2.25
S. tmolea	397.04 ± 4.72	80.07 ± 1.26	208.90 ± 3.84

Among the investigated species, S. tmolea exhibited the highest TPC (397.04 ± 4.72 mg_gae g_dwe ¹), TFC (80.07 ± 1.26 mg_qe g_dwe^-1), and TTC (208.90 ± 3.84 mg_tae g_dwe^-1). TPCs followed the order S. tmolea > S. annua > S. cretica. Although no significant difference was observed between TFCs of S. annua and S. cretica (p > 0.05), a slight but statically significant difference was found in their TTCs. TPCs determined for the three Stachys species examined in the present study were higher than these previously reported for S. byzantina, S. lavandulifolia Vahl., S. acerosa Boiss., S. obtusicrena Boiss., S. persica and S. cretica L. subsp. vacillans extracts.¹⁵ Stegăruş et al.³⁷ reported total polyphenol contents of 197 ± 0.27, 222 ± 0.34, and 232 ± 43 mg_gae g^-1 in for the ethanolic extracts of S. sylvatica, S. byzantine, S. officinalis, respectively, values that are generally comparable with those obtained in the present study. Similarly, the TTC of S. sieboldii Miq. from Korea was reported to be 40.41 ± 2.54 mg_tae g_dwe^-1.³⁸ Although studies investigating the TPC, TFC, and TTC of Stachys species remain limited, the values reported in the available literature are generally lower than those obtained in the present study.²⁷,³⁶,³⁷,³⁸

Antioxidant properties of Stachys extracts

In recent decades, increasing attention has been given to the studies on the antioxidant properties of phenolic compounds, which may synergistically, additively, or antagonistically in scavenging reactive radical species in biological systems.³,⁴ In the present study, the DPPH^•, HO^•, and NO^• radical scavenging activities, as well as metal chelating capacity, TRP, and FRAP of the Stachys extracts, were evaluated and presented in Table 5. Among the tested samples the highest DPPH^• scavenging activity (IC₅₀ = 5.53 ± 0.32 µg mL^-1) was observed for the S. annua FPA extract. In comparison, the IC₅₀ values for the S. cretica-AHPA and S. tmolea-flavan-3-ol-(AH) extracts were 29.62 ± 0.95, and 30.31 ± 0.92 µg mL^-1, respectively, with no statically significant difference (p > 0.05). The most active S. annua extract exhibited 3.39 and 4.87 fold higher activity than vitamin-C and BHT, respectively (p < 0.05), indicating a stronger radical scavenging capacity. In a previous study, the IC₅₀ values for the DPPH^• scavenging activity of aqueous and methanolic extracts of S. annua were reported as 8.9 ± 0.2 and 7.8 ± 0.2 µg mL ¹, respectively.¹² Similarly, methanolic extracts of several Stachys species from different geographical regions have shown higher IC₅₀ values, including S. anisochila (17.90 µg mL^-1), S. plumosa (101.61 µg mL^-1), S. alpina subsp. dinarica (26.14 µg mL^-1), and S. beckeana (20.90 µg mL^-1).³⁹ In another study IC₅₀ values for DPPH^• scavenging activity of methanolic extracts of nine Stachys species ranged from 454.4 ± 26.8 µg mL^-1 (S. spectabilis) to 135.1 ± 4.5 µg mL^-1 (S. byzantine).¹⁵ Overall, the IC₅₀ value obtained for S. annua-FPA in the present study was lower than most values reported in the literature, indicating a comparatively stronger DPPH^• scavenging capacity.¹²,¹⁵,³⁹

Table 5
Antioxidant activity results of obtained S. annua, S. cretica and S. tmolea extracts. Results were expressed as mean ± standard deviation of three independent replicates

Phenolic extracts	IC50 / (µg mL-1)					mgvce gdwe-1
	DPPH•	HO•	NO•	Metal chelating		TRP	FRAP
Flavanone
S. annua	29.16 ± 0.88	15.00 ± 0.79	58.72 ± 2.16	71.70 ± 1.18		0.98 ± 0.02	0.30 ± 0.01
S. cretica	36.76 ± 1.03	15.40 ± 0.77	57.10 ± 1.14	59.16 ± 1.11		0.71 ± 0.05	0.10 ± 0.002
S. tmolea	46.15 ± 1.09	1.25 ± 0.02	54.46 ± 1.10	86.84 ± 1.34		0.82 ± 0.03	0.17 ± 0.005
Flavonol
S. annua	37.32 ± 1.03	1.09 ± 0.02	44.08 ± 1.26	108.58 ± 1.55		1.02 ± 0.03	0.24 ± 0.01
S. cretica	72.67 ± 2.14	11.89 ± 0.07	78.89 ± 1.18	108.90 ± 2.72		0.64 ± 0.01	0.09 ± 0.003
S. tmolea	36.75 ± 1.03	13.90 ± 0.85	42.62 ± 1.10	106.80 ±1.61		0.85 ± 0.04	0.28 ± 0.01
Flavan-3-ol
S. annua	46.33 ± 1.18	13.15 ± 0.87	52.27 ± 1.13	108.70 ± 1.62		0.90 ± 0.08	0.23 ± 0.01
S. cretica	100.66 ± 1.52	13.03 ± 0.06	44.15 ± 1.10	89.40 ± 1.45		0.69 ± 0.02	0.06 ± 0.001
S. tmolea	53.10 ± 1.10	11.64 ± 0.28	58.62 ± 1.12	74.40 ± 1.16		0.90 ± 0.05	0.16 ± 0.007
Flavan-3-ol-(AH)
S. annua	6.55 ± 0.35	16.70 ± 0.80	57.45 ± 1.12	177.81 ± 2.75		1.85 ± 0.04	1.11 ± 0.02
S. cretica	57.30 ± 1.11	16.40 ± 1.04	102.02 ± 1.59	126.01 ± 1.75		0.79 ± 0.02	0.47 ± 0.01
S. tmolea	30.31 ± 0.92	17.24 ± 0.65	57.01 ± 2.26	184.16 ± 2.02		1.92 ± 0.11	1.13 ± 0.08
Flavone
S. annua	16.65 ± 2.55	28.86 ± 1.90	131.44 ± 1.72	50.94 ± 1.15		0.57 ± 0.01	0.05 ± 0.001
S. cretica	172.18 ± 2.62	15.64 ± 0.85	275.50 ±3.97	30.23 ± 0.90		0.52 ± 0.03	0.02 ± 0.001
S. tmolea	252.65 ± 3.92	30.50 ± 2.21	183.55 ± 2.65	27.73 ± 0.85		0.50 ± 0.02	0.004 ± 0.0001
Free phenolic acids (FPA)
S. annua	5.53 ± 0.32	13.27 ± 0.66	88.56 ±1.33	112.08 ± 1.53		2.08 ± 0.13	1.46 ± 0.07
S. cretica	120.74 ± 1.61	16.00 ± 0.74	50.30 ± 1.10	88.96 ± 1.35		0.61 ± 0.02	0.06 ± 0.002
S. tmolea	101.28 ± 1.52	12.28 ± 0.61	128.21 ± 1.70	95.71 ±1.44		0.77 ± 0.03	0.09 ± 0.003
Base-hydrolysed phenolic acids (BHPA)
S. annua	97.14 ± 1.45	13.40 ± 0.68	61.97 ± 1.13	60.83 ± 1.11		0.73 ± 0.03	0.09 ± 0.001
S. cretica	36.12 ± 1.01	14.40 ± 0.71	36.34 ± 1.01	86.50 ± 1.32		1.13 ± 0.08	0.36 ± 0.02
S. tmolea	94.39 ± 1.44	12.02 ± 0.65	46.18 ± 1.17	107.53 ± 1.60		0.72 ± 0.04	0.12 ± 0.05
Acid-hydrolysed phenolic acids (AHPA)
S. annua	80.21 ± 1.25	13.56 ± 0.70	60.66 ± 1.18	121.15 ± 2.67		0.96 ± 0.04	0.22 ± 0.01
S. cretica	29.62 ± 0.95	17.24 ± 0.46	58.64 ± 2.11	50.00 ± 1.10		1.03 ± 0.03	0.41 ± 0.03
S. tmolea	31.98 ± 1.22	12.48 ± 0.63	86.68 ± 1.33	108.44 ± 3.61		0.82 ± 0.05	0.29 ± 0.02
Phenolic acids of solid hydrolysis solid phase hydrolysis: first acid (A1) then base (B2)
PASA1
S. annua	58.17 ± 2.11	13.82 ± 0.71	86.10 ± 1.35	138.70 ± 2.29		1.49 ± 0.04	0.29 ± 0.02
S. cretica	93.93 ± 1.45	16.00 ± 0.75	77.08 ± 1.21	100.89 ± 1.58		1.28 ± 0.03	0.23 ± 0.01
S. tmolea	46.36 ± 1.19	12.08 ± 0.58	18.82 ± 0.75	158.78 ± 3.26		1.03 ± 0.01	0.39 ± 0.02
PASB2
S. annua	86.64 ± 2.34	12.40 ± 1.57	57.59 ± 2.25	91.00 ± 2.43		0.80 ± 0.05	0.14 ± 0.005
S. cretica	58.04 ± 1.10	1.18 ± 0.02	42.09 ± 1.12	71.68 ± 0.15		0.84 ± 0.04	0.20 ± 0.016
S. tmolea	67.93 ± 1.62	12.58 ± 0.55	44.24 ± 0.69	81.94 ± 1.30		0.94± 0.07	0.18 ± 0.010
Phenolic acids of solid phase hydrolysis: first base (B1), then acid (A2)
PASB1
S. annua	88.03 ± 1.35	1.09 ± 0.05	41.54 ± 1.10	75.77 ± 1.11		0.43 ± 0.03	0.08 ± 0.003
S. cretica	155.18 ± 2.45	10.76 ± 0.47	41.82 ± 2.32	62.70 ± 2.41		0.41 ± 0.01	0.06 ± 0.004
S. tmolea	70.11 ± 1.18	1.17 ± 0.01	22.29 ± 0.87	50.00 ±1.38		0.57 ± 0.04	0.13 ± 0.001
PASA2
S. annua	90.40 ± 1.42	12.76 ± 0.62	128.00 ± 1.71	74.25 ± 1.67		0.83 ± 0.06	0.17 ± 0.008
S. cretica	90.94 ± 2.48	12.68 ± 0.28	188.11 ± 2.67	107.00 ± 3.51		0.61 ± 0.02	0.01 ± 0.001
S. tmolea	78.86 ± 2.24	13.99 ± 0.64	58.23 ± 2.11	65.70 ± 1.13		0.88 ± 0.07	0.16 ± 0.010
Positive control
BHT	26.80 ± 1.40	5.10 ± 0.20	-	-		-	-
Vitamin-C	18.72 ± 0.17	-	36.76 ± 1.03	-		-	-
EDTA	-	-	-	5.50 ± 0.30		-	-

HO^• radicals are among the most reactive oxygen species and can cause severe damage to biomolecules; therefore, their neutralisation is of particular biological importance. In this study, the HO^• scavenging activities of the Stachys extracts were evaluated using the 2-deoxyribose assay, and the results are presented in Table 5. The highest HO^• scavenging activity was observed for the flavonol and PASB₁ extracts of S. annua, with identical IC₅₀ values of 1.09 ± 0.02 µg mL^-1, showing no statically significant difference (p > 0.05). Similarly, IC₅₀ values of 1.17 ± 0.01 and 1.18 ± 0.02 µg mL^-1 were obtained for S. tmolea PASB₁ and S. cretica-PASB₂ extracts, respectively, with no significant difference between them (p > 0.05). These values were considerably lower than that of BHT (5.10 ± 0.20 µg mL^-1), indicating a stronger HO^• scavenging activity of the investigated extracts (p < 0.05) compared with the literature, the HO^• scavenging activity of Stachys species has been relatively less studied.³⁹ In a previous report, the methanolic extract of S. anisochila showed the highest activity among tested species, with an IC₅₀ value of 6.25 µg mL^-1.³⁹ Overall, the Stachys extracts examined in the present study exhibited notably strong HO^• scavenging activities, suggesting their potential as effective natural antioxidants against highly reactive radical species. are promising.

NO^• is an important signalling molecule involved in various physiological and pathological processes; however, excessive production of NO^• may lead to the formation of cytotoxic reactive nitrogen species and contribute to the development of several diseases. In this study, the NO^• scavenging activities of the Stachys extracts were evaluated using the Griess assay, and the results are presented in Table 5. The highest NO^• scavenging activity was observed for the S. tmolea-PASA₁ extract, with an IC₅₀ value of 18.82 ± 0.75 µg mL^-1. In comparison, the IC₅₀ values for S. cretica- BHPA and S. annua-PASB₁ extracts were 36.34 ± 1.01 and 41.54 ± 1.10 µg mL^-1, respectively. When compared with the positive control (vitamin C, IC₅₀ = 36.76 ± 1.03 µg mL^-1), the S. tmolea-PASA₁ extract exhibited approximately two-fold higher activity, indicating a stronger NO^• scavenging effect (p < 0.05). Notably, no previous studies have reported the NO^• scavenging activity of Stachys species; therefore, direct comparison with the literature is not possible. Excessive levels of NO^• can react with biological macromolecules, leading to cellular damage and contributing to the progression of disorders such as neurodegenerative diseases, including Parkinson’s disease.⁴⁰

In the present study, isoquercitrin, isorhamnetin, rutin (flavonols), and rosmarinic, trans-cinnamic, and caffeic acids (phenolic acids) were among the predominant compounds detected in the extracts, all of which are known to possess strong antioxidant properties.⁴,⁶ A previous study⁴¹ reported that the order of phenolic acids with strong DPPH^• scavenging activity was rosmarinic acid > caffeic acid > chlorogenic acid > Α-tocopherol > ferulic acid. Overall, the radical scavenging activities observed in the present study were generally higher than those of the positive controls, highlighting the potential of these extracts for further applications, particularly in the food industry. Synthetic antioxidants such as BHT and butylated hydroxyanisole (BHA), which are widely used as food preservatives, have been associated with potential health risks.⁴² Consequently, interest in natural antioxidant alternatives has increased in recent years.⁴³ As shown in Table 5, the highest metal chelating activity (lowest IC₅₀ values) was observed for the flavone fractions of S. tmolea and S. cretica (27.73 ± 0.85 and 30.23 ± 0.90 µg mL^-1, respectively), with no statistically significant difference between them (p > 0.05). In contrast, S. annua-flavone extract showed a higher IC₅₀ value (50.94 ± 1.15 µg mL^-1), indicating lower activity. All extracts were less effective than the positive control EDTA (IC₅₀ = 5.50 ± 0.20 µg mL ¹) (p < 0.05). Ferhat et al.⁴⁴ reported 48.00 ± 1.71% metal chelating activity for the n-butanol extract of S. guyoniana aerial parts at 100 µg mL^-1. In another study, Kirkan³⁵ reported ferrous ion chelating capacities of methanolic and aqueous extracts of S. cretica as 17.53 ± 1.31 and 68.72 ± 0.80 mg_edtae g_extract^-1, respectively. Overall, the metal chelating activities of S. annua, S. cretica, and endemic S. tmolea observed in the present study are comparable to or higher than those reported in the literature.³⁶,⁴⁴

TRP and FRAP assays, which are based on the reduction of Fe^III to Fe^II, are widely used to evaluate the electron-donating capacity of antioxidant compounds. Iron also plays an essential role in various physiological processes, including neuronal function and cellular electron-transfer reactions.⁴⁵ Thus, these assays provide important insight into the reducing potential of plant extracts. As presented in Table 5, the highest TRP and FRAP values were obtained for S. annua-FPA extract (2.08 ± 0.13 and 1.46 ± 0.07 mg_vce g_dwe^-1, respectively). The best TRP values for the other species were 1.28 ± 0.03 mg_vce g_dwe^-1 for S. cretica-PASA₁ and 1.92 ± 0.11 mg_vce g_dwe^-1 for S. tmolea- flavan-3-ol-(AH), with no significant difference between the highest values of S. annua and S. tmolea (p > 0.05). For FRAP activity, the highest values for S. cretica and S. tmolea were observed in the flavan-3-ol-(AH) fractions (0.47 ± 0.01 and 1.13 ± 0.08 mg_vce g_dwe^-1, respectively). Bursal et al.¹² reported Fe^III reducing capacities of S. annua methanol and water extracts as 1.956 ± 0.004 and 1.731 ± 0.010, respectively, while the standard BHA showed a higher value (2.404 ± 0.013). In another study, methanolic extracts of S. persica and S. fruticulosa exhibited strong FRAP values (61.42 and 62.02 mmol Fe^II 100 g^-1, respectively).³⁶ Additionally, FRAP values of S. cretica methanol and water extracts were reported as 254.40 ± 8.58 and 160.01 ± 4.14 mg_TroloxE g_extract^-1, respectively.³⁶ The FRAP value of S. sieboldii extract (0.78 ± 0.04 mg_vce g_dwe^-1) was previously reported to be 1.87-fold lower than that of the best-performing S. annua-FPA extract in the present study.⁴⁶ These differences may be attributed to variations in phenolic content, particularly the number and position of hydroxyl groups, which directly influence electron-donating capacity and antioxidant activity. In general, comparison of TRP and FRAP values across studies remains limited due to differences in expression units and reference standards. Nevertheless, the results of the present study indicate reducing capacities of the investigated Stachys extracts.

Overall, S. annua-FPA showed superior performance in DPPH^• scavenging, TRP, and FRAP assays, while S. tmolea extracts exhibited stronger NO^• scavenging and metal chelating activities. No significant differences were observed between the three species for HO^• scavenging activity, nor between S. annua and S. tmolea for TRP (p > 0.05). Consequently, S. annua and S. tmolea emerged as the most active species in terms of four antioxidant parameters. These findings are consistent with the higher TPC, TFC, and TTC observed in S. annua and endemic S. tmolea compared with S. cretica. Overall, the antioxidant activities of Stachys extracts appear to associate well with their phenolic composition, supporting the role of phenolic compounds as key contributors to radical scavenging and reducing properties. Recent studies have highlighted the complex synergistic, additive, and antagonistic interactions of phenolic compounds in biological antioxidant systems.²,⁴⁷

Inhibition of AChE and tyrosinase activities

Enzyme inhibitors have attracted increasing attention due to their potential applications in pharmaceutical, food, and cosmetic industries. AChE inhibitors are particularly important for improving cognitive function and food related bioactivities, while tyrosinase inhibitors are widely used to prevent hyperpigmentation and to manage melanin-related disorders, including certain neurological conditions.¹³ The inhibitory effects of the Stachys extracts against AChE and tyrosinase are presented in Table 6. No inhibitory activity was observed for extracts not included in this table. The highest AChE inhibitory activities (IC₅₀ values) were obtained for S. annua, S. tmolea, and S. cretica FPA extracts, with values of 181 ± 9.21, 200 ± 8.73, and 210 ± 10.34 µg mL-¹, respectively.

Table 6
Enzyme inhibitory effects of the phenolic extracts of the Stachys species. Results were expressed as mean ± standard deviation of three independent replicates

Enzyme	Stachys species	IC50 / (µg mL-1)
		Flavanone	Flavan-3-ol	Flavan-3-ol-(AH)	Flavone	FPA	Positive control
AChE	S. annua	750 ± 15.72	ni	1073 ± 16.09	ni	181 ± 9.21	galantamine 8.12 ± 0.02
	S. cretica	983 ± 17.43	403 ± 11.64	611 ± 12.68	322 ± 9.46	210 ± 10.34
	S. tmolea	1104 ± 19.85	ni	ni	310 ± 8.27	200 ± 8.73
Tyrosinase	S. annua	107 ± 8.41	684 ± 10.72	531 ± 17.03	273 ± 6.85	1084 ± 17.96	kojic acid 94.70 ± 1.38
	S. cretica	201 ± 8.55	213 ± 7.83	402 ± 8.15	190 ± 7.09	170 ± 9.01
	S. tmolea	373 ± 7.13	ni	1224 ± 18.27	272 ± 5.16	233 ± 5.88

In a previous study, the IC₅₀ values for methanolic and aqueous extracts of S. annua were reported as 119.8 ± 2.4 and 150.1 ± 3.0 µg mL^-1, respectively, while tacrine, used as a positive control, showed an inhibitory effect (0.12 ± 0.02 µM).¹² Kocak et al.³⁴ reported AChE inhibitory activities of S. annua extracts (ethyl acetate, methanol, and water) as 1.89 ± 0.01, 1.58 ± 0.01, and 0.46 ± 0.02 mg_galae g_extract^-1, respectively. Similarly, Bahadori et al.¹³ reported AChE inhibition values for S. cretica extracts ranging from 21.54 ± 1.74 to 343.78 ± 10.79 µg_galae g_dryplant^-1. Overall, although the inhibitory activity observed in the present study is lower than that of galantamine, it is comparable with previously reported values for Stachys species.

Regarding tyrosinase inhibition, the highest IC₅₀ values were recorded for S. annua-flavanone (107 ± 8.41 µg mL ¹), S. cretica-FPA (170 ± 9.01 µg mL-¹), and S. tmolea FPA (233 ± 5.88 µg mL-¹). These activities correspond to approximately 88, 56, and 41% of the activity of the positive control kojic acid, respectively (Table 6), which is noteworthy. Interestingly, the most active fractions were flavanone and FPA extracts, which are rich in compounds containing Α-keto functional groups. Badria reported that flavonoids bearing Α-keto groups exhibit strong tyrosinase inhibitory activity, supporting the findings of the present study.⁴⁸

In a previous study, methanolic and aqueous extracts of S. cretica from Antalya, Türkiye, showed IC₅₀ values of 1.14 and 2.4 mg mL^-1 for tyrosinase inhibition, respectively.³⁶ Kocak et al.³⁴ reported tyrosinase inhibitory activities of S. annua extracts ranging from 8.13 ± 0.87 to 23.95 ± 1.34 mg_kae g_extract^-1, while Bahadori et al.¹³ reported values of 0.42 ± 0.07 to 2.45 ± 0.05 mg_kae g_dryplant^-1 for S. cretica extracts. Overall, the results obtained in the present study indicate that specific fractions of Stachys species exhibit measurable inhibitory activity against both AChE and tyrosinase enzymes.

Cytotoxic effects of Stachys extracts against HeLa, ACC 201, OE-33, HepG2, and MCF-7 cancer cell lines

The cytotoxic and antiproliferative effects of Stachys extracts were evaluated against HeLa, ACC-201, OE-33, HepG2, and MCF-7 cancer cell lines following 24 h and 48 h of incubation. The effectiveness of the extracts was expressed as IC₅₀ values, defined as the concentration required to reduce cell viability by 50%. According to the plant screening program of the United States National Cancer Institute (NCI), crude plant extracts are considered promising when IC₅₀ values are below 30 µg mL^-1 in in vitro cytotoxicity assays.⁴⁹ Therefore, in the present study, extract concentrations ranging from 10 to 150 µg mL^-1 were used.

As shown in Figure 1, the most potent cytotoxic activity against the HeLa cell line was observed for S. tmolea-PASA₁, with an IC₅₀ value of 24.5 ± 1.62 µg mL^-1 after 48 h of incubation. For the ACC-201 cell line, the highest cytotoxic effects were recorded for S. annua-FPA (IC₅₀ = 20.1 ± 1.25 µg mL^-1 at 24 h) and S. cretica-AHPA (IC₅₀ = 20.5 ± 1.35 µg mL^-1 at 48 h). Against the OE-33 cell line, S. annua-FPA and S. tmolea-PASA₁ exhibited IC₅₀ values of 22.0 ± 3.72 and 24.1 ± 2.02 µg mL^-1, respectively (24 h), with no statistically significant difference between them (p > 0.05). Overall, S. annua-FPA showed strong activity against both ACC-201 and OE-33 cell lines without significant variation in IC₅₀ values (p > 0.05), while S. tmolea-PASA₁ was active against both OE-33 and HeLa cell lines under different incubation periods. For the HepG2 cell line, the highest cytotoxic activity was observed for S. cretica-PASA₁ (IC₅₀ = 45.3 ± 1.35 µg mL ¹, 48 h), whereas for the MCF-7 cell line, the effect was recorded for S. tmolea-AHPA (IC₅₀ = 120.0 ± 2.50 µg mL^-1, 24 h). These values exceed the NCI threshold of 30 µg mL^-1, indicating relatively weak activity against these two cell lines.

Figure 1
The IC₅₀ values of the S. annua, S. cretica, and S. tmolea extracts against the HeLa, ACC-201, OE-33, HepG2, and MCF-7 cancer cell lines after 24 h (a) and 48 h (b) incubations. FPA: free phenolic acids; AHPA: acid-hydrolysed phenolic acids; flavan-3-ol-(AH); PASA₁, phenolic acid: solid phase-acid hydrolysis 1; PASA₂, phenolic acid: solid phase-acid hydrolysis 2; PASB₁, phenolic acid: solid phase-base hydrolysis 1. IC₅₀ values of 140 μg mL^-1 and above are not considered and symbolised (*). Results were expressed as mean ± standard deviation of three independent replicates.

Phenolic acid-rich extracts showing IC₅₀ values below 30 µg mL^-1 against HeLa, ACC-201, and OE-33 cell lines contained compounds such as trans-cinnamic, caffeic, vanillic, syringic, rosmarinic, 4-hydroxybenzoic, protocatechuic, gallic, and o-/p-coumaric acids. Notably, the first four of these compounds were consistently present in all active extracts. Previous studies have indicated that these phenolic acids may exert cytotoxic effects individually or in combination through synergistic mechanisms.²,⁵⁰

Literature data⁵¹ indicate that cytotoxic effects of Stachys extracts against HeLa, HepG2, and MCF-7 cell lines are generally moderate or weak. For instance, IC₅₀ values exceeding 500 µg mL^-1 were reported for ethanol and methanol extracts of S. lavandulifolia against HeLa cells, while a methanol extract of S. cretica subsp. vacillans showed an IC₅₀ value of 759 ± 1.53 µg mL ¹.⁵¹,⁵² Saravanakumar et al.¹¹ reported that ethyl acetate extract of S. riederi var. japonica, despite being rich in rosmarinic and caffeic acids, did not exhibit significant cytotoxicity against HepG2 or NIH3T3 cells. In MCF-7 cell line studies, hydroalcoholic extract of S. setifera showed an IC₅₀ value of 827.52 µg mL^-1, while other Stachys species such as S. recta, S. palustris, S. germanica, and S. byzantina showed inhibition rates between 28-55% at 10 mg mL-¹.⁵³,⁵⁴ Leporini et al.⁵⁵ reported inhibition rates of 23-37% at 200 µg mL^-1 for S. glutinosa ethanol extract against MCF-7 and HepG2 cell lines. Importantly, reports on the cytotoxic effects of Stachys species against OE-33 and ACC-201 cell lines are extremely limited. Therefore, the cytotoxic activities observed in the present study for S. annua, S. cretica, and S. tmolea against these cell lines are particularly noteworthy when compared with existing literature.⁵⁰ Overall, phenolic compounds derived from natural plant sources have attracted increasing attention as potential candidates for anticancer drug development, combination therapies, and chemotherapy strategies.⁵⁶,⁵⁷ However, further mechanistic studies are still required to fully elucidate their therapeutic potential.

Conclusions

In the present study, flavonoid subgroups, phenolic acids, and their acidand base-hydrolysed fractions (obtained from both liquid and solid phases) were extracted from three Stachys species using specific extraction procedures. RP-HPLC-DAD analysis revealed several phenolic compounds that are reported in the genus Stachys, including hesperetin (S. annua, S. cretica, and S. tmolea), myricetin, isoquercitrin, and quercitrin (S. annua and S. tmolea), epigallocatechin (S. cretica and S. tmolea), and diosmin (S. annua). Among the phenolic acids, benzoic, protocatechuic, and vanillic acids predominated in S. cretica and S. tmolea, whereas rosmarinic, trans-cinnamic, and protocatechuic acids were the major constituents of S. annua. The investigated extracts exhibited remarkable antioxidant activities. In particular, S. annua showed DPPH^• scavenging activity that was 4.87-fold stronger than that of BHT, while all three species displayed similarly high HO^• scavenging capacities. Furthermore, S. tmolea indicated NO^• scavenging activity that was approximately two-fold greater than that of vitamin C. These findings highlight the potential of Stachys extracts as natural antioxidant sources and suggest their possible application as alternatives to synthetic antioxidants in food-related products. The extracts also exhibited moderate inhibitory activities against AChE and tyrosinase, indicating their potential for further investigation in neuroprotective and cosmetic applications. In addition, several phenolic acid-rich fractions indicated promising cytotoxic activity, particularly against ACC-201 and OE-33 cancer cell lines, with IC₅₀ values below the threshold proposed by the NCI for promising crude plant extracts. Overall, the results indicate that S. annua, S. cretica, and especially the endemic species S. tmolea represent valuable natural sources of phenolic compounds with antioxidant, enzyme inhibitory, and cytotoxic properties. These findings provide a scientific basis for the further exploitation of Stachys species in food, cosmetic, and pharmaceutical applications.

Supplementary Information

Supplementary information is available free of charge at http://jbcs.sbq.org.br as PDF file.

Supplementary PDF

Supplementary PDF

Acknowledgments

This study was supported by the scientific research projects unit of Dokuz Eylül University (2019.KB.FEN.010).

Data Availability Statement

All data are available in the text.

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Edited by

• Editor handled this article:
  João Henrique Ghilardi Lago (Associate)

Publication Dates

• Publication in this collection
  31 Aug 2026
• Date of issue
  2026

History

• Received
  22 Dec 2025
• Published
  17 Aug 2026