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Article

Phytochemical Study of the Plant Centaurea bruguieriana (DC.) Hand.-Mazz. subsp. belangeriana (DC.) Bornm. of the Family Asteraceae

by
Kyriakos Michail Dimitriadis
1,
Olga Tsiftsoglou
1,
Dimitra Hadjipavlou-Litina
2,
Mohammad Arfan
3 and
Diamanto Lazari
1,*
1
Laboratory of Pharmacognosy, Faculty of Health Sciences, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
2
Laboratory of Pharmaceutical Chemistry, Faculty of Health Sciences, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
3
International Center for Chemical & Biological Sciences, HEJ Research Institute of Chemistry, University of Karachi, Karachi 75210, Pakistan
*
Author to whom correspondence should be addressed.
Separations 2024, 11(11), 319; https://doi.org/10.3390/separations11110319
Submission received: 30 September 2024 / Revised: 28 October 2024 / Accepted: 30 October 2024 / Published: 4 November 2024
(This article belongs to the Special Issue The 10th Anniversary of Separations: Advances in Separation and Analysis of Natural Products/Medicines)
Browse Figure
Versions Notes

Abstract

The aim of this study is to isolate and identify the secondary metabolites of the aerial part of the plant Centaurea bruguieriana (DC.) Hand. -Mazz. subsp. belangeriana (DC.) Bornm. (Centaurea phyllocephala) (Asteraceae), and to study the biological activities of the extracts and isolated compounds with in vitro tests. With the use of chromatography and spectroscopy we identified three elemanolides: 8α-O-(3,4-dihydroxy-2-methylenebutanoyloxy) dehydromelitensine (1), 8α-O-(3-hydroxy-4-acetoxy-2-methylene-butanoyloxy) dehydromelitensine (2) and methyl 6α,8α,15-trihydroxyelema-1,3,11(13)-trien-12-oate (3); two germacranolides: cnicin (4) and 4′-O-acetylcnicin (5); one eudesmanolide: malacitanolide (6); five flavonoids: cirsilineol (7), eupatorine (8), 5-hydroxy, 6,7,3′,4′-tetramethoxy-flavone (9), 3,4′,5,7-tetrahydroxy-6-methoxyflavone 3-O-β-D-glucopyranoside (10) and astragalin (11); and also p-OH-benzoic acid (12) and 3-hydroxy-2-methyl-butyrolactone (13). All the isolated compounds were evaluated in silico with the use of molinspiration, while the crude extract, the organic phase B and compounds 2, 4, 5 and 6 were tested as antioxidants and anti-inflammatories for the inhibition of lipid hyperoxide and the inhibition of lipoxygenase.

1. Introduction

The genus Centaurea is one of the largest genera of the family Asteraceae, encompassing approximately 500–600 species [1], which include annual, biennial or perennial herbs or shrubs [2]. Species of the genus Centaurea have been reported to possess several pharmacological activities, including antidiabetic, antioxidant, anti-inflammatory, antibacterial, antipyretic, antidandruff and cytotoxic, diuretic, hypotensive, cholagogue and choleretic effects [2,3]. Traditionally, these plants have been used for their antidiarrheal, antirheumatic, antipyretic, antibacterial, anti-inflammatory, cytotoxic, choleretic and cholagogue effects [4]. Regarding the phytochemistry of the genus Centaurea, sesquiterpene lactones, flavonoids, lignans, alkaloids and triterpenes have been isolated and identified [5,6,7].
Centaurea bruguieriana (synonym C. phyllocephala) is an erect annual herb, reaching a height of 15–35 cm, with white stems that bear both glandular and non-glandular hairs. The flowers are pinkish and appear, usually, in individual inflorescences (heads). The heads are relatively small compared to the large, straight, white thorns, which are 1.3–2.0 cm long and have glandular hairs. This species grows in moist plains or basins of the Caucasus region [8,9]. According to the Euro+Med plantbase database, C. bruguieriana is further divided into two subspecies: Centaurea bruguieriana (DC.) Hand. -Mazz. subsp. belangeriana (DC.) Bornm. found in central Asia (Pakistan, Iraq, Iran, India, etc.) and Centaurea bruguieriana (DC.) Hand.-Mazz. subsp. bruguieriana, found in central-East Asia (Iraq, Iran, Turkey, Saudi Arabia, etc.) [10].
In southern Iran, C. bruguieriana is commonly known as “Baad-Avard” and is traditionally used for its antidiabetic properties and to treat peptic ulcers [11]. Preparations of the plant are also used as a bitter tonic, a hepatoprotective agent to aid digestion, enhance resistance to infections, eye diseases and skin rashes [12,13]. Extracts and isolated compounds from Centaurea bruguieriana subsp. belangeriana have been investigated for their inhibitory effects on aldose reductase. Among the extracts studied, the polar extract exhibited the most potent inhibitory activity, while among the isolated compounds, eupatorine and cirsilineol demonstrated the strongest inhibitory effects [14]. The chemical composition of Centaurea bruguieriana and its subspecies C. bruguieriana subsp. belangeriana has been the subject of limited study. Noman et al. [15] studied the composition of the chloroform and ethyl acetate fractions of C. bruguieriana using gas chromatography (GC-MS) and high-pressure liquid chromatography (HPLC). The primary compounds identified in the chloroform fraction were methyl 8-oxooctanoate, 1,10-di-epi-cubenol, 7-oxabicyclo [2.2.1]heptane and 3-methyl-5-(2,6,6-trimethylcyclohex-1-enyl)pent-1-yn-3-ol. In the ethyl acetate fraction, chlorogenic acid, luteolin-7-O-glucoside, kaempferol and isorhamnetin were identified. Additionally, Rustaiyan et al. [16] isolated cnicin and 5-hydroxy-3′,4′6,7-tetramethoxy flavone from the aerial parts of C. bruguieriana, while Lazari et al. [14] isolated 8α-(3,4-dihydroxy-2-methylene-butanoyloxy), 8α-(3-hydroxy-4-acetoxy-2-methylene-butanoyloxy), malacitanolide, p-OH-benzoic acid, eupatorin, cirsilineol, 5-hydroxy,6,7,3′,4′-tetramethoxyflavone and β-sitosterol from the subspecies belangeriana. More recently, Mirzahosseini et al. [17] reported the isolation of cirsilineol, cirsimaritin and eupatilin from C. bruguieriana subsp. belangeriana.
The present study aims to isolate and identify secondary metabolites, specifically sesquiterpene lactones, from C. bruguieriana subsp. belangeriana, and to evaluate the biological activities of the plant extracts and isolated compounds using both in vitro and in silico methods. Previous studies suggest that extracts of C. bruguieriana are used for various medicinal purposes. This study seeks to determine whether sesquiterpene lactones, known for their anti-inflammatory properties, are responsible for the plant’s medicinal properties. Additionally, the molecular properties and the potential biological activities of all the isolated compounds will be analyzed to see their connection to the pharmacological properties of the plant.

2. Materials and Methods

2.1. Plant Material

The aerial parts of C. bruguieriana subsp. belangeriana were collected in May 2004 from the suburbs of Peshawar (Pakistan) and were authenticated by Dr. Shahid Farooq. A voucher specimen was deposited in the Herbarium at the PCSIR Laboratories, Peshawar, under the code CA-001-04.

2.2. Chromatographic Technics

Thin layer chromatography (TLC) was performed using silica gel (Kieselgel F254, Merck, Art. 5554, Merck GLOBAL, Athens, Greece) and cellulose (Merck, Art. 5552) plates. For the preparative thin layer chromatography (pTLC), silica gel (Kieselgel F254, Merck, Art. 5715, Merck GLOBAL, Athens, Greece) plates were used. Detection was carried out under ultraviolet (UV) light at wavelengths of 254 and 366 nm. The cellulose plates were treated with Naturstoffreagenz A, following the method of Neu [18] and the silica gel plates were treated with a vanillin–H2SO4 spray reagent.
Vacuum liquid chromatography (VLC) was carried on 10.0 × 7.0 cm silica gel (Merck 60H, Art. 7736), column chromatography (CC) was carried on silica gel 60 (Merck Art. 9385) with various solvent mixtures as well as Sephadex LH-20 with methanol as a mobile phase.
High-performance liquid chromatography (HPLC) was conducted using a LabAlliance Series III system (LabAlliance, Scientific Systems, Inc., 349 N Science Park Rd., State College, PA, USA) equipped with Clarity software (version 9.0.) and a reversed-phase semi-preparative column (Waters Spherisorb 10 μm ODS2 C18, 250 mm × 10 mm) (Milford, MA, USA). The separation was monitored using a Shodex refractive index (RI) detector (Kawasaki, Japan). The mobile phase employed consisted of various mixtures of methanol (MeOH) and water (H2O).

2.3. Spectroscopy

UV spectroscopy: Hitachi U-2000, 1212301-06 was used for the TLC analysis and a Shimadzu PharmaSpec UV-1700 spectrophotometer (Shimadzu, Kyoto, Japan) was used for the in vitro tests.
Nuclear magnetic resonance (NMR) spectroscopy was conducted using an Agilent DD2 500 spectrometer (Palo Alto, CA, USA) and a Bruker 200 MHz (Billerica, MA, USA). The 1H NMR (500 MHz) and 13C spectra (125 MHz) were recorded using deuterium methanol (CD3OD) and deuterium chloroform (CDCl3). Chemical shifts are reported in δ (ppm) values relative to tetramethylsilane (TMS). Two-dimensional NMR (COSY, HSQCAD and HMBCAD) were performed using standard Agilent microprograms (VnmrJ 2.2C).

2.4. In Silico Study

Molecular modeling and property prediction were conducted using Molispiration software version 2011.06 (accessed on 25 July 2024) (available at https://www.molinspiration.com/) [19]. The software was used to calculate various molecular properties such as molecular weight, volume, partition coefficient (logPP), topological polar surface area (TPSA), hydrogen bond (H-bond) acceptors and donors, violations of Lipinski’s rule of five and the number of rotatable bonds. The permeability through the blood–brain barrier was calculated using the Rishton equation: logBB = 0.155logP − 0.01TPSA + 0.164 [20]. The software also predicts if the tested molecule could act as a ligand for a G-protein-coupled receptor, a modulator of the ion channel, a kinase inhibitor, a ligand for nuclear receptor, a protease inhibitor or an enzyme inhibitor.

2.5. Inhibition of Linoleic Acid Lipid Peroxidation

The inhibition of linoleic acid lipid peroxidation was evaluated following the methodology described by Hodaj-Çeliku et al. [21] The test samples were prepared by dissolving them in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/mL for the extracts and 10 mM for the isolated compounds. The peroxidation process was initiated using 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH, Sigma-Aldrich, Darmstadt, Germany). The ability of the tested compounds to prevent lipid peroxidation in aqueous solution and the subsequent formation of conjugated diene hydroperoxide was recorded at 234 nm using a Shimadzu PharmaSpec UV-1700 spectrophotometer (Shimadzu, Kyoto, Japan). Trolox (Sigma-Aldrich, Darmstadt, Germany) served as the positive control for antioxidant activity.

2.6. Inhibition of Soybean Lipoxygenase (LOX)

The inhibition of soybean lipoxygenase S-LOX was conducted using the method described by Peperidou et al. [22]. The test samples were prepared by dissolving them in DMSO at a concentration of 5 mg/mL for the extracts and 10 mM for the isolated compounds. Then, they were incubated with sodium linoleate (0.1 mM) along with 0.2 mL of soybean lipoxygenase solution (Sigma-Aldrich, Darmstadt, Germany), which was prepared at 1/9 × 10−9 w/v in saline, in a Tris buffer of pH 9. The conversion of sodium linoleate to 13-hydroperoxylinoleic acid was recorded at 234 nm using a Shimadzu PharmaSpec UV-1700 spectrophotometer (Shimadzu, Kyoto, Japan). Nordihydroguaiaretic acid (NDGA) (Sigma-Aldrich, Darmstadt, Germany) was used as the reference inhibitor.

2.7. Statistical Analysis

For the assessment of the significant differences (p < 0.05) in the in vitro studies, Duncan analysis was employed using IBMSPSS Statistics for Windows v. 29.0.01 (IBMCorp., Armonk, NY, USA).

3. Results

3.1. Extraction and Isolation of Compounds

The extract was prepared according to the standard procedure for the isolation of sesquiterpene lactones [23]. The air-dried aerial parts of C. bruguieriana subsp. belangeriana (DC.) Bornm. (1.5 kg) were finely ground and extracted at room temperature with a mixture of cyclohexane—diethyl ether—methanol 1:1:1 which yielded 80.46 g of extract (Crude Extract). The extract was re-dissolved in the same mixtures of solvents and washed with brine. The organic phase was discarded, and the aqueous fraction was subsequently extracted with ethyl acetate yielding 25.81 g of extract (Organic Phase B). Organic Phase B was fractioned via VLC on silica gel with mixtures of solvents of increasing polarity [hexane (He), ethyl acetate (EA), acetone (Ac) and methanol (MeOH)], yielding 11 fractions of 300 mL each: CEP-A (He 100%, 126.1 mg), CEP-B (He:EA 75:25 14.6 mg), CEP-C (He:EA 50:50, 658.7 mg), CEP-D (He:EA 25:75, 865.7 mg), CEP-E (EA 100%, 3.2874 g), CEP-F (EA:Ac 90:10, 4.6052 g), CEP-G (EA:Ac 75:25, 1.4741 g), CEP-H (Ac 100%, 1.3307 g), CEP-I (MeOH 100%, 3.1299 g), CEP-K (MeOH 100%, 3.9256 g) and CEP-L (MeOH 100%, 61.0 mg).
CEP-D (865.7 mg) was subjected to CC on silica gel with a mixture of dichloromethane (DM):MeOH of increasing polarity resulting in 14 fractions (CEP-DA to DO). CEP-DC (202.8 mg) was fractionated on CC silica gel with a mixture of DM:MeOH of increasing polarity and yielded 11 fractions (CEP-DCA to DCL). CEP-DCE (59.2 mg) and CEP-DCF (27.0 mg) were combined and subjected to CC Sephadex LH-20 using MeOH of increasing polarity and yielded 7 fractions (CEP-DCE′A to DCE′G). Among these, CEP-DCE′E (2.2 mg) was identified as compound 9.
CEP-E (3.287 g) was subjected to VLC on silica gel with mixtures of solvents (He, DM and MeOH) of increasing polarity yielding 13 fractions (CEP-EA to EN). CEP-EC (166.2 mg) eluted with DM:MeOH 99:1, was fractionated on CC Sephadex LH-20 using MeOH resulting in 11 fractions (CEP-ECA to ECL). CEP-ECE (14.5 mg) was subjected to pTLC on silica gel with elution solvent DM:MeOH 98:2 and yielded four bands (CEP-ECEα to ECEδ). CEP-ECEα (5.5 mg) was identified as compound 9, CEP-ECEβ (4.0 mg) was identified as compound 8 and CEP-ECEγ (6.8 mg) was identified as compound 7. CEP-ECF (9.4 mg) was further subjected to pTLC on silica gel with elution solvent DM:MeOH 98:2 and yielded three bands (CEP-ECFα to ECF-γ). Band CEP-ECFα (3.2 mg) was identified as compound 9. CEP-ECG (32.5 mg) was subjected to pTLC on silica gel with elution solvent DM:MeOH 98:2 and yielded three bands (CEP-ECGα to ECGγ). Band CEP-ECGα (14.9 mg) was identified as compound 9. Fractions CEP-ECH (3.0 mg) and CEP-ECI (11.8 mg) were combined (CEP-ECH′) and subsequently subjected to pTLC on silica gel with elution solvent DM:MeOH 98:2 yielding three bands (CEP-ECH′1 to ECH′3). Band CEP-ECH′1 (3.2 mg) was identified as compound 9. CEP-ECK was subjected to pTLC on silica gel with elution solvent DM:MeOH 98:2 yielding two bands (CEP-ECK1 to ECK2). Band CEP-ECK1 (34.3 mg) was identified as compound 9. CEP-EF (712.7 mg) eluted with DM:MeOH 96:4 was subjected to CC on silica gel using a mixture of DM:MeOH of increasing polarity resulting in 15 fractions (CEP-EFA to EFP). CEP-EFE (31.6 mg) was subjected to HPLC with elution solvent MeOH:H2O 3:2 and flow rate 1 mL/min yielding 18 peaks (CEP-H2-1 to CEP-H2-18). CEP-H2-3 (t = 14.0 min, 8.6 mg) was identified as compound 13. CEP-EFG (50.7 mg) was subjected to HPLC with elution solvent MeOH:H2O 3:2, with a flow rate of 1 mL/min resulting in 15 peaks (CEP-H3-1 to CEP-H3-15). CEP-H3-11 (t = 32.5 min, 6 mg) was identified as compound 5. CEP-EG (1.3 mg) eluted with DM:MeOH 95:5 was subjected to CC silica gel with a mixture of He:DM:MeOH of increasing polarity yielding 17 fractions (CEP-EGA to EFR). CEP-EGE (14.0 mg) was identified as compound 2. CEP-EM (99.4 mg) was fractionated on CC Sephadex LH-20 using MeOH yielding 7 fractions (CEP-EMA to EMG). CEP-EMD (31.9 mg) was identified as compound 12.
CEP-F (4.6052 g) was subjected to VLC on silica gel using mixtures of He:DM:MeOH of increasing polarity and yielded 14 fractions (CEP-FA to FO). CEP-FH (1.5652 g) eluted with DM:MeOH 93:7 was fractionated on CC silica gel with DM:MeOH resulting in 20 fractions (CEP-FHA to FHU). CEP-FHI (304.7 mg), CEP-FHK (260.4 mg), CEP-FHL (162.6 mg) and CEP-FHM (127.0 mg) were all identified as compound 4. CEP-FHN (83.7 mg) was subjected to HPLC, with elution solvent MeOH:H2O 1:1 and flow rate 1mL/min yielding eight peaks (CEP-H7-1 to CEP-H7-8). CEP-H7-6 (t = 29.3 min, 1.8 mg) was identified as compound 3. CEP-FI (842,7 mg) eluted with DM:MeOH 92:8 was fractionated on CC silica gel with a mixture of DM:MeOH of increasing polarity yielding 25 fractions (CEP-FIA to FIZ). CEP-FIL (9.6 mg), CEP-FIN (114.4 mg), CEP-FIP (141.1 mg), CEP-FIQ (47.7 mg), CEP-FIR (25.5 mg), CEP-FIS (35.7 mg) and CEP-FIT (27.0 mg), as well as the precipitate (CEP-FI-PR, 44.7 mg) from the dilution of the fraction FI, were identified as compound 4.
CEP-G (1.4741 g) was fractionated on CC silica gel with a mixture of He:DM:MeOH of increasing polarity resulting in 14 fractions (CEP-GA to GO). CEP-GG (681.6 mg) was identified as compound 6 and CEP-GH (42.0 mg) as compound 1.
CEP-I (3.1299 g) was subjected to CC silica gel with a mixture of DM:MeOH:H2O of increasing polarity and yielded 23 fractions (CEP-IA to IX). CEP-IM (188.8 mg) was subjected to CC Sephadex LH-20 with MeOH giving 10 fractions (CEP-IMA to IMK). CEP-IMI (5.6 mg) was identified as compound 10 and CEP-IMK (20.0 mg) as compound 11.

3.2. Identification of the Isolated Compounds

All the isolated compounds (Figure 1) were identified via 1H NMR and in some cases 13C NMR and two-dimensional spectra (gDQCOSY, HSQCAD, HMBCAD). For some flavonoids, UV spectra under different conditions were used. Specifically, the UV spectra were recorded in Methanol, Sodium methoxide (MeONa), Aluminum chloride (AlCl3), Aluminum chloride with hydrochloric acid (AlCl3/HCl), Sodium acetate (NaOAc) and Sodium acetate with Boric acid (NaOAc/H3BO3). The data were compared to the literature. The NMR spectra can be found in the Supplementary Material.
Compound 1 (42.0 mg) was isolated as an oily compound and was identified as 8α-(3′,4′-dihydroxy-2′-methylene-butanoyloxy)-dehydro-melitensin or isocnicin. 1H-NMR (500 MHz, CD3OD) δ (ppm) 6.39 (1H, s, H-5′a), 6.08 (1H, s, H-5′b), 6.07 (1H, d, J = 2.8 Hz, H-13a), 5.87 (1H, dd, J = 10.9, 17.5 Hz, H-1), 5.62 (1H, d, J = 3.0 Hz, H-13b), 5.44 (1H, s, H-3a), 5.36 (1H, dt, J = 4.1, 10.8 Hz, H-8), 5.07-5.02 (2H, m, H-2), 5.03 (1H, s, H-3b), 4.61 (1H, t, J = 5.0 Hz, H-3′), 4.51 (1H, t, J = 11.5 Hz, H-6), 4.08 (1H, d, J = 14.9 Hz, H-15a), 3.98 (1H, d, J = 14.9 Hz, H-15b), 3.72 (1H, dd, J = 3.8, 11.2 Hz, H-4′a), 3.53 (1H, dd, J = −6.5, 11.2 Hz, H-4′b), 3.10 (1H, tt, J = 3.1, 10.9 Hz, H-7), 2.54 (1H, d, J = 11.8 Hz, H-5), 1.98 (1H, dd, J = 4.2, 12.9 Hz, H-9a), 1.77 (1H, t, J = 11.5 Hz, H-9b), 1.22 (3H, s, H-14) [24].
Compound 2 (14.0 mg) was isolated as white powder and was identified as 8α-(4′-acetoxy-3′hydroxy-2′-methylene-butanoyloxy)-dehydro-melitensin. 1H-NMR (500 MHz, CD3OD) δ (ppm) 6.38 (1H, s, H-5′a), 6.07 (1H, s, H-5′b), 6.05 (1H, d, J = 3.1 Hz, H-13a), 5.85 (1H, dd, J = 10.8, 17.6 Hz, H-1), 5.59 (1H, d, J = 2.9 Hz, H-13b), 5.41 (1H, s, H-3a), 5.34 (1H, dt, J = 4.2, 10.8 Hz, H-8), 5.02 (2H, m, H-2), 5.00 (1H, s, H-3b), 4.74 (1H, t, J = 4.8 Hz, H-3′), 4.48 (1H, t, J = 11.5 Hz, H-6), 4.19 (2H, m, H-4′), 4.05 (1H, d, J = 14.9 Hz, H-15a), 3.95 (1H, d, J = 14.9 Hz, H-15b), 3.09 (1H, tt, J = 3, 10.9 Hz, H-7), 2.52 (1H, d, J = 11.8 Hz, H-5), 2.03 (3H, s, 4′-OAc), 1.97 (1H, dd. J = 4.1, 12.9 Hz, H9a), 1.75 (1H, t, J = 11.9 Hz, H-9b), 1.19 (3H, s, H-14) [25].
Compound 3 (1.8 mg) was isolated as an oily compound and was identified as methyl 6α,8α,15-trihydroxyelema-1,3,11(13)-trien-12-oate. 1H-NMR (500 MHz, CD3OD) δ (ppm) 6.33 (1H, d, J = 1.3 Hz, H-13a), 5.76 (1H, dd, J = 11.3, 17.5 Hz, H-1), 5.74 (1H, d, J = 1.3 Hz, H-13b), 5.33 (1H, brs, H-3a), 4.92 (1H, brs, H-3b), 4.92 (1H, d, J = 1.0 Hz, H-2a), 4.91 (1H, d, J = 1.0 Hz, H2-b), 4.13 (1H, t, J = 10.6 Hz, H-6), 4.11 (1H, m, H-8), 3.99 (1H, d, J = 14.7 Hz, H-15a), 3.89 (1H, d, J = 14.7 Hz, H-15b), 3.75 (3H, s, 12-OCH3), 2.31 (1H, t, J = 10.4 Hz, H-7), 1.84 (1H, d, J = 10.8 Hz, H-5), 1.73 (1H, dd, J = 4.3, 12.6 Hz, H-9a), 1.49 (1H, t, J = 12.0 Hz, H-9b), 1.13 (3H, s, H-14) [5].
Compound 4 (845.96 mg) was isolated as white crystals and was identified as cnicin. 1H-NMR (500 MHz, CD3OD) δ (ppm) 6.38 (1H, brs, H-5′a), 6.18 (1H, d, J = 3.5 Hz, H-13a), 6.08 (1H, brs, H-5′b), 5.80 (1H, d, J = 3.1 Hz, H-13b), 5.20 (1H, t, J = 10 Hz, H-6), 5.08 (2H, m, H-8 and H-1), 4.95 (1H, d, J = 10 Hz, H-5), 4.57 (1H, s, 4′OH), 4.51 (1H, dd, J = 3.7, 6.6 Hz, H-3′), 4.26 (1H, d, J = 13.6 Hz, H-15a), 4.01 (1H, d, J = 13.5 Hz, H-15b), 3.71 (1H, dd, J = 3.7, 11.3 Hz, H-4′a), 3.48 (1H, dd, J = 6.5, 11.3 Hz, H-4′b), 3.30 (1H, os, H-7), 2.73-2.47 (3H, m, H-3b, H-9), 2.30 (1H, td, J = 5.2, 12.3 Hz, H-2a), 2.20 (1H, m, H-2b), 2.02 (1H, m, H-3a), 1.53 (3H, s, H-14). 13C-NMR (125 MHz, CD3OD) δ (ppm) 170.1 (C-12), 165.1 (C-1′), 144.1 (C-4), 140.9 (C-2′), 136.0 (C-11), 131.8 (C-10), 129.4 (C-1), 128.3 (C-5), 125.8 (C-5′), 123.8 (C-13), 77.2 (C-6), 73.0 (C-8), 70.5 (C-3′), 65.2 (C-4′), 59.4 (C-15), 59.4 (H-7), 48.2 (C-9), 33.8 (C-3), 25.5 (C-2), 15.6 (C-14) [26].
Compound 5 (11.6 mg) was isolated as white crystals and was identified as 4′-O-Acetyl-cnicin. 1H-NMR (500 MHz, CD3OD) δ (ppm) 6.41 (1H, brs, H-5′a), 6.18 (1H, d, J = 3.5 Hz, H-13a), 6.11 (1H, brs, H-5′b), 5.79 (1H, d, J = 3.1 Hz, H-13b), 5.21 (1H, t, J = 10.0 Hz, H-6), 5.08 (2H, m, H-1 and H-8), 4.95 (1H, d, J = 10.0 Hz, H-5), 4.68 (1H, t, J = 4.8 Hz, H-3′), 4.26 (1H, d, J = 13.6 Hz, H-15a), 4.18 (2H, m, H-4′), 4.01 (1H, d, J = 13.5 Hz, H-15b), 3.30 (1H, m, H-7), 2.64-2.52 (3H, m, H-3b and H-9), 2.31 (1H, td, J = 5.2, 12.3 Hz, H-2a), 2.20 (1H, m, H-2b), 2.03 (3H, s, 4′-OAc), 2.02 (1H, brs, H-3a), 1.54 (3H, s, H-14) [27].
Compound 6 (681.6 mg) was isolated as an oily compound and was identified as malacitanolide. 1H-NMR (500 MHz, CD3OD) δ (ppm) 9.92 (1H, s, H-15), 6.35 (1H, brs, H-5′a), 6.17 (1H, d, J = 3.0 Hz, H-13a), 6.03 (1H, brs, H-5′b), 5.54 (1H, d, J = 2.8 Hz, H-13b), 5.24 (1H, td, J = 4.1, 11.3 Hz, H-8), 4.59 (1H, dd, J = 3.8, 6.9 Hz, H-3′), 4.50 (1H, t, J = 11.8 Hz, H-6), 3.76 (1H, dd, J = 3.7, 11.0 Hz, H-4′a), 3.57 (1H, dd, J = 6.9, 11.1 Hz, H-4′b), 3.37 (1H, dd, J = 4.2, 10.9 Hz, H-1), 2.85 (1H, tt, J = 4.0, 11.2 Hz, H-7), 2.78 (1H, td, J = 1.5, 5.8 Hz, H-4), 2.51 (1H, m, H-3a), 2.46 (1H, dd, J = 4.0, 12.9 Hz, H-9a), 2.01 (1H, dd, J = 5.8, 11.7 Hz, H-5), 1.80-1.40 (3H, m, H-2 and H-3b), 1.30 (1H, m, H-9b), 0.89 (3H, s, H-14) [28].
Compound 7 (6.8 mg) was isolated as a yellow amorphous compound and was identified as cirsilineol. UV spectra MeOH: Band II (248sh, 274 nm), Band I (342 nm), MeONa: Band II (268.5nm), Band I (406 nm), AlCl3: Band II (261.5, 285, 297sh nm), Band I (372.5 nm), AlCl3/HCL: Band II (261.5, 289, 297sh nm), Band I (361 nm), NaOAc: Band II (275, 308sh nm), Band I (405), NaOAc/H3BO3: Band II (279 nm), Band I (346 nm). 1H-NMR (200 MHz, CD3OD) δ (ppm) 7.47 (1H, dd, J = 1.8, 8.4 Hz, H-6′), 7.30 (1H, d, J = 1.8 Hz, H-2′), 7.01 (1H, d, J = 8.4 Hz, H-5′), 6.56 (1H, s, H-8), 6.53 (1H, s, H-3), 3.99 (3H, s, -OCH3), 3.95 (3H, s, -OCH3), 3.90 (3H, s, -OCH3) [29].
Compound 8 (4.0 mg) was isolated as a yellowish amorphous compound and was identified as eupatorin. UV spectra MeOH: Band II (251sh, 273 nm), Band I (341 nm), MeONa: Band II (279, 307sh nm), Band I (390 nm), AlCl3: Band II (262.5, 281sh, 301sh nm), Band I (367 nm), AlCl3/HCL: Band II (260.5, 278.5sh, 297.5sh nm), Band I (362 nm), NaOAc: Band II (254sh, 274 nm), Band I (340), NaOAc/H3BO3: Band II (256sh, 272 nm), Band I (342 nm). 1H-NMR (200 MHz, CD3OD) δ (ppm) 7.49 (1H, dd, J = 2.1, 8.6 Hz, H-6′), 7.30 (1H, d, J = 2.1 Hz, H-2′) 6.95 (1H, d, J = 8.6 Hz, H-5′), 6.58 (1H, s, H-8), 6.56 (1H, s, H-3), 4.02 (3H, s, -OCH3), 3.95 (3H, s, -OCH3), 3.94 (3H, s, -OCH3) [30].
Compound 9 (69.3 mg) was isolated as a yellowish amorphous compound and was identified as 5-hydroxy, 6,7,3′,4′-tetramethoxy-flavone. 1H-NMR (500 MHz, CD3OD) δ (ppm) 7.67 (1H, dd, J = 2.2, 8.6 Hz, H-6′), 7.55 (1H, d, J = 2.1 Hz, H-2′), 7.13 (1H, d, J = 8.6 Hz, H-5′), 6.88 (1H, s, H-8), 6.75 (1H, s, H-3), 3.99 (3H, s, -OCH3), 3.95 (3H, s, -OCH3), 3.93 (3H, s, -OCH3), 3.84 (3H, s, -OCH3) [31].
Compound 10 (5.6 mg) was isolated as a yellowish amorphous compound and was identified as 6-methoxy kaempferol 3-O-β-glucopyranoside. 1H-NMR (500 MHz, CD3OD) δ (ppm) 8.05 (2H, d, J = 8.8 Hz, H-3′ and H-5′), 6.88 (2H, d, J = 8.8 Hz, H-2′and H-6′), 6.50 (1H, d, J = 6.3 Hz, H-8), 5.26 (1H, d, J = 6.3 Hz, H-1″), 3.87 (3H, s, 6-OCH3), 3.71-3.18 (6H, m, H-2″-H-6″) [32].
Compound 11 (20 mg) was isolated as a yellowish amorphous compound and was identified as astragalin. 1H-NMR (500 MHz, CD3OD) δ (ppm) 8.04 (2H, d, J = 8.8 Hz, H-3′ and H-5′), 6.87 (2H, d, J = 8.8 Hz, H-2′ and H-6′), 6.37 (1H, d, J = 2 Hz, H-8), 6.18 (1H, d, J = 2 Hz, H-6), 5.23 (1H, d, J = 7.2 Hz, H-1″), 3.18-3.71 (6H, m, H-2″-H-6″) [33].
Compound 12 (31.9 mg) was isolated as a white powder and was identified as p-OH benzoic acid. 1H-NMR (500 MHz, CD3OD) δ (ppm) 7.87 (2H, d, J = 8.8 Hz, H-2 and H-6), 6.78 (2H, d, J = 8.8 Hz, H-3 and H-5).
Compound 13 (8.6 mg) was isolated as an amorphous compound and was identified as 3-hydroxy-2-methyl-butyrolactone. 1H-NMR (500 MHz, CD3OD) δ (ppm) 4.52 (1H, dd, J = 5.9, 9.4 Hz, H-4a), 4.24 (1H, q, J = 5.6 Hz, H-3), 4.06 (1H, dd, J = 5.2, 9.4 Hz, H-4b), 2.55 (1H, qd, J = 5.4, 7.4 Hz, H-2), 1.32 (1H, d, J = 7.4 Hz, H-5). 13C-NMR (125 MHz, CD3OD) δ (ppm) 179.5 (C-1), 73.1 (C-3), 72.6 (C-4), 43 (C-2), 11.6 (C-5). With the gDQCOSY the couplings H-4/H-3, H-4a/H-4b, H-2/H-3 and H-2/H-5 are observed. With the gHSQCAD the correlations between protons and directly bonded carbons are observed. With the gHMBCAD the correlations H-2/C-5, H-2/C-3, H-2/C-1, H-5/C-2, H-5/C-3 and H-5/C-1 are observed [34,35].

3.3. In Silico Calculations of Physicochemical Parameters and Prediction of Biological Activities

To achieve good oral bioavailability, the TPSA value should be less than 160 Å2 [35,36]. Additionally, for a compound to cross the blood–brain barrier (BBB), the logBB value must exceed 0.30 [36,37]. Lipinski’s rule of five states that most of the drug-like molecules follow these criteria: LogP ≤ 5, molecular weight ≤ 500, no more than 10 hydrogen bond acceptors and no more than 5 hydrogen bond donors. Compounds that violate more than one of these guidelines will most likely encounter bioavailability issues [37,38]. As shown in Table 1, compounds 10 and 11, however, violate Lipinski’s rule of five, exhibit TPSA values greater than 160, and are therefore predicted to have low absorption and bioavailability following oral administration. The logBB values indicate that none of the compounds under investigation are likely to cross the BBB and exert effects on the Central nervous system (CNS). Imipramine was used as a reference compound because it is a tricyclic antidepressant known to cross the BBB, act on the CNS, and has a logBB value of 0.74 [20].
As shown in Table 2, sesquiterpenoid lactones (1–6) are likely to be effective ligands for nuclear receptors, with compounds 1 and 4 expected to exhibit the strongest activity, approaching that of the reference drug Corticosterone. Additionally, these compounds are likely to act as enzyme inhibitors, with compound 4 showing the greatest inhibition with values exceeding those of the reference drug Ebselen. Interestingly, compounds 1–6 showed higher scores as possible enzyme inhibitors than NDGA (0.13), a known S-LOX inhibitor. The remaining compounds do not appear to exhibit significant activity against the targets studied.

3.4. Results of In Vitro Studies

CEP-crude extract and CEP-Organic phase B were used in the in vitro studies to evaluate the antioxidant and anti-inflammatory activities of the plant. For the examination of anti-inflammatory activity, we applied the study of S-LOX inhibition, since LOX is one of the main enzymes implicated in the cascade of arachidonic acid, to start the inflammation phenomenon. Among the isolated compounds, only 1, 2, 4 and 6 were tested, as the remaining compounds were not available in sufficient quantities for the in vitro assays.
As shown in Table 3, CEP-crude extract exhibits very weak inhibition of lipid peroxidation and does not inhibit lipoxygenase. CEP-organic phase B shows very weak inhibition of both lipid peroxidation and lipoxygenase. Among the four sesquiterpene lactones studied, none inhibited lipid peroxidation. However, compounds 1 and 2 strongly inhibited lipoxygenase, however, less effectively than the reference compound NDGA.

4. Discussion

In this study, we have expanded the phytochemical knowledge of C. bruguieriana subsp. belangeriana, providing new insights that complement the limited existing research. Prior phytochemical investigations on C. bruguieriana have been sparse, with only two studies by Norman et al. [15] and Rustaiyan et al. [16]. Lazari et al. [14] investigated the phytochemical composition and the inhibitory effect of the extracts and the isolated compounds of the subspecies belangeriana against aldose reductase, while Mirzahosseini et al. [17] investigated the phytochemical composition and the cytotoxic activities of the subspecies belangeriana, identifying the chloroform fraction as the most active against four cell lines (K562, AGS, MCF-7 and SW742).
In our study, we isolated and identified six sesquiterpene lactones (three elemanolides, two germacranolides and one eudesmanolide), five flavonoids, as well as p-hydroxybenzoic acid and hydroxy-2-methyl-butyrolactone. These compound classes are consistent with those typically found in the Centaurea genus [27,38,39,40,41].
Among the isolated compounds, the germacranolide cnicin (4) (845.96 mg) and the eudesmanolide malacitanolide (6) (681.6mg) were the major constituents. Cnicin (4) and 5-hydroxy, 6,7,3′,4′-tetramethoxy-flavone (9) have previously been isolated from the species C. bruguieriana [16], while compounds 1 [14], 2 [14], 6 [14], 7 [14,17], 8 [14], 9 [14] and 12 [14] have been isolated from the subspecies belangeriana. Compounds 1, 2 [27,38,39], 3 [5,38,39], 5 [38,39], 6 [27,38,39], 8 [41,42], 10, 11 [42,43], 12 [43,44] and 13 [27] have been isolated from different Centaurea species. While Lazari et al. [14] isolated β-sitosterol and Mirzahosseini et al. [17] isolated also cirsimaritin and eupatilin, in our study we did not isolate them.
In silico studies were used to assess the bioactivity of the isolated compounds [44,45], since this methodology is of a great importance in preliminary screening for efficiency, speed and cost-effectiveness. In recent years, many studies have been conducted regarding the in silico evaluation of the biological activities/molecular docking of compounds of different Centaurea species. For example, Noman et al. [15] studied the molecular docking of compounds found in the chloroform extract of C. bruguieriana against three bacterial receptors (TyrRS, DNA gyrase and DHFR receptors), while Fatullayev et al. [45,46] studied the molecular docking of apigenin and myristoleic acid, found in C. lucaonica extracts, against α-glucosidase and α-amylase. Perveen et al. [47] reported that the presence of the α-methylene γ-lactone moiety of some guaianolides and eudesmanolides does not necessarily result in anti-inflammatory activity. Lazanaki et al. [48] discovered that several sesquiterpene lactones isolated from Staehelina uniflosculosa might be potential nuclear receptor ligands. Herein, we found that among all the isolated compounds, only compounds 10 and 11 violated Lipinski’s rule of five, indicating that most of the compounds have good bioavailability. However, none of the compounds were likely to cross the BBB. Notably, sesquiterpenoid lactones (1–6) exhibited promising potential as ligands for nuclear receptors and enzyme inhibitors, where they showed greater scores as potential enzyme inhibitors of enzymes (e.g., LOX), than the reference drug NDGA. This inhibition might explain their anti-inflammatory activity. The remaining compounds demonstrated low affinity for the targets tested, which indicates that their pharmacological activity is probably caused by a different mechanism of action. Therefore, further in vitro studies are required to identify the mechanism of action and the potential biological activities of all the isolated compounds.
Our findings on antioxidant activity diverge from prior studies. Most Centaurea species exhibit strong antioxidant properties due to their phenolic and lipid acid content [46,47,48,49,50,51]. Specifically, C. bruguieriana ethyl acetate fraction showed high antioxidant activity, while the crude extract showed the lowest (DPPH and ABTS tests) [15]. Similarly, the antioxidant activity of the methanol extracts of five Centaurea species showed high antioxidant activity among several assays, including DPPH, ABTS and inhibition of lipid peroxidation. These effects were closely related to the total phenolic content of the extracts, with higher phenolic content usually correlating with greater antioxidant activity [52]. However, in our case, CEP-crude extract and CEP-Organic phase B showed low antioxidant activity. This discrepancy could be attributed to the focus of our extraction method on sesquiterpene lactones rather than phenolic compounds, leading to lower phenolic content. Additionally, the use of different antioxidant assays, which proceed through different mechanisms, likely contributed to the difference in activity.
Regarding the anti-inflammatory activity expressed as S-LOX inhibition, our findings partially align with previous research on the Centaurea genus. The hexane extracts of C. adjarica, C. bracteata, C. cataonica, C. cynaroides, C. dealbata, C. indurata, C. macrocephala, C. melitensis, C. nigrescens and C. ruthenica showed non-selective inhibition of the enzymes COX-1 and COX-2 [49,53]. The methanol extracts of the aerial parts and capitula of C. cuneifolia, C. iberica, C. kilaea, C. solstitialis subsp. solstitialis and C. stenolepi were investigated for their potential to inhibit S-LOX. The extracts of the aerial parts showed lower inhibition of S-LOX (below 50%) than the capitula extracts (higher than 50%) [48,51]. In our study, CEP-crude extract did not inhibit S-LOX, and CEP-Organic phase B inhibited S-LOX weakly (18.4%), which aligns with the findings of Şen and Ali [48,51], who also observed lower inhibition of S-LOX in the aerial parts of Centaurea species.
Sesquiterpene lactones are known for their anti-inflammatory activity [38,39]. Isocnicin and cnicin possess strong anti-inflammatory activity by inhibiting TNF-α-induced ICAM-1 expression in HMEC-1 cells [51,54]. Additionally, cnicin inhibits the transcription factor NF-κB, reducing inflammation [52,55], and inhibits the pro-inflammatory mediator iNOS in LPS-induced macrophages [53,56]. In our study, all of the in vitro-tested compounds were sesquiterpene lactones. Isocnicin (1) and 8α-(4′-acetoxy-3′hydroxy-2′-methylene-butanoyloxy)-dehydro-melitensin (2) did not inhibit lipid peroxidation but exhibited strong inhibition of S-LOX. Their anti-inflammatory activity is in agreement with the in silico tests, which showed that sesquiterpene lactones are potential enzyme inhibitors. In contrast, cnicin (4) and malacitanolide (6) were inactive against both tests. None of the four sesquiterpene lactones showed antioxidant activity. Interestingly, while isocnicin (1) and 8α-(4′-acetoxy-3′hydroxy-2′-methylene-butanoyloxy)-dehydro-melitensin (2) showed strong anti-inflammatory activity, cnicin (4), despite its known anti-inflammatory activity, did not inhibit S-LOX. This is possibly because these compounds inhibit COX-2 and present their anti-inflammatory activity through this pathway. Regarding the antioxidant activity of the sesquiterpene lactones, several were tested for their radical scavenging capacity and their ability to inhibit lipid peroxidation. The results showed that these compounds moderately scavenge radicals and weakly inhibit lipid peroxidation. However, in some cases, sesquiterpene lactones act as prooxidants. In our study, the tested sesquiterpene lactones did not inhibit lipid peroxidation, which agrees with the finding of the previously mentioned study [57].

5. Conclusions

This study of Centaurea bruguieriana subsp. belangeriana makes a valuable contribution to both the phytochemistry and the potential biological activities of this species. The isolation and identification of 13 compounds, including six sesquiterpene lactones and five flavonoids, deepens the knowledge of the plant’s secondary metabolites, many of which have been linked to pharmacological activities in prior studies. The in vitro tests demonstrated strong lipoxygenase inhibition supporting anti-inflammatory activity and low inhibition of lipid peroxidation by some isolated sesquiterpene lactones, which aligns with the literature. The low antioxidant activity of the crude extract and the organic phase B was expected, given the fact that the extracts were low in phenolic content. The in silico prediction studies further enhance our understanding of the potential biological applications of this plant’s isolated compounds. This study broadens the phytochemical knowledge of the Centaurea genus and provides evidence for future investigations of the plant’s medicinal applications.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/separations11110319/s1. Figure S1: 1H NMR of compound 1 (CD3OD, 500 MHz); Figure S2: 1H NMR of compound 1 (CD3OD, 500 MHz); Figure S3: 1H NMR of compound 3 (CD3OD, 500 MHz); Figure S4: 1H NMR of compound 4 (CD3OD, 500 MHz); Figure S5: 13C NMR of compound 4 (CD3OD, 125 MHz); Figure S6: 1H NMR of compound 5 (CD3OD, 500 MHz); Figure S7: 1H NMR of compound 6 (CD3OD, 500 MHz); Figure S8: 1H NMR of compound 7 (CD3OD, 200 MHz); Figure S9: 1H NMR of compound 8 (CD3OD, 200 MHz); Figure S10: 1H NMR of compound 9 (CD3OD, 500 MHz); Figure S11: 1H NMR of compound 10 (CD3OD, 500 MHz); Figure S12: 1H NMR of compound 11 (CD3OD, 500 MHz); Figure S13: 1H NMR of compound 12 (CD3OD, 500 MHz); Figure S14: 1H NMR of compound 13 (CD3OD, 500 MHz); Figure S15: 13C NMR of compound 13 (CD3OD, 125 MHz); Figure S16: gDQCOSY NMR of compound 13 (CD3OD, 500 MHz); Figure S17: HSQCAD NMR of compound 13 (CD3OD, 500 MHz); Figure S18: HMBCAD NMR of compound 13 (CD3OD, 500 MHz).

Author Contributions

Conceptualization, D.L. and M.A.; methodology, D.L., K.M.D. and O.T.; software, D.L. and K.M.D.; validation, D.L., D.H.-L., O.T. and M.A.; formal analysis, D.L., K.M.D. and O.T.; investigation, D.L., K.M.D. and O.T.; resources, D.L. and M.A.; data curation, K.M.D., O.T. and D.L.; writing—original draft preparation, K.M.D., O.T. and D.L.; writing—review and editing, D.L., O.T. and D.H.-L.; visualization, K.M.D., O.T. and D.L.; supervision, D.L., O.T. and D.H.-L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

All data supporting the results of this study are included in the manuscript and the supplementary material, and the datasets are available upon request.

Acknowledgments

The authors would like to thank Tsioumela Chrysanthi for her valuable help with the first fractionations of the crude extract, Skaltsa Helen (professor at the University of Athens, School of Pharmacy, Laboratory of Pharmacognosy, Athens, Greece) and Karioti Anastasia (professor at the Aristotle University of Thessaloniki, School of Pharmacy, Laboratory of Pharmacognosy, Thessaloniki, Greece) for recording the 1H-NMR spectra of compounds 7 and 8 in the Bruker 200 MHz spectrometer and Krigas Nikolaos (a researcher at the National Agricultural Research Foundation, Laboratory for the Conservation and Evaluation of Native and Floricultural Species, Thermi, Thessaloniki Greece) for the identification of the subspecies of the plant material.

Conflicts of Interest

The authors declare no conflicts of interest.

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Separations 11 00319 g001
Figure 1. Structures of all the isolated compounds from C. bruguieriana subsp. belangeriana and imipramine.
Figure 1. Structures of all the isolated compounds from C. bruguieriana subsp. belangeriana and imipramine.
Separations 11 00319 g001
Table 1. In silico calculations of physicochemical parameters of the isolated compounds.
Table 1. In silico calculations of physicochemical parameters of the isolated compounds.
CompoundmiLogPTPSALogbbnatomsMWnONnOHONViolationsnrotbVolume
10.44113.29−0.9027378.427308349.47
21.15119.37−0.8530420.4682010385.99
31.5586.99−0.4721296.365306287.55
40.53113.29−0.8927378.427306349.12
51.24119.37−0.8430420.468208385.63
6−0.21130.37−1.1728394.428306352.61
72.6098.37−0.4225344.327204292.67
82.6098.37−0.4225344.327204292.67
92.9187.38−0.2626358.357105310.20
100.14199.51−1.8134478.4112725389.73
110.12190.28−1.7232448.3811724364.19
121.3757.53−0.2010138.123201119.06
13−1.0146.53−0.468116.123100104.80
Imipramine4.166.480.7421280.422004287.31
miLogP: logarithm of the partition coefficient according to molinspiration; TPSA: molecular polar surface area; logBB: calculated using Rishton’s equation; natoms: number of atoms; MW: molecular weight; nON: number of hydrogen bond acceptors; nOHON: number of hydrogen bond donors; Violations: number of violations of Lipinski’s rule; nrotb: number of rotatable bonds; Volume: molecular volume; Imipramine: reference drug, The red color indicates the violations of Lipinski’s rule of five and TPSA > 160 Å2.
Table 2. In Silico Prediction of Biological Activities and Potential Targets of Pharmacophores.
Table 2. In Silico Prediction of Biological Activities and Potential Targets of Pharmacophores.
GPCR LigandIon Channel ModulatorKinase InhibitorNuclear Receptor LigandProtease InhibitorEnzyme Inhibitor
10.14−0.07−0.180.900.130.61
20.07−0.08−0.210.760.110.54
3−0.02−0.07−0.430.750.120.43
40.330.12−0.130.980.060.76
50.250.10−0.170.830.040.68
60.12−0.04−0.290.750.180.61
7−0.09−0.230.200.13−0.290.14
8−0.09−0.230.200.13−0.290.14
9−0.10−0.230.180.12−0.270.12
100.01−0.140.090.07−0.160.35
110.06−0.050.100.20−0.050.41
12−0.98−0.39−1.21−0.62−1.19−0.41
13−2.52−2.44−3.42−2.91−2.47−2.24
Adenosine1.10
Capsazepine −0.03
K-252α 1.27
Corticosterone 1.02
Z-VAD-(OMe)-FMK 1.03
Ebselen 0.65
NDGA 0.13
GPCR ligands: G-protein-coupled receptors. Green color indicates possible biological activities. Adenosine, Cepsazepine, K-252α, Corticosterone, Z-VAD-(Ome)-FMK, Ebselen were used as reference drugs for each respective target.
Table 3. Results of Lipid Peroxidation Inhibition and Lipoxygenase Inhibition (mean ± SD; n = 3).
Table 3. Results of Lipid Peroxidation Inhibition and Lipoxygenase Inhibition (mean ± SD; n = 3).
% Lipid Peroxidation Inhibition% Lipoxygenase Inhibition
CEP-Crude extract22.1 a ± 0.23n.a.
CEP-Organic phase Β6.1 b ± 0.2918.4 c ± 0.37
1n.a.74.4 a ± 0.65
2n.a.73.3 b ± 0.31
42.7 c ± 0.06n.a.
6n.a.n.a.
NDGAn.m.93.0
TROLOX95.0n.m.
n.a.: not active, n.m.: not measured, NGDA: Nordihydroguaiaretic acid; Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. a–c values with the same letter are not significantly different (p < 0.05).
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Dimitriadis, K.M.; Tsiftsoglou, O.; Hadjipavlou-Litina, D.; Arfan, M.; Lazari, D. Phytochemical Study of the Plant Centaurea bruguieriana (DC.) Hand.-Mazz. subsp. belangeriana (DC.) Bornm. of the Family Asteraceae. Separations 2024, 11, 319. https://doi.org/10.3390/separations11110319

AMA Style

Dimitriadis KM, Tsiftsoglou O, Hadjipavlou-Litina D, Arfan M, Lazari D. Phytochemical Study of the Plant Centaurea bruguieriana (DC.) Hand.-Mazz. subsp. belangeriana (DC.) Bornm. of the Family Asteraceae. Separations. 2024; 11(11):319. https://doi.org/10.3390/separations11110319

Chicago/Turabian Style

Dimitriadis, Kyriakos Michail, Olga Tsiftsoglou, Dimitra Hadjipavlou-Litina, Mohammad Arfan, and Diamanto Lazari. 2024. "Phytochemical Study of the Plant Centaurea bruguieriana (DC.) Hand.-Mazz. subsp. belangeriana (DC.) Bornm. of the Family Asteraceae" Separations 11, no. 11: 319. https://doi.org/10.3390/separations11110319

APA Style

Dimitriadis, K. M., Tsiftsoglou, O., Hadjipavlou-Litina, D., Arfan, M., & Lazari, D. (2024). Phytochemical Study of the Plant Centaurea bruguieriana (DC.) Hand.-Mazz. subsp. belangeriana (DC.) Bornm. of the Family Asteraceae. Separations, 11(11), 319. https://doi.org/10.3390/separations11110319

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