Chemical profiling of essential oils and evaluation of enzyme inhibition, antioxidant, and antimicrobial activities in both methanol extract and essential oils of Centaurea oltensis

Abstract

The phytochemical composition and biological activities of Centaurea oltensis, an endemic species in Türkiye, were studied. GC–MS/FID analysis identified 128 compounds representing 99.2% of the EO, with decanoic acid (15.1%), α-pinene (7.9%), and sabinyl acetate (5.4%) as the principal constituents. GC–MS/FID analysis identified 128 compounds representing 99.2% of the essential oil (EO), with decanoic acid (15.1%), α-pinene (7.9%), and sabinyl acetate (5.4%) as the principal constituents. LC–MS/MS analysis of the methanol extract (ME) revealed high concentrations of ferulic acid (2410 µg/g dw) and chlorogenic acid (1170 µg/g dw), together with caffeic, salicylic, and 4-hydroxybenzoic acids. The ME exhibited potent carbonic anhydrase-II inhibition (IC₅₀ = 0.028 µg/mL) and significant α-glucosidase inhibition (IC₅₀ = 0.255 mg/mL). The EO showed strong acetylcholinesterase and butyrylcholinesterase inhibitory activities, with IC₅₀ values of 0.0145 µg/mL and 0.204 µg/mL, respectively. Antioxidant assays, including DPPH, ABTS, and FRAP, demonstrated superior activity of the ME, correlating with its higher phenolic and flavonoid contents. The EO displayed remarkable antibacterial activity against Staphylococcus aureus (MIC = 0.02 mg/mL) and moderate antifungal activity against Candida albicans. Overall, C. oltensis demonstrated considerable chemical diversity and promising antioxidant, antimicrobial, neuroprotective, and enzyme inhibitory potential for future pharmacological and therapeutic applications.

Full Article

Chemical Profiling of Essential Oils and Evaluation of Enzyme Inhibition, Antioxidant, and Antimicrobial Activities in Both Methanol Extract and Essential Oils of Centaurea oltensis

İmdat Aygül ^a,* and Berfin Akşit ^b

The phytochemical composition and biological activities of Centaurea oltensis, an endemic species in Türkiye, were studied. GC–MS/FID analysis identified 128 compounds representing 99.2% of the EO, with decanoic acid (15.1%), α-pinene (7.9%), and sabinyl acetate (5.4%) as the principal constituents. GC–MS/FID analysis identified 128 compounds representing 99.2% of the essential oil (EO), with decanoic acid (15.1%), α-pinene (7.9%), and sabinyl acetate (5.4%) as the principal constituents. LC–MS/MS analysis of the methanol extract (ME) revealed high concentrations of ferulic acid (2410 µg/g dw) and chlorogenic acid (1170 µg/g dw), together with caffeic, salicylic, and 4-hydroxybenzoic acids. The ME exhibited potent carbonic anhydrase-II inhibition (IC₅₀ = 0.028 µg/mL) and significant α-glucosidase inhibition (IC₅₀ = 0.255 mg/mL). The EO showed strong acetylcholinesterase and butyrylcholinesterase inhibitory activities, with IC₅₀ values of 0.0145 µg/mL and 0.204 µg/mL, respectively. Antioxidant assays, including DPPH, ABTS, and FRAP, demonstrated superior activity of the ME, correlating with its higher phenolic and flavonoid contents. The EO displayed remarkable antibacterial activity against Staphylococcus aureus (MIC = 0.02 mg/mL) and moderate antifungal activity against Candida albicans. Overall, C. oltensis demonstrated considerable chemical diversity and promising antioxidant, antimicrobial, neuroprotective, and enzyme inhibitory potential for future pharmacological and therapeutic applications.

DOI: 10.15376/biores.21.4.11085-11114

Keywords: Centaurea oltensis; Enzyme inhibition; Phenolic compounds; Antioxidant activities; Antimicrobial activities; Essential oil

Contact information: a: Department of Nutrition and Dietetics, Faculty of Health Sciences, Gümüşhane University, Gümüşhane, 29100, Türkiye; b: Department of Chemistry, Postgraduate Education Institute, Gümüşhane University, Gümüşhane, 29100, Türkiye;

* Corresponding author: [email protected]

Graphical Abstract

INTRODUCTION

In the southwest, center, and east of Türkiye, there are representatives of the genus Centaurea L. (Asteraceae). In addition, this genus includes roughly 500 to 600 annual, biannual, and perennial herbaceous plants that are found worldwide, but exclusively north of the equator. The majority of these plants are found in the Eastern Hemisphere, with the Middle East serving as the center of diversification (Djeddi et al. 2011; Carev et al. 2022). With 112 endemic species out of 181 total, Centaurea has one of Türkiye’s highest rates of endemism. Türkiye is one of the gene centers of this genus, as evidenced by the high endemism ratio (Yayli et al. 2005; Aydin et al. 2013; Kilic 2013). In Centaurea species, essential oils are commonly obtained from aerial organs, particularly flowers, leaves, and stems, although their yield and chemical composition may vary considerably among plant organs, species, developmental stages, and environmental conditions. Flowers and leaves are often of particular interest because of their abundance of secretory structures and volatile secondary metabolites. Therefore, the selection and clear identification of the plant organs used for essential-oil extraction are important for both chemical characterization and comparison among Centaurea species.

The majority of terpenes and terpenoids found in EO are naturally occurring combinations that are derived from fragrant and medicinal plants. Each species or subspecies has a different essential oils chemical makeup, which is unique to that species. Several approaches must be used in order to identify the various components of complicated combinations, such as terpenes and terpenoids in essential oils. Gas chromatography–mass spectrometry (GC–MS), one of the most widely used techniques for analyzing the composition of essential oils, enables the identification of the particular natural compounds present in an essential oil by comparing their mass spectra and relative retention times/indices (Yayli et al. 2005).

According to earlier chemical research on the genus Centaurea, the most distinctive and consistently significant ingredients are the sesquiterpene lactones. Triterpenes, steroids, hydrocarbons, polyacetylenes, flavonoids, anthocyanins, lignans, and alkaloids are some of the additional secondary metabolites found in plants in this genus (Kilic 2013).

Phytopharmaceuticals from Centaurea species are recognized to have both positive and negative effects on human health. Research on herbal therapy has grown in emerging nations as a means of reviving long-standing customs and as a substitute for urban health issues. Therefore, with the increasing popularity of traditional medicine as an alternative type of health treatment, the screening of plants for active chemicals has become highly significant. A number of Centaurea members biological actions, including cytogenetic, antimicrobial, antifungal, antiulcerogenic, antioxidant, antiviral, antiplasmodial, antiprotozoal, anti-colon cancer, and cytotoxic activity, have been documented elsewhere. Additionally, folk medicine uses the aerial parts of numerous Centaurea species as antibiotics, diuretics, choleretics, antibacterials, antipyretics, antidiarrheics, and anti-inflammatory agents (Kilic 2013; Carev et al. 2022).

Given the considerable interspecific variation in essential-oil and phytochemical composition previously reported among Centaurea species, it was hypothesized that C. oltensis may possess a distinctive phytochemical profile and exhibit significant biological activities. Specifically, the high phenolic and terpenoid content of C. oltensis was expected to contribute to strong antioxidant, antimicrobial, and enzyme inhibitory properties. Therefore, this study was designed to test whether the EO and ME of C. oltensis demonstrate distinct compositional characteristics and multifunctional bioactivities that could support their potential use as natural therapeutic and preservative agents. Compared with previous studies on other Centaurea species, the significance of the present work lies in the integrated evaluation of the chemical composition and multiple biological activities of C. oltensis. By combining essential-oil profiling, phenolic characterization, enzyme inhibition, antioxidant, and antimicrobial analyses, this study provides a broader assessment of the phytochemical and biological potential of this endemic species and contributes to the comparative understanding of chemical and functional diversity within the genus Centaurea.

EXPERIMENTAL

Plant Material

In this study, samples of Centaurea oltensis were collected from the Bayburt province (Türkiye), Kop Pass, near the Coşan Mountain and the border of Erzincan (coordinates: 39°58ꞌ37ꞌꞌN, 40°22ꞌ17ꞌꞌE, altitude: 2220 m, date: 28 June 2024, Habitat: roadsides, sandy places). Figure 1 illustrates the habitus, flowers, and leaves of the plant. A total of 1000 g of plant material was collected for analysis. The taxonomic identification of the plant was verified by Assoc. Prof. 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 1049a.

Morphologically, C. oltensis is a perennial herb bearing solitary or up to six capitula. Scapes are sessile or up to 2 cm long. Rosette leaves are conspicuously covered with simple hairs and are pinnatipartite to pinnatifid or sublyrate, with linear to lanceolate lateral segments, occasionally up to 15 pairs, and a slightly larger, triangular terminal segment. Phyllaries bear brownish-yellow, lanceolate, usually curved appendages, and the florets are golden yellow. Achenes are 4 to 5 mm long, with a pappus 1 to 2 mm long. The species typically occurs on rocky slopes.

Fig. 1. Centaurea oltensis: Habitus and capitula with appendages

Extraction Procedure

Essential oil extraction

A 500 g portion of the collected aerial parts (stems, leaves, and flowers) was dried in the shade and finely ground. After homogenization and sieving through a 250-µm screen, 100 g of the processed aerial plant material was used for essential-oil extraction. Using a modified Clevenger apparatus with both internal and exterior cooling systems, the extraction was performed by hydrodistillation at 100 °C. To maintain its integrity, the resultant EO was filtered through a 0.45- μm membrane, dissolved in hexane, and stored in amber-colored vials (Oz et al. 2023, 2025).

Extraction with methanol

A 3 L, 320 W ultrasonic bath (Bandelin Ultrasonic Bath) was used for the extraction procedure. First, 50 mL of a 30% aqueous methanol (MeOH) solution was added to 10 g of the ground plant material after weighing. The mixture was extracted using ultrasonic assistance for 60 min at a controlled temperature of 40 °C. To separate the plant extracts, the solution was centrifuged at 4000 rpm for 10 min after being filtered twice through Whatman No. 1 filter paper. The final extract was obtained by carefully transferring the resulting supernatant to a beaker and completely evaporating the methanol–water mixture at 40 °C (Oz et al. 2023). The dried extract was dissolved in hexane, filtered through a 0.45 μm membrane, and securely stored in amber-colored vials to preserve its integrity (Oz et al. 2023).

Enzyme Inhibitory Activities of the Extracts

Activity assay for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes

The Ellman method was used to spectrophotometrically evaluate the activity of the AChE and BChE enzymes. Acetylthiocholine iodide, a thioester, is utilized as a substrate in this experiment rather than acetylcholine. AChE hydrolyzes it enzymatically, releasing thiocholine, which then interacts with DTNB (5,5′-dithio-bis-(2-nitrobenzoic acid)). The production of TNB (5-thio-2-nitrobenzoic acid), a yellow-colored molecule produced by this reaction, is tracked by measuring absorbance at 412 nm (Ellman et al. 1961). The process is the same for BChE, with the exception that the substrate is butyrylthiocholine iodide. At different inhibitor doses, enzyme activities were measured and the % activity was computed. Lineweaver–Burk plots were used to extract IC₅₀ values from the inhibition curves (Lineweaver and Burk 1934).

Carbonic anhydrase enzyme activity assay

Using p-nitrophenyl acetate as the substrate, this approach uses the esterase activity of carbonic anhydrase to hydrolyze it producing p-nitrophenol or p-nitrophenolate, both of which absorb at 348 nm. The dissociation of a proton from the phenolic OH group has no effect on the measurement because these two forms show the same absorbance at this wavelength (Kandel et al. 1970; Armstrong et al. 1966; Landolfi et al. 1997).

α-Glucosidase enzyme activity assay

α-Glucosidase activity was measured using p-nitrophenyl-α-D-glucopyranoside (p-NPG) as the substrate, in accordance with the procedure outlined by Akmese et al. (2025). Absorbance at 405 nm was used to track the enzymatic process. Plotting the proportion of residual activity against inhibitor concentration allowed for the evaluation of inhibitory effects at different concentrations. The obtained inhibition curves were analyzed using the Lineweaver–Burk model to determine the IC₅₀ values (Lineweaver and Burk 1934).

α-Amylase enzyme activity assay

A modified version of the Caraway–Somogyi iodide/potassium iodide (IKI) method described by Kilinc et al. (2025) was used to evaluate the α-amylase inhibitory activity. 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 incubation at 37 °C for 10 min, 50 µL of a 0.05% starch solution was added. In parallel, an enzyme-free blank was prepared. The plate was then incubated for an additional 10 min at 37 °C. The reaction was stopped by adding 25 µL of 1 M HCl, after which 100 µL of IKI reagent was added to develop the 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

A Thermo Scientific Dionex Ultimate 3000 UHPLC system in conjunction with a TSQ Quantum Access Max tandem mass spectrometer was used to conduct the LC-MS/MS analysis. The liquid chromatography system has a column compartment, dual pump, autosampler, and degasser. A C18 reversed-phase Inertsil ODS HYPERSIL analytical column (250 mm × 4.6 mm, 5 μm) was used for the chromatographic separation, which was kept at a steady temperature of 30 °C.

Phase A was ultrapure water with 0.1% formic acid, while phase B was methanol. These two substances made up the mobile phase. The following gradient elution program was used: 0% B for 0 to 1 min, 95% B for 1 to 22 min, 95% B for 22 to 25 min, and 100% B for 25 to 30 min. 34 min was the allotted run time, including re-equilibration. The flow rate was set at 0.7 mL/min, and the injection volume was 20 μL.

This mobile phase composition and gradient program were chosen after a number of optimization trials to guarantee efficient ionization and separation of the target phenolic chemicals. This LC-MS/MS method was used to examine the phenolic compounds indicated in Tables 2 and 3. A chromatogram of the standard phenolic compounds is also included. The protocol created by Kayir et al. (2023) served as the model for the analytical technique used in this investigation.

Determination of Antioxidant Capacity

Several techniques were used to assess the antioxidant capacity of the methanol extract and essential oil of Centaurea oltensis. The FRAP assay was used to analyze the ferric ion’s (Fe³⁺) reducing capability, and the ABTS and DPPH assays were used to evaluate its capacity to neutralize free radicals. Additionally, the total antioxidant capacity (TAC), total flavonoid content (TFC), and total phenolic content (TPC) were measured in order to quantify the extract’s bioactive composition. The antioxidant qualities of the extract and its underlying bioactive components were thoroughly understood thanks to these analyses.

The ferric ion reducing antioxidant power (FRAP) of the methanol extracts and essential oil was measured using the method described by Fidan et al. (2023). First, 500 µL of distilled water was used as the blank, and a FRAP reagent was created. The same conditions were used to process standard solutions (250 µL). A calibration curve (y = 0.0115x + 0.0521, R² = 0.998) produced from FeSO₄ solutions was used to calculate the samples’ FRAP values. The entire iron-reducing ability was represented by the results, which were given as milligrams of FeSO₄ equivalents per 100 grams of material.

The method outlined by Kobya et al. (2024) was used to assess the ABTS radical scavenging activity. Methanol (150 µL) was utilized as the blank in an ABTS solution. The sample extract and standard ascorbic acid solutions (150 µL) underwent the same processing. Using a spectrophotometer, the absorbance of the resultant mixes was determined at 734 nm. The ABTS cation radicals’ scavenging activity was measured using a calibration curve made from Trolox standards (y=-0.0144x+0.615, R² = 0.997). According to Eq. 1, the results were reported as mg of ascorbic acid equivalents (AAE) per 100 g, mg of Trolox equivalents per gram, and the percentage of free radical scavenging.

(1)

The ME and EO extracted from the plant were tested for their ability to scavenge DPPH radicals using the method described by Yilmaz et al. (2023). This method involved mixing the extract with solutions of 2,2-diphenyl-1-picrylhydrazyl (DPPH) at specific quantities. After giving the mixtures a thorough vortex, they were allowed to sit at room temperature for 30 min in the dark. Following the incubation period, the samples’ absorbance at 517 nm was evaluated. Equation 1 was used to calculate the DPPH radicals’ % inhibition. The findings were presented as the percentage of free radical scavenging activity and as milligrams of ascorbic acid (AA) equivalents per 100 grams (based on the calibration curve y=-0.0093x+0.945, R² = 0.998).

Following the procedure described by Gedikli et al. (2024), a molybdate reagent was used to assess the TAC of the plant’s ME and EO. Pure water (250 µL) was used as the blank in this process instead of the sample. A spectrophotometer was used to measure the reaction mixtures’ absorbance at 695 nm. First, 500 µL aliquots of the standard solutions were made and treated in the same way. Using a calibration curve (y=0.0022x-0.057, R² = 0.998) produced from ascorbic acid standards, the TAC content in the methanol extract was measured in milligrams of gallic acid (GA) equivalents per gram (Gedikli et al. 2024).

The Yilmaz et al. (2023) technique was used to calculate the TFC of the EO and ME. A spectrophotometer was used to measure the reaction mixture’s absorbance at 506 nm, using 500 µL of distilled water as the blank. The same procedure was applied to standard catechin solutions made in ethanol. TFC values were calculated using the calibration curve y=0.0036x+0.0168 (R² = 0.999) and reported as mg catechin equivalents (CAE) per 100 g of sample.

The Folin-Ciocalteu reagent was used to assess the TPC, a crucial bioactive component in the ME and EO, in accordance with the protocol described by Gedikli et al. (2024). The reaction mixture was prepared, vortexed completely, and then allowed to sit at room temperature for 120 min in the dark. At 760 nm, the mixture’s absorbance was then measured. 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 were combined to create a blank. The phenolic content of the samples was measured using a calibration curve (y=0.0052x+0.0074, R² = 0.997) created from GA standards, and expressed as milligrams of GA equivalents (GAE) per 100 grams.

Determination of Antimicrobial Activity

Agar diffusion method

Essential oils from C. oltensis were evaluated for their antibacterial activity using a modified agar diffusion method based on CLSI criteria (Clinical & Laboratory Standards 2017). The test organisms included gram-positive bacteria (Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 9634), gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 13883, Escherichia coli ATCC 25922), and the yeast Candida albicans ATCC 18804. Fresh microbiological cultures were grown on Müller-Hinton Agar (MHA), and they were standardized to 0.5 McFarland turbidity (about 1.5 x 10⁸ CFU/mL) using 0.9% sterile saline. The inoculum was uniformly spread across MHA plates using sterile swabs.

Sterile 5.5 mm disks were used to cover the inoculated agar surfaces, and 15 µL of each sample (25 mg/mL in DMSO) was applied. To allow for compound diffusion, plates were kept at 4 °C for two h. DMSO served as the negative control, while nalidixic acid (512 µg/mL) and chloramphenicol served as the positive controls. Following diffusion, bacterial and yeast plates were incubated for 24 and 48 h at 37 and 28 °C, respectively. The inhibitory zones were measured using a digital caliper. Each experiment was conducted in triplicate, and the results were statistically analyzed.

Determination of MIC and MBC values

Centaurea oltensis essential oils’ minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against particular pathogens were calculated in accordance with CLSI criteria (Clinical & Laboratory Standards 2017). Using Müller-Hinton broth (MHB) as the culture medium, MIC testing was carried out in sterile 96-well plates using the broth microdilution method. Double-strength MHB was included in the first wells, and normal MHB was added to the following wells. Sample solutions (25 mg/mL) were added to the first wells following successive dilutions in order to reach an initial concentration of 12.5 mg/mL. Positive controls included chloramphenicol (256 µg/mL in ethanol) and nalidixic acid. First, 10 µL of a microbiological suspension standardized to 0.5 McFarland turbidity were added to each well as an inoculant. Negative controls were wells that contained either medium alone or medium with extracts. For 24 h, plates were incubated at 37 °C for bacteria, and for 48 h, plates were incubated at 28 °C for fungus. The lowest concentration that totally prevented observable growth was known as the minimum inhibitory concentration, or MIC.

The MBC values of essential oils of C. oltensis against particular infections were calculated in accordance with CLSI recommendations (Clinical & Laboratory Standards 2017). Using Müller-Hinton Broth (MHB) as the culture medium, MIC testing was carried out in sterile 96-well plates using the broth microdilution method. Double-strength MHB was included in the first wells, and normal MHB was added to the following wells. Sample solutions (25 mg/mL) were added to the first wells following successive dilutions in order to reach an initial concentration of 12.5 mg/mL. Positive controls included chloramphenicol (256 µg/mL in ethanol) and nalidixic acid. Ten microliters of a microbiological suspension standardized to 0.5 McFarland turbidity were added to each well as an inoculant. Negative controls were wells that contained either medium alone or medium with extracts. 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 that completely inhibited visible growth.

To estimate MBC, 50 µL samples from wells corresponding to MIC, 2×MIC, and 4×MIC were spread onto MHA plates using a Drigalski spatula. The plates were then incubated in the same way. After incubation, colonies were counted, and the MBC was calculated by selecting the lowest concentration that resulted in a decrease of at least 99.9% in viable cells.

GC–MS/FID Analysis of the Essential Oil

Helium (>99.999%) was used as the carrier gas at a constant flow rate of 1 mL/min to inject 1 µL of the EO solution with a 1:5 split ratio for GC–MS/FID analysis. The analyses were performed using an Agilent 7890A gas chromatograph coupled with an Agilent 5975C mass spectrometer (GC–MS/FID; Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an HP-5 apolar capillary column (30 m × 0.32 mm, 0.25 µm film thickness). The injector, ion source, and quadrupole temperatures were maintained at 250 °C, 230 °C, and 150 °C, respectively. The GC oven temperature was initially held at 60 °C for 2 min, then increased to 240 °C at a rate of 3 °C/min. The FID detector was operated at 250 °C with hydrogen and air flows of 35 mL/min and 350 mL/min, respectively. Mass spectra were acquired in the range of 45 to 450 m/z with a solvent delay of 4.0 min (Oz et al. 2023). By comparing their mass spectra with reference data from the NIST and Wiley libraries, the constituents of essential oils were identified. Furthermore, in accordance with earlier research, the compounds’ identities were verified using the Kovats indices (Adams 2007).

RESULTS AND DISCUSSION

Enzyme Inhibitory Activities

With low IC₅₀ values underscoring their potential, the enzyme inhibitory results of Centaurea oltensis ME and EO demonstrate exceptional bioactivity. ME inhibited CA-II very well (IC₅₀ = 0.0283 µg/mL), but acetazolamide (0.0067 µg/mL) was still more effective. Since glaucoma, epilepsy, and similar conditions can be effectively treated with carbonic anhydrase inhibitors (Supuran 2016), the ME may be a viable natural source of CA-II inhibitors because of its high phenolic content (Poulsen and Davis 2013).

In terms of cholinesterase inhibition in Table 1, the EO was less effective than tacrine (0.038 µg/mL), although it showed better activity against AChE (IC₅₀ = 0.0145 µg/mL) than the ME (0.111 mg/mL). This is in accordance with other research showing that EO’s terpenoids and volatile chemicals work well as cholinesterase inhibitors (Loizzo et al. 2008; Mukherjee et al. 2007). The importance of lipophilic components was further shown by the EO’s greater BChE inhibition (IC₅₀ = 0.204 µg/mL) in comparison to the ME’s (0.422 mg/mL). The EO of C. oltensis may be a viable natural neuroprotective candidate because both AChE and BChE inhibitors are important for treating Alzheimer’s disease (Darvesh et al. 2003; Greig et al. 2001).

The ME exhibited considerable α-glucosidase inhibition (IC₅₀ = 0.255 mg/mL) with respect to carbohydrate-hydrolyzing enzymes, whereas the EO remained inactive. Despite having a lower activity than acarbose (IC₅₀ = 0.059 µg/mL), the presence of this inhibition points to possible antidiabetic benefits associated with flavonoids and phenolics (Tadera et al. 2006; Van de Laar et al. 2005). The ME (IC₅₀ = 18.52 mg/mL) and EO (IC₅₀ = 11.20 mg/mL) showed comparatively weaker α-amylase inhibition than acarbose (33.20 µg/mL). This finding is consistent with previous studies reporting that plant extracts generally show greater inhibitory activity against α-glucosidase than against α-amylase (Tadera et al. 2006).

Overall, the findings show that C. oltensis has strong enzyme inhibitory qualities, especially when it comes to cholinesterase and CA-II inhibition. The EO works better against cholinesterase, while the ME seems more promising for inhibiting carbonic anhydrase. According to Khammar and Djeddi (2012), these results are consistent with prior publications on bioactive chemicals in Centaurea species and similar taxa, indicating that both fractions merit more phytochemical and pharmacological research. Isolating the accountable secondary metabolites and validating the therapeutic potential in vivo should be the main goals of future research.

Table 1. Enzyme Activity Results of Centaurea oltensis ME and EO

Phenolic Compounds

Figures 2 and 3 present the chromatograms of the 33 standards used in the analysis and the chromatogram of the ME obtained from the aerial parts of C. oltensis. This comprehensive analysis, conducted using LC-MS/MS, highlights the extract’s rich phenolic profile, providing detailed insights into its chemical composition.

The current study indicates that C. oltensis‘s aerial parts have a distinct phenolic profile. Despite the absence of most flavonoids, these sections have a particularly high phenolic acid content (Table 2). This suggests that the species may have a distinct phenolic metabolic pathway compared to other species in the genus Centaurea. The most common of these compounds (2410 µg/g dw) was ferulic acid, which is an essential part of the species’ phenolic composition. This observation highlights C. oltensis as a species particularly rich in ferulic acid.

In C. oltensis, chlorogenic acid represents the second most abundant phenolic component, with a concentration of 1170 µg/g dw, highlighting a notable species-specific enrichment. Although chlorogenic acid has been regularly reported in other Centaurea taxa, the relatively high levels observed in C. oltensis suggest that genetic factors may strongly influence phenolic composition within the genus. For instance, Centaurea gigantea contains chlorogenic acid as a dominant phenolic in its aerial parts, where it exhibits significant anti-colon cancer activity (Shoeb et al. 2007). Similarly, Centaurea paphlagonica harbors chlorogenic acid and its methyl ester in the flowers and aerial tissues, with the latter showing antiproliferative activity against cancer cell lines (Tufekci et al. 2023). In Centaurea bruguieriana, chlorogenic acid has been quantified at 3.2 µg/mg in the ethyl acetate fraction, further confirming its consistent occurrence across the genus (Noman et al. 2022). Taken together, these findings indicate that chlorogenic acid is a ubiquitous but variably abundant phenolic in Centaurea taxa. Its presence contributes substantially to the phytochemical profile and pharmacological potential of the genus, underscoring both its taxonomic relevance and its importance as a bioactive compound with potential applications in health, nutrition, and biotechnology.

Caffeic acid (79.97 µg/g dw) and 4-hydroxybenzoic acid (79.80 µg/g dw) were among the compounds found in moderate amounts. Both substances are known to contribute to the genus’s phenolic diversity. In the genus Centaurea, caffeic acid has frequently been reported as one of the dominant phenolic acids, whereas 4-hydroxybenzoic acid is considered a marker of phenolic variation across species. For instance, in C. solstitialis, caffeic acid was identified as the most abundant phenolic compound (24.1 µg/g extract), while 4-hydroxybenzoic acid was present at 2.08 µg/g (Alper et al. 2021). Similarly, C. paphlagonica was shown to contain caffeic acid, 3,4-dihydroxybenzoic acid, and various caffeic acid derivatives (Tufekci et al. 2023). In polyherbal mixtures involving C. erythraea, both caffeic acid and p-hydroxybenzoic acid, along with their derivatives, were also detected (Petrovič et al. 2023). Additional studies reported the presence of 4-hydroxybenzoic acid in C. urvillei and C. jacea while C. horrida growing in Lebanon contained caffeic acid together with flavonoids such as fisetin, hispidulin, and quercetin (Boukhary et al. 2017). These findings confirm that both compounds, although varying in concentration among species, play a central role in shaping the antioxidant and cytotoxic activities attributed to Centaurea (Khan et al. 2021; Pavlikova 2022).

Fig. 2. Chromatogram of standard phenolic compounds analyzed by LC/MS/MS

Fig. 3. Chromatogram of extracts of aerial parts of Centaurea oltensis by LC/MS/MS

Furthermore, significant concentrations of protocatechuic acid (56.15 µg/g dw), salicylic acid (56.65 µg/g dw), and protocatechuic aldehyde (28.53 µg/g dw) were found. Previous observations of salicylic acid in C. behen and protocatechuic acid in C. cyanus (Bahadori et al. 2020) indicate that these phenolics are common constituents across multiple Centaurea species. Together with the frequently reported caffeic acid and 4-hydroxybenzoic acid profiles in the genus (Alper et al. 2021), these findings underscore a shared yet quantitatively variable phenolic backbone that likely contributes to the antioxidant and cytotoxic capacities noted for Centaurea extracts. Beyond chemotaxonomic value, protocatechuic acid and protocatechuic aldehyde have been associated with broad pharmacological actions—including antioxidant, anti-inflammatory, and cytoprotective effects—supporting their functional relevance when present at moderate-to-high levels (Song et al. 2020; Zhang et al. 2021). Overall, the relative abundances of these acids and aldehydes vary among species, highlighting intra-generic chemical diversity with potential implications for bioactivity and standardization of Centaurea-derived preparations (Alper et al. 2021). The profile was further complicated by compounds that were present at lower amounts, such as luteolin-7-glucoside (9.32 µg/g dw), vanillin (9.68 µg/g dw), and rosmarinic acid (34.1 µg/g dw). Particularly, luteolin derivatives have been found in C. cyanus and C. stoebe, suggesting that C. oltensis and other genus members have similarities in phenolic variety.

Interestingly, C. oltensis did not contain any of the phenolic compounds that are frequently described in many Centaurea species, such as catechin, epicatechin, rutin, quercetin, oleuropein, naringenin, taxifolin, resveratrol, and ellagic acid. Other congeners, including C. horrida, C. castriferrei, C. parviflora, C. lycaonica, and C. glastifolia, have consistently shown flavonoids such as quercetin, apigenin, luteolin, kaempferol, and chlorogenic acid (Boukhary et al. 2017; Gozcu et al. 2024; Hechaichi et al. 2023). The absence of these compounds in C. oltensis highlights the uniqueness of its phenolic profile and may be explained by both methodological and biological factors. Variations in extraction method, solvent type, or analytical technique can substantially affect phenolic detection (Zengin et al. 2018), while genetic and environmental influences such as climate, soil characteristics, and altitude are also known to modulate secondary metabolite synthesis in Centaurea species (Salachna et al. 2021). This pattern underscores the remarkable chemical diversity within the genus and suggests that C. oltensis may represent a chemotaxonomically distinctive lineage.

Overall, ferulic acid and chlorogenic acid predominate in C. oltensis’s phenolic profile, which is reinforced by the moderate and low amounts of a number of phenolic acids and aldehydes. Although there are clear similarities in the types of compounds when compared to other Centaurea species, significant quantitative differences show that C. oltensis has distinct phytochemical properties. These results offer fresh perspectives on the phytochemical uniqueness of C. oltensis and advance our knowledge of the chemical diversity within the genus. Taken together, these findings indicate that the phenolic profile of C. oltensis is particularly remarkable for its exceptionally high levels of ferulic and chlorogenic acids, while the presence of compounds such as protocatechuic acid, salicylic acid, 4-hydroxybenzoic acid, and rosmarinic acid further underscores the species’ chemical diversity. Comparative evaluations with other Centaurea taxa reveal clear qualitative and quantitative distinctions, confirming the phytochemical uniqueness of C. oltensis. This distinctiveness not only highlights its significance for chemotaxonomic classifications but also points to its potential value in biological activity, pharmacological applications, and natural product research, warranting further detailed investigations in future studies

Table 2. The Amount of Phenolic Compound in the Extracts of Aerial Parts of C. oltensis

FTIR Analysis

As shown in Fig. 4, the FTIR spectrum of the methanol extract of C. oltensis exhibited several characteristic absorbance bands, confirming the chemically diverse nature of the extract. The broad and intense band at 3252 cm⁻¹ was attributed to hydrogen-bonded O–H stretching vibrations, indicating the presence of phenolic and alcoholic hydroxyl groups. The weak band at 2932 cm⁻¹ corresponded to aliphatic C–H stretching vibrations. The prominent band at 1587 cm⁻¹ was assigned mainly to aromatic C=C stretching and/or asymmetric carboxylate vibrations, whereas the band at 1397 cm⁻¹ may be associated with O–H bending, aliphatic C–H deformation, and symmetric carboxylate stretching (Coates 2000; Stuart 2004).

Fig. 4. FTIR spectrum of the methanol extract obtained from the aerial parts of Centaurea oltensis

The strong absorbance band at 1056 cm⁻¹ was consistent with C–O stretching vibrations of alcohols, phenols, ethers, or glycosidic structures. Bands observed at 818 and 776 cm⁻¹ were attributable to out-of-plane aromatic C–H bending, while the lower-frequency band at 611 cm⁻¹ was associated with skeletal and ring-deformation vibrations. Overall, the FTIR profile indicates that the methanol extract is rich in hydroxylated aromatic and oxygen-containing constituents. These spectral features support the LC–MS/MS findings, particularly the predominance of ferulic and chlorogenic acids and the presence of other phenolic acids, including caffeic, protocatechuic, salicylic, and 4-hydroxybenzoic acids (Socrates 2001; Stuart 2004).

Antioxidant Capacity and Bioactive Component

The present study provides a comparative evaluation of the antioxidant activity and bioactive components of the ME and EO of C. oltensis. The findings clearly demonstrate significant differences between the two extracts, which can be attributed to their chemical composition, extraction efficiency, and solubility of bioactive compounds.

The DPPH assay is widely used to assess the hydrogen‐donating ability of compounds, which directly reflects their capacity to neutralize free radicals. Although the EO exhibited a higher DPPH value (83.8 mg AAE/100 g), the ME displayed a much lower IC₅₀ value (172 mg/mL) compared to the EO (674 mg/mL), suggesting that the ME is more efficient at lower concentrations (Table 3). This result is consistent with the notion that lower IC₅₀ values correspond to stronger antioxidant activity (Prior et al. 2005). Ascorbic acid, used as a positive control, exhibited an even lower IC₅₀ value (92.6 µg/mL), confirming its exceptional radical scavenging efficiency.

The ABTS assay, which measures the ability of antioxidants to quench the ABTS radical cation, also highlighted significant differences between the two extracts. The ME (975 mg TRE/100 g) exhibited a nearly fivefold stronger scavenging activity compared to the EO (205 mg TRE/100 g) (Table 3). These findings are in line with previous reports indicating that extracts rich in polar phenolic compounds exhibit superior ABTS radical scavenging activity (Re et al. 1999). Moreover, the FRAP assay demonstrated the superior electron-donating ability of the ME (74.1 mg FeSO₄/100 g) relative to the EO (17.2 mg FeSO₄/100 g), supporting earlier findings that high ferric ion reducing power correlates with phenolic content (Benzie and Strain 1996).

The antioxidant assays strongly correlate with the bioactive composition of the extracts. The ME was found to contain significantly higher levels of TPC (281 mg GAE/100 g) compared to the EO (49.9 mg GAE/100 g). Phenolic compounds are widely acknowledged as major contributors to antioxidant activity because of their ability to donate hydrogen atoms and electrons to stabilize free radicals (Huang et al. 2006). Similarly, the TFC of the ME (33.4 mg CAE/100 g) exceeded that of the EO (20.7 mg CAE/100 g). Flavonoids not only act as radical scavengers but also possess metal chelation properties, which reduce oxidative stress by limiting the availability of transition metals that catalyze free radical formation (Middleton et al. 2000).

Interestingly, the TAC values of both extracts were relatively close (364 mg AAE/100 g for ME and 353 mg AAE/100 g for essential oil). This suggests that while phenolics and flavonoids contribute substantially to antioxidant activity, other non-phenolic compounds present in the EO may also play a role in its antioxidant potential.

Similar observations have been reported in other aromatic plants, where terpenoids and other volatile constituents contribute to moderate antioxidant activity despite lower phenolic content (Bakkali et al. 2008).

The superior performance of the ME can largely be explained by solvent polarity. Methanol is a highly effective solvent for extracting polar compounds such as phenolics and flavonoids, which are largely responsible for antioxidant activity (Dai and Mumper 2010). In contrast, essential oils obtained through hydrodistillation or steam distillation are primarily composed of volatile terpenoids, which may not exhibit as strong antioxidant effects as phenolics. Therefore, the differences in activity between the two extracts are directly linked to their chemical profiles.

The antioxidant values observed in the ME of C. oltensis are comparable to those reported for other members of the Centaurea genus, where methanol extracts have consistently shown higher phenolic content and stronger radical scavenging activity compared to essential oils (Kedare and Singh 2011). This consistency suggests that methanolic extracts of Centaurea species can serve as promising natural sources of antioxidants for food and pharmaceutical industries. Essential oils, while less effective in radical scavenging assays, may provide complementary benefits through antimicrobial and anti-inflammatory properties, as previously demonstrated (Bakkali et al. 2008).

Table 3. Antioxidant Activity and Bioactive Component Profile of Centaurea oltensis ME and EO

Different letters within the same column in the table indicate significant statistical differences in pairwise comparisons (p<0.05). n:3. Duncan’s test was applied. AAE: Ascorbic Acid Equivalent, TRE: Trolox Equivalent, GAE: Gallic Acid Equivalent, CAE: Catechin Equivalent

Overall, the results indicate that the methanol extract of C. oltensis has strong potential as a natural antioxidant source due to its high phenolic and flavonoid content. Such properties make it suitable for incorporation into functional foods, nutraceuticals, and natural preservatives in the food industry. The essential oil, despite its lower antioxidant efficiency, may still be valuable in pharmaceutical or cosmetic formulations where antimicrobial or anti-inflammatory effects are desired. Future studies could focus on optimizing extraction conditions, identifying specific active compounds, and exploring the synergistic effects of combining methanol extracts with essential oils to enhance bioactivity.

Antimicrobial Activities

The 30% ME and EO of C. oltensis were tested against gram-positive bacteria (Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis), gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa), and the yeast Candida albicans, with chloramphenicol and nystatin used as reference standards (Table 4).

The EO showed strong antimicrobial activity against gram-positive strains. For Staphylococcus aureus, the inhibition zone was 14.4 mm, with an MIC of 0.02 mg/mL and MBC of 0.05 mg/mL, representing the lowest MIC among all tested microorganisms. In comparison, chloramphenicol exhibited superior potency (DDT = 19.0 mm, MIC = 0.001 mg/mL, MBC = 0.005 mg/mL). Similarly, Enterococcus faecalis was sensitive to the EO (DDT = 14.2 mm, MIC = 0.07 mg/mL, MBC = 0.14 mg/mL), whereas Bacillus cereus showed inhibition of 15.0 mm with MIC 0.14 mg/mL and MBC 0.28 mg/mL. These results clearly indicate that the EO is more active against gram-positives, consistent with earlier findings for Centaurea sessilis and C. armena, where essential oils exhibited higher efficacy against gram-positive bacteria (Yayli et al. 2005). The activity is generally attributed to lipophilic sesquiterpenes such as β-caryophyllene, spathulenol, and caryophyllene oxide, which destabilize bacterial membranes through synergistic mechanisms (Carev et al. 2023).

Against gram-negatives, EO activity was comparatively weaker. Escherichia coli was inhibited at 13.3 mm (MIC = 0.14 mg/mL, MBC = 0.28 mg/mL), while Klebsiella pneumoniae showed an inhibition zone of 13.2 mm (MIC = 0.48 mg/mL, MBC = 0.56 mg/mL).

Pseudomonas aeruginosa was the least sensitive (DDT = 12.2 mm, MIC = 0.28 mg/mL, MBC = 0.56 mg/mL). These findings are in agreement with reports that gram-negative bacteria are less susceptible to plant essential oils due to their outer membrane structures (Yayli et al. 2005). Nonetheless, the ability of lipophilic EO components to disrupt membrane integrity provides moderate inhibitory effects. A noteworthy observation is that the methanol extract of K. pneumoniae presented an unusual profile, with MIC 3.20 mg/mL but a lower MBC (0.41 mg/mL), suggesting either bactericidal effects at reduced concentrations or possible experimental variability that requires further study.

The EO exhibited moderate antifungal activity against C. albicans, with DDT 13.4 mm, MIC 0.57 mg/mL, and MBC 1.13 mg/mL. By contrast, nystatin produced markedly stronger inhibition (DDT = 27.2 mm, MIC 0.008 mg/mL, MBC 0.016 mg/mL). The antifungal activity of the EO is likely linked to terpenoids that interfere with fungal cell wall integrity and ergosterol biosynthesis (Serra et al. 2018). Comparable results have been reported for other Centaurea species: the methanol extract of C. baseri inhibited Candida utilis (MIC = 60 µg/mL) (Kose et al. 2016); the EO of C. pulcherrima showed activity against C. albicans (Kahriman et al. 2012); and C. solstitialis EO displayed potent antifungal effects attributed to its high germacrene D content (Carev et al. 2016).

In contrast, the ME exhibited very weak antimicrobial activity, with inhibition zones ranging from 6.80 to 7.73 mm, MIC values of 0.20 to 3.25 mg/mL, and MBC values of 0.41 to 6.50 mg/mL. The limited activity can be explained by the extract’s chemical profile, which is dominated by polar phenolic acids and flavonoids. These compounds possess antioxidant capacity and only modest antimicrobial properties compared to the more membrane-active, lipophilic sesquiterpenes in the EO (Albayrak et al. 2017; Sharonova et al. 2021).

Taken together, the EO of C. oltensis demonstrated remarkable antimicrobial potential, particularly against gram-positive bacteria (S. aureus, E. faecalis, B. cereus) and moderate antifungal activity against C. albicans. Activity against gram-negatives was limited but meaningful, in agreement with earlier studies on C. pumilio EO, which showed inhibitory effects even against multidrug-resistant strains (Naeim et al. 2020). The observed activities across Centaurea species are thought to arise from synergistic interactions among multiple terpenoid constituents (Carev et al. 2023).

These findings support the potential of C. oltensis EO as a complementary or alternative antimicrobial agent in contexts of rising antibiotic resistance. However, further in vivo research and detailed phytochemical isolation are necessary to identify the key active components and evaluate their clinical applicability.

Table 4. Antimicrobial Activities of Centaurea oltensis ME and EO

GC-MS/FID Conditions for Essential Oil Analysis

The EO yield calculated as a result of the hydrodistillation process carried out with C. oltensis (100 g dried aerial parts) was determined as 0.17±0.03% w/w. Djeddi et al. (2011) reported that the yields of essential oils from Centaurea pullata, C. grisebachii subsp. grisebachii and C. affinis were approximately 0.03%, 0.01% and 0.02% (v/w) on a dry weight basis, respectively. By hydrodistillation with clevenger type apparatus, EO yields of 0.25% and 0.18% (v/w) on dry weight basis were obtained from whole Centaurea sessilis and C. armena plants, respectively (Yayli et al. 2005). When the EO yield obtained in the study is compared, it can be said that it is more or similar to some Centaurea species.

The structures of 128 natural compounds identified in the analyzed EO were determined by comparing their retention times and mass spectra with those of compounds in the NIST and Wiley libraries using a GC-MS/FID instrument, as well as with data reported in the literature. The structures of two compounds were not determined. 99.2% of the compounds identified in the EO analysis were identified. The GC-MS/FID analysis results of C. oltensis EO are shown in Table 5.

The main components of the EO were determined to be 15.1% decanoic acid, 7.93% α-pinene, and 5.40% sabinyl acetate. Some terpene and terpenoid compounds, whose structures are identified and listed in Table 4, are α-pinene, p-cymene, dihydroedulane-I, α-copaene, and limonene; some aldehyde compounds are duraldehyde, nonanal, and hexanal; some alcohol compounds are nonanol, dihydromyrcenol, and 1-decanol; and some ester compounds are sabinyl acetate and 4-tert-butylcyclohexyl acetate.

A total of 53 compounds were identified, representing 93.6% of the EO of C. pullata. Oxygenated monoterpenes (45.2%) were the dominant components, among which caryophyllene oxide (38.5%) was the main compound. Other major components were hydrocarbons (36.5%), aldehydes (16.3%), followed by alkanes and alkenes (16.1%). Twenty-six different substances were identified, representing 82.4% of C. grisebachii subsp. grisebachii; oxygenated sesquiterpenes were the dominant components with 47.3%, among which spathulenol (12.2%) and β-eudesmol (10.1%) were the main components, followed by hydrocarbons (28.6%), and 6,10,14-trimethyl pentadecan-2-one (12.9%) was found as the main compound. The EO of C. affinis evidenced 62 compounds, constituting 92.9% of the total extract, with oxygenated sesquiterpenes (15.5%) being detected as the dominant components (Djeddi et al. 2011).

Table 5. GC-MS/FID Analysis Results of The EO Obtained from Centaurea oltensis

Yayli et al. (2005) determined that the major components of the EO of C. sessilis were β-eudesmol (12.4%), caryophyllene oxide (10.0%), phytol (6.4%), spathulenol (4.9%) and 6,10,14-trimethyl-2-pentadecanone (3.1%). The major components of the EO of C. armena were β-eudesmol (19.3%), calarin (10.3%), 6,10,14-trimethyl-2-pentadecanone (5.7%) and β-caryophyllene (5.4%). The chemical composition of the EO of dried aerial parts of C. iberica, C. solstitialis subsp solstitialis, and C. virgata was analyzed using the HS-SPME/GC-MS extraction technique and the GC-MS system. Thirty-nine, forty-two, and forty-two components were identified, representing 90.8%, 91.6%, and 92.5% of the oil, respectively. The main compounds of C. iberica, C. solstitialis subsp. solstitialis and C. virgata were determined as germacrene D (20.3%), caryophyllene oxide (10.7%), β-caryophyllene (10.5%), β-eudesmol (15.5%), bicyclogermacrene (14.2%), spathulenol (11.3%) and germacrene D (21.4%), β-caryophyllene (16.5%), caryophyllene oxide (9.5%), respectively (Kilic 2013). The predominant chemical compounds in the EO obtained by hydrodistillation from air-dried aerial parts of C. scabiosa using a Clavenger apparatus were heptacosane (19.6%), caryophyllene oxide (10.90%), alloaromadendrene epoxide (10.27%), β-cyperone (8.16%), and α-bisabolol (4.99%) (Carev et al. 2022).

The chemical classification, number of compounds, main components, and percentage amounts of the compounds identified in C. oltensis EO are shown in Table 6.

When the chemical classification results of the compounds identified in the EO obtained from C. oltensis are examined in Table 6, it can be seen that the 128 compounds identified were divided into 12 groups.

When the chemical component classes identified in the EO were evaluated, it was apparent that the most abundant compound class in terms of percentage was monoterpenes at 21.3%, with α-pinene as the main component. Other compound classes included monoterpenoids at 16.5%, with dihydroedulane-I as the main component; oil acids at 15.2%, with decanoic acid as the main component; and aldehydes at 11.3%, with duraldehyde as the main component. The most abundant compound classes in these classes were monoterpenoids (31), aldehydes (18), monoterpenes (14), esters (12), and sesquiterpenoids (11). There was a total of 64 compounds in the essential oil samples and 51.1% were terpene or terpenoid class compounds (Table 6).

Sesquiterpenoids are the main components of C. sessilis and C. armena essential oils at 35.1% and 34.8%, respectively. A total of 28 components and 59.4% and 15 components and 70.7% of terpenes or terpenoid class compounds were found in the EO samples, respectively (Yayli et al. 2005). Carev et al. (2022) grouped the chemical compounds of the EO extracted from C. scabiosa as terpenes (43.7%), including deoxygenated sesquiterpenes (1.23%), sesquiterpenoids (41.1%), and diterpenes (1.41%); and non-terpene compounds, including hydrocarbons (31.1%), aldehydes (3.67%), acids (7.00%), esters (4.03%), and other compounds (0.68%).

Table 6. Chemical Classification, Number of Compounds, Main Components, and Percentage Amounts of the Compounds Identified in Centaurea oltensis EO

Comparison of the volatile components identified in studies of essential oils from C. oltensis and other Centaurea species reveal similar compounds. Sesquiterpenoids are prominent among the chemical classes of volatile components in these studies, and monoterpenes predominate in this study. The differences in the chemical classification and volatile compound amounts identified can be attributed to many factors, including species differences, climatic conditions, different growing locations, natural diversity, collection times, storage conditions, and analysis parameters (Öz et al. 2023).

Further investigations involving compound isolation, mechanistic studies, and in vivo validation are recommended to elucidate the therapeutic potential and industrial applicability of this endemic Turkish species.

Compared with previous studies on Centaurea species, the present findings indicate that C. oltensis may have potential for multiple biological applications rather than a single functional property. Previous studies have demonstrated the antioxidant, antimicrobial, cytotoxic, and enzyme inhibitory potential of different Centaurea species, whereas the present study integrates these functional evaluations with detailed volatile and phenolic profiling. In particular, the strong cholinesterase inhibitory activity of the EO suggests potential relevance to further research on neuroprotective applications, while the CA-II and α-glucosidase inhibitory activities and antioxidant capacity of the ME indicate additional potential for pharmacological and nutraceutical investigations. Moreover, the antimicrobial activity of the EO, particularly against Staphylococcus aureus, supports its further evaluation as a source of natural antimicrobial agents. However, these potential applications should be considered preliminary, and further isolation, mechanistic, toxicity, and in vivo studies are required before practical therapeutic or industrial applications can be proposed.

CONCLUSIONS

1. The present study provides the first comprehensive chemical and biological characterization of Centaurea oltensis. Gas chromatography-mass spectroscopy/flame ionization detector (GC–MS/FID) analysis revealed a complex essential oil (EO) composition dominated by decanoic acid, α-pinene, and sabinyl acetate, while liquid chromatography – tandem mass spectrometry (LC–MS/MS) profiling of the methanol extract (ME) demonstrated exceptionally high levels of ferulic and chlorogenic acids. These findings confirm that C. oltensis possesses a distinctive phenolic and terpenoid signature compared with other Centaurea taxa.
2. Biological activity assays indicated that both extracts exhibit notable pharmacological potential. The ME showed remarkable carbonic-anhydrase and α-glucosidase inhibitory effects and superior antioxidant activity, directly associated with its rich phenolic and flavonoid content. Conversely, the EO demonstrated pronounced acetylcholinesterase (AChE) and butylcholinesterase (BChE) inhibition, together with strong antibacterial and moderate antifungal properties, suggesting possible neuroprotective and antimicrobial applications.
3. Overall, C. oltensis represents a valuable natural source of bioactive compounds with multifunctional properties relevant to the development of antioxidant, antimicrobial, and enzyme-inhibitory agents.

ACKNOWLEDGEMENTS

This study is a part of a master thesis entitled “Determination of Antioxidant, Antimicrobial, Anticholinesterase, Antiglaucoma and Antidiabetic Activities of Methanol Extract and Essential Oils of Centaurea Oltensis Sosn Plant” prepared by Berfin AKŞİT under the supervision of Assistant Professor Dr. İmdat AYGÜL in Gümüşhane University.

Conflict of Interest

The authors report no conflicts of interest.

Data Availability Statement

The original contributions presented in this study are included in the article.

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Article submitted: May 8, 2026; Peer review completed: August 8, 2026; Revised version received and accepted: September 8, 2026; Published: September 25, 2026.

DOI: 10.15376/biores.21.4.11085-11114