A. ÖZKAN, A. ERDOĞAN

Turk J Biol 35 (2011) 735-742 © TÜBİTAK doi:10.3906/biy-1011-170

A comparative evaluation of antioxidant and anticancer activity of essential oil from onites () and its two major phenolic components

Aysun ÖZKAN, Ayşe ERDOĞAN Department of Biology, Faculty of Science, Akdeniz University, 07058 Antalya - TURKEY

Received: 23.11.2010

Abstract: Th e objectives of the present study were to determine the eff ects of essential oil from Turkish (Origanum onites L.) and its 2 major phenolic components, carvacrol and thymol, on cell viability, and to investigate their cytoprotective (antioxidant) eff ects against hydrogen peroxide-induced cytotoxicity and membrane damage in hepatoma G2 (Hep G2) cells. Th eir antioxidant properties were also evaluated with 2 in vitro complementary test systems: DPPH radical scavenging activity and linoleic acid oxidation inhibition. Th e aerial parts of the essential oil were isolated using a Clevenger-type apparatus (hydrodistillation) and its components were analyzed by gas chromatography/mass spectrometry (GC/MS). Th e essential oil of O. onites decreased cancer cell viability at higher concentrations. Th e essential oil was found to be less cytototoxic (IC50: 149.12 μg/mL) than carvacrol and thymol (IC50: 53.09 and 60.01 μg/mL, respectively) for Hep G2 cells. On the other hand, with the O. onites essential oil, carvacrol and thymol protected the cells against H2O2-induced cytotoxicity when the cells were preincubated with the oil and its components at a lower concentration (

Key words: O. onites, essential oil, antioxidant, anticancer

Origanum onites (Lamiaceae)’den elde edilen uçucu yağın ve iki önemli fenolik bileşeninin antioksidan ve antikanser aktivitelerinin karşılaştırılması

Özet: Bu çalışmanın amacı, uçucu yağın ve onun iki önemli bileşeni olan karvakrol ve timol’ün hücre canlığı ve hepatoma G2 (Hep G2) hücrelerinde hidrojen peroksidin indüklediği sitotoksisite ve membran hasarına karşı koruyucu etkisinin araştırılmasıdır. Uçucu yağın ve bileşenlerinin antioksidan özelliği, birbirini tamamlayan iki in vitro test sistemi ile ortaya konmuştur, bu testler; DPPH radikalini ortadan kaldırma ve linoleik asit oksidasyonu inhibisyonu testidir. Bitknin toprak üstü organlarının uçucu yağı Clevenger (su buharı distilasyon) cihazı ile izole edilmiş, bileşenler ise gaz kromotografi si/kütle spektrometresi (GC/MS) yöntemi ile analiz edilmiştir. Çalışılan yağ, Türk kekiğinden elde edilmiştir (Origanum onites L.). O. onites’ten elde edilen uçucu yağ yüksek konsantrasyonlarda, kanser hücrelerinin

735 A comparative evaluation of antioxidant and anticancer activity of essential oil from Origanum onites (Lamiaceae) and its two major phenolic components

canlılığını azaltmıştır. Hep G2 hücrelerinde, uçucu yağ (IC50: 149,12 μg/mL), karvakrol ve timol’den (IC50: 53,09 ve

60.01 μg/mL) daha az toksik bulunmuştur. Diğer taraft an, Hep G2 hücrelerinin H2O2 uygulamasından önce, düşük konsantrasyonlarda O. onites uçucu yağı, karvakrol ve timol (

H2O2 tarafından indüklenen sitotoksisiteye karşı korumuştur. Malondialdehit miktarı, H2O2’e (IC50 ve IC70) maruz

bırakılan hücrelerde artmış, fakat H2O2’e maruz bırakmadan önce uçucu yağ ve bileşenleri ile ön-uygulamaya maruz bırakılan hücrelerde azalmıştır. Uçucu yağ, timol ve karvakrolden daha yüksek membrane koruyucu etki göstermiştir. Uçucu yağın en önemli üç bileşeni sırasıyla linalool (% 50,53), carvacrol (% 24,52) ve thymol (% 15,66) olarak tanımlanmıştır. Uçucu yağın DPPH radikali süpürücü aktivite (EC50: 80 μg/mL) karvakrol ve timolden daha yüksek bulunmuştur. Uçucu yağın linoleik asit oksidasyonunu inhibisyon oranı (% 40) diğer iki majör bileşene yakındır. Bu bulgular, O. onites’den elde edilen uçucu yağın ve onun iki önemli bileşeninin antioksidan aktiviteye ve karsinogenezi azaltıcı potansiyele sahip olduğunu göstermektedir.

Anahtar sözcükler: O. onites, uçucu yağ, antioxidan, antikanser

Introduction 2-(1-methylethyl)phenol) is an isomer of carvacrol, Natural antioxidants are considered to be useful having the hydroxyl group at a diff erent location agents for the prevention of diseases (1-4). Many on the phenolic ring. Th e hydrophobic nature of studies have shown that phenolic compounds in carvacrol and thymol enables them to react with essential oils display antioxidant activity as a the lipids of the cell membrane and mitochondria, result of their capacity to scavenge free radicals (5,6). rendering them permeable and leading to leakage of It has been shown that many species of Origanum the cell components (18). have a high amount of phenolic contents in their We tried to prove the essential oil’s ability essential oils (7-9). Herbal parts of the Origanum to prevent the cytotoxicity of Hep G2 cells and species are aromatic and are used as condiments or membrane damage induced by the oxidative agent herbal tea. Dried Origanum species are also used for H2O2, as compared to its 2 major components, the production of essential oil (Origanum oil) and an because many plant oils and their components have aromatic water or hydrosol (Origanum water) (10). been shown to possess various protective activities in Identifying the main components of the essential oil, diff erent biological systems. testing their safety, and uncovering their medicinal value in disease treatment will provide an important Materials and methods means for drug development and drug targeting therapy. Being lipophilic in nature, essential oils will Plant material cross the plasma membrane and may exert their eff ect Origanum onites samples were collected during by interacting with intracellular proteins and/or the fl owering season from Elmalı, Antalya, Turkey, intraorganelle sites (11-13). Monoterpenes are highly in August 2006. Th ey were identifi ed by senior hydrophobic substances present in plant essential taxonomist Dr. Orhan Ünal. Th e voucher specimen oils. Th ey exert a wide spectrum of biological actions was deposited at the Herbarium of the Department of great importance in many diff erent areas (14). Th e of Biology, Akdeniz University, Antalya, Turkey major compounds of O. onites’ essential oil, as with (Vou cher No: 325-OO). many herb oils, are carvacrol (71.22%) and thymol Th e essential oil of O. onites was obtained by (5.97%) (15). Carvacrol (2-methyl-5-(1-methylethyl) hydrodistillation in 100 mL of H2O for 3 h with a phenol), a cyclic monoterpene, is a constituent of Clevenger apparatus (İLDAM Ltd., Ankara, Turkey) the essential oil of oregano. Th e mode of action of in the Molecular Biology Department of Akdeniz carvacrol has received considerable attention from University. Th e major components of the essential researchers because of its use in fl avoring, and oil were purchased from diff erent manufacturers: also as an antibacterial or antifungal agent in food carvacrol from Aldrich at 98% purity and thymol preservation methods (16,17). Th ymol (5-methyl- from Sigma at 99.5%.

736 A. ÖZKAN, A. ERDOĞAN

Gas chromatography/mass spectrometry (GC/ of these values, Microsoft Excel was used. Values are MS) analysis presented as means ± SD of 5 parallel measurements. Th e oils, dried over anhydrous sodium sulfate, β-Carotene/linoleic acid assay: In this assay, the were subsequently analyzed by GC/MS and stored at antioxidant capacity was determined by indirectly –20 °C. Th e composition of the volatile constituents measuring the inhibition of the volatile organic was established by GC/MS quadruple detector compounds and the conjugated diene hydroperoxides analyses, using the Shimadzu QP 5050 system fi tted arising from linoleic acid oxidation (21). A stock with a free fatty acid phase (FFAP) capillary column solution of a β-carotene/linoleic acid mixture was (50 m × 0.32 mm (i.d.), fi lm thickness: 0.25 mm). prepared as follows: fi rst, 0.5 mg of β-carotene was Th e detector and injector temperatures were set at dissolved in 1 mL of chloroform (HPLC grade), and 240 °C and 250 °C. Th e temperature program for the then 25 μL of linoleic acid and 200 mg of Tween FFAP column went from 60 °C (1 min) to 220 °C at 40 were added. Th e chloroform was completely a rate of 2 °C/min, and was then held at 220 °C for evaporated using a vacuum evaporator, and then 20 min. Helium was used as a carrier gas at a fl ow of 100 mL of distilled water, saturated with oxygen (30 14 psi (split 1:10) and the injection volume of each min at a fl ow rate of 100 mL/min), was added with sample was 1 mL. Th e percentage composition was vigorous shaking. A 2.5-mL amount of this reaction computed from the GC peak areas according to the mixture and a 350-μL portion of the samples (2 mg/ 100% method, without using any correction factors. mL) in ethanol were dispensed into each test tube and Th e identifi cation of the components was based on incubated for up to 48 h at room temperature. Aft er the comparison of their mass spectra with those of this incubation period, the absorbance of the mixture Wiley and NIST tutorial libraries. Th e ionization was measured at 490 nm . Values are presented as energy was set at 70 eV. means ± SD of 5 parallel measurements. Measurement of antioxidant capacities using Cancer cell culture the two in vitro assays Th e hepatoma G2 cell (Hep G2) line was 2,2’-Diphenyl-1-picrylhydrazyl (DPPH) assay: purchased from the American Type Culture Th e hydrogen atoms or the electron donation abilities Collection (Rockville, MD, USA). Cells were of the corresponding samples were measured from routinely cultured in Dulbecco’s Modifi ed Eagle’s the bleaching of a purple-colored methanol solution Medium (DMEM), supplemented with 10% fetal calf of DPPH. Th is assay was carried out following the serum, and 1% antibiotic-antimycotic solution in a same method as reported elsewhere (19,20). Added humidifi ed atmosphere containing 5% CO at 37 °C. to 5 mL of a 0.004% methanol solution of DPPH 2 For subculturing, cells were harvested aft er a trypsin/ (2,2-diphenyl-1-picrylhydrazyl, free radicals) were EDTA treatment at 37 °C. Cells were used when the 50 μL of various concentrations of the samples monolayer confl uence had reached 75%. dissolved in methanol. Aft er a 30-min incubation period at room temperature, the absorbance was Cell viability assay: Th e cancer cells (500 cells/ read against a blank at 517 nm. Th e inhibition free well, monolayer) were plated in a 96-well plate. radical DPPH, in percentages (I%), was calculated in Th e next day, the cells were treated with diff erent concentrations of essential oil, carvacrol, and thymol the following way: I% = (Acontrol – Asample/Acontrol) × 100, (10-3000 μg/mL) in the medium for 24 h. At the where Ablank is the absorbance of the control reaction, containing all reagents except the test compound, end of the incubation period (24 h), the cytotoxicity of the essential oil and its 2 major components on and Asample is the absorbance of the test compound. An extract concentration providing 50% inhibition cancer cells was determined with the CellTiter-Blue®

(EC50) was calculated from the linear regression Cel l Viability Assay (Promega, Madison, WI, USA). algorithm of the graph-plotted inhibition percentage Th e assay is based on the ability of living cells to against the extract concentration. For the calculation convert a redox dye (resazurin) into a fl uorescent

737 A comparative evaluation of antioxidant and anticancer activity of essential oil from Origanum onites (Lamiaceae) and its two major phenolic components end product (resorufi n). Nonviable cells rapidly Results and discussion lose metabolic capacity and thus do not generate a Antioxidant activity fl uorescent signal (22). Following cellular reduction, fl uorescence was recorded at 560 nm (excitation) Th e components included in the essential oil and 590 nm (emission). Th e data were expressed investigation were classifi ed into 3 groups depending as average values obtained from 8 wells for each on their chemical nature. Group 1 includes alcohols, concentration. Th e IC value was calculated from the group 2 includes hydrocarbons, and group 3 includes 50 phenols such as carvacrol and thymol. GS/MS graphical equation. H2O2 cytotoxicity on the cancer cells was measured the same way. For measuring the analysis of the crude oil isolated from dried aerial antioxidant eff ect of the essential oil and its 2 major parts of O. onites resulted in the identifi cation of some compounds, as shown in Table 1. According to the components against H2O2 cytotoxicity, the cells were GS/MS analysis results, 12 (99.44%) compounds were preincubated at diff erent con centrations (

738 A. ÖZKAN, A. ERDOĞAN

by employing 2 complementary tests: free radical- Antioxidant eff ect of the essential oils on Hep scavenging activity (DPPH) and β-carotene/linoleic G2 cells acid antioxidant assays. Th eir antioxidant capacities We measured the protective (antioxidant) eff ect of were determined by comparison with the activities of the essential oil and its 2 main phenolic components known antioxidants, such as BHT, ascorbic acid, and against strong oxidant H2O2 in Hep G2 cells. Figure α-tocopherol. 1 shows the levels of H2O2-induced cytotoxicity Free radical scavenging capacities of the in Hep G2 cells, preincubated with diff erent corresponding samples were measured by DPPH concentrations of essential oil. Th e essential oil has assay and the results are shown in Table 2. According a strong antioxidant eff ect (<90 μg/mL) against H2 O2 cytotoxicity. Th e maximum protective concentrati on to the results, essential oil (EC50: 80.00 μg/mL) was less active than the positive controls of BHT (EC : of essential oil was found to be 20 μg/mL. Under 50 the same conditions, carvacrol and thymol reduced 18.45 μg/mL), ascorbic acid (EC50: 3.21 μg/mL), and α-tocopherol (EC : 7.25 μg/mL), but more active the cytotoxic eff ects and induced the strong oxidant 50 H O in Hep G2 with 10.62 μg/mL (IC ) and 24μg/ than carvacrol (EC : 248 μg/mL) and thymol (EC : 2 2 10 50 50 mL (IC ), respectively, for maximum levels (Figures 163 μg/mL). Th e essential oil of O. onites had higher 20 2 and 3). antiradical activity than some other plant essential oil (30). As shown in Table 2, oxidation of the linoleic Th e essential oil and its 2 major components acid was eff ectively inhibited by the essential oil of signifi cantly decreased membrane damage in H2O2- O. onites (40.00%). On the other hand, the oxidation treated Hep G2 cells (Table 3). Th e Hep G2 cells, capacity of the essential oil was not superior to preincubated with 20 μg/mL of essential oil, 24 μg/ positive controls BHT (95.02%), ascorbic acid mL of carvacrol, and 10.62 μg/mL of thymol for 1

(92.92%), or α-tocopherol (96.20%), but was close to h bef ore hydrogen peroxide treatment (IC10, IC20, carvacrol (52.02%) and thymol (57.11%). Moreover, it was found to be close to the essential oils of Salvia limbata C.A.Mey. and Salvia sclarea L. (31). 120 IC70 (H2O2) IC (H O ) 100 50 2 2 IC (H O ) 80 20 2 2 IC10 (H2O2) 60 Table 2. Antioxidant capacities of the essential oil of O. onites, its 2 major components, and positive controls. 40

Cell viability (%) 20 0 β-Carotene/ 300 200 150 100 90 80 70 60 40 20 10 Samples DPPH1 Essential oil concentration (μg/mL) linoleic acid2 Figure 1. Antioxidant eff ect of essential oil of O. onites on Hep d x Essential oil 80.00 ± 2.11 40.00 ± 1.07 G2 cancer cells against H2O2 cytotoxicity.

Carvacrol 248 ± 31.11f 52.02 ± 1.30b

e bc Th ymol 163 ± 25.11 57.11 ± 1.27 IC10 (H2O2) IC50 (H2O2) 100 IC20 (H2O2) IC70 (H2O2) c d 90 BHT 18.45 ± 1.07 95.02 ± 1.61 80 70 Ascorbic acid 3.21 ± 0.19a 92.92 ± 1.72d 60 50 40 α-Tocopherol 7.25 ± 1.130ab 96.20 ± 1.81d 30

Cell viability (%) 20 10 Results are the means of 5 diff erent experiments. Values that are 0 followed by diff erent letters within each column are signifi cantly 10.62 mμ/mL 21.24 mμ/mL 31.85 mμ/mL (IC ) (IC ) (IC ) diff erent (P ≤ 0.05). 10 20 30 Carvacrol concentration 1EC value of DPPH assay (as μg/mL). 50 Figure 2. Antioxidant eff ect of carvacrol on Hep G2 cancer cells 2Given as percentage of % inhibition of the linoleic acid. against H2O2 cytotoxicity.

739 A comparative evaluation of antioxidant and anticancer activity of essential oil from Origanum onites (Lamiaceae) and its two major phenolic components

IC (H O ) 120 10 2 2 the IC50 H2O2-treated cells, and by 48%, 41%, and 34%, IC20 (H2O2) IC (H O ) respectively, in the IC70 H2O2-treated Hep G2 cells. 100 50 2 2 IC (H O ) 70 2 2 We assume that decreasing H O levels induced 80 2 2 a cytotoxic eff ect; membrane damage can be 60 accompanied by antioxidant action of the essential 40 oil and its 2 major components with concentrations Cell viability (%) 20 lower than IC50. Th e essential oil from O. onites, 0 carvacrol, and thymol showed a protective 12 mμ/mL 24 mμ/mL 36 mμ/mL (antioxidant) eff ect on Hep G2 cells, depending on (IC10) (IC20) (IC30) Thymol concentrations the concentrations. Aydin et al. (32) also observed that the phenolic compounds thymol and carvacrol, Figure 3. Antioxidant eff ect of thymol on Hep G2 cancer cells at concentrations below 0.2 and 0.1 mM, respectively, against H O cytotoxicity. 2 2 signifi cantly reduced the oxidative damage in human lymphocytes. In another study, the incubation

IC50, and IC70) for 24 h had lower MDA levels than of Hep G2 and Caco-2 cells in the presence of the unpreincubated cells. Th e amount of MDA increased whole scale of concentrations of carvacrol or thymol almost 2.8-fold in the IC50 H2O2-treated Hep G2 cells led, in both cases, to a signifi cant protection of the and 4. 7-fold in the IC70 H2O2-treated Hep G2 cells cells studied from DNA strand breaks induced by compared to the control cells. Th e preincubation of the potent oxidant hydrogen peroxide (33). Further cells with the essential oil and its 2 major components understanding of the underlying mechanism of decreased the MDA amount compared to the Hep their protective eff ects in reducing intracellular

G2 cells treated with H2O2 alone. Th e membrane- oxygen radicals in Hep G2 cell death may lead to the protective eff ect of the essential oil was found to development of new therapeutic treatments for cancer, be higher than that of carvacrol and thymol at the since the essential oil and its main components have highest cytoprotective concentration. Th e data in protection against H2O2 insult. Th eir hepatoprotective

Table 3 reveal a membrane-protective eff ect wit h the eff ects against H2O2 toxicity might be of importance essential oil decreasing the MDA amount by 54%, and may contribute in part to their clinical effi cacy carvacrol decreasing it by 38%, and thymol by 29% in for the treatment of hepatocellular carcinoma. Th ese

Table 3. Amount of malondialdehyde in Hep G2 cells preincubated with O. onites essential oil and its 2 major components before hydrogen peroxide treatment.

Groups MDA levels (nmol/mg protein ± SD)

Control 0.25 ± 0.11a 0.5% DMSO 0.22 ± 0.20a

H2O2 (IC50) 0.69 ± 0.55ef

EsO pretreated (20 μg/mL) + H2O2 (IC50) 0.32 ± 0.10b

Carvacrol pretreated (10.62 μg/mL) + H2O2 (IC50) 0.43 ± 0.13c

Th ymol pretreated (24 μg/mL) + H2O2 (IC50) 0.49 ± 0.15cd

H2O2 (IC70) 1.18 ± 1.42jk

EsO pretreated (20 μg/mL) + H2O2 (IC70) 0.61 ± 0.44e

Carvacrol pretreated (10.62 μg/mL) + H2O2 (IC70) 0.70 ± 0.56ef

Th ymol pretreated (24 μg/mL) + H2O2 (IC70) 0.78 ± 0.86fg

Values that are followed by diff erent letters within column are signifi cantly diff erent (P ≤ 0.05). Each datum in the table is an average of 5 repetitions of MDA amount. MDA: malondialdehyde, EsO: essential oil, SD: standard deviation.

740 A. ÖZKAN, A. ERDOĞAN

results suggest that O. onites may be a potentially cancer treatments. Th ey induced membrane damage valuable source of natural therapeutic agents. Th us, and cytotoxicity in Hep G2 cells at relatively higher it is becoming increasingly evident that certain concentrations than those that mediate its anticancer phytochemicals, particularly those included in our activities. Th e induction of cytotoxic cell death can daily diet, have important cancer chemopreventive be accompanied by membrane damage. Koparal and properties (34). Zeytinoglu (35) also observed that carvacrol was Cytotoxic eff ect of essential oils on Hep G2 cells a very potent inhibitor of cell growth in the A549 cancer cell line. In another study, carvacrol and Th e cytotoxicity of the essential oil and its 2 major thymol had dose-dependent antiproliferative eff ects components, thymol and carvacrol, studied in Hep on human uterine carcinoma cells (36). G2 cells, was evaluated with the CellTiter-Blue® Cell Viability Assay aft er 24-h treatment of the cells with Taken together, these fi ndings suggest that the the compounds given in Figure 4. Hep G2 cells were essential oil and its 2 main components exhibit much more sensitive to the toxic eff ect of carvacrol antioxidant activity at lower concentrations and and thymol, with IC values of 53.09 and 60.01 μg/ anticancer eff ects at higher concentrations in cells. 50 Further studies will be needed to identify the active mL, respectively, than the essential oil (IC50: 149.12 μg/mL). Th e essential oil was found to be less to xic compounds in plant extracts that have antioxidant than carvacrol and thymol for Hep G2 cells. Th e and/or carcinogenesis-reducing potential. viability of the Hep G2 cells decreased when the cells were exposed to the essential oil, carvacrol, and Acknowledgements thymol at increasing concentrations between 20-170 μg/mL, and did not change at concentrations of 170 Th is work was supported by the Scientifi c Research μg/mL or higher (Figure 1). Th e essential oil from Projects of the Administration Unit of Akdeniz O. onites and its 2 components had dose-dependent University (2007.06.0105.008-015). Th e authors antiproliferative eff ects on Hep G2 cells, which makes thank Assoc. Prof. Dr. Th omas Zimmermann, them potentially interesting for adjuvant experimental University of the Virgin Islands, and Dr. Tomas Ayala-Silva, Agricultural Research Service, United States Department of Horticulture (USDA-ARS, FL, 100 USA) for their critical review of this manuscript. 90 Thymol 80 Carvacrol 70 Essential oil 60 Corresponding author: 50 40 Aysun ÖZKAN

Cell viability (%) 30 Department of Biology, 20 10 Faculty of Science, 0 0 20 40 60 80 100 120 140 160 180 200 Akdeniz University, Concentration (mμ/mL) 07058 Antalya - TURKEY Figure 4. Dose-dependent cytotoxicity of essential oil from O. onites, carvacrol, and thymol on Hep G2 cells. E-mail: [email protected]

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