Isolation and identification of chemical constituents from Origanum majorana and investigation of antiproliferative and antioxidant activities.

Isolation and identification of chemical constituents from Origanum majorana and investigation of antiproliferative and antioxidant activities

Ramazan Erenlera,*, Ozkan Sena, Huseyin Aksita, Ibrahim Demirtasb,*, Ayse Sahin Yaglioglub, Mahfuz Elmastasa, İsa Telcic

a

Department of Chemistry, Faculty of Art and Science, Plant Research Laboratory-A, Gaziosmanpasa

University, Taslıciftlik Campus, 60240 Tokat, Turkey b

Department of Chemistry, Faculty of Natural Sciences, Plant Research Laboratory-B, Cankiri Karatekin

University, 18100, Cankiri, TURKEY c

Department of Field Crops, Faculty of Agriculture, Gaziosmanpasa University, 60240, Tokat, Turkey

*Corresponding authors Dr. Ramazan Erenler Tel:+90-3562521585/3055 Fax: +90-3562521585 E-mail: [email protected]

Dr. Ibrahim Demirtas Tel: +90-3762189538 Fax: +90-3762189541 E-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.7155

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Abstract BACKGROUND: The Origanum majorana L. belonging to the Lamiaceae family has a great potential and used as folk medicine against asthma, indigestion, headache, and rheumatism; in addition, the essential oils of this plant have been used widely in food industries. The plant materials have been harvested from the Medicinal and Aromatic Plant Field of Gaziosmanpasa University. Air-dried plant materials were boiled in water, filtered then solvent part was extracted subsequently with hexane and ethyl acetate. The chromatographic method was applied for ethyl acetate extract to isolate bioactive secondary metabolites of which the structures were elucidated by spectroscopic techniques, basically 1D-NMR, 2D-NMR and LC-QTOF. Antiproliferative and antioxidant activities were carried out of isolated secondary metabolites. RESULTS: 5,6,3'-trihydroxy-7,8,4'-trimethoxyflavone, hesperetin, hydroquinone, arbutin and rosmarinic acid were isolated from the water-soluble ethyl acetate extract of aerial parts of Origanum majorana. Antioxidant activities of isolated compounds and water-soluble ethyl acetate extract were investigated using the assays of DPPH•, ABTS·+, reducing power and total phenolic contents. The antiproliferative activities of the isolated compounds and plant extracts were investigated against C6 and HeLa cell lines using BrdU Cell Proliferation ELISA and xCELLigence assays, respectively. Both hesperetin and hydroquinone were determined to have stronger antiproliferative activities against C6 and HeLa cells than the other isolated compounds and 5-FU. CONCLUSION: The results showed that the extract and isolated compounds exhibited significant antioxidant activities. Hence, this plant has a potential to be a natural antioxidant in food industries and an anticancer drugs. Key Words: Origanum majorana; chemical constituents; antioxidant activity, structure elucidation, antiproliferative activities

INTRODUCTION

Origanum genus belonging to the Lamiaceae family is represented by 23 species and six hybrids in the flora of Turkey, 14 of which are endemic.1 The species are important aromatic plants widely used in many countries in food industries.2 They are also employed as powerful disinfectants, flavouring agents, in perfumes and in scenting soaps.3 Origanum, well known for their essential oils has been applied in the flavouring of various


foods, particularly soups, sauces, meat, fish, canned foods, liqueurs, vermouths and bitters.4 Some species of these plants are well known in Anatolian folk medicine and widely used as spices and herbal tea.5 The effectiveness is attributed to the specific composition of essential oils,6 flavonoids,7 phenolic acids, and other chemical constituents.8 O. majorana is useful as health complement and in food preservation.9 Traditionally, O. majorana has been used as a remedy against asthma, indigestion, headache, and rheumatism.10 Many phytochemical studies have been conducted so far to investigate the chemical composition of the essential oil of O. majorana.11-13 The essential oil obtained from the flowering heads of O. majorana has aromatic smell and contains high percentage of polyphenols and monoterpenes, which are established antioxidants.14 The essential oil of O. majorana has a great potential for use in food industry since it has antimicrobial properties against food borne bacteria and mycotoxigenic fungi.15 Antioxidants play significant functions in eliminating free radicals, quenching singlet oxygen, disintegrating peroxides, donating hydrogen and chelating metal ion. These properties enable antioxidants to decrease DNA damage, reduce lipid peroxidation and inhibit cell proliferations.16 During the storage and processing, enzymatic oxidation and also auto-oxidation of lipids is the reaction responsible for the deterioration of food quality affecting the flavour, texture, color, and degree of nutrition of the foods.17 Antioxidants are frequently added to foods to prevent the radical chain reactions of oxidation.18 However, synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxy toluene (BHT), are suspected to have some toxic effects and possible carcinogens.19 Hence, there has been a considerable growing trend in consumer priority for natural antioxidants over synthetic compounds, and elimination of synthetic antioxidants in food has given more impetus to explore natural source of antioxidants. Thus, antioxidants are of interest to chemists, biologists, pharmacologists and researches have been carried out to find the natural antioxidants from the plants.20 Although reports on the essential oil compositions and biological activities of O. majorana are relatively common, isolation of the secondary metabolites and investigation of antioxidant activities are rare. In addition, cultivation conditions affect the chemical constituent of the plants. Since the plant we work on, Origanum majorana, is cultivated, its chemical contents differ than the wild one. The isolated compounds were obtained from this plant at first and their structures were elucidated by spectroscopic techniques based on 1D-, 2D-NMR, IR, UV, and HPLC-TOF/MS. The antioxidant activities were carried out of isolated compounds and plant extracts with various methods including total phenolic compound, DPPH• free radical scavenging activity, ABTS cation radical scavenging activity, and reducing power. The results were compared using BHT, BHA and


trolox as standards. The isolated compounds and extracts exhibited high antioxidant activities. The antiproliferative activities of the isolated compounds and plant extracts were investigated against C6 and HeLa cell lines using BrdU Cell Proliferation ELISA and xCELLigence assays, respectively. Hesperetin and hydroquinone were determined to have higher antiproliferative activities than the other isolated compounds and 5-FU.

MATERIALS AND METHODS

General experimental procedures All NMR spectra were measured on a Bruker-400 MHz spectrometer and TMS was used as internal standard. Shimadzu UV-260 UV–Vis spectrometer was used for UV spectra. IR spectra were recorded on a Jasko Spectrometer. EI-MS were measured in a Perkin Elmer. Silica gel (200–300 mesh) for column chromatography and GF254 for TLC were produced by Qingdao Ocean Chemical Group Co. of China. Ammonium thiocyanate and BHT were purchased from E. Merck (Darmstadt, Germany). Ferrous chloride, α-tocopherol, polyoxyethylenesorbitanmonolaurate (Tween-20), 1,1-diphenyl-2-picryl-hydrazyl (DPPH•), 3-(2-pyridyl)-5,6bis (4-phenyl-sulphonic acid)-1,2,4-triazine (Ferrozine), nicotinamide adenine dinucleotide (NADH), BHA, and trichloracetic acid (TCA) were purchased from Sigma (Sigma-Aldrich GmbH, Sternheim, Germany). HPLCTOF/MS analysis was performed on Agilent 6210 TOF-LC/MS.

Plant material The samples of Origanum majorana L. used in the research were grown in experimental area of Gaziosmanpasa University in Tokat, Turkey, and harvested in 2012 at flowering periods. Aerial part of the harvested plant material was dried at room temperature and powdered before extraction procedure.

Extraction and isolation

The aerial parts of the plant material (500 g) were boiled with distilled water for 2 h.21 The aqueous part was sequentially extracted with hexane and ethyl acetate. The ethyl acetate extract was chromatographed on silica gel, eluted with hexane, followed by increasing concentrations of ethyl acetate and methanol in hexane (0-100% ethyl acetate and methanol), respectively to yield 250 fractions. The fractions were checked by TLC (Thin layer


chromatography) to determine retention values (Rf) of the compounds. A particular compound will travel the same distance along the stationary phase by a specific solvent. Therefore the fractions including compounds which have the same Rf value were combined. Fractions 5-15 (35 mg) yielded the compound of hydroquinone, fractions 61-72 (50 mg) were combined and identified as 5,6,3'-trihydroxy-7,8,4'-trimethoxyflavone . The compound in fractions 120-140 (150 mg) was detected as arbutin (alpha and beta as a mixuture) and rosmarinic acid was obtained from the fractions of 170-210 (200 mg). The fractions of 75-100 were combined and subjected to column chromatography, eluted with dichloromethane, followed by increasing concentrations of ethyl acetate (0-100%), respectively to yield 40 fractions. The hesperetin was identified from fractions 15-25 (35 mg). After mixing the fractions to obtain same pure compounds, some impurities have been detected. To eliminate these impurities, preparative thin layer chromatography was executed.

Cell culture and cell proliferation assays ELISA assay Antiproliferative effects of the compounds were investigated on C6 (rat brain tumor) cell lines using proliferation BrdU ELİSA assay.22, 23 5-Fluorouracil (5-FU) was used as positive controls. C6 cells were grown in Dulbecco's modified eagle’s medium (DMEM, Sigma), supplemented with 10% (v/v) fetal bovine serum (Sigma, Germany) and PenStrep solution (Sigma, Germany) at 37°C in a 5% CO2 humidified atmosphere. For proliferation assay, cells were plated in 96-well culture plates (COSTAR, Corning, USA) at a densitiy of 30.000 cells per well. 5-FU was used as a standard. The activities of samples and standard were investigated on eight concentrations (5, 10, 20, 30, 40, 50, 75 and 100 µg/mL). Cells were then incubated overnight before applying the BrdU Cell Proliferation ELISA assay reagent (Roche, Germany) according to the manufacturer’s procedure. Briefly, cells were pulsed with BrdU labeling reagent for 4 h followed by fixation in FixDenat solution for 30 min at room temperature. Thereafter, cells were incubated with 1:100 dilution of anti- BrdU-POD for 1.30 h at room temperature. The amount of cell proliferation was assessed by determining the A450 nm of the culture media after addition of the substrate solution by using a microplate reader (Awareness Chromate, USA). Results were reported as percentage of the inhibition of cell proliferation, where the optical density measured from vehicle-treated cells was considered to be 100% of proliferation. All assays were repeated at least twice using against HeLa and C6 cells. Percentage of inhibition of cell proliferation was calculated as follows: [1-(Atreatments /Avehicle control) × 100. The stock solution of samples and 5-FU were prepared in DMSO and diluted with


Dulbecco’s modified eagle medium (DMEM). DMSO final concentration is below 0.1% in all tests. The half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a compound in inhibiting biological function. In this paper, IC50 and IC75 values were determined using ED50 plus v1.0. xCELLigence assay The Real Time Cell Analyzer-Single Plate (RTCA-SP) instrument (Roche Applied Science, Basel, Switzerland) was used to analyse the proliferation effects of the ethyl acetate and hexane extracts and isolated compounds from O. majorana on human cervical cancer (HeLa) cells. A newly developed electronic cell sensor array – the xCELLigence real-time cell analyser- was used with recently published literature method at the concentrations of 100, 50 and 10 µg mL-1.24 Ferric ions (Fe3+) reducing antioxidant power assay (FRAP) The reducing powers of extract and isolated compounds were determined by given method.25 Sodium phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 mL, 1%) were mixed with different concentrations of samples (1.25-5 µg/mL) in 1 mL of distilled water. The incubation was carried out at 50 ºC for 20 min. The sample (2.5 mL) of trichloroacetic acid (10%) was added to the mixture. The solution was mixed with distilled water (2.5 mL) and FeCl3 (0.5 mL, 0.1%), and the absorbance was measured at 700 nm in a spectrophotometer. Increase absorbance of the reaction mixture indicates an increase of reduction capability.

DPPH• free radical scavenging activity The donation ability of electron or hydrogen atom of compounds was measured by the bleaching of a purple colored methanol solution of DPPH•. The free radical scavenging activities of crude extract, isolated compounds (1, 2, 3, 4, 5) and standards were measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH• ).26 Within a stable free radical bleaching rate, DPPH• is monitored at a characteristic wavelength in the presence of the sample. In its radical form, DPPH• absorbs at 517 nm, but upon reduction by an antioxidant or a radical species its absorption decreases. When a hydrogen atom or electron was transferred to the odd electron in DPPH•, the absorbance at 517 nm decreased proportionally to the increases of non-radical forms of DPPH•. Briefly, 0.1 mM solution of DPPH• in methanol was prepared and 1 mL of this solution was added 3 mL of samples at different concentrations (2.5-10 µg/mL). The mixture was stirred and allowed at room temperature for 30 minutes. Then the absorbance was measured at 517 nm in a spectrophotometer. The ability of scavenge the DPPH• radical was calculated with the equation: DPPH• scavenging effect (%) = [(Ac – As) / Ac] 100 Where Ac is the absorbance of the control and As is the absorbance in the presence of samples or standards.21


ABTS radical cation decolorization assay

ABTS·+ scavenging activity was determined according to the literature. 27 This method is based on the ability of antioxidants to quench the long-lived ABTS radical cation, a blue/gren chromophore with characteristic absorption at 734 nm, in comparison to that of BHA, BHT, and trolox. The ABTS·+ was produced by reacting 2 mM ABTS in H2O with 2.45 mM potassium persulfate (K2S2O8), stored in the dark at room temperature for four hours. Before usage, the ABTS·+ solution was diluted to get an absorbance of 0.750 ±0.025 at 734 nm with sodium phosphate buffer (0.1 M, pH 7.4). Then, 1 mL of ABTS·+ solution was added to 3 mL of each samples solution in ethanol at different concentrations (1.25–10 µg/mL). After 30 min, the percentage inhibition of at 734 nm was calculated for each concentration relative to a blank absorbance. Blank solvents were run in each assay. The extent of decolorization is calculated as percentage reduction of absorbance. For preparation of a standard curve, different concentrations of ABTS·+ was used. The ABTS·+ concentration (mM) in the reaction medium was calculated from the following calibration curve, determined by linear regression (r2:0.9841): Absorbance = 4.6788 ×[ ABTS·+] + 0.199 The scavenging capability of ABTS·+ radical was calculated using the following equation: ABTS·+ scavenging effect (%) = [(Ac – As) / Ac] × 100 where, Ac is the initial concentration of the ABTS·+ and As is absorbance of the remaining concentration of ABTS·+ in the samples.28 Determination of Total Phenolic Compounds Total soluble phenolic compounds in extract of leaves and seeds were determined with Folin-Ciocalteu reagent29 using gallic acid as a standard phenolic compound. Briefly, 1 mL of sample solution (containing 1000 mg sample) was placed in a volummetric flask diluted with distilled water (46 mL), 1 mL of Folin-Ciocalteu reagent was added, and the content of the flask was mixed thoroughly. After 3 minutes, 3mL of Na2CO3 (2%) were added, then the mixture was allowed to stand for 2 hours with intermittent shaking. The absorbance was measured at 760 nm in a spectrophotometer. The concentrations of total phenolic compounds in the samples were determined as micrograms of gallic acid equivalent by using an equation that was obtained from standard gallic acid graph: Absorbance = 0.0053 × Total phenols [Gallic Acid Equivalent (µg)] – 0.0059.


HPLC-TOF/MS Analysis Agilent 6210 HPLC-TOF/MS with column Poroshell 120 EC-C18, 3.0x50 mm, 2.7 µm (Agilent Technologies, USA) with an injection volume of 10 µL was used. The mobile phase consisted of the eluent A - water with 0.1% formic acid and 5 mM ammonium formate and B - acetonitrile. The flow rate was 0.7 mL/min at 35 °C. The gradient program was fixed as follows: 0-1 min, 10% B; 1-8 min, 10% B; 8-11.1 min, 95% B; 11.1-13 min, 10% B; 13-14 min, 10% B. Total time of evaluation was 14 min. TOF analyses were executed in negative ion mode; gas temperature, 325 °C and column temperature, 35 °C; drying gas flow, 0.7 mL/min; fragmentor voltage, 175 V.

UV Analysis The phenolics in complex plant extracts determined with classical ultraviolet (Shimadzu UV-260 UV–Vis spectrometer). To obtain qualitative analysis, ultraviolet (at 280 and 340 nm) detection was used.

Statistical Analysis The experimental results were performed in triplicate. The data were recorded as mean ± standard deviation and analyzed by SPSS (version 11.5 for Windows 2000, SPSS Inc.). The associations between the inhibition and concentrations of samples were analyzed by the Pearson correlation coefficient. RESULTS AND DISCUSSION

Extraction of polyphenols Origanium species are mostly consumed by dissolving in boiling water.3 Therefore investigation of watersoluble extracts is crucial for determination of bioactive compounds causing the pharmaceutical effects in these extracts. Treatment of plant with boiling water before extraction with organic solvents is an essential part of the isolation of phenolic contents. It is responsible for softening of plant tissue and reducing the separation steps, resulting in a better water soluble extract. Phenolic compounds can be found in boiling waters particularly due to solution of hydrolysable tannins and flavonoids and water-soluble lignin fragments solved under acidic conditions. Based on this knowledge, we performed the extraction by treatment of plant with boiling water, then extracting of the aqueous parts with organic solvent.

Phytochemical content


5,6,3'-trihydroxy-7,8,4'-trimethoxyflavone (1) (Fig. 1)was obtained as yellow prism crystals. Its molecular formula was established as C18H16O8 by HPLC-TOF/MS (m/z 359.0873 [M-H]) (Fig. 1). The 13C (APT) NMR spectrum confirmed the presence of 18 carbons consisting of three methoxyl, four methines and eleven quaternary carbons, verifying the flavonoid structure. The UV spectrum also showed presence of a flavone at 288, 340 nm. IR absorptions suggested the presence of hydroxy groups (3451 cm-1), a carbonyl group (1641 cm1

). The 1H-NMR spectrum showed signals at δ 7.56 (1H, dd, J = 8.4 Hz, J = 2.1 Hz), and 6.90 (1H, d, J = 8.4

Hz), methoxy singlet δ 3.93 (3H, s) are characteristic for the B ring, along with two methoxy singlets at δ 3.90 (3H, s), δ 3.96 (3H, s). Based on this data (Table 1), compound can be assigned as 5,6,3'-trihydroxy-7,8,4'trimethoxyflavone.30 Hesperetin (2) was obtained as white needles. Its molecular formula was based on C16H14O6 by HPLC-TOF/MS (m/z 301.1042 [M-H]) (Fig. 1). The 13C (APT) NMR spectrum confirmed the presence of 16 carbons comprising of a methoxyl, a methylene, five methines and eight quaternary carbons. The UV spectroscopy showed presence of a flavonone at 288, 330 nm. IR absorptions suggested the presence of a hydroxy groups (3475 cm-1), a carbonyl group (1641 cm-1). The 1H-NMR spectrum showed the signals at δ 2.71 (1H, dd, J = 3.1 Hz, J = 17.1 Hz), indicating the existence of single bond and H3 proton coupled with H3' and H2. The signals observed at δ 5.43 (dd, J = 3.1 Hz, J = 17.1 Hz) belonged to H2, which coupled with H3 and H3'. H6 resonated at δ 6.08 (1H, d, J = 2.2 Hz) which coupled with H8 as meta coupling (Table 1). The signals appeared at δ 6.10 as a doublet which coupled with H6 belonged to H8. Based on this data, compound can be assigned as hesperetin.31 Hydroquinone (3) was obtained as white solid. The UV spectroscopy showed presence of an aromatic ring at 286, 331 nm. IR absorptions suggested the presence of a hydroxy group (3320 cm-1), and an aromatic ring (1608, 1517, and 1465 cm-1). In 1H-NMR spectrum, a singlet was observed at δ 6.55 (4H) due to the high symmetrical structure. Moreover, appearance of two lines in 13C-NMR spectrum accord with the proposed structure32 (Table 2). Arbutin (4) was obtained as white needles. Its molecular formula was established as C12H16O7 by HPLCTOF/MS (m/z 271.0641 [M-H]). The UV spectroscopy showed presence of aromatic ring at 292, 336 nm. IR absorptions suggested the presence of hydroxy groups (3370 cm-1), an aromatic ring (1610, 1513, and1411 cm1

). The 1H-NMR spectrum showed signals at δ 6.87 (1H, d, J = 9.0 Hz), and 6.66 (1H, d, J = 8.9 Hz), as well as

13

C-NMR signals at δ 102.1 (C-1') and 61.2 (C-6') suggesting the presence of a glucoside moiety. The

comparison of the spectral data with the literature confirmed the compound as arbutin. 33 It is impossible to


distinguish the alpha arbutin and beta arbutin by 1H- and 13C-NMR spectrum, since chemical shifts of both arbutins are the same in 1H- and 13C-NMR (Table 2). These two compounds (α-arbutin and β-arbutin) were detected by HPLC-TOF/MS analysis by using chiral column. In HPLC-TOF chromatogram of extract, each peak corresponds to one compound. Two peaks were observed at the acquisition time of 2.5 s and 3.5 s belonging to the α-arbutin and β-arbutin (Fig 2 A). In addition, in HPLC-TOF chromatogram of arbutin compound, two peaks were observed corresponding to the same molecular weights. Therefore it is clear that the compound consists of α-arbutin and β-arbutin (Fig. 3). Rosmarinic acid (5) was obtained as white needles. Its molecular formula was established as C18H16O8 by HPLC-TOF/MS (m/z 359.0873 [M-H]) (Fig. 3). The UV spectroscopy showed presence of aromatic ring at 288, 328 nm. IR absorptions suggested the presence of hydroxy groups (3405 cm-1), a carbonyl group (1600 cm-1). Caffeic acid and β-(3,4-dihydroxyphenyl) glyceric acid were linked together to form compound 5, which was established by application of a long-range coupling experiment (HMBC), which showed coupling between H-8' (δ 5.08) and C-9 (δ 166.1). The 1H-NMR spectrum showed signals at δ 6.24 (H8) and δ 7.46 (H7) as a doublet with coupling constant 15.8 Hz. This could be attributed to the alpha-beta unsaturated carbonyl moiety. The 13C (APT) NMR spectrum confirmed the presence of 18 carbons comprising of a methylene, nine methines and eight quaternary carbons. Upon the basis of all spectral data, the compound was elucidated as rosmarinic acid (Table 2).34, 35 As the separation of hydroquinon, 5,6,3'-trihydroxy-7,8,4'-trimethoxyflavone, hesperetin, rosmarinic acid and arbutin in complex plant extracts yields some problems such as slight differences in retention times among several components in this study a classical ultraviolet (UV, Fig. 4), together with a infrared (FTIR) and mass (HPLC-TOF/MS) were used for monitoring of phenolics in samples examined. To obtain precise, accurate and validated results of qualitative analysis, ultraviolet (at 280 and 340 nm) detection was used.

Antiproliferative activities C6 cells Antiproliferative activities of samples were determined against C6 cells. The IC50 and IC75 values of the the compounds against C6 were given at Table 3. The antiproliferative activities of EtOAc and hexane extracts were shown to increase of the activities depending to dose increasing against C6 cells. The hexane extracts were determined to have the higher antiproliferative activity than the EtOAc extract and 5-FU at 75 and 100 µg/mL concentrations (Fig. 5). The potency of inhibitions (at 100 µg/mL) against C6 cells were: Hexane extracts > 5-


FU > EtOAc extract. The antiproliferative activities of EtOAc extracts and isolated compunds were shown to increase of the activities depending to dose increasing aganist C6 cells. Hesperetin and hydroquinone were determined to have stronger antiproliferative activity than the other isolated compounds and 5-FU at 75 and 100 µg/mL concentrations (Fig.6). Hesperetin has been shown to inhibit mammary,36 urinary bladder,37 and colon38, 39

carcinogenesis in laboratory animals. Treatment of human gastrointestinal carcinoid (BON) cells with

hesperetin inhibits cell growth and decrease to the expression of tumor markers.39 The antiproliferative activity of arbutin was not found to have at all doses. The potency of inhibitions (at 100 µg/mL) against C6 cells were: Hesperetin> Hydroquinone> 5-FU > EtOAc extract> Rosmarinic acid ~ 5,6,3'trihydroxy-7,8,4'-trimethoxyflavone > Arbutin.

HeLa cells Figure 7 shows the results of real time cell monitoring of the proliferation of HeLa cells treated with hexane, ethyl acetate extracts and hydroquinone isolated from ethyl acetate extract as a the highest antiproliferative activities. Differences in antiproliferative activities between hexane and ethyl acetate extracts are dependent on their phytochemical composition. The cell index measurements provide a clear indication that the anticancer activities of the solvent extracts are not similar. The higher activities of the ethyl acetate extract could be due to the lower quantity (0.053756 g kg-1) of the hydroquinone (see Table 3 and Fig. 7). However, the major compounds (hesperetin and 5,6,3'-trihydroxy-7,8,4'-trimethoxyflavone see Table 3) and other isolated compounds have the lower activities in the present work (Fig. 8). The compounds with higher quantities (hesperetin and 5,6,3'-trihydroxy-7,8,4'-trimethoxyflavone) exhibited the higher activities at higher concentrations (100 and 50 µg mL-1) as seen in Figure 8 and Table 3. There was a strong positive correlation between the inhibition and concentrations of samples (except arbutin) by the Pearson correlation coefficient. Arbutin has a moderate negative correlation. The correlation between inhibition and concentrations of samples is shown in Table 3. Antioxidant activities Antioxidant capacity is widely used as a parameter for medicinally bioactive and functional components in food. In the present study, the antioxidant activities of crude extract of Origanum majorana and isolated compounds from this extract were analyzed and compared to BHA, BHT and trolox as positive control. The mechanisms of antioxidant activity of flavonoids are well discussed but the mechanisms and structural requirements have not been fully understood 40. Flavonoids behave as antioxidants by free radical scavenging mechanism to form less


reactive phenoxyl radicals. The antioxidant capacities of flavonoids as scavenging free radicals may be defined as the ability to donate hydrogen atoms from their hydroxyl groups, therefore flavonoid phenoxyl radicals gaining the resonance stability and a stable molecule are forming.40 The hydroxyl groups of isolated molecules donating hydrogen lead to the formation of less reactive flavonoid phenoxyl radicals. This is the basis of its ability to prevent lipid peroxidation chain reactions. The prevention process of lipid peroxidation leads to the identification of antioxidants as reducing agents that prevent oxidative reactions, often by scavenging ROS before damaging cells.41 The antioxidant effects of phenolic compounds have been studied in relation to the prevention of coronary diseases and cancer, as well as age-related degenerative brain disorders.

42

In addition,

phenolic compounds were associated with antioxidant activity and play an important role in stabilizing lipid peroxidation.43 Previous work revealed that O. majorana essential oils exhibited the antioxidant,44 antifungal,45 antimicrobial,46 antibacterial47, 48 activities. O. marjorana ethanolic extract also showed the anti-metastatic and anti-tumor growth effects on human breast cancer cells.49 Ethanol extract of O. majorana leaves exhibited antiproliferative effect and high antioxidant activity.50 Quantitative analysis of arbutin in O.majorana was presented.51 The salt reduced the aerial part growth but increased the fatty acid content of O. majorana.52 The scientific works were carried out on the other species of Origanum, such as: A correlation was identified between the total phenolic content of the essential oils and DPPH• radical scavenger capacity of O. glandulosum.53 The essential oil composition, phenolic constituents and antioxidant activities of Turkish Origanum onites leaves harvested during the months of June to September were determined.54 The essential oil of O. minutiflorum exhibited the strong antimicrobial activities.55 O. syriacum essential oil and extract revealed the high antioxidant and antimicrobial activities.

56

Herein, we revealed antioxidant activities of extracts and

isolated compounds from O.majorana and presented the responsible compound for activity at the plant extract. The isolated compounds have a pharmaceutical and medicinal importance. Arbutin, isolated from ethyl acetate extracts is a hydroguinone glycoside that has the anti-inflammatory,57 anti-ulcerogenic,58 antityrosinase,59 activities. It has also been used as whitening agent in cosmetic products.60, 61 The hydrolyze of arbutin lead to hydroquinone. The most antioxidant active compound, hydroquinone has been used of a cholesterol biosensor.61 Hydroquinone derivatives revealed the activities against P388 lymphocytic leukemia, inhibits superoxide anion production in rat, exibited antiproliferative activity, displayed significant cytotoxicity against tumor cell lines.62 According to these results, it may be concluded that O. majorana should be acknowledged as a promising source of non-toxic natural antioxidants, which could be used for cultivation and for breeding programs.


Ferric ions (Fe3+) reducing antioxidant power assay (FRAP) The presence of reductants such as antioxidant materials in the antioxidant samples brings about the reduction of the Fe3+/ferricyanide complex to the ferrous form. Therefore, Fe2+ can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm.63 The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. In this assay, the yellow colour of the test solution changes to various shades of green and blue depending on the reducing power of antioxidant samples. Figure 9 illustrated reduction power activities of crude extract, isolated compounds (1, 2, 3, 4, 5), BHA, BHT and trolox. At different concentrations (5-20 μg/mL), crude extract, isolated compounds, BHA, BHT and trolox demonstrated reducing ability. The reducing power of crude extract, compounds (1, 2, 3, 4, 5), BHA, BHT and trolox increased steadily with increasing concentrations. Reducing power of crude extract, isolated compounds, and standards exhibited the following order (5 µg/ml): hydroquinone (0.92) > BHA (0.91) > EtOAc extract (0.79) > 5,6,3'-trihydroxy-7,8,4'-trimethoxyflavone (0.65) > BHT (0.62) > arbutin (0.53) > hesperetin (0.47) > trolox (0.46) > rosmarinic acid (0.4) > hexane extract (0.1). The results on reducing power present the electron donor properties of ethyl acetate extract and isolated compounds, thereby neutralizing free radicals by forming stable products. The isolated compounds reduced the Fe3+/ferricyanide complex to the Fe+2/ferrous form. All isolated compounds and EtOAc extract have the hydroxyl groups which have the ability of formation complexes with iron metal. Hydroxyls groups of the molecules donated the electron pairs to the iron metal with coordinate covalent bond to form the metal compexes. The antioxidant activity of isolated compounds and EtOAc extract could attribute to the hydroxyl groups of the molecules that donate the electron pairs to the metal.

DPPH• free radical scavenging activity

The DPPH• assay was based on the measurement of altering color of DPPH• radical from purple to yellow at 517 nm after reaction with antioxidant compound. The effect of antioxidants on DPPH• radical was thought to be due to their hydrogen donating ability. DPPH• is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule.64 The isolated compounds and water-soluble extract exhibited very high activities. Water soluble ethyl acetate extract exhibited higher activity than the standards. In comparison standards and isolated compounds, the order of activity is as follows: (IC50, µg/ml), hydroquinone


(1.92) > EtOAc (2.77) > BHA (3.47) > Trolox (4.88) > Hesperetin (6.48) > rosmarinic acid (8.89) > 5,6,3'trihydroxy-7,8,4'-trimethoxyflavone (14.50) > BHT (14.78) > Arbutin (45.02) (Fig. 9).

ABTS radical cation decolorization assay The antioxidant ability of extracts and isolated compounds to scavenge the blue-green colored ABTS radical cation was measured relative to the radical scavenging ability of BHA, BHT and Trolox. The result indicates that water soluble ethyl acetate extract and isolated compounds (1, 2, 3, 4, 5) have high ABTS radical cation scavenging activity, which exhibited the following order (IC50, µg/ml): hydroquinone > hesperetin > EtOAc extract > 5,6,3'-trihydroxy-7,8,4'-trimethoxyflavone > BHA > BHT > rosmaniric acid > trolox > arbutin > hexane extract (Fig. 9). ABTS·+ scavenging method has been used to carry out the antioxidant activity of compounds due to its simple, rapid and sensitive procedure. In this method, antioxidants oxidize the ABTS to its radical cation form, ABTS·+ which is deeply coloured and antioxidant capacity of compounds are measured according to the decreasing colour of ABTS·+ due to the reaction of ABTS·+ with antioxidant compounds leading to the formation of ABTS cation. ABTS·+ is applicable for both lipophilic and hydrophilic compounds.65 In this assay, hydroquinone bearing two hyroxyl groups donates the hydrogen atom to the ABTS·+ so hydroquinone exhibited the most antioxidant activities.

Determination of total phenolic compounds Phenols and related compounds have antioxidant potentials due to the hydroxyl groups which the acidic protons could be donated easily. 111.8 and 8485.9 g gallic acid equivalent of phenols were detected in hexane extract and ethyl acetate extract respectively. Due to the lack of phenolic contents of hexane extract, it does not exhibit the antioxidant activity whereas the ethyl acetate extract containing significant phenolic compounds displays strong antioxidant activity. The total phenolic contents determination in plant was carried out by Folin-Ciocalteu method, which depended on electrons trasfer from phenolic compounds to the Folin-Ciocalteu in alkaline medium. As the reference standard compound, gallic acid is used, and result was described as gallic acid equivalents.

CONCLUSION


The application of the effective extraction procedure for the water soluble phenolic compounds in combination with variable (column chromatography, flash chromatography, PTLC) separation techniques made the superior to traditional separation techniques used for the water soluble phenolics. The water-soluble phenolics extracted with ethyl acetate and isolated compounds (1, 2, 3, 4, 5) exhibited strong antioxidant activity, therefore Origanum majorana can be used as a natural antioxidant in food industry. It was presented that hydroquinone is the responsible antioxidant compound at the ethyl acetate extract. Hesperetin and hydroquinone were determined to have high antiproliferative activities. The strong correlations with conventional method (ELISA) and a new method (xCELLigence) imply a similar observation of cell behavior and higher activities with hydroquinone at different cells (C6 and HeLa, respectively). Pharmaceutically and medicinally valuable 5,6,3'trihydroxy-7,8,4'-trimethoxyflavone and hesperetin were isolated for the first time from this plant.

ACKNOWLEDGMENTS This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK, 113Z195). References

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OH

OCH3

OCH3 O

OCH3 HO

O HO

OH OH

O

OH

OH

O

3

1 2

OH

OH O OH OH HO

O O

O

O

OH

HO OH

4 Figure 1. Structures of compounds isolated from O. majorana


5

OH

OH

A

Rosmarinic acid

5,6,3'-trihydroxy-7,8,4'trimethoxyflavone

Arbutin

Hydroquinone

Hesperetin

OH OCH3

OCH3 H3CO

O

OH OH

O 1

OH OCH3 HO

O

OH

O 2

Figure 2. HPLC-TOF/MS chromatogram of extract (A) and spectrums of 5,6,3'-trihydroxy-7,8,4'trimethoxyflavone (1) and Hesperetin (2)


OH OH OH O

HO

OH

O 4

OH O

O

OH

OH

O HO OH

5

Figure 3. HPLC-TOF/MS spectrums of arbutin (4) and rosmarinic acid (5)


Figure 4. UV spectrum of 5,6,3'-trihydroxy-7,8,4'-trimethoxyflavone, hesperetin, hydroquinone, arbutin and rosmarinic acid

Figure 5. The antiproliferative activity of O. majorana extracts against C6 cell. *each substance was tested twice in triplicates against cell lines. Data show average of two individual experiments (p

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