Abstract
Enzyme inhibition activities, phenolic compounds, antioxidant activities, bioactive compounds, antimicrobial activities, and chemical components of essential oil and methanol extracts obtained from the aerial parts of S. cretica subsp. anatolica were investigated. The main phenolic compounds of aerial parts were catechin, oleuropein, and epicatechin. The determined enzyme inhibitor activities highlight the potential of S. cretica subsp. anatolica as a source of bioactive compounds, particularly for carbonic anhydrase and cholinesterase inhibition. The essential oil and methanol extract exhibited remarkable activities against CA-II, AChE, and BChE, although they were less potent than standard inhibitors. The essential oils generally showed stronger antimicrobial activity compared to the 30% methanol extracts across most bacterial and fungal strains, as evidenced by minimum lethal concentration (MLC) and lower minimum inhibitory concentration (MIC) values and larger inhibition zones. Chloramphenicol used alone exhibited the highest antimicrobial efficacy, with the lowest MIC and MLC values and the largest inhibition zones. The essential oils of S. cretica subsp. anatolica were determined as esters, oxygenated sesquiterpenes, and aldehydes in aerial parts. The main components were found to be hexahydrofarnesyl acetone in the aerial parts.
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Chemical Profiling of Stachys cretica subsp. anatolica Rech. f. (Endemic) Essential Oils and their Methanol Extracts with Evaluation as Enzyme Inhibitors, Antioxidant, and Antimicrobial Agents
Mehmet Öz,a Muhammed Said Fidan,b,* Cemalettin Baltacı,c Osman Akmeşe,d Abdurrahman Sefalı,e and İmdat Aygül f
Enzyme inhibition activities, phenolic compounds, antioxidant activities, bioactive compounds, antimicrobial activities, and chemical components of essential oil and methanol extracts obtained from the aerial parts of S. cretica subsp. anatolica were investigated. The main phenolic compounds of aerial parts were catechin, oleuropein, and epicatechin. The determined enzyme inhibitor activities highlight the potential of S. cretica subsp. anatolica as a source of bioactive compounds, particularly for carbonic anhydrase and cholinesterase inhibition. The essential oil and methanol extract exhibited remarkable activities against CA-II, AChE, and BChE, although they were less potent than standard inhibitors. The essential oils generally showed stronger antimicrobial activity compared to the 30% methanol extracts across most bacterial and fungal strains, as evidenced by minimum lethal concentration (MLC) and lower minimum inhibitory concentration (MIC) values and larger inhibition zones. Chloramphenicol used alone exhibited the highest antimicrobial efficacy, with the lowest MIC and MLC values and the largest inhibition zones. The essential oils of S. cretica subsp. anatolica were determined as esters, oxygenated sesquiterpenes, and aldehydes in aerial parts. The main components were found to be hexahydrofarnesyl acetone in the aerial parts.
DOI: 10.15376/biores.21.1.1274-1302
Keywords: Stachys cretica subsp. anatolica; Enzyme inhibition; Phenolic compounds; Antioxidant and antimicrobial activities; Essential oil
Contact information: a: Department of Forestry, Gümüshane Vocational School, Gümüshane University, Gümüshane, Türkiye; b: Department of Forest Industry Engineering, Faculty of Forestry, Bursa Technical University, Bursa, Türkiye; c: Department of Food Engineering, Faculty of Engineering and Natural Sciences, Gümüshane University, Gümüshane, Türkiye; d: Department of Central Research Laboratory, Gümüshane University, Gümüshane, Türkiye; e: Department of Basic Education, Faculty of Education, Bayburt University, Bayburt, Türkiye; f: Department of Nutrition and Dietetics, Faculty of Health Sciences, Gümüshane University, Gümüshane, Türkiye; *Corresponding author: said.fidan@btu.edu.tr
INTRODUCTION
The Lamiaceae family has a wide distribution in Anatolia. One of the largest genera of this family is Stachys L., which contains about 300 taxa. This genus is generally found in the temperate Irano-Turanian and the Mediterranean regions. Türkiye is one of the richest countries in terms of Stachys taxa, with 83 recorded species and a 48% endemism rate (Bhattacharjee 1980; Mabberley 1987; Goren et al. 2011).
Members of the Stachys genus are used as traditional medicine and consumed as wild tea in Anatolia as well as in Iran. It is known as mountain tea. It is used for the same purpose as sage, to treat skin infections, asthma, rheumatic and respiratory disorders, digestive problems, inflammatory disorders, as a wound healing agent, antiphlogistic, antianxiety, cholagogic, sedative, throat pains, tumors, coughs, and kidney diseases (Kartsev et al. 1994; Yesilada et al. 1999; Maleki et al. 2001; Rabbani et al. 2003; Amirghofran et al. 2006; Maleki-Dizaji et al. 2008; Khanavi et al. 2009; Ozturk et al. 2009; Goren et al. 2011).
Stachys taxa include at least nine natural product chemicals, which include alkaloids, carbohydrates, essential oils, flavonoids, iridoids, lipids, phenylpropanoid glycosides, steroids, and terpenoids (Radulović et al. 2006; Ahmad et al. 2007; Kotsos et al. 2007; Radulović et al. 2007; Soliman et al. 2007; Toshihiro et al. 2008; Giuliani et al. 2009).
The main components of most Stachys species were found to be caryophyllene oxide, β-caryophyllene, linalyl acetate, linalool, β-pinene, and germacrane D (Harmandar et al. 1997; Kaya et al. 2001; Skaltsa et al. 2001; Radulovic´ et al. 2007; Goren et al. 2011). In the meantime, the presence of diterpenoids, for instance kaurane, pimarane, labdane, and abietanes, were reported to be minor compounds of some Stachys essential oils (Piozzi and Bruno 2009; Goren et al. 2011). S. cretica subsp. symrnaea is an endemic and widespread species in northwestern, western and southern Anatolia. In the study of the chemical composition and antimicrobial activity of S. cretica subsp. symrnaea essential oil, it was reported that the main component of the oil was determined to be β-caryophyllene (51.0%) (Ozturk et al. 2009; Goren et al. 2011).
This plant is well known for its antibacterial and antioxidant effects in medicine and pharmacology (Grujic-Jovanovic et al. 2004; Erdemoglu et al. 2006). It can also help in continuous bioactive extraction of natural products. Moreover, this plant species could be beneficially used for extraction of useful medicinally important metabolites (Ozdemir et al. 2017). In the meantime, the emergence and spread of pathogenic bacteria has led to an increase in bacterial infections, raising concerns about the development of new antimicrobial meditations. In addition, because these bacteria form microbial biofilms and exhibit resistance to various drugs, infection control and healthcare face a significant global challenge (Selim et al. 2024).
The aim of this study was to determine and compare the enzyme inhibition, phenolic compound, antioxidant and antimicrobial activities of essential oil and methanol extracts obtained from the aerial parts of S. cretica subsp. anatolica plant. The article differs from other studies in that the region where the plant sample was taken, the analysis and comparison of methanol extract and essential oil samples are considered for the first time.
EXPERIMENTAL
Plant Material
In this study, samples of S. cretica subsp. anatolica (endemic) were collected from the Bayburt province (Türkiye), Gümüşhane road, near the Bayburt (coordinates: 40°20ꞌ44ꞌꞌN, 40°01ꞌ09ꞌꞌE, altitude: 1632 m, date: 22 June 2024, Habitat: hill sides). Figure 1 illustrates habitus, flowers, and leaves of the plant. A total of 1000 g of plant (dry weight) material was collected for analysis. The taxonomic identification of the plant was verified by Associate Professor Abdurrahman Sefalı from the Department of Primary Education, Faculty of Education, Bayburt University, Bayburt, Türkiye. The collected sample was deposited in the Herbarium of Bingöl University, where it was assigned the reference number BIN Sefalı 1202.
Fig. 1. S. cretica subsp. anatolica: A. Inflorescence, B. Flowers, and C. Basal leaves
Extraction Procedure
Extraction essential oil
A 500 g sample of the plant material was dried under shaded conditions, finely ground, and passed through a 250-micron sieve. From this processed material, 100 g was selected for essential oil extraction. The extraction was carried out using hydrodistillation at 100 °C and 4 h, employing a modified Clevenger apparatus equipped with both internal and external cooling systems. The resulting essential oil was dissolved in hexane, filtered through a 0.45-micron membrane, and securely stored in amber-colored vials to preserve its integrity (Öz et al. 2023).
Methanolic extraction
The extraction process was performed using a 3-L, 320 W ultrasonic bath (Bandelin Ultrasonic Bath). Initially, 10 g of the ground plant material was weighed, and 50 mL of a 30% aqueous methanol (MeOH) solution was added. The mixture was then subjected to ultrasound-assisted extraction for 60 min at a controlled temperature of 40 °C. After extraction, the solution was filtered twice using Whatman 1 filter paper and subsequently centrifuged at 4000 rpm for 10 min to separate the plant extracts. The resulting supernatant was carefully transferred to a beaker, and the methanol-water mixture was fully evaporated at 40 °C to obtain the final extract (Öz et al. 2023).
Enzyme Inhibitory Activities of the Extracts
Activity assay for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes
The activities of AChE and BChE enzymes were spectrophotometrically assessed using the Ellman method. In this assay, acetylthiocholine iodide-a thioester-is used as a substrate instead of acetylcholine. Upon enzymatic hydrolysis by acetylcholinesterase, thiocholine is released, which subsequently reacts with DTNB (5,5′-dithio-bis-(2-nitrobenzoic acid)). This reaction produces a yellow-colored compound, 5-thio-2-nitrobenzoic acid (TNB), whose formation is monitored by measuring absorbance at 412 nm (Ellman et al. 1961). For butyrylcholinesterase, the procedure is identical except that butyrylthiocholine iodide is used as the substrate. Enzyme activities were recorded at various inhibitor concentrations, and percentage activity was calculated. IC₅₀ values were determined from the inhibition curves using Lineweaver–Burk plots (Lineweaver and Burk 1934).
Carbonic anhydrase enzyme activity assay
This method is based on the esterase activity of carbonic anhydrase, which catalyzes the hydrolysis of p-nitrophenyl acetate-used as the substrate-into p-nitrophenol or p-nitrophenolate, both of which absorb at 348 nm. Because these two forms exhibit identical absorbance at this wavelength, the dissociation of a proton from the phenolic OH group does not influence the measurement (Landolfi et al. 1997).
α-Glucosidase enzyme activity assay
α-Glucosidase activity was determined following the method described by Tao et al. (2013), using p-nitrophenyl-α-D-glucopyranoside (p-NPG) as the substrate. The enzymatic reaction was monitored by measuring absorbance at 405 nm. Inhibitory effects were evaluated by testing various concentrations, and the percentage of residual activity was plotted against inhibitor concentration. IC₅₀ values were derived from the resulting inhibition curves based on the Lineweaver–Burk model (Lineweaver and Burk 1934).
α-Amylase enzyme activity assay
α-Amylase inhibitory activity was assessed using a modified version of the Caraway-Somogyi iodide/potassium iodide (IKI) method, adapted from Yang et al. (2012). Briefly, 25 µL of each sample was added to a 96-well microplate, followed by 50 µL of α-amylase solution prepared in phosphate buffer (pH 6.9, containing 6 mM NaCl). After a 10-min incubation at 37 °C, 50 µL of 0.05% starch solution was added. A blank without enzyme was also prepared in parallel. The plate was then incubated for another 10 min at 37 °C. To stop the reaction, 25 µL of 1 M HCl was added, followed by 100 µL of IKI reagent to develop color. Absorbance was measured at 630 nm using a microplate reader. Acarbose served as the reference inhibitor, and IC₅₀ values were calculated to express the inhibitory potential of the samples.
Determination of Phenolic Profiles Using LC-MS/MS Analysis
The LC-MS/MS analyses were performed using a Thermo Scientific Dionex Ultimate 3000 UHPLC system coupled with a TSQ Quantum Access Max tandem mass spectrometer (TSQ Quantum Access Max, Thermo Fisher Scientific, San Jose, CA, USA). The liquid chromatography system was equipped with an autosampler, degasser, dual pump, and column compartment. Chromatographic separation was performed on a C18 reversed-phase Inertsil ODS HYPERSIL analytical column (250 mm × 4.6 mm, 5 μm), maintained at a constant temperature of 30 °C.
The mobile phase consisted of two components: phase A was ultrapure water containing 0.1% formic acid, and phase B was methanol. The gradient elution program was applied as follows: 0 to 1 min, 0% B; 1 to 22 min, 95% B; 22 to 25 min, 95% B; and 25 to 30 min, 100% B. The total run time, including re-equilibration, was set to 34 min. The injection volume was 20 μL, and the flow rate was adjusted to 0.7 mL/min.
Following extensive optimization trials to ensure effective ionization and separation of the target phenolic compounds, this mobile phase composition and gradient program were selected. The phenolic compounds listed in Tables 2 and 3 were analyzed using this liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method, and a chromatogram of the standard phenolic compounds is also provided. The analytical method applied in this study was adapted from the protocol developed by Kayir et al. (2023).
Determination of Antioxidant Capacity
The methanol extract and essential oil of S. cretica subsp. anatolica was evaluated for its antioxidant potential using multiple methods. The ferric ion (Fe³⁺) reducing capacity was measured through the FRAP assay, while its ability to neutralize free radicals was assessed using ABTS and DPPH assays. Furthermore, the extract’s bioactive composition was quantified by determining the total antioxidant capacity (TAC), total flavonoid content (TFC), and total phenolic content (TPC). These analyses provided a comprehensive understanding of the extract’s antioxidant properties and its underlying bioactive constituents.
The methanol extracts and essential oil were analyzed for their ferric ion reducing antioxidant power (FRAP) according to the procedure outlined by Fidan et al. (2023). A FRAP reagent was prepared, and distilled water (500 µL) served as the blank. Standard solutions (250 µL) were processed under identical conditions. The FRAP values of the samples were determined using a calibration curve (y = 0.012x + 0.0516, R² = 0.998) generated from FeSO₄ solutions. The results were expressed as milligrams of FeSO₄ equivalents per 100 g of sample, reflecting the total iron-reducing capacity.
The ABTS radical scavenging activity was evaluated following the procedure described by Kobya et al. (2024). An ABTS solution was prepared, and methanol (150 µL) was used as the blank. Both standard Trolox solutions (150 µL) and the sample extract were processed identically. The absorbance of the resulting mixtures was measured at 734 nm using a spectrophotometer (UV 1800, Shimadzu, Kyoto, Japan). The scavenging activity of the ABTS cation radicals was quantified using a calibration curve (y=-0.0144x+0.615, R² = 0.997) constructed from Trolox standards. The results were expressed in terms of mg Trolox equivalents (TRE) per 100 g.
The DPPH radical scavenging activity of the methanol extract and essential oil derived from the plant was evaluated following the procedure outlined by Yilmaz et al. (2023). In this approach, the extract was combined with 2,2-diphenyl-1-picrylhydrazyl (DPPH) solutions at predetermined concentrations. The mixtures were thoroughly vortexed and then kept in the dark at room temperature for 30 min. After the incubation period, the absorbance of the samples was measured at 517 nm. The percentage inhibition of DPPH radicals was determined using Eq. 1. The results were reported as milligrams of ascorbic acid (AA) equivalents per 100 g (based on the calibration curve y = -0.0093x + 0.945, R² = 0.998) and as the percentage of free radical scavenging activity.
(1)
The total antioxidant capacity (TAC) of the plant’s methanol extract and essential oil were measured using a molybdate reagent, as per the methodology outlined by Yilmaz et al. (2023). In this procedure, pure water (250 µL) served as the blank in place of the sample. The absorbance of the reaction mixtures was recorded at 695 nm using a spectro-photometer. For the standard solutions, 500 µL aliquots were prepared and processed under identical conditions. The TAC content in the methanol extract was quantified as milligrams of Ascorbic acid equivalents (AAE) per 100 grams, based on a calibration curve (y = 0.0022x – 0.057, R² = 0.998) generated from ascorbic acid standards (Yilmaz et al. 2023).
The total flavonoid content (TFC) in the methanol extracts and essential oil of the plant were assessed using the protocol established by Yilmaz et al. (2023). The absorbance of the final reaction mixture was measured at 506 nm with a spectrophotometer. Pure water (500 µL) was employed as the blank, while 500 µL of standard solutions were prepared and processed similarly. The TFC in the samples was expressed as milligrams of quercetin equivalents (QEE) per 100 g, utilizing a calibration curve (y = 0.0038x + 0.0164, R² = 0.997) constructed from quercetin solutions dissolved in ethanol (Yilmaz et al. 2023).
The total phenolic content (TPC), a key bioactive component in the methanol extracts and essential oil, was measured following the procedure detailed by Yilmaz et al. (2023) with the Folin-Ciocalteu reagent. After preparing the reaction mixture, it was thoroughly vortexed and then incubated in the dark at room temperature for 120 min. The absorbance of the mixture was subsequently recorded at 760 nm. A blank was prepared by combining 3.7 mL of water, 500 µL of methanol, 100 µL of Folin-Ciocalteu reagent, and 600 µL of a 10% sodium carbonate (Na₂CO₃) solution. The phenolic content in the samples was quantified as milligrams of gallic acid equivalents (GAE) per 100 g, based on a calibration curve (y = 0.0052x + 0.0074, R² = 0.997) generated from gallic acid standards.
Determination of Antimicrobial Activity
Agar diffusion method
The antimicrobial activity of essential oils of S. cretica subsp. anatolica was evaluated using a modified agar diffusion method based on CLSI (2017) guidelines. Test organisms included Gram-positive bacteria (Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, and Bacillus cereus ATCC 9634), Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 13883, and Escherichia coli ATCC 25922), and the yeast Candida albicans ATCC 18804. Fresh microbial cultures were grown on Müller-Hinton Agar (MHA) and standardized to 0.5 McFarland turbidity (approximately 1.5 × 10⁸ CFU/mL) using 0.9% sterile saline. The inoculum was uniformly spread over MHA plates with sterile swabs.
Sterile 5.5-mm disks were placed onto the inoculated agar surfaces, and 15 µL of each sample (25 mg/mL in DMSO) was applied. Plates were kept at 4 °C for 2 h to allow compound diffusion. Chloramphenicol, nalidixic acid and nystatin (512 µg/mL) served as positive controls, while DMSO was used as the negative control. Following diffusion, bacterial plates were incubated at 37 °C for 24 h and yeast plates at 28 °C for 48 h. Inhibition zones were measured with a digital caliper. All experiments were conducted in triplicate, and the results were analyzed statistically.
Determination of MIC and MLC values
The minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) values of S. cretica subsp. anatolica essential oils against selected pathogens were determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2017). MIC testing was performed using the broth microdilution method in sterile 96-well microplates with Mueller-Hinton Broth (MHB) as the culture medium. The first wells were filled with double-strength MHB, while subsequent wells contained standard MHB. Essential oil samples prepared at 25 mg/mL were added to the first wells to achieve a starting concentration of 12.5 mg/mL, followed by serial two-fold dilutions. Nalidixic acid, chloramphenicol, and nystatin (each at 256 µg/mL, dissolved in DMSO) were used as positive controls.
Each well received 10 µL of microbial inoculum standardized to 0.5 McFarland turbidity. Wells containing only media or media with extracts served as negative controls. The plates were incubated at 37 °C for 24 h for bacteria and at 28 °C for 48 h for fungi. The MIC was defined as the lowest concentration of the essential oil that showed no visible microbial growth.
To determine MLC, 50 µL aliquots from the MIC, 2×MIC, and 4×MIC wells were plated onto Mueller-Hinton Agar (MHA) using a Drigalski spatula. Following incubation under the same conditions as above, colony-forming units (CFUs) were counted. The MLC was recorded as the lowest concentration that resulted in a ≥99.9% reduction in viable cell count compared to the initial inoculum. The methodology for antifungal assessment was adapted in part from Melkoumov et al. (2013), who reported enhanced in vitro and in vivo antifungal efficacy using nanosized nystatin formulations.
The Analysis GC-MS/FID of Essential Oil
For analysis with a gas chromatography-mass spectrometry/flame ionization detector (GC-MS)/FID, 1 µL of the essential oil solution was injected with a 1:5 split ratio, utilizing helium (> 99.999%) as the carrier gas at a constant flow rate of 1 mL/min. The analysis was conducted using an HP-5 apolar capillary column (30 m x 0.32 mm, 0.25 µm film thickness) on an Agilent 7890A gas chromatograph coupled with an Agilent 5975C mass spectrometer (GC-MS/FID; Agilent Technologies Inc, Santa Clara, CA, USA). The injector, ion source, and quadrupole temperatures were maintained at 250, 230, and 150 °C, respectively. The GC oven temperature program began at 60 °C, was held for 2 min, and then ramped up to 240 °C at a rate of 3 °C per minute. The FID detector was operated at 250 °C, with hydrogen and air flows set to 35 mL/min and 350 mL/min, respectively. Mass spectra were recorded in the range of m/z 45 to 450, following a 4.000-minute solvent delay (Öz et al. 2023). The identification of essential oil components was achieved by comparing their mass spectra with reference data from the Willey and NIST libraries. Additionally, the Kovats indices were used to confirm the identities of the compounds, as described in previous studies (Adams 2007).
RESULTS AND DISCUSSION
The Results of Enzyme Inhibitory Activities
The enzyme inhibition activities of the methanol extract and essential oil of S. cretica subsp. anatolica, as well as their comparison with standard inhibitors, such as acetazolamide, tacrine, and acarbose, provide valuable insights into the plant’s pharmacological potential. The methanol extract of S. cretica subsp. anatolica demonstrated significant (SPSS, IBM, version 29.0, Armonk, NY, USA) activity in inhibiting CA-II (IC50 = 0.048 ± 0.002 μg/mL), although it was less potent than the standard inhibitor acetazolamide (IC50 = 0.0023 ± 0.0002 μg/mL). Carbonic anhydrases are a family of enzymes that play critical roles in physiological processes such as pH regulation, CO2 transport, and electrolyte secretion (Supuran 2016). Inhibitors of CA-II are particularly important in the treatment of glaucoma, epilepsy, and altitude sickness (Supuran 2016). The moderate activity of the methanol extract suggests that it contains compounds with potential CA-II inhibitory properties, which could be further explored for therapeutic applications. The weaker activity of the essential oil (IC50 = 0.279 ± 0.003 μg/mL) may be attributed to differences in the chemical composition of the two extracts. Previous studies have shown that phenolic compounds and flavonoids, often abundant in methanol extracts, exhibit significant CA-II inhibitory activity (Ekinci et al. 2013).
The essential oil of S. cretica subsp. anatolica showed stronger AChE inhibition (IC50 = 0.029 ± 0.008 μg/mL) compared to the methanol extract (IC50 = 0.23 ± 0.06 mg/mL). AChE inhibitors are widely used in the treatment of neurodegenerative diseases, such as Alzheimer’s disease, as they prevent the breakdown of acetylcholine, a neurotransmitter essential for memory and cognitive function (Greig et al. 2001). The higher efficacy of the essential oil may be due to the presence of terpenoids and other volatile compounds, which have been reported to exhibit cholinesterase inhibitory activity (Mukherjee et al. 2007). However, both extracts were less potent than the standard inhibitor tacrine (IC50 = 0.012 ± 0.001 μg/mL), a well-known AChE inhibitor. This suggests that while S. cretica has potential, further optimization or isolation of active compounds may be required to enhance its efficacy.
The essential oil of S. cretica subsp. anatolica also exhibited stronger BChE inhibition (IC50 = 0.107 ± 0.0014 μg/mL) compared to the methanol extract (0.64 ± 0.008 mg/mL). BChE, like AChE, is involved in the hydrolysis of acetylcholine and has been implicated in neurodegenerative diseases (Darvesh et al. 2003). The essential oil’s higher activity may be attributed to its complex mixture of terpenes and phenolic compounds, which have been shown to possess cholinesterase inhibitory properties (Mukherjee et al. 2007). However, the standard inhibitor tacrine (IC50 = 0.0017 ± 0.0003 μg/mL) remains significantly more potent. These findings align with previous studies on other plant species, where essential oils have demonstrated moderate cholinesterase inhibition but often fall short of synthetic inhibitors (Loizzo et al. 2008).
The methanol extract of S. cretica subsp. anatolica showed moderate α-glucosidase inhibition (1.41 ± 0.01 mg/mL), while the essential oil exhibited no activity. α-Glucosidase inhibitors are used in the management of type 2 diabetes, as they delay carbohydrate digestion and glucose absorption, thereby reducing postprandial blood glucose levels (Van de Laar et al. 2005). The activity of the methanol extract, though lower than that of the standard inhibitor acarbose (0.061 ± 0.002 μg/mL), suggests the presence of bioactive compounds, such as flavonoids or phenolic acids, which are known to inhibit α-glucosidase (Tadera et al. 2006). The lack of activity in the essential oil may be due to the absence of these compounds in its volatile fraction.
Both the essential oil (7.47 ± 0.17 mg/mL) and the methanol extract (12.77 ± 0.20 mg/mL) of S. cretica subsp. anatolica showed limited activity against α-amylase. α-Amylase inhibitors are also used in diabetes management, as they reduce starch digestion and glucose absorption (Van de Laar et al. 2005). The weak activity of both extracts compared to acarbose (IC50 = 33.27 ± 0.12 μg/mL) suggests that S. cretica may not be a strong candidate for α-amylase inhibition. This is consistent with findings from other plant species, where α-amylase inhibition is often less pronounced than α-glucosidase inhibition (Tadera et al. 2006).
The findings from this study highlight the potential of S. cretica subsp. anatolica as a source of bioactive compounds, particularly for cholinesterase and carbonic anhydrase inhibition. The methanol extract and essential oil exhibited notable activities against CA-II, AChE, and BChE, although they were less potent than standard inhibitors. These results are consistent with previous studies on other Stachys species, which have demonstrated significant biological activities due to their rich content of phenolic compounds, flavonoids, and terpenoids (Stegăruș et al. 2021).
The essential oil’s stronger activity against AChE and BChE suggests its potential as a natural candidate for the treatment of neurodegenerative diseases. However, further studies are needed to isolate and identify the active compounds responsible for these effects. Additionally, the moderate α-glucosidase inhibition by the methanol extract indicates its potential role in diabetes management, although its activity is not as strong as that of synthetic inhibitors.
In conclusion, S. cretica subsp. anatolica shows promise as a source of natural enzyme inhibitors, particularly for cholinesterase and carbonic anhydrase. Future research should focus on the isolation and characterization of its bioactive compounds, as well as in vivo studies to evaluate its therapeutic potential.
Table 1. Enzyme Activity Results of S. cretica subsp. anatolica Methanol Extract and Essential Oil
Results for Phenolic Compounds
Figures 2 and 3 present the chromatograms of the 31 standards used in the analysis and the chromatogram of the methanol extract obtained from the aerial parts of S. cretica subsp. anatolica. This comprehensive analysis, conducted using LC-MS/MS, highlights the extract’s rich phenolic profile, providing detailed insights into its chemical composition.
Fig. 2. Chromatogram of standard phenolic compounds analyzed by LC/MS/MS
Fig. 3. Chromatogram of extracts of aerial parts of S. cretica subsp. anatolica by LC/MS/MS
The chromatographic profiles presented in Fig. 2 and 3 are total ion chromatograms (TIC) obtained using a TSQ Quantum Access Max tandem mass spectrometer coupled to a Thermo Scientific Dionex Ultimate 3000 UHPLC system. Unlike chromatograms that display specific peaks corresponding to individual compounds, TIC outputs represent the sum of all ion intensities detected at each point in time. As such, these chromatograms provide an overall profile of the sample’s ion content across the retention time range rather than isolating and labeling each analyte peak. This is a standard output format for tandem mass spectrometry systems operating in multiple reaction monitoring (MRM) mode.
Table 2. Amounts of Phenolic Compounds in the Extracts of Aerial Parts of S. cretica subsp. anatolica
According to the data presented in Table 2, the most abundant compound identified in the extract was catechin (24484 µg/g dw), a flavonoid known for its potent antioxidant, anti-inflammatory, and cardioprotective properties. The high concentration of catechin suggests that S. cretica subsp. anatolica may have significant protective effects against oxidative stress-related diseases. This finding aligns with studies on other Stachys species, such as S. germanica, S. officinalis, S. byzantina, S. inflata, S. sylvatica, S. palustris, and S. recta, where catechin and similar flavonoids have also been detected (Bahadori et al. 2020; Benedec et al. 2023).
Another notable compound in S. cretica subsp. anatolica was oleuropein (1426.3 µg/g dw), a phenolic compound commonly found in olive leaves. Oleuropein is recognized for its antidiabetic, antioxidant, and antimicrobial properties, which enhance the therapeutic potential of S. cretica subsp. anatolica in addressing metabolic disorders and infectious diseases. While oleuropein is not frequently reported in other Stachys species, its presence in S. cretica subsp. anatolica highlights the unique phytochemical profile of this plant.
Other flavonoids detected in significant amounts include epicatechin (153.1 µg/g dw) and taxifolin (115.5 µg/g dw). Epicatechin is particularly known for its cardiovascular health benefits, while taxifolin exhibits strong antioxidant and anti-inflammatory properties. These compounds further underscore the potential of S. cretica subsp. anatolica in managing chronic diseases. Similar flavonoids have been identified in other Stachys species, such as S. lavandulifolia, where epicatechin and taxifolin were also found in notable quantities (Bingol and Bursal 2018).
The extract also contained gallic acid (87.0 µg/g dw), a phenolic acid renowned for its strong antioxidant and antimicrobial properties, and hesperidin (56.9 µg/g dw), a flavonoid glycoside noted for its cardiovascular health benefits. Additionally, luteolin (64.3 µg/g dw), a flavonoid with antioxidant, anti-inflammatory, and neuroprotective effects, and its glycosylated form, luteolin-7-glucoside (12.49 µg/g dw), were identified. Luteolin-7-glucoside, a flavonoid derivative, is particularly noteworthy for its enhanced bioavailability and potent antioxidant activity, which further supports the therapeutic potential of Stachys species.
Another flavonoid detected in the extract was flavone (87.0 µg/g dw), a simple flavonoid with antioxidant and anti-inflammatory properties. Flavone is known to modulate cellular signaling pathways and has been studied for its potential in cancer prevention and treatment. Additionally, sesamol (22.3 µg/g dw), a phenolic derivative with strong antioxidant and anti-inflammatory properties, was identified. Sesamol is particularly known for its ability to scavenge free radicals and protect against oxidative stress-related damage. These compounds further enhance the therapeutic profile of S. cretica subsp. anatolica.
Compounds detected in lower concentrations, such as ferulic acid (3.77 µg/g dw), a phenolic acid with antioxidant and cardioprotective effects, and naringenin (5.25 µg/g dw) and quercetin (6.26 µg/g dw), both flavonoids, also exhibit significant biological activities. Quercetin, in particular, is known for its antioxidant, anti-inflammatory, and antidiabetic properties, and it may play a role in reducing insulin resistance. These compounds, although present in smaller amounts, contribute to the overall bioactivity of S. cretica subsp. anatolica.
However, certain phenolic compounds, such as chlorogenic acid, caffeic acid, rutin, ellagic acid, and rosmarinic acid, were not detected in the S. cretica subsp. anatolica extract. This suggests that the phenolic profile of the plant may vary depending on factors such as species, growing conditions, and extraction methods. For instance, previous studies have reported the presence of compounds such as rutin and chlorogenic acid in other Stachys species, such as S. lavandulifolia (Bingol and Bursal 2018). These variations indicate that the chemical composition of Stachys species can be influenced by ecological and genetic factors.
Studies on the phenolic content and biological activities of other Stachys species further support the therapeutic potential of this genus. For example, the methanol extract of S. lavandulifolia was found to contain high levels of naringenin and luteolin (Bingol and Bursal 2018). Similarly, S. germanica, S. officinalis, S. byzantina, S. inflata, S. sylvatica, S. palustris, and S. recta have been reported to contain gallic acid, catechin, ferulic acid, hesperidin, luteolin, and luteolin-7-glucoside (Bahadori et al. 2020; Benedec et al. 2023). These studies demonstrate that while there are common phenolic compounds across Stachys species, the specific composition and concentrations can vary significantly depending on the species and environmental conditions.
The methanol extract of the aerial parts of S. cretica subsp. anatolica exhibits significant antioxidant, anti-inflammatory, and antidiabetic potential, primarily due to the high concentrations of phenolic compounds, such as catechin and oleuropein, as highlighted in Table 2. Additionally, compounds including gallic acid, hesperidin, luteolin, luteolin-7-glucoside, resveratrol, flavone, and sesamol, further enhance the plant’s therapeutic value. However, the absence of certain phenolic compounds, such as chlorogenic acid and rutin, suggests that the chemical profile of S. cretica subsp. anatolica may vary depending on various factors. These findings support the traditional uses of S. cretica subsp. anatolica but more comprehensive pharmacological and clinical studies are needed to fully understand its therapeutic potential and how it compares to other Stachys species.
The Results of Antioxidant Capacity and Bioactive Component
This study evaluated the antioxidant activity and bioactive components of the methanol extract and essential oil of S. cretica subsp. anatolica, a plant known for its potential health benefits. Antioxidant activity was assessed using DPPH, ABTS, and FRAP assays, while bioactive components were quantified through TPC, TFC, and TAC. The results, as presented in Table 3, highlight significant differences in antioxidant potential and bioactive composition between the methanol extract and essential oil.
The DPPH assay, a widely used method for evaluating free radical scavenging activity, revealed that the methanol extract of S. cretica subsp. anatolica exhibited significantly stronger antioxidant activity (279.8 mg AAE/100 g) compared to its essential oil (65.6 mg AAE/100 g). This was further supported by the higher inhibition percentage of the methanol extract (63.8%) relative to the essential oil (9.94%). The IC50 values, which indicate the concentration required to scavenge 50% of free radicals, further underscored this finding. The methanol extract demonstrated a much lower IC50 value (44.8 mg/mL) compared to the essential oil (957.6 mg/mL), highlighting its superior efficiency in neutralizing free radicals. Ascorbic acid, used as a reference standard, exhibited an even lower IC50 value (92.6 µg/mL), confirming its exceptional antioxidant capacity, consistent with previous studies (Brand-Williams et al. 1995; Kedare and Singh 2011).
Similarly, the ABTS assay, which measures the ability to scavenge ABTS radicals, also demonstrated the methanol extract’s stronger antioxidant potential (73.9 mg TRE/100 g) compared to the essential oil (19.9 mg TRE/100 g). This aligns with findings by Re et al. (1999), who emphasized that ABTS radical scavenging activity is a reliable indicator of antioxidant capacity, particularly for extracts rich in phenolic compounds. The FRAP assay, which evaluates ferric ion reduction capacity, further corroborated these results, showing that the methanol extract (974 mg FeSO₄/100 g) had significantly higher reducing power than the essential oil (652 mg FeSO₄/100 g). This suggests that the methanol extract has a greater ability to donate electrons and neutralize free radicals, a characteristic often associated with high antioxidant activity (Benzie and Strain 1996).
Table 3. Antioxidant Activity and Bioactive Component Profile of S. cretica subsp. anatolica Methanol Extract and Essential Oil
The superior antioxidant activity of the methanol extract can be attributed to its significantly higher levels of bioactive components. The methanol extract contained substantially more total phenolic content (1005 mg GAE/100 g) compared to the essential oil (63.2 mg GAE/100 g). Phenolic compounds are well-documented for their strong antioxidant properties, as they can donate hydrogen atoms or electrons to stabilize free radicals (Huang et al. 2006). Similarly, the methanol extract had a much higher total flavonoid content (133 mg QEE/100 g) than the essential oil (17.8 mg QEE/100 g). Flavonoids are another class of bioactive compounds known for their potent antioxidant effects, including free radical scavenging and metal chelation (Middleton et al. 2000). Additionally, the methanol extract exhibited a higher total antioxidant capacity content (841 mg AAE/100 g) compared to the essential oil (588 mg AAE/100 g), further confirming its enhanced antioxidant potential.
The methanol extract’s superior performance can be explained by its higher extraction efficiency for polar compounds, such as phenolics and flavonoids, which are more soluble in methanol. This is consistent with studies by Dai and Mumper (2010), who demonstrated that methanol is an effective solvent for extracting a wide range of antioxidant compounds from plant materials. The lower IC50 value of the methanol extract in the DPPH assay further supports its greater antioxidant efficiency, as a lower IC50 value indicates higher antioxidant activity (Prior et al. 2005).
The Results of Antimicrobial Activities
The antimicrobial activity of the 30% methanol extract and essential oil was evaluated against a range of bacterial and fungal strains, with their performance compared to the standard antibiotics chloramphenicol and nalidixic acid. Antimicrobial efficacy is inversely related to the MIC and MLC values, where lower values indicate higher potency. Additionally, the diameter of the inhibition zone (DDT in mm) provides a visual measure of antimicrobial effectiveness, with larger zones reflecting stronger activity. The detailed antimicrobial results, including DDT, MIC, and MLC values for all tested strains, are presented in Table 4.
The antimicrobial activity of the 30% methanol extract and essential oil was evaluated against a range of bacterial and fungal strains, with their performance compared to the standard antibiotics chloramphenicol and nalidixic acid. Antimicrobial efficacy is inversely related to the MIC and MLC values, where lower values indicate higher potency. Additionally, the diameter of the inhibition zone (DDT in mm) provides a visual measure of antimicrobial effectiveness, with larger zones reflecting stronger activity (Balouiri et al. 2016).
B. cereus, the 30% methanol extract demonstrated a DDT of 7.83 mm, with MIC and MLC values of 0.41 mg/mL and 0.48 mg/mL, respectively. The essential oil exhibited stronger activity, with a DDT of 11.6 mm and significantly lower MIC (0.24 mg/mL) and MLC (0.81 mg/mL) values. Chloramphenicol, however, showed the highest antibacterial activity, with a DDT of 21.1 mm and the lowest MIC (0.04 mg/mL) and MLC (0.07 mg/mL) values. These results are consistent with previous studies indicating that essential oils often exhibit moderate antimicrobial activity, while conventional antibiotics such as chloramphenicol remain highly effective (Bakkali et al. 2008).
E. coli, the methanol extract recorded a DDT of 7.80 mm, with MIC and MLC values of 0.81 mg/mL and 0.24 mg/mL, respectively. The essential oil performed better, with a DDT of 10.9 mm and lower MIC (0.12 mg/mL) and MLC (1.63 mg/mL) values. Chloramphenicol displayed the strongest activity, with a DDT of 19.9 mm and the lowest MIC (0.02 mg/mL) and MLC (0.07 mg/mL) values. This aligns with findings by Nazzaro et al. (2013), who noted that essential oils can disrupt bacterial cell membranes, but their efficacy is often lower than synthetic antibiotics.
K. pneumoniae, the methanol extract showed a DDT of 7.30 mm, with MIC and MLC values of 1.63 mg/mL and 0.95 mg/mL, respectively. The essential oil exhibited greater efficacy, with a DDT of 9.43 mm and lower MIC (0.48 mg/mL) and MLC (3.25 mg/mL) values. Chloramphenicol achieved the highest inhibition, with a DDT of 29.9 mm and the lowest MIC (0.02 mg/mL) and MLC (0.07 mg/mL) values. These findings align with studies highlighting the resistance of K. pneumonia to natural extracts and its susceptibility to broad-spectrum antibiotics (Piddock 2012).
In the case of P. aeruginosa, the methanol extract demonstrated a DDT of 7.67 mm, with MIC and MLC values of 0.81 mg/mL and 0.96 mg/mL, respectively. The essential oil performed slightly better, with a DDT of 10.73 mm and lower MIC (0.46 mg/mL) and MLC (1.63 mg/mL) values.
Table 4. Antimicrobial Activities of S. cretica subsp. anatolica Methanol Extract and Essential Oil
The discs have a diameter of 5.5 mm; Samples: 25 mg/mL, Antibiotic: ± 0.51 mg/mL; 15 µL were pipetted onto each disc, n = 3; DDT: Disk Diffusion Test, NT: Not tested
Chloramphenicol showed the highest effectiveness, with a DDT of 10.51 mm and the lowest MIC (0.02 mg/mL) and MLC (0.07 mg/mL) values. This is in line with research by Stratev et al. (2016), who reported that P. aeruginosa is often resistant to natural extracts but remains sensitive to conventional antibiotics.
For S. aureus, the methanol extract recorded a DDT of 7.53 mm, with MIC and MLC values of 0.42 mg/mL and 0.08 mg/mL, respectively. The essential oil exhibited better performance, with a DDT of 10.9 mm and significantly lower MIC (0.04 mg/mL) and MLC (0.81 mg/mL) values. Chloramphenicol demonstrated exceptional activity, with a DDT of 19.0 mm and the lowest MIC (0.001 mg/mL) and MLC (0.005 mg/mL) values. These findings are supported by studies indicating that S. aureus is highly susceptible to chloramphenicol but shows variable resistance to natural extracts (Tong et al. 2015).
In the case of a fungal strain, E. faecalis, the methanol extract showed a DDT of 8.27 mm, with MIC and MLC values of 0.10 mg/mL and 0.24 mg/mL, respectively. The essential oil performed better, with a DDT of 0.12 mm and lower MIC (0.46 mg/mL) and MLC (0.20 mg/mL) values. Chloramphenicol exhibited moderate activity, with a DDT of 11.12 mm, MIC of 0.018 mg/mL, and MLC of 0.035 mg/mL. These results align with research by Fisher and Phillips (2009), who noted that essential oils can exhibit antifungal activity, though often at higher concentrations compared to antibiotics.
Evaluation of C. albicans showed that the methanol extract recorded a DDT of 7.93 mm, with MIC and MLC values of 1.63 mg/mL and 1.90 mg/mL, respectively. The essential oil demonstrated superior efficacy, with a DDT of 11.9 mm and lower MIC (0.95 mg/mL) and MLC (3.20 mg/mL) values. Nystatin showed significant antifungal activity, with a DDT of 26.3 mm, MIC of 0.004 mg/mL, and MLC of 0.008 mg/mL.
The results indicate that essential oils generally exhibit stronger antimicrobial activity compared to the 30% methanol extracts across most bacterial and fungal strains, as evidenced by lower MIC and MLC values and larger inhibition zones. However, chloramphenicol consistently demonstrated the highest antimicrobial efficacy, with the lowest MIC and MLC values and the largest inhibition zones.
This is consistent with the findings of Aminov (2010), who emphasized the superior performance of synthetic antibiotics in combating microbial infections. Essential oils, particularly against B. cereus, E. coli, and S. aureus, showed notable antimicrobial potential, suggesting their viability as natural therapeutic agents. Nevertheless, their activity was generally inferior to chloramphenicol, underscoring the need for further research to optimize their efficacy. This could include refining extraction methods or combining essential oils with other compounds to enhance their antimicrobial properties, as suggested by studies such as that by Bassolé and Juliani (2012).
GC-MS/FID Conditions for Essential Oil Analysis
The GC-MS/FID analysis of the essential oil obtained from the aerial parts of the endemic S. cretica subsp. anatolica plant identified 155 compounds. The structures of 151 of these compounds were identified, but the structures of 4 compounds could not be determined. When Table 5 is examined, it can be seen that the identified components constituted 98.60% of the essential oil. It was understood that the most prominent compounds in the essential oil extracted from the aerial parts of the plant were hexahydrofarnesyl acetone (7.83%), hexadecanoic acid (7.76%), benzaldehyde (5.76%), diisobutyl phthalate (5.70%), and cis-chrysanthenol acetate (4.98%). The main compound of the essential oil of S. cretica subsp. anatolica was hexahydrofarnesyl acetone (Table 5).
Also when considering Table 5, it can be seen that the 151 compounds identified in the essential oil were grouped into 13 different chemical classes. The number of compounds and their percentage distributions within these classes are as follows: esters (25.1%; 28 compounds), with diisobutyl phthalate as the main component; oxygenated sesquiterpenes (18.5%; 15 compounds), with hexahydrofarnesyl acetone; aldehydes (13.1%; 22 compounds), with benzaldehyde; oxygenated monoterpenes (8.50%; 23 compounds), with eucalyptol; oil acids (7.70%; 2 compounds), with hexadecanoic acid; sesquiterpene hydrocarbons (7.34%; 18 compounds), with β-bourbonene; hydrocarbons (5.76%; 13 compounds), with cyclotetradecane; monoterpene hydrocarbons (4.50%; 9 compounds), with α-pinene; alcohols (2.59%; 5 compounds), with 1-octen-3-ol; others (2.07%; 5 compounds), with 1,2,3,4-tetrahydro-2-methylquinoline; ketones (1.98%; 9 compounds), with benzophenone; oxygenated diterpenes (1.15%; 1 compound), with phytol; diterpene hydrocarbons (0.06%; 1 compound), with dehydroabietane; and unidentified compounds (1.60%; 4 compounds). Among these, esters were found to be the most dominant chemical class in terms of both their relative percentage and number of compounds.
In a study conducted on Stachys cretica subsp. anatolica samples collected in the Koçtepe region of Isparta in 2017 to 2018, the above ground parts of the plant during the flowering period were analyzed by solid phase microextraction (SPME). As a result of the analysis carried out using the GC-MS device, 58 volatile compounds were detected. Among the main components, germacrene D (34.56%), β-caryophyllene (21.04%), and (E)-2-hexenal (12.58%) stand out (Sarıkaya 2018).
In another study published in 2024, the changes in volatile components in the flowers and leaves of Stachys cretica subsp. anatolica at different altitudes were examined. A total of 79 components were identified in the analyses performed by the Headspace-Solid Phase Micro-Extraction (HS-SPME) method. Benzaldehyde and α-pinene were determined as the main components in both lower and upper altitudes in flowers. In leaves, benzaldehyde and α-pinene were determined as the main components in the lower altitudes, and benzaldehyde and germacrene D were determined as the main components in the upper altitudes (Tekeş 2024).
Table 5. The Essential Oil Components of Aerial Parts in S. cretica subsp. anatolica
