Antioxidant activity of an anatolian herbal tea—Origanum minutiflorum: isolation and characterization of its secondary metabolites

ABSTRACT

Origanum species are significant aromatic and medicinal plants used in food and pharmaceutical industries. Isolation of bioactive compounds was executed on n-butanol extract to yield the compounds responsible for the activities. Tricosan-1-ol (1), (8E,16E)-tetracosa-8,16-diene-1,24-diol (2), azepan-2-one (3), 3,4-dihydroxybenzoic acid (4), apigenin (5), eriodictyol (6), globoidnan-A (7), luteolin (8), rosmarinic acid (9), apigenin-7-O-glucuronide (10), and vicenin-2 (11) were isolated by chromatographic methods (column chromatography and semi-preparative High Performance Liquid Chromatography (HPLC) and structures were elucidated on the basis of spectroscopic techniques including 1D/2D nuclear magnetic resonance (NMR) and Liquid chromatography/Time-of-flight/Mass spectrometry (LC-TOF/MS). The isolated compounds and extracts were applied for antioxidant assays including 1,1-diphenyl-2-picrylhydrazyl (DPPH^•) scavenging, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS^•+) scavenging, reducing power, and cuprac techniques. 3,4-Dihydroxy benzoic acid (4), eriodictyol (6), luteolin (8), and rosmarinic acid (9) revealed the considerable antioxidant activities.

KEYWORDS:

• Herbal tea
• Origanum minutiflorum
• Antioxidant activity
• Chromatography
• Spectroscopy

Introduction

Oxygen is the final electron acceptor in the electron flow system that produces energy in the form of adenosine triphosphate (ATP) lipid peroxidation and thereby stabilizing fat-containing food-stuffs.^[1,2] It constitutes most of the mass of living organisms, because water is their major constituent. It is very important for the continuity of vital functions and has unpaired two electrons and tends to form oxygen-centered free radicals, known as reactive oxygen species (ROS).^[3,4] ROS include free radicals such as hydroxyl radicals (OH•), superoxide anion radicals (O₂^•−), and non-free radical species such as singlet oxygen (¹O₂) and hydrogen peroxide (H₂O₂).^[5–7] ROS have been implicated in a lot of diseases, including malaria, acquired immunodeficiency syndrome, stroke, heart disease, arteriosclerosis, diabetes, and cancer. The oxidative deterioration of fats and oils in foods is responsible for rancid odors and flavors, with a consequent decrease in nutritional quality and safety caused by the formation of secondary, potentially toxic compounds.^[8,9] Antioxidants are molecules capable of inhibiting the oxidation of other molecules. In terms of food, an antioxidant has been defined as any substance that when present in low concentrations compared to that of an oxidizable substrate significantly delays or inhibits the oxidation of that substrate.^[10,11] Antioxidants have been widely used as food additives to protect food quality mainly by the prevention of oxidative deterioration of lipid constituents. Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are widely used as potential inhibitors of lipid peroxidation and thereby stabilizing fat-containing food-stuffs.^[12–14] However, these synthetic antioxidants have been restricted for use in foods as they are suspected to be carcinogenic.^[15–17] The use of spices and herbs as antioxidants in processed foods is a promising alternative to the use of synthetic antioxidants.^[18,19] In this context, natural products appear as healthier as and safer than synthetic antioxidants. Hence, there is a growing interest in exploring, purifying, and characterization of natural sources of natural and safer antioxidants for food and pharmaceutical applications.^[20–22]

Origanum minutiflorum is an endemic plant found in the mountains of southern Turkey. It is used as a spice for seasoning, as a herbal tea, and also as a medicinal herb for curing stomach-aches and respiratory colds.^[23] Natural products have been the source of many pharmaceutically and medicinally active compounds.^[24–28] Origanum genus consists of forty three species and eighteen hybrids, sixteen of which are endemic for Turkey.^[29] Origanum species have been used in folk medicine to alleviate the various diseases. Due to the fragrance, Origanum has been widely used in food products as flavoring agent. This genus has gained the great importance in pharmaceutical and medicinal industries due to their high amount of essential oil concentration as well as including bioactive secondary metabolites.^[30] Origanum species exhibit high antioxidant activities; therefore, they can be potentially replaced with existing synthetic antioxidant.^[31] Some species of these plants are well known in Anatolian folk medicine and widely used as spices and herbal tea.^[32] The effectiveness is attributed to the specific composition of essential oils,^[33] flavonoids,^[34] phenolic acids,^[35] and other chemical constituents of Origanum.

Previous phytochemical and pharmaceutical studies on this species have resulted in the isolation of phenolic compounds, which are responsible for antioxidant and anticancer activities.^[36] Recent works on Origanum species exhibited a wide range of biological activities such as antimicrobial,^[37] anticancer,^[38] antioxidant,^[39] antidiabetes,^[40] antibacterial,^[41] antifungal,^[42] antinociceptive,^[43] and antilipase^[44] properties.

Although extensive knowledge on the chemical composition of essential oil on Origanum species is available, the antioxidative activity of secondary metabolites isolated from Origanum minutiflorum O. Schwarz & P.H has not been investigated previously. Herein, bioactivity-guided isolation of secondary metabolites from O. minutiflorum was achieved. In addition, antioxidant activities of isolated compounds were investigated. Antioxidant activities of extracts and isolated compounds were evaluated using the assays of DPPH^• scavenging, ABTS^•+ scavenging, cuprac, and ferric reducing power.

Materials and methods

General experimental procedures

NMR spectra were carried out on Bruker-400 MHz spectrometer (400 MHz for proton and 100 MHz for carbon analysis). UV measurements were performed on Hitachi U-290 UV–VIS spectrophotometer. Silica gel (60–200 mesh) was used as stationary phase for column chromatography. GF254 was used for TLC application purchased commercially. Ferrous chloride, α-tocopherol, DPPH, ferrozine, trichloroacetic acid (TCA), and butylated hydroxyanisole (BHA) were bought from Sigma (Sigma-Aldrich GmbH, Sternheim, Germany). Butylated hydroxytoluene (BHT) was supplied by E. Merck (Darmstadt, Germany). High Resolution Mass Spectrometry (HRMS) analyses were carried out on Agilent 6210 LC-TOF/MS. Other chemicals for antioxidant activity were supplied by Sigma-Aldrich (Germany).

Plant material

Origanum minutiflorum was collected from Isparta, Turkey in September 2015 and identified by Prof. Dr. Ahmet İlcim. A voucher specimen was deposited in the Herbarium Laboratory of Mustafa Kemal University, Faculty of Art and Science, Department of Biology (Herbarium No: MKUH 1321).

Extraction and isolation

Aerial part of the plant material (0.7 kg) was dried at shade and then boiled in water (5.0 L) for an hour (h). After cooling at room temperature (rt), it was filtered to yield the aqueous phase, which was partitioned with hexane, ethyl acetate, and n-butanol sequentially for seven days (3 × 5.0 L for each). The solvents were removed under reduced pressure to yield the hexane extract, ethyl acetate extract, and n-butanol extract which were subjected to antioxidant activities. The highest phenolic compound content was found in n-butanol extract, therefore chromatographic techniques were executed on this extract to isolate the bioactive compounds. The crude n-butanol extract (40 g) was subjected to silica gel (60–200 mesh) column (120 cm×5 cm) eluting with hexane, hexane-dichloromethane, dichloromethane, dichloromethane-methanol, and methanol. The polarity was increased with the ration of 10%. 800 fractions (each, 50 mL) were collected. After TLC checking, fractions consisting of same compounds were combined. Tricosan-1-ol (1) was isolated from the 1–25 fractions. (8E,16E)-tetracosa-8,16-diene-1,24-diol (2) was isolated from the 25–58 fractions and fractions 58–112 contained the azepan-2-one (3). 3,4-Dihydroxybenzoic acid (4) was generated from the fractions 112–186. Fractions 186–356 were observed to have apigenin (5) and eriodictyol (6). Therefore, these fractions were combined and applied to preparative HPLC analysis. After HPLC analysis, apigenin (5) and eriodictyol (6) were isolated. TLC and HPLC analyses revealed that the fractions 356–567 included two compounds. So, these fractions were combined and subjected to preparative HPLC to isolate the pure globoidnan-A (7) and luteolin (8). Rosmarinic acid (9) and apigenin-7-O-glukoronid (10) were found in 567–648 fractions as a mixture. Hence, rosmarinic acid (9) and apigenin-7-O-glukoronid (10) were isolated by preparative HPLC from the combined fractions 567–648. Vicenin-2 (11) was isolated from the fractions 648–718.

Compound (1): Tricosan-1-ol: (20 mg), Anal. Calcd for C₂₃H₄₈O: C, 81.10, H, 14.57, O, 4.70. Found: C, 80.77, H, 14.57, O, 4.72. ¹H-NMR (400 MHz, CDCl₃), δ: 3.67 (m, 2H), 1.59 (m, 4H), 1.27 (m, 38H), 0.90 (t, J = 8.0 Hz, CH₃). ¹³C-NMR (100 MHz, CDCl₃), δ: 62.1, 32.8, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 25.7, 22.7, 14.1.^[45]

Compound (2): (8E,16E)-tetracosa-8,16-diene-1,24-diol: (25 mg). Anal. Calcd for C₂₄H₄₆O₂: C, 78.63, H, 12.65, O, 8.73. Found: C, 78.01, H, 12.88, O, 8.73. ¹H-NMR (400 MHz, CDCl₃), δ: 8.12 (2H, OH), 4.29 (m, 2H, H8, H17), 3.67 (m, 2H, H9, H16), 0.9–2.0 (m, 40H). ¹³C-NMR (100 MHz, CDCl₃), δ: 129.49, 63.11, 32.83, 31.93, 29.70, 29.67, 29.62, 29.61, 29.44, 29.37, 28.99, 25.75.

Compound (3): Azepan-2-one: (15 mg). ¹H-NMR (400 MHz, DMSO-d6), δ: 7.74 (s, N-H), 3.02 (m, H7), 2.03 (m, H3), 1.36 (m, H6), 1.21 (m, H5), 1.19 (m, H4). ¹³C-NMR (100 MHz, DMSO-d6), δ: 172.5 (C2), 38.5 (C7), 35.7 (C3), 28.9 (C6), 26.0 (C5), 25.3 (C4).^[46]

Compound (4): 3,4-dihydroxybenzoic acid (4): (18 mg). ¹H-NMR (400 MHz, DMSO-d6), δ: 7.34 (d, J = 2.1 Hz, H2), 7.28 (dd, J = 8.2, 2.1, H6), 6.79 (d, J = 8.2 Hz, H5). ¹³C-NMR (100 MHz, DMSO-d6), δ: 167.8 (C7), 150.5 (C4), 145.3 (C3), 122.3 (C1), 122.1 (C6), 117.0 (C5), 115.6 (C2).^[47]

Compound (5): Apigenin (5): Isolated as a white solid (15 mg). ¹H-NMR (400 MHz, DMSO-d6), δ: 7.93 (d, J = 8.80 Hz, H2ʹ, H6ʹ), 6.93 (d, J = 8.80 Hz, H3ʹ, H5ʹ), 6.78 (s, H3), 6.48 (d, J = 2.0 Hz, H8), 6.19 (d, J = 2.0 Hz, H6). ¹³C-NMR (100 MHz, DMSO-d6), δ: 182.2 (C4), 164.9 (C2), 164.2 (C7), 161.9 (C5), 161.7 (C4ʹ), 157.8 (C9), 128.9 (C2ʹ, C6ʹ), 121.6 (C1ʹ), 116.4 (C3ʹ, C5ʹ), 104.1 (C10), 103.3 (C3), 99.4 (C6), 94.5 (C8) (Owen et al., 2003).^[48]

Compound (6): Eriodictyol (6): Isolated as a solid (25 mg). ¹H-NMR (400 MHz, DMSO-d6), δ: 6.88 (brs, H5ʹ), 6.75 (brs, H2ʹ, H6ʹ), 5.88 (brs, H6, H8), 5.38 (dd, J = 12.5, 2.9 Hz, H2), 3.20 (dd, J = 17.1, 12.6 Hz, H3α), 2.68 (dd, J = 17.1, 2.9 Hz, H3β). ¹³C-NMR (100 MHz, DMSO-d6), δ: 196.8 (4), 167.1 (C7), 163.9 (C5), 163.4 (C9), 146.2 (C3ʹ), 145.7 (C4ʹ), 129.9 (C1ʹ), 118.4 (C6ʹ), 115.8 (C2ʹ), 114.8 (C5ʹ), 102.3 (C10), 96.2 (C8), 95.2 (C6), 78.9 (C2), 42.5 (C3).^[49]

Compound (7): Globoidnan-A (7): Isolated as a liquid (20 mg). ¹H-NMR (400 MHz, DMSO-d6), δ: 8.23(s, H5), 7.51 (s, H2), 7.36 (s, H5), 7.27 (s, H8), 6.92 (d, J = 8.0 Hz, H5ʹ), 6.85 (s, H2ʹ), 6.73 (dd, J = 1.7, 8.0 Hz, H6ʹ), 6.68 (brs, H6ʹ’), 6.67 (brs, H3ʹ’), 6.64 (brs, H2ʹ’), 5.23 (dd, j = 4.4, 7.7 Hz, H8ʹ’), 3.03–3.10 (m, H7ʹ’). ¹³C-NMR (100 MHz, DMSO-d6), δ: 171.6 (C9ʹ’), 166.2 (C9), 149.9 (C7), 147.9 (C6), 145.6 (C4ʹ), 145.5 (C3ʹ), 144.5 (C5ʹ’), 144.4 (C4ʹ’), 138.6 (C8α), 131.7 (C1ʹ), 130.1 (C4α), 128.8 (C1), 128.1 (C4), 127.9 (C1ʹ’), 123.6 (C3), 123.0 (C2), 120.9 (C6ʹ), 120.6 (C2ʹ’), 117.3 (C2ʹ), 117.1 (C3ʹ’), 116.2 (C5ʹ), 115.8 (C6ʹ’), 112.0 (C5), 108.5 (C8), 74.2 (C8ʹ’), 36.8 (C7ʹ’).^[49]

Compound (8): Luteolin (8): Isolated as a white solid (25 mg). ¹H-NMR (400 MHz, DMSO-d6), δ: 7.43 (dd, J = 8.3 Hz, 2.2, H6ʹ), 7.40 (d, J = 2.2 Hz, H2ʹ), 6.90 (d, J = 8.3 Hz, H5ʹ), 6.67 (s, H3), 6.45 (d, J = 2.0 Hz, H8), 6.19 (d, J = 2.0 Hz, H6). ¹³C-NMR (100 MHz, DMSO-d6), δ: 182.1 (C4), 164.6 (C2), 164.4 (C7), 161.9 (C5), 157.7 (C9), 150.2 (C4ʹ), 146.2 (C3ʹ), 121.9 (C1ʹ), 119.5 (C6ʹ), 116.5 (C5ʹ), 113.8 (C2ʹ), 104.2 (C10), 103.3 (C3), 99.3 (C6), 94.3 (C8).^[50]

Compound (9): Rosmarinic acid (9): Isolated as a white solid (30 mg). LC-TOF/MS m/z: 359.0824 [M-H]⁻¹ (Calcd. 359.0777 for C₁₈H₁₅O₈). IR (KBr), λ_max, cm⁻¹:3326, 1706, 1604, 1517, 1448, 1267. ¹H-NMR (400 MHz, DMSO-d6), δ: 7.46 (d, J = 15.6 Hz, H7), 7.07 (d, J = 1.4 Hz, H2), 7.01 (d, J = 8.0 Hz, H6), 6.78 (d, J = 8.0 Hz, H5), 6.69 (d, J = 1.5 Hz, H2ʹ), 6.65 (d, J = 7.9 Hz, H6ʹ), 6.54 (d, J = 7.9 Hz, H5ʹ), 6.25 (d, J = 15.6 Hz, H8), 5.05 (dd, J = 4.0, 8.0 Hz, H8ʹ), 3.01 (dd, J = 4.0, 8.0 Hz, H7ʹα), 2.92 (dd, J = 4.0, 8.0 Hz, H7ʹβ). ¹³C-NMR (100 MHz, DMSO-d6), δ: 171.3 (C9ʹ), 166.4 (C9), 149.1 (C3), 146.3 (C7), 146.1 (C4), 145.4 (C4ʹ), 144.5 (C3ʹ), 127.7 (C1ʹ), 125.8 (C1), 122.1 (C6), 120.5 (C5ʹ), 117.1 (C2ʹ), 116.2 (C5), 115.8 (C6ʹ), 115.3 (C2), 113.7 (C8), 73.3 (C8ʹ), 36.6 (C7ʹ).^[36]

Compound (10): Apigenin-7-O-β-glucuronide (10): Isolated as a white solid (14 mg). LC-TOF/MS/MS m/z 445.0888 [M-H]⁻ (clcd for C₂₁H₁₇O₁₁ 445.0776). ¹H-NMR (400 MHz, DMSO-d6) δ: 6,86 (s, H3), 6,46 (d, J = 1.5 Hz, H6), 6.86 (d, J = 1.5 Hz, H8), 7.95 (d, J = 8.5 Hz, H2′, H6ʹ), 6.95 (d, J = 8.5 Hz, H3ʹ, H5ʹ), 5.26 (d, J = 6.9 Hz, H1ʹ’), 4.02 (d, J = 9.2 Hz, H-4ʹ’), 3.38 (m, H2ʹ’), 3.33 (m, H5ʹ’), 3.29 (m, H3ʹ’). ¹³C-NMR (100 MHz, DMSO-d6) δ: 182.5 (C3), 170.8 (C6ʹ’), 164.8 (C2), 163.7 (C7), 161.9 (C5), 161.6 (C4ʹ), 157.4 (C9), 129.1 (C-2ʹand C-6ʹ), 121.4 (C1ʹ), 116.5 (C3ʹ and C5ʹ), 105.9 (C10), 103.6 (C3), 99.9 (C6), 99.7 (C1ʹ’), 95.1 (C8), 76.2 (C5ʹ’), 75.7 (C4ʹ’), 73.2 (C3ʹ’), 71.8 (C2ʹ’).^[51]

Compound (11): Vicenin-2 (11): Isolated as a white solid (20 mg). LC-TOF/MS/MS m/z 593.1672 [M-H]⁻ (clcd for C₂₇H₂₉O₁₅ 593.1506). IR (KBr), λ_max, cm⁻¹: 3434, 1631, 1025. ¹H-NMR (400 MHz, DMSO-d6) δ: 8.04 (d, J = 8.5 Hz, H2ʹ, H6ʹ), 6.90 (d, J = 8.5 Hz, H3ʹ, H5ʹ), 6.83 (s, H3), 4.80 (d, J = 9.8 Hz, H1ʹ’’), 4.75 (d, J = 9.8 Hz, H1ʹ’), 3.41 (m, H2ʹ’’), 3.88 (m, H4ʹ’), 3.78 (m, H6ʹ’’β), 3.70 (m, H6ʹ’β), 3.58 (m, H6ʹ’α, H6ʹ’’α), 3.52 (m, H4ʹ’’), 3.39 (m, H2ʹ’), 3.33 (m, H5ʹ’), 3.29 (m, H3ʹ’’), 3.28 (m, H3ʹ’), 3.23 (m, H5ʹ’’). ¹³C-NMR (100 MHz, DMSO-d6) δ: 182.8 (C4), 164.5 (C2), 161.7 (C4ʹ), 161.4 (C7), 159.0 (C5), 155.5 (C9), 129.5 (C2ʹ, C6ʹ), 116.3 (C3ʹ, C5ʹ), 121.9 (C1ʹ), 107.9 (C6), 105.8 (C8), 104.3 (C10), 103.05 (C3), 82.4 (C5ʹ’’), 81.3 (C5ʹ’), 79.3 (C3ʹ’’), 78.3 (C3ʹ’), 74.5 (C1ʹ’’), 73.8 (C1ʹ’), 72.4 (C4ʹ’’), 71.38 (C4ʹ’, C2ʹ’’), 69.5 (C2ʹ’), 61.8 (C6ʹ’’α), 61.26 (C6ʹ’α).^[52]

Total phenolic contents

Total phenolic contents of hexane, ethyl acetate, and n-butanol extracts were determined with Folin-Ciocalteu reagent using gallic acid as standard ^[53] as described previously.^[54] The method is based on the formation of colored complex by treatment of phenolic compounds with Folin-Ciocalteu reagent in alkaline medium. Total phenolic compounds of extracts are determined by measuring the absorbance of purple complex at 760 nm by UV-VIS spectrophotometer.^[55] Each plant extract was dissolved in water and an aliquot (100 µL) was treated with Folin-Ciocalteu (100 µL) for 5 min vigorously. After adding sodium carbonate (2%, 300 μL), the reaction mixture in tube was vortexed for 2 min; then, distilled water (4.5 mL) was added into the reaction tube. Each tube was vortexed again and then kept at 25°C for 90 min. The absorbance was measured for each tube with UV-VIS spectrophotometer at 760 nm.^[56]

DPPH^• free radical assay

DPPH^• free radical activity of extracts, isolated compounds, and standards was executed. The method is based on the electron- or hydrogen-donating ability from compound to DPPH^•, resulting in the bleaching purple color solution of DPPH^•.^[57] Absorbance decrease of the reaction mixtures revealed the high antioxidant activity. The various concentrations of each compound (1.0 mL, 0.1–0.5 µg/mL) was reacted with DPPH^• solution (0.1 mM, 4.0 mL) for 30 sec via vortex and kept at dark for 60 min. Spectroscopic measurement was executed at 517 nm. DPPH^• solution and methanol were used as a control and blank, respectively. Activity of DPPH^• scavenging was calculated by following equation: DPPH^• scavenging effect$\left(\mathrm{\%}\right)=\left[\left(Ac-As\right)/Ac\right]\times 100$. In which Ac is the absorbance of the control and As is the absorbance of the sample.^[58]

ABTS^+• scavenging assay

This method is based on the ability of quenching ABTS radical cation by antioxidant materials.^[59] The reaction of ABTS (2 mM) in water with potassium persulfate (K₂S₂O₈) (2.3 mM) yielded the ABTS cation radical, which was kept at room temperature for 4h in dark. ABTS^•+ was diluted with sodium phosphate buffer (0.1 mM, pH 7.4) to adjust the absorbance at 734 nm. Subsequently, each sample solution (3.0 mL) at various concentrations (2.5–20.0 µg/mL) was treated with ABTS^•+ (1.0 mL). The percent inhibition was calculated at 734 nm for each concentration compared to a blank absorbance.^[60] ABTS^•+ concentration (mM) in the reaction medium was calculated by calibration curve. The capability of ABTS^•+ was calculated by the equation: ABTS^•+ scavenging effect$\left(\mathrm{\%}\right)=\left(\left(Ac-As\right)/Ac\right)\times 100$. In which, Ac is ABTS^•+ initial concentration and As is ABTS^•+ remaining concentration in the sample.^[61,62]

Reducing power

Antioxidant materials reduce Fe³⁺ ion to Fe^2+.[63] After addition of FeCl₃ to the reaction medium, Prussian blue complex had been formed, then the absorbance of the complex was measured. Phytochemical properties of Origanum minutiflorum were presented. To a reaction flask, 0.1 mL of each sample (50–200 µg/mL, individually), phosphate buffer (1.15 mL, 0.2 M, pH 6.7), and potassium ferricyanide (K₃Fe(CN)₆) (1.25 mL, 1%) were added and reaction mixture was incubated at 50°C for 20 min. After cooling to room temperature, trichloroacetic acid (1.25 mL, 10%) and FeCl₃ (0.25 mL, 0.1%) were added to the reaction mixture and then vortexed for 1 min; afterward, the absorbance was measured at 700 nm in a spectrophotometer.^[64] High absorbance value of the reaction mixture indicated high reducing capability.^[65]

Cupric ion (cu⁺²) reducing power

Cupric ion reducing power assay was executed on extracts, isolated compounds, and standards.^[66] Antioxidant sample reduced the Cu²⁺ to Cu⁺. After addition of CuCl₂ to the reaction medium, a yellow color complex was formed; then, the absorbance of this complex was measured. High absorbance value indicated the high antioxidant activity. In this assay, extract (50–200 µg/mL, 1.0 mL), isolated compounds, and standards (0.01 M, 1.0 mL) were treated with CuCl₂ (0.01 M, 1 mL), 1 mL neocuproine (7.5 × 10⁻³ M), and 1.0 mL acetate tampon solution. The reaction mixture was incubated for 30 min. Then, absorbance was measured at 450 nm. The result was calculated according to the Trolox equivalence (μmol Trolox/g sample).

Results and discussion

Phytochemical properties of Origanum minutiflorum were presented. Water extract of O. minutiflorum was partitioned with hexane, ethyl acetate, and n-butanol sequentially. The chromatographic techniques were applied on n-butanol extract, which revealed the most antioxidant activity and including highest phenolic contents among the extracts. n-Butanol extract included phenolic compounds (131.3 g/kg extract) equivalent to gallic acid. In addition, this extract displayed the high DPPH^• (IC₅₀: 24.54µg/mL), ABTS^•+ (IC₅₀: 10.70 µg/mL), reducing power (4.63 TEA μmol/g extract), cupric ion reducing power (10.89 TEA μmol/g extract) activities (Table 1). Hence, isolation of bioactive compounds was executed on n-BuOH extract. After a series of chromatographic techniques (column chromatography, semi-preparative HPLC), eleven compounds were isolated and identified (Figure 1). Antioxidant activity assays were carried out on isolated compounds. 3,4-Dihydroxybenzoic acid (4) displayed the outstanding DPPH^• activity. Also, globoidnan-A (7), luteolin (8), rosmarinic acid (9), apigenin-7-O-glucuronide (10) showed the good DPPH^• effect (Table 1). The ability of DPPH^• Activity of these compounds resulted from the acidic hydrogen bearing of the molecules. Luteolin (8), eriodictyol (6), 3,4-dihydroxybenzoic acid (4), apigenin (5) exhibited the excellent ABTS^•+ scavenging. These molecules scavenged the cation radical by donating the hydrogen easily. Additionally, 3,4-dihydroxybenzoic acid (4) was revealed the excellent reducing power activity. Besides, eriodictyol (6), globoidnan-A (7), rosmarinic acid (9) showed the good activity on this assay. In cuprac assay, 3,4-dihydroxy benzoic acid (4), eriodiktiol (6), globoidnan-A (7), luteolin (8), rosmarinic acid (9), apigenin-7-O-glucuronide (10) displayed the better activity than the other isolated compounds. 3,4-Dihydroxy benzoic acid (4) revealed the antioxidant effect in all assays (Table 2). Moreover, compounds 6, 7, 8, 9 inhibited radical formation on three assays. Consequently, O. minutiflorum has a potency to be a natural antioxidant due to presence of antioxidant compounds. It was reported that the composition of O. minutiflorum extracts include thymol, carvacrol, pinene, terpinene, and cymene which are known to have antimicrobial, antifungal, and antioxidant properties.^[65,67–69]

Table 1. Antioxidant activities of Origanum minutiflorum extracts.

Samples	Total phenolics (g GAE/kg extract)	DPPH^• scavenging IC50 (µg/mL)	Reducing power (μmol TE/g extract)	ABTS^•+ scavenging IC50 (µg/mL)	Cuprac assay (μmol TE/g extract)
Hexane extract	28.5 ± 1.7	229.55 ± 9.12	0.58 ± 0.01	65.34 ± 2.48	5.80 ± 0.03
EtOAc extract	102.27 ± 6.88	51.61 ± 1.32	5.68 ± 0.85	11.72 ± 0.17	7.88 ± 0.01
n-BuOH extract	131.32 ± 5.81	24.54 ± 0.56	4.63 ± 0.57	10.70 ± 0.09	10.89 ± 0.16
BHT	-	5.42 ± 0.41	4.33 ± 0.23	5.03 ± 0.01	4.82 ± 0.38
BHA	-	8.20 ± 0.68	7.26 ± 0.26	7.56 ± 0.04	8.14 ± 0.19
Trolox	-	17.26 ± 0.32	3.96 ± 0.26	13.80 ± 0.13	4.13 ± 0.43

Figure 1. Isolated compounds from Origanum minutiflorum.

3,4-Dihydroxybenzoic acid (protocatechuic acid) is a phenolic compound found in many food plants such as Olea europaea (olives), Hibiscus sabdafiffa (rosella), Eucommia ulmoides, Citrus microcarpa, and Vitis vinifera. Protocatechuic acid was reported to exhibit a broad spectrum of biological activities such as antioxidant, antiinflammatory, antihyperglycemia, antiapoptosis, and atimicroabial.^[70]

Rosmarinic acid, a commonly used phenolic acid, is a well-known hydroxycinnamic acid ester, widely found in Lamiaceae family, such as marjoram, basil, mint, sage, rosemary, and perilla.^[71] Rosmarinic acid possessed a large variety of biological activities, including antiinflammatory, antiviral, and antibacterial effect. In addition, it has been used as a fragrance in cosmetic and food additive to prevent food spoilage. Hence, rosmarinic is accepted to be one of the most promising food-functional polyphenol.^[72]

Apigenin (5), a well-known metabolite found in many fruits, vegetables, and herbs, has a broad variety biological effects including, various cancer types, antioxidant, and antiinflammatory.^[73] Origanum minutiflorum has a potency to be a promising medicinal plant for food and pharmaceutical industries on account of including significant bioactive compounds.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

The authors thank TUBITAK for its contributions and to Prof. Dr. Ahmet İLÇİM for the identification of plant species.

Additional information

Funding

This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK project number 114Z020)