Article pubs.acs.org/JAFC
Assessment of Antioxidative, Chelating, and DNA-Protective Effects of Selected Essential Oil Components (Eugenol, Carvacrol, Thymol, Borneol, Eucalyptol) of Plants and Intact Rosmarinus officinalis Oil Eva Horvathova,*,† Jana Navarova,‡ Eliska Galova,§ Andrea Sevcovicova,§ Lenka Chodakova,§ Zuzana Snahnicanova,§ Martina Melusova,†,⊥ Katarina Kozics,† and Darina Slamenova† †
Department of Genetics, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovak Republic Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Dubravska cesta 9, 841 04 Bratislava, Slovak Republic § Department of Genetics, Faculty of Natural Sciences, Comenius University, Mlynska dolina, 842 15 Bratislava, Slovak Republic ⊥ Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinskeho 9, 812 37 Bratislava, Slovak Republic ‡
ABSTRACT: Selected components of plant essential oils and intact Rosmarinus officinalis oil (RO) were investigated for their antioxidant, iron-chelating, and DNA-protective effects. Antioxidant activities were assessed using four different techniques. DNA-protective effects on human hepatoma HepG2 cells and plasmid DNA were evaluated with the help of the comet assay and the DNA topology test, respectively. It was observed that whereas eugenol, carvacrol, and thymol showed high antioxidative effectiveness in all assays used, RO manifested only antiradical effect and borneol and eucalyptol did not express antioxidant activity at all. DNA-protective ability against hydrogen peroxide (H2O2)-induced DNA lesions was manifested by two antioxidants (carvacrol and thymol) and two compounds that do not show antioxidant effects (RO and borneol). Borneol was able to preserve not only DNA of HepG2 cells but also plasmid DNA against Fe2+-induced damage. This paper evaluates the results in the light of experiences of other scientists. KEYWORDS: comet assay, DNA topology assay, DPPH radicals scavenging assay, Fe2+-chelating assay, hydroxyl radicals scavenging assay, plant essential oil components, reducing power assay, Rosmarinus officinalis oil
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INTRODUCTION In recent decades plant essential oils (EOs) and their components have attracted increased interest and consequently have been extensively investigated mainly in in vitro systems. Their effectiveness against a wide range of microorganisms is related to their hydrophobicity, which enables them to integrate into the lipids of the cell membrane and mitochondria, rendering them permeable and leading to leakage of cell contents.1,2 Many EOs and their ingredients exhibit also antiinsecticidal, antifungal, antiarthropodal, and antiprotozoal effects. For more data see the paper by Slamenova and Horvathova.3 Besides these activities different EOs have been qualified as natural antioxidants due to their ability to reduce free radical formation and scavange free radicals. They were proposed as potential substitutes of synthetic antioxidants in specific sectors of food preservation.4 From the chemical point of view essential oils represent complex mixtures. They are constituted from hydrocarbons (monoterpenes and sesquiterpenes) and oxygenated compounds (alcohols, esters, ethers, aldehydes, ketones, lactones, phenols, and phenol ethers). EOs comprise a large number of components; therefore, it is likely that their mode of action involves several targets. The antioxidant activities of putative antioxidants have been attributed to various mechanisms; among these are prevention of chain initiation, binding of transmission metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, and radical scavenging.5 It is therefore evident that the antioxidative nature © 2014 American Chemical Society
of EOs needs to be examined with the help of a wide range of assays. One procedure is not sufficient to identify all aspects characterizing a substance. In the present study we investigated the antioxidative activity, Fe2+-chelating properties, and DNA-protective effects of selected essential oil components (eugenol, which represents a derivative of benzene, and four oxygenated monoterpenes, carvacrol, thymol, borneol, and eucalyptol) and intact Rosmarinus officinalis oil (RO). Reducing power, 1,1-diphenyl2-picrylhydrazyl (DPPH•) or hydroxyl (•OH) radical scavenging, and Fe2+-chelating assays were used for an evaluation of antioxidative and chelating effects. The DNA-protective potential of EO components and RO against hydrogen peroxide (H2O2)- or Fe2+-induced DNA damage was assessed by single-cell gel electrophoresis (SCGE; comet assay) and by DNA topology assay, respectively. Experimental techniques used in this study could facilitate the monitoring of antioxidative, chelating, and DNA-protective effects of plant compounds that occur very often in the environment. We believe that the findings gained in this study could expand global knowledge in the field of essential oil research. This kind of investigation is important mainly for the growing interest in Received: Revised: Accepted: Published: 6632
November 21, 2013 June 19, 2014 June 21, 2014 June 21, 2014 dx.doi.org/10.1021/jf501006y | J. Agric. Food Chem. 2014, 62, 6632−6639
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Hydroxyl Radical (•OH) Scavenging Activity. For the determination of •OH scavenging activity, hydroxyl radicals (•OH) were generated in an L-ascorbic acid/CuSO4 system. The assay is based on quantification of cytochrome c oxidation.8 In this experiment, OH radicals were generated in 1 mL of 0.15 mM sodium phosphate buffer (pH 7.4) containing 100 μM L-ascorbic acid, 100 μM CuSO4, 12 μM cytochrome c, and the samples of plant volatiles to be tested at different concentrations. The mixture was incubated at 25 °C for 90 min. The change in transmittance caused by the color change of cytochrome c was measured at 550 nm using a spectrophotometer (GENESYS 10 Bio, Spectronic). The inhibition of •OH generation by 500 μg/mL thiourea was taken as 100%. BHT was used as a positive control. The inhibition ratio was calculated using the formula
ingredients obtained from natural sources, which can often fully replace potentially dangerous synthetic additives.
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MATERIALS AND METHODS
Chemicals. The constituents of plant EOs studied included eugenol (Eug; 4-allyl-2-methoxyphenol), carvacrol (Ca; 5-isopropyl2-methylphenol), thymol (T; 2-isopropyl-5-methylphenol), borneol (B; endo-(1S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol), and eucalyptol (Euc; 1,8-cineole; 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane). The DNA-damaging agent used was hydrogen peroxide (H2O2). Positive controls included gallic acid (GA), ascorbic acid (AA), butylated hydroxytoluene (BHT), quercetin, and ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA·2H2O), and reagents 1,1-diphenyl2-picrylhydrazyl radical (DPPH), dimethyl sulfoxide (DMSO), cremophor EL, ethidium bromide (EtBr) were obtained from Fluka or Sigma, Sigma-Aldrich Co. (Steinheim, Germany, or St. Louis, MO, USA). R. of f icinalis essential oil (Ph. Eur. 4) was purchased from Calendula Inc., Nova Lubovna, Slovakia (lot 5-014-009-12-06, containing approximately 25% 1,8-cineole, 19% α-pinene, 19% camphor, 17% p-cymene, 9% camphene, 5% β-pinene, 2% borneol, and 4% unidentified compounds as specified by the manufacturer). All plant volatiles were kept at room temperature and diluted in appropriate reaction mixtures just before the experiments. William’s medium was purchased from PAN-Biotech GmbH (Aidenbach, Germany), fetal bovine serum (FBS) and antibiotics were purchased from GIBCO BRL (Paisley, UK), and phosphate-buffered saline (PBS; Ca2+- and Mg2+-free) was purchased from Oxoid Limited (Basingstoke, UK). All other reagents and chemicals used were of analytical grade. Reducing Power Assay. The reducing capacities of the plant volatiles were determined according to the method of Zhao et al.6 In this assay, the yellow color of the test solution is changed to various shades of green and blue, depending on the reducing power of each compound. The presence of reducing agents (i.e., antioxidants) induces the conversion of the Fe3+/ferricyanide complex into the ferrous forms. In brief, different concentrations of selected essential oil components, intact RO, and GA, used as a positive control, were mixed in 1 mL of methanol with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide [K3Fe(CN)6] (1%). The mixtures were incubated at 50 °C for 20 min. Trichloroacetic acid (2.5 mL, 10%) was added to each mixture and centrifuged at 3000 rpm for 10 min. Finally, the upper layer of supernatants (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl3 (0.1%), and the absorbances were recorded at 700 nm (GENESYS 10 Bio, Spectronic). The higher absorbance of the reaction mixture indicates the higher reducing power. 1,1-Diphenyl-2-picrylhydrazyl Radical Scavenging Activity. Selected plant volatiles were investigated for their radical scavenging activity using a modified DPPH assay.7 DPPH is a stable free radical. The methanolic solution of this compound is dark purple (absorbs light at a wavelength of 517 nm) at the radical state. When DPPH reacts with an antioxidant, by providing hydrogen atoms or by electron donation, it is reduced to the molecular form, which is yellow. In brief, methanol (100 μL) solution of DPPH radical at the concentration of 0.05 mg/mL was added to 50 μL of various concentrations of plant volatiles or AA, used as a positive control. The experiments were carried out at room temperature. The decrease in absorbance at 500 nm was measured at 0, 15, 30, and 60 min using a spectrophotometer (Multiscanreader RC, Labsystems, Vantaa, Finland), and the DPPH• scavenging activities of plant volatiles were then expressed as percentage of DPPH• scavenging activity using the formula
scavenging of OH radicals (%) = [(T − T2)/(T − T1)] × 100 where T is the transmittance of the •OH generation system and T1 and T2 are the transmittances of the control (no •OH generation) and test systems, respectively. DNA Topology Assay. The method of electrophoretically monitored DNA-damaging activity and DNA protectivity was described in detail by Cipak et al.9 In brief, the reaction mixture (final volume of 10 mL) contained 200 ng of plasmid DNA in buffer and either Fe2+ alone, tested compounds alone, or combinations of tested compounds with Fe2+. Specific details of agent concentrations are given in the caption of Figure 6. Single- (ss) and double-strand (ds) DNA breaks were assayed by measuring the conversion of supercoiled DNA, form I, to relaxed forms (II + III, Figure 1).
Figure 1. Electrophoretic changes induced in the plasmid DNA by the influence of Fe2+ (form I = supercoiled DNA, form II = ssDNA breaks, form III = dsDNA breaks). Topological changes of DNA molecules correspond with the electrophoretical mobility of DNA topoisomers. Analysis of DNA modifications was made by agarose gel electrophoresis (1.5% agarose, 60 min, 60 V). The DNA was visualized by staining with EtBr (1 mg/ mL) and UV illumination (UV Transilluminator MiniBISPro, DNR Bio Imaging Systems Ltd.). Fe2+-Chelating Activity Assay. Some results obtained by the above-mentioned techniques were verified by Fe2+-chelating activity assay, as one of the mechanisms of antioxidant action is a chelation of transition metals, which prevents decomposition of hydroperoxide and Fenton-type reactions. Fe2+-chelating assay uses an iron reagent, ferrozine, which forms complexes with Fe2+. In the presence of chelating agents, the complex formation is disrupted with the result of color reduction. The chelating activity of borneol and two positive controls (quercetin and Na2EDTA·2H2O) toward ferrous ions was studied by using the method described by Rajic et al.10 HepG2 Cell Line and Treatment of Cells. Malignant human hepatoma HepG2 cell line was obtained from Prof. A. R. Collins (University of Oslo, Norway) and cultured in William’s medium, supplemented with 10% FBS and antibiotics (penicillin, 200 U/mL; streptomycin and kanamycin, 100 μg/mL). Cells were cultured on plastic Petri dishes at 37 °C in a humidified atmosphere of 5% CO2. Eugenol, carvacrol, and eucalyptol were dissolved immediately before use in complete culture medium to final concentrations of 0.05−0.6 (Eug), 0.025−0.2 (Ca), and 0.5−5 (Euc) mM, respectively. Thymol (T) was disolved in DMSO and diluted to final concentrations of 0.1−0.5 mM in complete culture medium. RO was dissolved in cremophor EL and 70 °C William’s medium to 1.25%
scavenging of DPPH radicals (%) = [(Acontrol − A sample)/Acontrol ] × 100 where Acontrol is the absorbance of the control reaction, containing all reagents except the tested compounds, and Asample is the absorbance of tested compounds. Methanol was used as a blank. 6633
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Figure 2. Reducing power of eugenol (A), carvacrol (B), thymol (C), Rosmarinus of f icinalis oil (D), borneol (E), and eucalyptol (F). Gallic acid (GA) is shown as a positive control.
Figure 3. DPPH radical scavenging activity of eugenol (A), carvacrol (B), thymol (C), Rosmarinus of f icinalis oil (D), borneol (E), and eucalyptol (F). Ascorbic acid (AA) is shown as a positive control. stock solution and further diluted to final concentrations of 0.00625− 0.0625‰ in complete culture medium. Borneol (B) was disolved in ethyl alcohol (500 mM) and diluted to final concentrations of 0.5−3 mM in complete culture medium. Appropriate control cells were kept in complete William’s medium with or without the addition of solvents. HepG2 cells were treated for 24 h. Single-Cell Gel Electrophoresis (SCGE; Comet Assay). Assessment of DNA damage in HepG2 cells was performed by the use of the SCGE (comet assay), which represents a sensitive method for
measuring DNA damage at the level of single cells. The method of SCGE according to Singh et al.11 was followed with minor modifications made by Gabelova et al.12 In brief, appropriate controls and HepG2 cells preincubated with volatiles for 24 h were trypsinized, centrifuged, and resuspended in 0.75% low melting point agarose in PBS. A volume of 85 μL of the cell suspension (approximately (2−2.5) × 104 cells) was spread on a base layer (100 μL of 1% normal melting point agarose in PBS) on a microscopic slide. When the agarose had solidified, half of the samples (slides) was submerged for 5 min in 250 6634
dx.doi.org/10.1021/jf501006y | J. Agric. Food Chem. 2014, 62, 6632−6639
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Figure 4. Hydroxyl radicals scavenging activity of eugenol (A), carvacrol (B), thymol (C), Rosmarinus of f icinalis oil (D), borneol (E), and eucalyptol (F). Butylated hydroxytoluene (BHT) is shown as a positive control.
Figure 5. Effects of 24 h of pretreatment of HepG2 cells with eugenol (A), carvacrol (B), thymol (C), Rosmarinus of f icinalis oil (D), borneol (E), and eucalyptol (F) on DNA damage induced by 250 μM H2O2. Black columns represent the level of DNA strand breaks induced by 24 h of treatment with EOs or RO; white columns represent the level of DNA strand breaks induced by H2O2 alone (0) or after pretreatment with different concentrations of the EOs or RO studied and then with H2O2. (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001 indicate significant differences between HepG2 cell cultures treated with H2O2 alone and cultures pretreated with volatiles and then exposed to H2O2. HCl, pH 10.0, 1% Triton X-100) for 1 h at 4 °C to remove cellular proteins. The second part of the samples was placed in a lysis mixture
μM H2O2 in PBS on ice for induction of DNA damage and then placed in a lysis mixture (2.5 M NaCl, 0.1 M Na2EDTA, 10 mM Tris6635
dx.doi.org/10.1021/jf501006y | J. Agric. Food Chem. 2014, 62, 6632−6639
Journal of Agricultural and Food Chemistry
Article
immediately after 5 min in cold PBS. After lysis, the slides were transferred to an electrophoresis buffer (0.3 M NaOH, 1 mM Na2EDTA, pH >13.0) for unwinding (40 min at 4 °C). A current of 25 V (0.3 A) was then applied for 30 min. The slides were neutralized with 0.4 M Tris-HCl (pH 7.5) and stained with EtBr (5 μg/mL). For each sample, 100 EtBr-stained nucleoids were evaluated and scored with an Olympus fluorescent microscope and computerized image analysis (Komet 5.5, Kinetic Imaging, Liverpool, UK) for determination of the percentage of DNA in the tail. Statistical Analysis. The results are expressed as means ± standard deviations (SD) from at least three independent experiments carried out in triplicates. Student’s t test was performed to determine the significance of differences between HepG2 cell cultures treated with H2O2 and cultures pretreated with volatiles and then exposed to H2O2 in the comet assay. Differences with p values
