Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(3): 71-88, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-260 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: S. Gateva, G. Jovtchev, C. Chanev, A. Georgieva, A. Stankov, A. Dobreva, M. Mileva (2020) Assessment of anti-cytotoxic, anti-genotoxic and anti- oxidant potentials of Bulgarian Rosa alba L. essential oil. Caryologia 73(3): 71-88. doi: 10.13128/caryologia-260 Received: May 13, 2019 Accepted: June 02, 2020 Published: December 31, 2020 Copyright: © 2020 S. Gateva, G. Jovtch- ev, C. Chanev, A. Georgieva, A. Stank- ov, A. Dobreva, M. Mileva. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of Bulgarian Rosa alba L. essential oil Svetla Gateva1,*, Gabriele Jovtchev1, Christo Chanev2, Almira Geor- gieva3, Alexander Stankov1, Anna Dobreva4, Milka Mileva5,* 1 Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Str., Bulgarian Academy of Sciences, Sofia 1113, Bulgaria 2 University of Sofia, Department of Chemistry, 1 J. Bourchier Str., Sofia 1164, Bulgaria 3 Institute of Neurobiology, Bulgarian Academy of Sciences, 23 Acad. G. Bonchev Str., Sofia 1113, Bulgaria 4 Institute for Roses and Aromatic Plants, 49 Osvobojdenie Blvd., Kazanlak 6100, Bul- garia 5 The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. G. Bonchev Str., Sofia 1113, Bulgaria *Corresponding author. E-mail: spetkova2002@yahoo.co.uk; milkamileva@gmail.com Abstract. Bulgarian Rosa alba L. essential oil is widely used in perfumery, cosmetics and pharmacy. The scarce data about its cytotoxic/genotoxic effect and anti-cytotox- ic/anti-genotoxic potential gave us a reason to set our aim: i) to study its cytotoxic/ genotoxic activities, iii) to explore its cytoprotective/genoprotective potential against the experimental mutagen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) in two experimental test-systems - barley and human lymphocytes using appropriate end- points and iii) to assess its antioxidant properties. Findings about chemical compo- sition of rose essential oil would help us to explain its activities. Chromatogaphic profile of rose essential oil was obtained by Gas Chromatography-Mass Spectrometry and quantificaton of particular constituents was done with a Gas Chromatography- FID system. Superoxide anion radical scavenging, DPPH inhibition and iron ion chelating activity were used to study a possible antioxidant potential of the rose oil. Its defense potential was investigated by induction of chromosome aberrations and micronuclei in both test-systems. Cytogenetic analysis showed a low cytotoxic effect in both test-systems and no high genotoxic effect in human lymphocytes in vitro in a dose-dependent manner. Rose oil possessed a well-expressed anti-cytotoxic/anti- genotoxic potential against MNNG manifested by decreasing both of chromosome aberrations and micronuclei regardless of the experimental schemes used. A well- expressed concentration-depended free radical scavenging activity of the essential oil was obtained. Current data suggest a promising ethnopharmacological potential of Bulgarian white rose essential oil. Keywords: Rosa alba L. essential oil, rose oil components, genotoxicity, anti-cytotoxic- ity, anti-genotoxicity, antioxidant effect. 72 Svetla Gateva et al. INTRODUCTION Аs a general rule, living organisms exist in condi- tions of continuous attack by various environmental pol- lutants such as alkylating agents, oxidative stress induc- ers, etc. As a result serious alteration in the main heredi- tary molecule DNA could be induced. There is a constant need to obtain products, which could decrease or elimi- nate the harmful effects of the genotoxins. Plants are known as a rich source of various bioactive natural com- pounds, which are widely used in various areas of human life. A cursory look at the literature cited in relation to plants’ essential oils in recent years indicates that there is a growing interest in evaluation of the biological activi- ties of various extracts of essential plants, their antimuta- genic and antigenotoxic potential (Blasiak et al. 2002; Mezzoug et al. 2007; Bakkali et al. 2008; Vicuña et al. 2010; Siddique et al. 2010; Arumugam et al. 2010; Leffa et al. 2012; Gokbulut et al. 2013; Madrigal-Santillán et al. 2013; Oyeyemia and Bakare 2013; Reddy and Devi 2014; Shohayeb et al. 2014; Laribi et al. 2015), and presum- ing their role in the prevention of degenerative diseases and other human ailments including cancer (Hajhashe- mi et al. 2002; Raut and Karuppayil 2014; Horváth and Ács 2015). The genus Rosa is one of the largest and most important aromatic and medicinal genera of the Rosace- ae family. Numerous rose phytocomplexes, including essential oils isolated from Rosa damascena Mill., Rosa x centifolia L., Rosa gallica L., Rosa alba L. and Rosa rugo- sa Thunb. have been identified and used for therapeutic purposes as well (Rangaha 2001; Degraf 2003; Moein et al. 2010). The rose extracts help in the reduction of thirst, healing of old cough, special complaints of women, abdominal and chest pain, digestive problems and show skin health effects (Tabrizi et al. 2003; Boskabady et al. 2006; EMA/HMPC 2013). The rose essential oils and by-products of Rosa damascena, from Shafaa, Taif, Sau- di Arabia (Shohayeb et al. 2014) and from Amman and Ajloun areas, Jordan (Talib and Mahasneh 2010) have antimicrobial activity against various Gram-positive, Gram-negative, acid-fast bacteria and fungi. Absolute oil of Rosa damascena trigintipetala Dieck has an antimuta- genic activity against mitomycin C in normal human blood lymphocytes (Hagag et al. 2014). Methanolic and aqueous extracts of Rosa damascena white variety from Iran show better anti-radical activity than some synthetic antioxidants (Kashani et al. 2011). Unfortunately, little is known about single- and repeat-dose cytotoxicity, geno- toxicity, carcinogenicity, reproductive and developmental toxicity, local tolerance or other special studies of prepa- rations from rosae flos in animals and humans according to current state-of-the-art standards (Hagag et al. 2014). Rosa alba L. is the second most important plant for Bulgarian rose production. Kovatcheva et al. (2011) and Dobreva (2010) demonstrated that Bulgarian Rosa alba L. has a similar oil composition but some of com- pounds are with lower content to that of Rosa dama- scena Mill. collected from Bulgaria. Rosa alba L. essen- tial oil, known as Bulgarian rose oil of white rose, has been defined as “original, exclusively fine, only suitable for the highest perfumery” (Degraf 2003). Fukada et al. (2011) found that in an experimental model of acute stress in rats, inhalation with Rosa alba L. essential oil (supplied by Kanebo Cosmetics) lowered corticosterone levels almost twice. Water extract of calyces of Rosa alba from India might be a useful memory restorative agent in the treatment of cognitive disorders (Naikwade et al. 2009). Our previous study indicated well-expressed anti- microbial activities of Bulgarian Rosa alba L. essential oil (Mileva et al. 2014). Insufficient data exist about cyto- toxic/genotoxic effect and anti-cytotoxic/anti-genotoxic potential of essential oil from Bulgarian Rosa alba L. This gave us a reason to set our aim in the present paper: i) to study cytotoxic and genotoxic activities of Bulgar- ian Rosa alba L. essential oil, ii) to explore its cytopro- tective and genoprotective potential against well known experimental mutagen – alkylating agent N-methyl- N’-nitro-N-nitrosoguanidine (MNNG) in two types of widely used experimental test-systems (higher plant and human lymphocytes in vitro) using appropriate for this purpose endpoints, and iii) to assess its antioxidant properties. Phytochemical analysis of rose essential oil would help us to explain its cytotoxic/genotoxic and anti-cytotoxic/anti-genotoxic effects. MATERIAL AND METHODS Chemicals Used All chemicals, standards and solvents used for anal- ysis of rose essential oil (GC-MS, GC-FID), methods for testing of the antioxidant activity and cytogenetic analysis were of high purity (>99%). Tetracosane [646- 31-1] GA14075, EC 2114745, free puriss. p.a. > 99.5 % (GC) used as a reference compound in Predicted Rela- tive Response Factors calculations was purchased from Fluka, USA. Nitroblue tetrazolium (NBT), xanthine, α-tocopherol, butylated hydroxytoluene (BHT), ascor- bic acid, ferrous chloride, ferrozine, EDTA, dimetylsul- foxyde (DMSO) used for examination of antioxidant properties and the chemicals used for cell cultivation and cytogenetical analysis (RPMI 1640, bovine serum, phytohaemagglutinin PHA, Giemsa) were purchased from Sigma-Aldrich, Germany. α-bromonaphthaline, 73Assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of Bulgarian Rosa alba L. essential oil colchicine, KCL were purchased from Merck, Germany, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) from Fluka – AG, Switzerland and Schiff ’s reagent from Rie- del-De Haen AG, Germany. Plant material and distillation of Rosa alba L. essential oil Fresh flowers of Rosa alba L., from the experimen- tal field of the Institute of Rose and Essential Oil Plants (IREOP), in Kazanlak, Bulgaria were used as raw material in the study. The flowers were picked up in the mornings in May/June 2009, from 8 to 10 am, in a phase of semi- blooming – blooming. Roses were distilled immediately by water-steam distillation using IREOP’s semi-industri- al processing line. Process parameters of the distillation were as follows: a raw material for charge – 10 kg; hydro module 1:4, rate of 8-10% and duration – 150 min. The aromatic water was re-distilled in the same apparatus. The essential oil, obtained of each charge is the sum of primary and secondary oil in their natural ratio. Total oil is a mixture of distillates collected over 15 days – the time of the collection campaign of Rosa alba L. for 2009. Final- ly, it was dried with sodium sulfate (Merck, Germany), fil- tered and stored at 4 °C for further use. The concentration of the main compounds as well as the physicochemical parameters and characteristics of the rose essential oil are controlled through implemen- tation of national (BDS ISO9842:2006) and international Standards (ISO 9842:2003, www.iso.org) only for oil obtained from Rosa damascena Mill. So, in our study we used the essential oil from Rosa damascena Mill. culti- vated in Bulgaria, Kazanlak as one of the controls in the tests for antioxidant activities. Gas Chromatography-Mass Spectrometry (GC-MS) Chromatogaphic profile of rose essential oil was obtained by Gas Chromatography-Mass Spectrometry. GC-MS analysis was carried out on a HP 6890 “PLUS” gas chromatograph interfaced with a 5975 mass selective detector. Separations were performed using a HP-5MS silica-fused capillary column – 30 m × 0.25 mm coated with 0.25 μm film of (5%-phenyl)- methylpolysiloxane as the stationary phase (Agilent Technologies, USA). The flow rate of carrier gas (helium) was maintained at 0.8 ml/min. The injector and the transfer line temperature were kept at 250 and 300 °C respectively. The oven tem- perature program used was 60 °C for 2 min then 3 °C/ min to 300 °C for 8 min, total run time - 90 min. The injections were carried out in a split mode with a split ratio of 25:1. The mass spectrometer was scanned from 30 to 550 m/z. The injection volume was 1 µl. The quantitative analysis was carried out on a HP 5890 “SERIES II” gas chromatograph equipped with a FID detection system. We analysed the same sample and the separation was performed at the same chro- matographic conditions, column, carrier gas and tem- perature program. GC-FID – eluted constituents were identified on the basis of a Kovats Retention Index (RI), determined with reference to a homologous series of n-alkanes (C10-C28), under identical experimental con- ditions. GC-MS – eluted constituents were identified using MS Library search (NIST version 2.1), as well as by comparison with the fragmentation pattern of the mass spectra with data published in the literature (Adams 2007). For each identified constituent, from GC-MS analysis was obtained its Kovats RI. Every particular val- ue for these indices was confirmed by the literature. The differences between the measured and published Kovats RI exceeding 10 units were not reported, except these for n-alcanes and a few unsaturated hydrocarbons. The per- centage compositions of Rosa alba L. essential oil were determined from the GC-FID peak ’s areas corrected with Predicted Relative Response Factors for the constit- uents calcutated by formula given by Tissot et al. (2012). Examination of antioxidant properties Superoxide scavenging properties The generation of superoxide anion radical O2•- in the model system xanthine-xanthine oxidase (XO) and the changes occurring upon the Rosa alba L. oil effect were investigated by the nitroblue tetrazolium (NBT) test. The detailed procedure was described elsewhere (Mileva et al. 2000). Briefly, the spectrophotometric reg- istration of superoxide was carried out measuring the amount of formazan generated by O2•- induced reduction of NBT. The investigated samples of a volume 1 ml PBS contained: 1 mmol/l xanthine, 2.10-3 IU XO, 0.04 mmol/1 NBT, as well as oil at concentrations from 0 to 100 μg/ ml. Samples were incubated at 37 oC and the amount of the formed formazan was measured by absorption at 560 nm. The time of incubation was selected so that the absorption for the controls was 0.2. The decrease of absorbance in the presence of oil indicated the con- sumption of superoxide anion in the reaction mixture. Data were calculated in percentage as spectrophotomet- ric scavenger index (SpSI) - the ratio of the absorption at 560 nm for the sample with oil, and the same absorption for the controls (without oil). The scavenger activity of oil was compared with that of α-tocopherol – commonly used as superoxide radical scavenger. 74 Svetla Gateva et al. DPPH Test Hydrogen atoms and electron-donating potential of essential oils were measured from the bleaching of the purple-colored ethanol solution of DPPH (Sigma- Aldrich, Germany). All compounds were dissolved in ethanol to a concentration of 100 mg/ml stock solutions for the follow dilutions. DPPH assay was performed as follows: freshly prepared ethanolic solution of DPPH (100 mM) was incubated with tested substances in the concentration of 10 to 100 μg/ml, and the absorbance (A) monitored spectrophotometrically at 517 nm after 30 min incubation in dark at room temperature. Inhibition of DPPH in percentage (DPPH inhibition, %) was calcu- lated as given below: DPPH inhibition (%) = [(A control - A sample)/(A con- trol)] X 100 BHT and ascorbic acid served as positive controls. Each experiment was carried out in triplicate and data were presented as a mean of the three values (Singh et al. 2008). Iron binding capacity of essential oil Metal chelating activity of Rosa alba L. essential oil on ferrous ions was determined according to the method of Decker and Welch (1990). The percentage of inhibi- tion of ferrozine - Fe2+ complex formation was calculated using the formula: Chelating activity (%) = [(A control – A sample)/A con- trol] × 100 Where A control is the absorbance of the ferrozine - Fe2+ complex, and A sample is the absorbance of essen- tial oil. As a positive control EDTA was used. All results for antioxidant properties are presented as mean ± SD, and compared against routinely used ref- erence positive controls. The data were collected from three independent experiments with three parallel meas- urements for each experiment. Analysis of cytotoxic/anti-cytotoxic and genotoxic/anti-gen- otoxic effects of Rosa alba L. essential oil Rose essential oil and chemicals preparation Rose essential oil was dissolved in 1% dimetylsul- foxyde (DMSO). A standard well-known experimen- tal mutagen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) 50 μg/ml was used as DNA damage agent in the chromosome aberrations and micronucleus assays. MNNG was dissolved in bidistilled water. Test-systems Two types of experimental test-systems were used – Hordeum vulgare (barley) and human lymphocytes in vitro. Barley seed meristems and human lymphocyte cultures preparation as well as the schemes of treatment are given below. Hordeum vulgare (barley) meristem cells as a test- system. Seeds of reconstructed karyotype of Hordeum vulgare MK14/2034, 2n = 14 (Künzel and Nicoloff 1979) presoaked for 1 hour in tap water were germinated for 18h in Petri dishes on moist filter paper at 24oC. Well- synchronized seeds with a root meristem size of 1-2 mm were selected for further treatment. Experimental designs used for cytogenetic analysis. Three types of experimental schemes were applied. First to assess cytotoxic/genotoxic effects of rose essential oil, whole germinated seeds of barley with root meristems were treated with essential oil in concentrations from 250 to 1000 μg/ml. To assess the protective potential of rose oil germinated seeds were conditioning treated with 250 and/or 500 μg/ml for 60 min followed by 4h inter- treatment time and subsequent challenge treatment with 50 μg/ml MNNG (60 min). Third part of germinated seeds was pretreated with 250 and/or 500 μg/ml of rose oil (60 min), followed immediately by 50 μg/ml MNNG (60 min) without any inter-treatment time. For chromo- some aberrations evaluation the germinated seeds were treated at 24 oC for 2 hours with 0.025% colchicine in a saturated solution of α-bromonaphthaline after the treat- ment and recovery times for 18, 21, 24, 27 and 30 hours. The extracted embryos were fixed in a mixture of etha- nol and acetic acid (3:1), hydrolyzed in 1N HCl at 60oC for 9 min and stained with Schiff ’s reagent at room tem- perature for 1h. The root tips were macerated in a 4% pectinase solution for 12 min and squashed onto slides for scoring of metaphases with chromatid aberrations. For scoring of micronuclei, the root tips were fixed after 30h recovery time without colchicine treatment. Human lymphocytes in vitro as a test-system. Lym- phocy te cultures (1x106 mol/l) were prepared from venous blood of three healthy nonsmoking/nondrinking donors (men and women) aged between 33 to 50 years according to the standard method of Evans (1984). Each 75Assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of Bulgarian Rosa alba L. essential oil culture contained 3.5 ml RPMI 1640 medium, heat- inactivated fetal bovine serum (20%), phytohemaggluti- nin PHA (0.1%) and 40 mg/ml gentamycin (Pharmacia, Bulgaria). The study complies with the Declaration of Helsinki. Voluntary written informed consent was taken from all study participants. Experimental designs used for cytogenetic analy- sis. Lymphocyte cultures were treated with rose oil in a range of concentrations from 50 to 500 μg/ml to assess cytotoxic and genotoxic effect of Rosa alba L. essen- tial oil. To study its protective potential, non/or low cytotoxic concentrations of rose essential oil - 50 and/ or 200 μg/ml were applied as a conditioning treatment (60 min) followed by 4 hours inter-treatment time and a challenge treatment (60 min) with 50 μg/ml MNNG. Another part of the lymphocyte cultures was pretreat- ed with 50 and/or 200 μg/ml of rose oil (60 min), fol- lowed immediately by MNNG 50 μg/ml (60 min) with- out any inter-treatment time. After each treatment the lymphocyte cells were washed with a fresh RPMI medi- um. Untreated cells were used as a negative control. At the 72nd hour of cultivation to each culture was added 0.02% colchicine, then the cells were hypotonized in 0.56 % KCl; afterwards fixed in a mixture of methanol: glacial acetic acid (3:1, v/v) and stained in 2% Giemsa for assessment of chromosome aberrations. Lympho- cytes were directly fixed without colchicine for assess- ment of micronuclei. Cytogenetic analysis. Cytotoxicity of Bulgarian Rosa alba L. essential oil for both test-systems mentioned above was assessed by mitotic index (MI) using a formu- la: MI‰=A/1000, where A is a number of dividing cells. Genotoxic effect was evaluated by chromosome aberrations (CA) and micronuclei (MN) induction. Percentage of metaphases with chromosome aberra- tions (MwA % ± SD) was calculated. 1000 well spread metaphases (in M1 mitosis) of each treatment variant in both test-systems were assessed. Chromatid breaks (B’), isochromatid breaks (B”), translocations (T), intercalary deletions (D), duplication-deletions (DD) and dicentrics (DC) were determined. “Aberration hot spots” in the plant chromosomes were determined to obtain information about the DNA segments with higher susceptibility to DNA damage. For analyzing the locus specificity of aberration induction the metaphase chromosomes of H. vulgare were subdi- vided into 48 segments of approximately equal sizes. The segments are numbered with respect to their position in the standard karyotype as described earlier by Künzel and Nicoloff (1979). Percentage of micronuclei was calculated (MN % ± SD) where 4000 nuclei per experiment and treatment variant were assessed. Data analysis The results were calculated statistically by Student’s t-test and chi-square method. All experiments were repeated three times. An adapted formula was used for comparison of the upper limit of the confidence interval of the expected and observed chromatid aberrations in individual loci and evaluation of aberration „hot spots” in barley (Rieger et al. 1975; Künzel and Nicoloff 1979; Jovtchev et al. 2010). For multiple comparisons, a one- way analysis of variance (ANOVA) was employed, fol- lowed by Bonferroni s̀ correction. RESULTS Chromatogaphic profile of Rosa alba L. essential oil The chromatographic composition of the essen- tial oil of Bulgarian Rosa alba L. is presented in Table 1. The values were compared with literature data. The main groups are aliphatic hydrocarbons (AH) – 40.92% with high molecular weight (C15 – C27) as henei- cosane (12.75%), tricosane (2.69%), eicosane (1.46%), pentacosane (1.0%), heptacosane – 0.86%, (Z)-3-hene- icosene-0.64%, etc., followed by oxygenated monoter- penes (OM) – 40.35% as citronellol – 17.69 %, geraniol –16.64 %, trans-citral – 2.12%, linalool – 1.37%, β-citral – 0.68%, etc., sesquiterpenes hydrocarbons (SH) – 5.68% as caryophyllene – 1.52%, α-muurolene – 0.41%, b-cube- bene – 0.14%, etc., and oxygenated sesquiterpenes (OS) – 1.72%. The remaining 11.27% of the constituents were not reported due to their low content, less than 0.05% by mass, and/or not sufficiently reliably identification. After squalene some constituents leaving the column were in fact overlay with the column bleed and were also dis- carded from the final results. Superoxide scavenging analysis of Rosa alba L. essential oil The decrease of absorbance at 560 nm in semples with antioxidant supplements indicates the consump- tion of superoxide anion radical in the reaction mixture. As can be seen from Figure 1, Rosa alba L. essential oil exhibits moderate activity in superoxide scavenging assay in concentration range of 0–500 μg/ml. It is sig- 76 Svetla Gateva et al. Table 1. Volatile oil constituents of Bulgarian Rosa alba L. essential oil № Name Class R. alba oil, relative percetage Kovats RI (measured) Kovats RI (literature) Reference 1 Linalool OM 1.37 1092 1099 Babushok et al. 2011 2 cis Rose oxide HM 0.06 1124 1128 Babushok et al. 2011 3 a-Terpineol OM 0.27 1186 1190 Babushok et al. 2011 4 Citronellol OM 17.69 1227 1228 Babushok et al. 2011 5 β-Citral OM 0.68 1244 1249 Jalali-Heravi et al. 2006 6 Geraniol OM 16.64 1256 1255 Babushok et al. 2011 7 trans-Citral OM 2.12 1269 1276 Bertuzzi et al. 2013 8 Citronellyl format OM 0.23 1277 1277 Babushok et al. 2011 9 Geranyl formate OM 0.31 1289 1303 Babushok et al. 2011 10 Citronellol acetate OM 0.12 1346 1352 Babushok et al. 2011 11 3,7-dimethyl-2,6-Octadien-1-ol acetate OM 0.92 1380 1385 Wannes et al. 2009 12 Tetradecane AH 0.05 1400 1400 13 Caryophyllene SH 1.51 1419 1420 Babushok et al. 2011 14 b-Cubebene SH 0.14 1430 1434 Facey et al. 2005 15 Humulene SH 0.16 1453 1453 Babushok et al. 2011 16 Naphthalene, 1,2,3,4,4a,5,6,8a-oct SH 0.33 1479 1480 Marongiu et al. 2006 17 α-Muurolene SH 0.41 1504 1498 Babushok et al. 2011 18 g-Cadinene SH 0.19 1528 1523 Babushok et al. 2011 19 Salvial-4(14)-en-1-one SH 0.19 1557 1563 Pavlovic et al. 2006 20 Caryophyllene oxide SH 2.75 1580 1580 Babushok et al. 2011 21 3-Octadecine AH 0.15 1615   22 Heptadecane AH 0.54 1700 1700 23 (2Z,6E) Farnesol OS 1.72 1722 1722 Babushok et al. 2011 24 Z-5-Nonadecene AH 5.20 1880 1885 Tigrine-Kordiani et al. 2006 25 Nonadecane AH 12.18 1900   26 3-Nonadecyne AH 0.57 1911   27 3-Eicosene, (E)- AH 0.28 1953   28 Eicosane AH 1.46 2000   29 10-Heneicosene (c,t) AH 0.34 2070   30 (Z)-3-Heneicosene AH 0.64 2085   31 1-Heneicosene AH 0.55 2091   32 Heneicosane AH 12.75 2100   33 Docosene AH 0.41 2200   34 9-Tricosene, (Z)- AH 0.74 2290   35 Tricosane AH 2.69 2300 2300 36 1-Pentacosene AH 0.19 2490   37 Pentacosane AH 1.00 2500 2500 38 Hexacosane AH 0.09 2600 2600 39 1-Heptacosene AH 0.08 2697   40 Heptacosane AH 0.86 2700 2700 41 Octacosane AH 0.15 2800 2800 Heterocyclic monoterpenes (HM) – 0.06 % Oxygenated monoterpenes (OM) – 40.35% Sesquiterpenes hydrocarbons (SH) – 5.68% Oxygenated sesquiterpenes (OS) – 1.72% Aliphatic hydrocarbons (AH) – 40.92% Total identified - 88.73% 77Assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of Bulgarian Rosa alba L. essential oil nificantly lower than that of alpha tocopherol, but close to that of the Rosa damascena Mill. Оur previous works have shown that essential oils of rose species from dif- ferent origins in principal does not have high potential as a scavenger of superoxide anion radical (Mileva et al. 2014). DPPH – Radical scavenging assay Rosa alba L. oil, as well as citronellol and geraniol as main aromatic components of the essential oil were test- ed in terms of their DPPH – radical scavenging activity. The DPPH assay usually involves a hydrogen atom trans- fer reaction (Li et al. 2009). DPPH radical scavenging test is a sensitive antioxidant assay and depends on sub- strate polarity. As can be seen from the chromatographic composition of the essential oil, it is rich of components which possess ideal structural chemistry for DPPH radi- cal scavenging activity (Table 1). The presence of multi- ple hydroxyl functions could be considered as an option for hydrogen donation and/or radical scavenging activ- ity. As can be seen in Figure 2, we found that Bulgar- ian Rosa alba L. essential oil exhibits higher potency in scavenging DPPH radicals than geraniol and citronellol in the applied system, but significantly lower than the benchmark substances (BHT and ascorbic acid). In the concentrations in range of up to 50 μg/ml, Rosa alba L. and Rosa damascena Mill. rose oils have close activity, but in higher concentracions, over than 50 μg/ml, Rosa damascena Mill. oil exhibits about 10% higher activity. Iron binding capacity of Rosa alba L. essential oil One of the possible mechanisms of the antioxidant activity of essential oils is the chelation of transition metals. Among the transition metals, iron is known as the most active pro-oxidant, due to its high reactivity. The ferrous form of iron accelerates lipid peroxidation by breaking down hydrogen peroxide and lipid perox- ides to reactive free radicals by Fenton’s reaction (Li et al. 2010). The products of these reactions are able to oxi- dise cell lipid membranes, modify proteins, as well as to damage DNA. Chelating agents may inactivate metal ions and potentially inhibit the metal-dependent pro- cesses (Li et al. 2010). Ferrous ion chelating activities of the oil, citronellol and EDTA are shown in Figure 3. The chelating test provided that Rosa alba L. essential oil inhibits lipid peroxidation up to 75%. The antioxi- dant activity obtained in this test is significantly lower than the activity of EDTA but similar to this of citron- ellol. In the concentration in range of up to 50 μg/ml the two rose oils have close activity, but in higher than 50 μg/ml concentrations Rosa alba L. exhibits increas- ing chelating activity. Figure 1. Superoxide scavenging activity of Rosa alba L. essential oil. Data were calculated in percentage as spectrophotometric scav- enger index (SpSI) – the ratio of the absorption at 560 nm for the sample with oil, and the same absorption for the controls (without oil). The essential oil from Rosa damascena Mill. was used as a con- trol. Data are expressed as mean ± SD of three independent experi- ments. ***p<0.001 compared with alpha -tocopherol. Figure 2. DPPH scavenging activity (%) at various concentrations (μg/ml) of Bulgarian Rosa alba L. essential oil and its main ingredi- ents geraniol and citronellol. The essential oil from Rosa damascena Mill. was used as a control. Data are expressed as mean ± SD of three independent experiments. ***p<0.001 compared with citron- ellol (a), compared with BHT and ascorbic acid (b). 78 Svetla Gateva et al. Cytotoxic and genotoxic effects of Rosa alba L. essential oil Rosa alba L. essential oil didn’t show any cytotoxic effect in barley (Figure 4A). Here, human lymphocytes were found to be more susceptible than barley to rose oil in a concentration range (50-500 μg/ml) used in the present study. The rose oil decreased in a low extent the value of mitotic activity (Figure 4B) compared with the negative control in the lymphocyte cells (p < 0.01), whereas after treatment with MNNG (50 μg/ml) a well- expressed cy totoxic effect in both test-systems was observed (p < 0.001) (Figure 4A, 4B). Rosa alba L. essential oil treatment enhanced the induction of chromosome aberrations (MwA) compared to non-treated cells in both test – systems. These results showed its genotoxic effect (p < 0.05; p < 0.01, p < 0.001). The effect is clearly depended on the concentration applied (Figure 5A, 5B). Human lymphocyte cultures were more sensitive to Rosa alba L. oil than Hordeum vulgare. Genotoxic effect of rose oil was much lower (p < 0.001) than the alkylating agent MNNG (50 μg/ml) in both test-systems (Figure 5A, 5B). Analysis of the chromosome aberrations distribu- tion showed that in barley, rose essential oil induced only isochromatide breaks (data not shown). In human lymphocytes the observed chromosome aberrations were preferably B” 92.0 %, followed by B’ 8.0 %. In comparison with the rose oil, MNNG treatment (50 μg/ml) induced more diverse spectrum of chromosome disturbances in both test-systems. In Hordeum vulgare were obtained predominantly B”97.0 %, followed by B’ 3.0%, whereas in lymphocyte cultures B” were 88.6%, B’- 10.4%, DC-1%, respectively (data not shown) (Figure 6A, 6B). Treatment with rose oil enhanced the frequency of micronuclei (MN) clearly depending on the test-system and the concentration applied (Figure 6A, 6B; Figure 7A, 7B). No increase of the frequencies of this endpoint were observed in barley meristem cells (Figure 7A). The formation of micronuclei increased (p < 0.001) above two-fold (1.1%±0.3 for 50 μg/ml to 1.7%±0.2 for 500 μg/ ml) compared to the negative control (0.5%±0.2) in lym- phocyte cultures. The genotoxic effect of rose oil assessed as micronuclei is much lower than MNNG in the concen- tration applied in our study (p < 0.001) (Figure 7B). Anti-cytotoxic and anti-genotoxic potentials of Rosa alba L. essential oil Anti-cytotoxic potential Two types of experimental schemes were applied: i) conditioning treatment with non- toxic or low toxic Figure 3. Chelating activity of Bulgarian Rosa alba L. essential oil and its main constituent citronellol. The essential oil from Rosa damascena Mill. was used as a control. Data are expressed as mean ± SD of three independent experiments. ***p<0.001 compared with EDTA. Figure 4. Value of mitotic activity (MI) observed after Rosa alba L. essential oil treatment in Hordeum vulgare (A) and in human lym- phocyte cultures (B). Mitotic activity is calculated as a percent of the control. Data are expressed as mean ± SD of three independ- ent experiments. **p<0.01; ***p<0.001 compared with the negative control. Figure 5. Frequency of chromosome aberrations (MwA) calculated after Rosa alba L. essential oil treatment in Hordeum vulgare (A) and human lymphocyte cultures (B). Data are expressed as mean ± SD of three independent experiments.*p<0.05; **p<0.01 and ***p<0.001 compared with the negative control. 79Assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of Bulgarian Rosa alba L. essential oil concentrations of rose essential oil followed by chal- lenge treatment with alkylating agent MNNG (50 μg/ ml) and 4 hours inter-treatment time, ii) treatment without any inter-treatment time between treatments (Figure 8A, 8B). Mitotic activity (MI) obser ved after treatment showed clear dependence on the experimental design and test-systems (Figure 8A, 8B). The value of mitotic index was significantly increased (p < 0.01) after treat- ment applying both schemes of experimental design, compared to those obtained after MNNG (50 μg/ml) treatment alone in both test-systems (Figure 8A, 8B). A lack of any difference was obtained between the values of mitotic activity after rose essential oil conditioning treatment (250, 500 μg/ml for barley and 50, 200 μg/ml for human lymphocytes, respectively) followed by chal- lenge treatment with MNNG (50 μg/ml) with 4 hours Figure 6. Types of chromosome aberrations (CA) and micronuclei (MN) observed after treatment with Rosa alba L. essential oil in Hor- deum vulgare (A) and in human lymphocyte cultures (B). Figure 7. Induction of MN observed after Rosa alba L. essential oil treatment in Hordeum vulgare (A) and in human lymphocyte cul- tures (B). Data are expressed as mean ± SD of three independent experiments. **p<0.01 and ***p<0.001 compared with the negative control. 80 Svetla Gateva et al. inter-treatment time and treatment without any inter- treatment time. Anti-genotoxic potential Significantly (p < 0.05 till p < 0.001) lower frequency of chromosome aberrations was observed in Hordeum vulgare after conditioning treatment with rose essential oil (250, 500 μg/ml) prior to MNNG challenge (50 μg/ ml) with 4 hours inter-treatment time, compared to that induced after treatment with alkylating agent only (Fig- ure 9A). The reduction of MNNG induced chromosome aberrations was nearly three times lower in samples after rose oil 500 μg/ml conditioning (7.5%±1.6) compared to MNNG single treatment (20.7%±2.0). Similar anti-genotoxic effect (p < 0.001) was observed in human lymphocytes after applying the same experimental scheme of treatment – condition- ing with rose essential oil (50, 200 μg/ml) followed by challenge with MNNG (50 μg/ml) with 4 hour inter- treatment time (Figure 9B). Chromosome injuries were decreased approximately three times in samples condi- tioned with rose oil 50 μg/ml (5.6%±1.7) and with 200 μg/ml (6.0%±1.3) compared to MNNG treatment alone (16.3%±1.9). Lower structural chromosome disturbances were also calculated after treatment with rose essential oil and alkylating agent without any inter-treatment time (Figure 9A). The frequencies of chromosome aber- rations were decreased between 2.2-times for samples conditioned with 50 μg/ml rose oil to 1.8–times for samples conditioned with 200 μg/ml. Reduction of the frequencies of chromosome aberrations was observed in barley cells but to a lower extent (p < 0.01) also after treatment following the scheme without any inter-treat- ment time compared to those after MNNG treatment alone (Figure 9A). The spectrum of chromosome disturbances induced after applying experimental schemes for assessing anti- genotoxic potential of rose essential oil (250, 500 μg/ml) in H. vulgare showed predominantly B” 93%, followed by T 4% and B’ 3% in samples with 4 hours inter-treatment time, and B” 93%, T-5%, D-1%, B’-1% in samples without any inter-treatment time (data not shown). In human lymphocytes conditioning treated with rose oil (50, 200 μg/ml) followed by MNNG and inter-treatment time of 4 hours were obtained B” 84.4%, B’-11.8%, T-1.9%, RC-1% respectively, whereas in samples treated with rose oil and MNNG without any inter-treatment tine were observed mainly B” 85.5%, B’ 12.1% and T 2.4% (data not shown). By calculating the frequency of micronuclei as another endpoint for genotoxicity, it was observed that rose essential oil showed similar anti-genotoxic effect (p < 0.01, p < 0.001) in both test-systems (Figure 10A, 10B). The effect was obtained applying both schemes of exper- imental design. In Hordeum vulgare conditioning treat- ment with rose essential oil decreased MN approximate- ly two-times (0.97%±0.12 for 250 μg/ml and 1.02%±0.10 for 500 μg/ml) compared to that induced after treatment with the alkylating agent (1.95%±0.18). Similarly, lower MN frequency was obtained after treatment of barley cells without any inter-treatment time (Figure 10A). Fre- quency of micronuclei was decreased roughly 2 times – 1.0%±0.11 for 250 μg/ml and 1.04%±0.14 for 500 μg/ml. In human lymphocytes conditioning treatment with rose Figure 8. Anti-cytotoxic potential of rose essential oil assessed by mitotic index (MI) applying experimental schemes with: - Rosa alba L. essential oil conditioning treatment prior to MNNG challenge (50 μg/ml) with 4 hours inter-treatment time and, - without any inter-treatment time in Hordeum vulgare (A) and in human lym- phocyte cultures (B). Mitotic activity is calculated as a percent of the control. Data are expressed as mean ± SD of three independent experiments. **p<0.01 compared with MNNG. Figure 9. Anti-genotoxic potential of rose essential oil assessed by induction of chromosome aberrations (MwA) applying experimen- tal schemes with: - Rosa alba L. essential oil conditioning treatment prior to MNNG challenge (50 μg/ml) with 4 hours inter-treatment time and, - without any inter-treatment time in Hordeum vulgare (A) and in human lymphocyte cultures (B). Data are expressed as mean ± SD of three independent experiments. *p<0.05, **p<0.01 and ***p<0.001 compared with MNNG. 81Assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of Bulgarian Rosa alba L. essential oil oil decreased MN approximately four-times 1.0%±0.2 for 50 μg/ml and 1.6%±0.3 for 200 μg/ml compared with those induced by MNNG alone (3.9%±0.4). The frequen- cy of micronuclei was from 3-fold (for 50μg/ml) to 2.4- fold (200 μg/ml) lower than that of MNNG after treat- ment without any inter-treatment time (Figure 10B). “Aberration Hot Spots” in Barley. “Aberration hot spots” are a good expression tool to investigate genotoxic activity as well as anti-genotoxic potential of Rosa alba L. essential oil. They give additional information about the effect of the essential oil on Hordeum vulgare root meristem cells. The potential of MNNG to induce “aberration hot spots” in plant chromosomes was analyzed in the recon- structed barley karyotype MK14/2034. Seven out of the 48 inspected segments showed significant deviation from a random distribution of isochromatid breaks. Aber- ration clustering of isochromatid breaks (Table 2) was found in segment 10 and segment 14 of chromosome 2 (4.5% and 5.8%, respectively), in segment 17 of chromo- some 34 (6.4%), in segment 21 of chromosome 43 (4.2%), in segment 30 chromosome 5 (9.1%), and in segments 44 and 48 of chromosome 71 (7.3%, resp. 6.7%). All of these segments are located directly adjacent to the centromeres in the heterochromatin rich regions and are not connected with the regions of chromosome reconstruction. After R. alba L. essential oil treatment alone concentration dependent aberration hot spots (2 or 3, resp.) were observed (see Table 2), namely: after treatment with R. alba L. oil 250 µg/ml – segment 30 of chromosome 5 (8.2%) and segments 41 of chromo- some 6 (14.2%); after treatment with R. alba oil 500 µg/ ml – segment 14 of chromosome 2 (6.2%), segment 21 of chromosome 43 (6.2%) and segment 30 of chromosome 5 (10.0%)and after treatment with R. alba L. essential oil 1000 µg/ml – segment 17 of chromosome 34 (7.2%), Figure 10. Anti-genotoxic potential of rose essential oil assessed by induction of micronuclei (MN) applying experimental schemes with: - Rosa alba L. essential oil conditioning treatment prior to MNNG challenge (50 μg/ml) with 4 hours inter-treatment time and, - without any inter-treatment time in Hordeum vulgare (A) and in human lymphocyte cultures (B). Data are expressed as mean ± SD of three independent experiments. **p<0.01; ***p<0.001 com- pared with MNNG. Table 2. Observed aberration “hot spots” in chromosomes of barley root tip meristem cells of the reconstructed karyotype MK14/2034 after different treatment procedures Treatment variants Non-Spot segments Hot spot segments Chr.17 Chr.2 seg. 10 Chr.2 seg. 14 Chr.34 seg. 15 Chr.34 seg. 17 Chr.43 seg. 21 Chr.5 seg. 30 Chr.6 seg. 41 Chr.71 seg. 1 Chr.71 seg. 44 Chr.71 seg. 48 1. control 100% 2. MNNG (50 µg/ml) 56.0% 4.5% 5.8% 6.4% 4.2% 9.1% 7.3% 6.7% 3. Rosa alba oil (250 µg/ml) 77.6% 8.2% 14.2% 4. Rosa alba oil (500 µg/ml) 77.6% 6.2% 6.2% 10.0% 5. Rosa alba oil (1000 µg/ml) 72.2% 7.2% 10.3% 10.3% 6. Rosa alba oil (250 µg/ml) → MNNG (50 µg/ml) 60.3% 5.6% 6.3% 6.3% 6.9% 14.6% 7. Rosa alba oil (250 µg/ml) → 4h IT → MNNG (50 µg/ml) 60.6% 10.5% 11.4% 7.9% 9.6% 8. Rosa alba oil (500 µg/ml) → MNNG (50 µg/ml) 73.0% 8.5% 8.5% 10.0% 9. Rosa alba oil (500 µg/ml) → 4h IT → MNNG (50 µg/ml) 81.5% 7.6% 10.9% 82 Svetla Gateva et al. segment 30 of chromosome 5 (10.3%) and segment 41 of chromosome 6 (10.3%). Conditioning treatment with R. alba L. essential oil in concentrations of 250 µg/ml and 500 µg/ml, with- out any inter-treatment time prior to MNNG challenge decreased the aberration “hot spots” to five or three out of the 48 inspected segments (Table 2): R. alba L. essen- tial oil 250 µg/ml – segment 15 chromosome 34 (5.6%), segment 21 of chromosome 43 (6.3%), segment 30 of chromosome 5 (6.3%), segments 1 and 48 of chromo- some 71 (6.9% and 14.6%, resp.); R. alba L. oil 500 µg/ ml – segments 10 and 14 chromosome 2 (both 8.5%) and segment 48 of chromosome 71 (10.0%). After inter- treatment time of 4 hours between conditioning and challenge treatment only 4 aberrations “hot spots” for conditioning with R. alba L. oil 250 µg/ml showed a sig- nificant deviation from a random distribution of isoch- romatid breaks – segment 14 of chromosome 2 (10.5%), segment 15 of chromosome 34 (11.4%), segment 41 of chromosome 6 (7.9%) and segment 48 of chromosome 71 (9.6%). Two aberrations “hot spots” were found after conditioning with R. alba L. essential oil 500 µg/ml, namely segment 30 of chromosome 5 (7.7%) and seg- ment 44 of chromosome 71 (10.9%) (Table 2). “Aberration hot spots” were found in all treated var- iants in barley chromosomes – karyotype MK 14/2034, with exception of chromosome 17 (Table 2). In summary, applying both experimental schemes for assessing anti-genotoxic effect of R. alba L. essential oil the frequency of aberration “hot spots” was statisti- cally significant decreased compared to MNNG 50 µg/ ml (7 aberration “hot spots”) (see Table 2). After con- secutive treatment R. alba L. oil 500 µg/ml – 4 h inter- treatment time – MNNG the best result was achieved – reduction of aberration “hot spots” to 2 (Table 2). DISCUSSION In the current study valuable information was obtained about cytoprotective/genoprotective and anti- oxidant potentials of Rosa alba L. essential oil. The use of two types of test-systems – barley and human lym- phocytes in vitro, which are widely applied in the geno- toxic studies makes evaluation more representative. Our results showed that rose essential oil possessed good expressed DPPH radical scavenging activity; the effect increased with the increasing of the concentra- tion. It confirmed the data of Hatano et al. (1989) who proposed that rose’s extract radical scavenging ability is due to the polar-bounded hydrogen. The compounds, which are able to donate hydrogen, are able to break the chain reaction of lipid peroxidation at the first initiation step, and/or to produce redox-silent compounds. Gor- don (1990) reported that the chelating agents are effec- tive as secondary antioxidants because they reduce the redox potential thereby stabilizing the oxidized form of the metal ion. The data shown in Figure 3 reveal that the Bulgarian Rosa alba L. oil demonstrates an effective capacity as iron binding agent, depending on the con- centration applied, so its behavior as liposomal mem- brane peroxidation protector could be due to its iron binding capacity (Mileva et al. 2014). Moreover, in con- centrations higher than 400μg/ml Rosa alba L. oil has a higher activity than Rosa damascena Mill. In the present study Bulgarian Rosa alba L. essential oil didn’t show significant cytotoxic effect in barley but it has relatively low cytotoxicity in human lymphocytes in vitro depending on the concentration. Sinha et al. (2014) demonstrate that essential oils may be safe at low concen- trations, but show toxicity to humans at high concentra- tions represented as lethal doses. As typical lipophiles, essential oils can pass through the cell membrane and cytoplasmic membrane. They disrupt the structure of the different layers of polysaccharides, fatty acids and phos- pholipids and permeabilize through them (Bakkali et al. 2008). Their mode of action affects several targets at the same time. The cytotoxic activity of some essential oils for example of Ocotea quixos and others is mostly due to the presence of phenols, aldehydes and alcohols (Bruni et al. 2003; Sacchetti et al. 2005). In our study aliphatic hydrocarbons (AH) are the major compounds of Rosa alba L. essential oil. Here heneicosane (12.75%) and tri- cosane (2.69%) are representatives of the main compo- nents of this group (Table 1). Similar oil composition but in higher concentrations for some ingredients was detected for Rosa damascena Mill. by Kovacheva et al. (2010; 2011) and Mileva et al. (2014). The essential oils of plants such as Ceratonia siliqua (Hsouna et al. 2011), Ailanthus altissima (Albouchi et al. 2013) and Viscum album leaves (Cebovic et al. 2008), contain the same compounds in similar concentrations as in the white rose oil. They showed obvious cytotoxic effects, high antioxi- dant and phytotoxic activities. Shokrzadeh et al. (2017) reported a sensitivity of cancer cell line (A549) to high concentrations of rose oil obtained from Rosa damascene Mill. from Kashan. Probably these substances play a role in the cytotoxicicity of rose essential oil that we obtained for human lymphocytes. The higher resistance of Horde- um vulgare cells compared to the human lymphocytes is probably due to their different cell permeability and a cell wall existence compared to lymphocyte cells. The current investigation detects genotoxic activ- ity of Bulgarian Rosa alba L. essential oil depending on 83Assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of Bulgarian Rosa alba L. essential oil the concentrations in both test-systems applied. Shokr- zadeh et al. (2017) also reported that at concentrations of 50-200 μg/ml Rosa damascena Mill. oil significantly increased the frequency of micronuclei in human lym- phocytes. According to Bakkali et al. (2008) in the case of cytotoxicity and genotoxicity, essential oils can dam- age the cellular and organelle membranes and can act as pro-oxidants on proteins and DNA via production of reactive oxygen species (ROS). As it is known numerous plant extracts or phyto- chemicals have dual aspects, showing both genotoxicity and anti-genotoxicity against mutagens and carcinogens in vivo and in vitro test-systems (Kopaskova et al. 2012; Reddy et al. 2014; Gateva et al. 2015). Here we made an attempt to study the defense potential of Bulgarian Rosa alba L. essential oil and to determine the experimental conditions under which it can occur against alkylating agent MNNG. This is a typical mutagenic agent, which damages DNA. As a result it induced intra-strand, inter- strand crosslinks and double-strand breaks as well as base methylations, respectively (Kinzella and Radman 1980; Black et al. 1989). Extracts and essential oils of various plants were used to decrease cytotoxicity and genotoxicity induced by numerous genotoxins includ- ing alkylating agents (Vicuña et al. 2010; Mezzoug et al. 2007; Leffa et al. 2012; Madrigal-Santillán et al. 2013; Agabeyli 2012; Kuzilet et al. 2013; Matić et al. 2015). The current results clearly show anti-genotoxic potential of Bulgarian Rosa alba L. essential oil mani- fested by decreasing of the frequencies of chromosome aberrations and micronuclei after conditioning treat- ment with rose oil before MNNG challenge and 4 hours inter-treatment time in both experimental test-systems used by us. This is in agreement with our study (Gat- eva et al. 2019) where the preventive effect of acyclic monoterpenoid geraniol (which is one of the major rose essential oil compounds) was obtained against MNNG in human lymphocytes in vitro and Hordeum vulgare. Geraniol was found to be effective in limiting the geno- toxic effect of MNNG applied as conditioning treatment (4h inter-treatment time) prior challenge with MNNG compared to the samples treated with MNNG alone. Our current study also showed an increased resistance to the damaging effect of MNNG both in barley and in human lymphocytes after treatment with rose essen- tial oil and MNNG without any inter-treatment time. Hence the defence potential of Bulgarian Rosa alba L. essential oil is manifested regardless of the experi- mental conditions. The results about anti-cy totoxic and anti-genotoxic effect of the rose oil corresponded with those for antioxidant activity of rose essential oil obtained by DPPH test and iron chelating analysis. As a rule, the anti-cytotoxic, anti-genotoxic, and antioxidant properties of the plant extracts cannot be attributed of activities to single constituents. Their biological activ- ity could be explained with the combination of effects to one another. As can be seen on chromatographic profil of Rosa alba L. oil (Table 1), geraniol and citron- ellol are in major amounts in oil, so they could have an over additive participation in total antioxidant and iron chelating properties. Ruberto and Baratta (1999) demonstrated that most radical scavenging activities of essential oils are mainly due to the cumulative effect of ingredients nerol, citronellol and geraniol, within whose structure polar bounded hydrogen has been observed. Undoubtedly, DPPH radicals have little relevance as presence in biological systems, but the results are indic- ative of the capacity of the Bulgarian white rose oil to scavenge free radicals which relate to hydrogen atom or electron donation ability. There are data indicating that not only geraniol and citronellol, but citral (which belongs to the bioactive com- pounds of the Bulgarian Rosa alba L. essential oil) also possess antioxidant activity (Raut and Karuppayil 2014). Madankumar et al. (2013) showed that geraniol has a potent antioxidant effect by scavenging oxygen-free radi- cals and increasing the level of total glutathione content (GSH) in murine skin. It could modulate the activity of enzymatic and non-enzymatic antioxidants to exert its chemopreventive activity against 4-Nitroquinoline 1-oxide induced oral cancer in rats. Manoharan and Selvan (2012) proposed that geraniol inhibits abnormal cell proliferation occurring in skin carcinogenesis by modulating the activ- ities of Phase II detoxification agents and through free radical scavenging potential. Geraniol and camphene were found to significantly decrease lipid peroxidation, inhibit Nitric oxide release (83.84% and 64.61%) and ROS genera- tion in the pre-treated cells as compared to stressed cells (Tiwari and Kakkar 2009). Kashani et al. (2011) described a significant correlation between the phenolic content and DPPH scavenging capacity of white rose extracts. Our results indicate good iron chelating and DPPH radical- scavenging activities, medium superoxide scavenging abil- ity of Bulgarian R. alba L. essential oil, which is probably due to its chemical composition. Some authors reported that α-terpineol (which is one of the white rose essential oil compounds obtained by us) has remarkable ferrous ions chelating agent and possees antimutagenic activity against 2-aminoanthracene in S. typhimurium TA100 (Di Sotto et al. 2013). According to Bakkali et al. (2008) the mechanism of the decrease of mutagenicity did not depend only of the type of essential oil but on the type of mutagen, thus on the type of lesions and consequently on the DNA repair 84 Svetla Gateva et al. or lesion avoidance system involved. The protective effect obtained by us for Bulgarian Rosa alba L. essen- tial oil against alkylating agent MNNG suggests an acti- vation of repair pathways in addition to antioxidant and scavenging activity of rose essential oil. It is well known that N7-alkylG is responsible for about 90% of the total frequency of alkylation events among the alkyla- tion damages induced by alkylating agents. Quantity of O6-alkylG (DNA adduct formed by the alkylating agent) is less, but if not repaired could led to DNA damage such as cross-linking, strand breaks and modification of bases (Kondo et al. 2009). However N7-alkylG is consid- ered to be as mutagenic as O6-alkylG because it is effi- ciently repaired by base excision repair (BER) pathway (Drablos et al. 2004). O6-methylG lesions are repaired by direct damage reversal repair carried out by the enzyme O6-methylguanine-DNA methyltransferase (MGMT) also referred to as alkylguanine transferase (AGT). MGMT efficiently repairs O6-methylG before replica- tion, through direct transfer of the adducted methyl group from the oxygen in the guanine to a cysteine resi- due in the catalytic site of MGMT (Ramos et al. 2011). Niture et al. (2006; 2007) showed that both the ethanolic and aqueous extracts and compounds of some medical plants increased MGMT expression and its activity in lymphocytes and cancer cell lines. To understand the real mechanism of protective potential of rose essential oil when applied in combination with alkylating agents more studies are needed. CONCLUSION Bulgarian Rosa alba L. essential oil has low cyto- toxicity in barley and human lymphocytes in vitro in a dose-dependent manner, as well a good cytoprotec- tive/genoprotective effect against DNA damaging agent MNNG when applied both with 4 hours between treat- ments and without any inter-treatment time. Anti-geno- toxic potential of rose essential oil was manifested by the decrease of the frequency of chromosome aberrations and the micronuclei in both test-systems. Something more, white rose’s oil demonstrated well-pronounced anti-oxidant potential and very good metal chelating activity. The results show that rose oil contained protec- tive compounds that can decrease DNA damage. Data suggest a promising ethnopharmacological potential of Bulgarian white rose essential oil. It could serve in medi- cal cosmet as a prophylactic agent, and as an adjuvant in cancer prevention and therapy. GEOLOCATION Fresh flowers of Rosa alba L., from the experimen- tal field of the Institute of Rose and Essential Oil Plants (IREOP), in Kazanlak, Bulgaria were used. All studies were conducted in the laboratories of the Republic of Bulgaria. ACKNOWLEDGMENTS This work was supported by the project “Environ- mental and genetic assessment of the state of the envi- ronment, management and strategies for overcoming the risk” – Bulgarian Academy of Sciences, Sofia as well as partially supported by the Project of Bulgarian National Science Fund № KP-06 N36/17 (granted to M. Mileva). REFERENCES Adams RP. 2007. Identification of essential oil compo- nents by gas Chromatography quadropole mass spec- trometry; Allured Publishing Co. Carol Stream, IL: USA,; pp. viii + 804 pp. Agabeyli RA. 2012. Antimutagenic Activities Extracts from Leaves of the Morus alba, Morus nigra and Their Mixtures. Int. J. Biol. 4 (2): 166–172. doi:10.5539/ijb.v4n2p166 Albouchi F, Hassen I, Casabianca H, Hosni K. 2013. Phy- tochemicals, antioxidant, antimicrobial and phyto- toxic activities of Ailanthus altissima (Mill.) Swingle leaves. S. Af. J. Bot. 87: 164–174. Arumugam P, Ramamurthy P, Ramesh A. 2010. Anti- oxidant and Cytotoxic Activities of Lipophilic and Hydrophilic Fractions of Mentha Spicata L. (Lamiaceae). Int. J. Food Prop. 13 (1): 23-31. doi: 10.1080/10942910802144329. Babushok VI, Linstrom PJ, Zenkevich IG. 2011. Reten- tion indices for frequently reported compounds of plant essential oils. J. Phys. Chem. Ref. Data. 40, 4: 043101-1-47. https://doi.org/10.1063/1.3653552. Bakkali F, Averbeck S, Averbeck D, Idaomar M. 2008. Biological effects of essential oils--a review. Food Chem.Toxicol. 46: 446–475. doi: 10.1016/j. fct.2007.09.106. Bertuzzi G, Tirillini B, Angelini P, Venanzoni R. 2013. Antioxidative Action of Citrus limonum Essential Oil on Skin. Eur. J. Medicinal Plants. 3: 1, 1–9. Black KA, McFarland RD, Grisham JW, Smith GJ. 1989. Cell cycle perturbation and cell death after exposure of a human lymphoblastoid cell strain to N-methyl- 85Assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of Bulgarian Rosa alba L. essential oil N’-nitro-N-nitrosoguanidine. Am. J. Pathol. 134 (1): 53–61. Blasiak J, Trzeciak A, Gasiorowska AG, Drzewoski J, Maleska-Panas E. 2002. Vitamin C and quercetin modulate DNA-damaging effect of N-methyl-N'- nitro-N-nitrosoguanidine (MNNG). Plant Food. Hum. Nutr. 57: 53–61. Boskabady MH, Kiani S, Rakhshandah H. 2006. Relax- ant effects of Rosa damascena on guinea pig tracheal chains and its possible mechanism(s). J. Ethnophar- macol. 106: 377–382. doi: 10.1016/j.jep.2006.01.013. Bruni R, Medici A, Andreotti E, Fantin C, Muzzoli M, Dehesa M. 2003. Chemical composition and biologi- cal activities of Ishpingo essential oil, a traditional Ecuadorian spice from Ocotea quixos (Lam.) Kosterm (Lauraceae) flower calices. Food Chem. 85: 415–421. doi: 10.1080/14786419.2017.1402310. Cebovic T, Spasic S, Popovic M. 2008. Cytotoxic Effects of the Viscum album L. Extract on Ehrlich Tumour Cells In Vivo. Phytotherapy Research. 22: 1097–1103. doi: 10.1002/ptr.2464 Decker EA, Welch B. 1990. Role of ferritin as a lipid oxi- dation catalyst in muscle food. J. Agri. Food Chem. 38: 674–677. doi: 10.1021/jf00093a019. Degraf K. 2003. Bulletin of the Bulgarian National Asso- ciation of essential oil perfumery and cosmetics (BNAEOPC), 75, 4–5. Di Sotto A, Durazzi F, Sarpietro MG, Mazzanti G. 2013. Antimutagenic and antioxidant activities of some bioflavours from wine. Food Chem. Toxicol. 60: 141– 146. https://doi.org/10.1016/j.fct.2013.07.042 Dobreva A. (2010). Technological explorations on the yield and chemical composition of essential oil from white oil-bearing rose (Rosa alba L.). PhD disserta- tion. University of food technologies – Plovdiv, Bul- garia (in Bulgarian). Drablos F, Feyzi E, Aas PA, Vaagbo CB, Kavli B, Bratlie MS, Pena-Diaz J, Otterlei M, Slupphaug G, Krokan HE. 2004. Alkylation damage in DNA and RNA repair mechanisms and medical significance. DNA Repair (Amst). 3: 1389-1407. doi: 10.1016/j.dnarep.2004.05.004 EMA/HMPC/137298/2013 2013. European Medicines Agency, Committee on Herbal Medicinal Products (HMPC). 1–21. Evans H. 1984. Handbook of mutagenicity test proce- dures. B. Kilbey, M. Legator, W. Nicols, C. Ramel, (eds.), Elsevier Science Publishers BV: Amsterdam, 405–427. Facey PC, Porter RBR, Reese PB, Williams LAD. 2005. Biological activity and Chemical composition of the Essential Oil from Jamaican Hyptis verticillata Jacq., J. Agric. Food Chem. 53, 12: 4774-4777. Fukada M, Kano E, Miyoshi M, Komaki R, Watanabe T. 2011. Effect of “Rose Essential Oil” Inhalation on Stress-Induced Skin-Barrier Disruption in Rats and Humans. Chem. Senses. 37(4): 347-356. https://doi. org/10.1093/chemse/bjr108. Gateva S, Jovtchev G, Stankov A, Gregan F. 2015. Sal- via Extract Can Decrease DNA Damage Induced by Zeocin. Int. J. Pharm. Med. Biol. Sci. 4 (1): 1-10. doi: 10.12720/ijpmbs.4.1.1-10 Gateva S, Jovtchev G, Stankov A, Georgieva A, Dobre- va A, Mileva M. 2019. The potential of geraniol to reduce cytotoxic and genotoxic effects of MNNG in plant and human lymphocyte test-systems. S. Afr. J. Bot. 123: 170-179 doi:10.1016/j.sajb.2019.03.005. Gokbulut I, Bilenler T, Karabulut I. 2013. Determination of Chemical Composition, Total Phenolic, Antimicrobial, and Antioxidant Activities of Echinophora tenuifolia Essential Oil. Int. J. Food Prop. 16 (7): 1442-1451. Gordon MH. 1990. The mechanism of antioxidant action in vitro. B.J.F. Hudson, (ed.). Food antioxidants, Else- vier Applied Science: London: UK, 1–18. Hagag HA, Bazaid SA, Abdel-Hameed El-SS, Salman M. 2014. Cytogenetic, cytotoxic and GC-MS studies on concrete and absolute oils from Taif rose, Saudi Arabia. Cytotechnology 66: 913–923. doi: 10.1007/ s10616-013-9644-5. Hajhashemi V, Ghannadi A, Sharif B. 2003. Anti-inflam- matory and analgesic properties of the leaf extracts and essential oil of Lavandula angustifolia Mill. J. Ethnopharmacol. 89: 67–71. Hatano T, Edamatsu R, Mori A, Fujita Y, Yasuhara E, Okuda T. 1989. Effects of the interaction of tannins with co-existing substances. VI. Effects of tannins and related polyphenols on superoxide anion radical, and on 1,1-diphenyl-2-picrylhydrazyl radical. Chem. Pharm. Bull. 37: 2016–2021. Horváth G, Ács K. 2015. Essential oils in the treatment of respiratory tract diseases highlighting their role in bacterial infections and their anti-inflammatory action: a review. Flavour and Fragrance J. 30: 331– 341. |https://doi.org/10.1002/ffj.3252. Hsouna AB, Trigui M., Mansour RB, Jarraya RM, Damak M, Jaoua S. 2011. Chemical composition, cytotox- icity effect and antimicrobial activity of Ceratonia siliqua essential oil with preservative effects against Listeria inoculated in minced beef meat. Int. J. Food Microbiol. 148: 66–72. doi: 10.1016/j.ijfoodmi- cro.2011.04.028. http://www.iso.org/ ISO 9842:2003. Oil of rose (Rosa x damascena Miller). Jalali-Heravi M, Zekavat B, Sereshti H. 2006. Characteri- zation of essential oil components of Iranian gerani- 86 Svetla Gateva et al. um oil using gas chromatography-mass spectrometry combined with chemometric resolution techniques. J. Chromatogr. A, 1114, 1: 154–163. DOI: 10.1016/j. chroma.2006.02.034. Jovtchev G, Gateva S, Stergios M, Kulekova S. 2010. Cytotoxic and genotoxic effects of paraquat in Hor- deum vulgare and human lymphocytes in vitro. Envi- ron Toxicol. 25: 294-303. doi: 10.1002/tox.20503. Kashani AD, Rasooli I, Rezaei MB, Owlia P. 2011. Anti- oxidative properties and toxicity of white rose extract. Iran. J. Toxicol. 5 (1-2): 415–425. Kinzella AR, Radman M. 1980. Inhibition of carcinogen- induced chromosomal aberrations by an anticarcino- genic protease inhibitor. Proc. Natl. Acad. Sci. 77 (6): 3544–3547. Kopaskova M, Hadjo L, Yankulova B, Jovtchev G, Galo- va E, Sevcovicova A, Mucaji P, Miadokova E, Bryant P, Chankova S. 2012. Extract of Lillium candidum L. Can Modulate the Genotoxicity of the Antibiotic Zeocin. Molecules. 17(1): 80–97. doi:10.3390/mol- ecules17010080 Kondo N, Takahashi A, Ono K, Ohnishi T. 2010. DNA Damage Induced by Alkylating Agents and Repair Pathways. J. Nucleic Acids. Article ID 543531, 7 pag- es, doi: 10.4061/2010/543531 Kovacheva N, Rusanov K, Atanassov I. 2010. Industrial Cultivation of Oil Bearing Rose and Rose Oil Pro- duction in Bulgaria During 21ST Century, Directions and Challenges. Biotechnol. Biotechnol. Equip. 24 (2): 1793-1798. Kovatcheva N, Zheljazkov VD, Astatkie T. 2011. Pro- ductivity, Oil Content, Composition, and Bioactivity of Oil-bearing Rose Accessions. Hortscience. 46 (5): 710–714. Kuzilet H, Kasimoglu C, Uysal H. 2013. Can the Rosa canina Plant be Used Against Alkylating Agents as a Radical Scavenger? Pol. J. Environ. Stud. 22 (4): 1263–1267. Künzel G, Nicoloff H. 1979. Further results on karyotype reconstruction in barley. Biol. Zentralbl. 98: 587-592. Laribi B, Kouki K, M’Hamdi M, Bettaieb T. 2015. Cori- ander (Coriandrum sativum L.) and its bioactive constituents. Fitoterapia 103: 9–26. doi: 10.1016/j. fitote.2015.03.012. Leffa DD, Rosa R, Munhoz BP, Mello AAM, Mandelli FD, Amaral PA, Rossatto AE, Andrade VM. 2012. Geno- toxic and antigenotoxic properties of Calendula offici- nalis extracts in mice treated with methyl methane- sulfonate. Adv. Lif. Sci. 2 (2): 21-28. Li W, Hosseinian FS, Tsopmo A, Friel JK, Beta T. 2009. Evaluation of antioxidant capacity and aroma quality of breast milk. Nutrition. 25: 105–114. Li J, Nie S, Qiu Z, Che M, Li C, Xie M. 2010. Antimi- crobial and antioxidant activities of the essential oil from Herba Moslae. J. Sci. Food Agric. 90: 1347– 1352. https://doi.org/10.1002/jsfa.3941. Matić S, Katanić J, Stanić S, Mladenović M, Stanković N, Mihailović V, Boroja T. 2015. In vitro and in vivo assessment of the genotoxicity and antigenotoxicity of the Filipendula hexapetala and Filipendula ulmaria methanol extracts. J. Ethnopharmacol. 174: 287–292. doi: 10.1016/j.jep.2015.08.025 Madankumar A, Jayakumar S, Gokuladhas K, Rajan B, Raghunandhakumar S, Asokkumar S, Devaki T. 2013. Geraniol modulates tongue and hepatic phase I and phase II conjugation activities and may contrib- ute directly to the chemopreventive activity against experimental oral carcinogenesis. Eur. J. Pharmacol. 705: 148-155. doi: 10.1016/j.ejphar.2013.02.048 Madrigal-Santillán E, García-Melo F, Morales-González JA, Vázquez-Alvarado P, Muñoz-Juárez S, Zuñiga- Pérez C, Sumaya-Martínez MT, Madrigal-Bujaidar E, Hernández-Ceruelos A. 2013. Antioxidant and Anti- clastogenic Capacity of Prickly Pear Juice. Nutrients. 5 (10): 4145–4158. doi:10.3390/nu5104145. Manoharan S, Selvan MV. 2012. Chemopreventive poten- tial of geraniol in 7,12-dimethylbenz(a) anthracene (DMBA) induced skin carcinogenesis in Swiss albino mice. J. Environ. Biol. 33 (2): 255–260. Marongiu B, Porcedda S, Piras A, Sanna G, Murreddu M, Loddo R. 2006. Extraction of essential oil by super- critical carbon dioxide Juniperus communis L. ssp. nana Willd. Flavour Fragance J. 21 (1): 148-154. htt- ps://doi.org/10.1002/ffj.1549. Mezzoug N, Elhadri A, Dallouh A, Amkiss S, Skali NS, Abrini J, Zhiri A, Baudoux D, Diallo B, Jaziri MEl, Idaomar M. 2007. Investigation of the mutagenic and antimutagenic effects of Origanum compactum essen- tial oil and some of its constituents. Mutat. Res. 629: 100–110. Mileva M, Hadjimitova V, Tantcheva L, Traykov T, Galabov AS, Savov V, Ribarov S. 2000. Antioxidant properties of rimantadine in influenza virus infected mice and in some model systems. Z. Naturforsch C. 55 (9-10): 824–829. Mileva M, Krumova E, Miteva-Staleva J, Kostadi- nova N, Dobreva A, Galabov AS. 2014. Chemical Compounds, In Vitro Antioxidant and Antifungal Activities of Some Plant Essential Oils Belonging to Rosaceae Family. Compt. Rend. Acad. Bulg. Sci. 67: 1363–1368. Moein M, Karami F, Tavallali H, Ghasemi Y. 2010. Com- position of the essential oil of Rosa damascena Mill. Iran J. Pharm. Sci. 6: 59–62. 87Assessment of anti-cytotoxic, anti-genotoxic and antioxidant potentials of Bulgarian Rosa alba L. essential oil Naikwade NS, Mule SN, Adnaik RS, Magdum CS. 2009. Memory-enhancing activity of Rosa alba in mice. Int. J. Green Pharm. 3 (3): 239–242. Niture SK, Rao US, Srivenugopal KS. 2006. Chemopre- ventative strategies targeting the MGMT repair pro- tein: augmented expression in human lymphocytes and tumor cells by ethanolic and aqueous extracts of several Indian medicinal plants. Int. J. Oncol. 29: 1269-1278. doi: 10.3892/ijo.29.5.1269 Niture SK, Velu CS, Smith QR, Bhat GJ, Srivenugopa KS. 2007. Increased Expression of the MGMT Repair Protein Mediated by. Cysteine Prodrugs and Chemo- preventative Natural Products in Human. Lympho- cytes and Tumor Cell Lines. Carcinogenesis. 28: 378- 389. doi: 10.1093/carcin/bgl155 Oyeyemi IT, Bakare AA. 2013. Genotoxic and anti-gen- otoxic effect of aqueous extracts of Spondias mom- bin L., Nymphea lotus L. and Luffa cylindrica L. on Allium cepa root tip cells. Caryologia: Internat J. Cytol, Cytosyst. Cytogen. 66 (4): 360–367, doi: 10.1080/00087114.2013.857829. Pavlovic M, Kovacevic N, Tzakou O, Couladis M. 2006. Essential oil composition of Anthemis triumfetti (L.) DC. Flavour Fragrance J. 21 (2): 297–299. doi: 10.1002/ffj.1592. Ramos AA, Lima CF, Pereira-Wilson C. 2011. DNA dam- age protection and induction of repair by dietary phytochemicals and cancer prevention: What do we know? Chapter 11. C.C. Chen (ed.). Selected Topics in DNA Repair. University of California, San Diego: USA, 237–270. doi: 10.5772/22125 Rangaha MK. 2001. Rose – Rosa damascena ‘Trigintipe- tala.’ Crop Food Res. Broadsheet, 29. Raut JS, Karuppayil SM. 2014. A status review on the medicinal properties of essential oils. Ind. Crop. Prod. 62: 250–264. doi: 10.1016/j.ind- crop.2014.05.055. Reddy ChP, Devi KR. 2014. Evaluation of Antigenotoxic Effects of Aeges Marmalos Leaf Extract in Bone Mar- row Erythrocytes of Mice. Int. J. Pharm. Tech. Res. 6 (5): 1533-1538. Rieger R, Michaelis A, Schubert I, Döbel P, Jank HW. 1975. Non-random intrachromosomal distribution of chromatid aberrations induced by X-rays, alkylat- ing agents and ethanol in Vicia faba. Mutat. Res. 27: 69–79. Ruberto G, Baratta TM. 1999. Antioxidant activity of selected essential oil components in two lipid mod- el systems. Food Chem. 69: 167-174. doi:10.1016/ S0308-8146(99)00247-2 Sacchetti G, Maietti S, Muzzoli M, Scaglianti M, Man- fredini S, Radice M, Bruni R. 2005. Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobi- als in foods. Food Chem. 91: 621–632. doi:10.1016/j. foodchem.2004.06.031 Shohayeb M, Abdel-Hameed El-SS, Bazaid SA, Maghrabi I. 2014. Antibacterial and Antifungal Activity of Rosa damascena MILL. Essential Oil, Different Extracts of Rose Petals. Global J. Pharmacol. 8 (1): 1-7. doi: 10.5829/idosi.gjp.2014.8.1.81275. Shokrzadeh M, Habibi E, Modanloo M. 2017. Cytotoxic and genotoxic studies of essential oil from Rosa dam- ascena Mill., Kashan, Iran. Med Glas (Zenica) 14 (2): 152-157. Siddique YH, Ara G, Beg T, Afzal M. 2010. Anticlasto- genic effect of apigenin in human lymphocytes treat- ed with ethinylestradiol. Fitoterapia 81: 590–594. doi: 10.1016/j.fitote.2010.02.003. Singh HP, Kaur S, Mittal S, Batish DR, Kohli RK. 2008. Phytotoxicity of Major Constituents of the Volatile Oil from Leaves of Artemisia scoparia Waldst. & Kit. Z. Naturforsch. C. 63: 663–666. Sinha S, Jothiramajayam M, Ghosh M, Mukherjee A. 2014. Evaluation of toxicity of essential oils palma- rosa, citronella, lemongrass and vetiver in human lymphocytes. Food Chem. Toxicol. 68: 71–77. doi: 10.1016/j.fct.2014.02.036. Tabrizi H, Mortazavi A, Kamalinejad M. 2003. An in vitro evaluation of various Rosa damascena flower extracts as a natural antisolar agent. Int. J. Cosmet. Sci. 25: 259–265. doi: 10.1111/j.1467- 2494.2003.00189.x. Talib WH, Mahasneh AM. 2010. Antimicrobial, Cyto- toxicity and Phytochemical Screening of Jordanian Plants Used in Traditional Medicine. Molecules, 15: 1811–1824. doi:10.3390/molecules15031811. Tissot E, Rochat S, Debonneville C. A. 2012. Rapid GC- FID quantification technique without authentic sam- ples using predicted response factors. Flavour Fra- grance J. 27: 290–296. doi: 10.1002/ffj.3098. Tigrine-Kordiani N, Meklati BY, Chemat F. 2006. Abaly- sis by gas chromatography – mass spectrometry of the essential oil of Zygophyllum album L., an aro- matic and medicinal plant growing in Algeria. Int. J. Aromatherapy, 16 (3-4): 187–191. doi:10.1016/j. ijat.2006.09.008. Tiwari M, Kakkar P. 2009. Plant derived antioxidants – geraniol and camphene protect rat alveolar mac- rophages against t-BHP induced oxidative stress. Toxicol. In Vitro 23 (2): 295–301. doi: 10.1016/j. tiv.2008.12.014 Vicuña GC, Stashenko EE, Fuentes JL. 2010. Chemical composition of the Lippia origanoides essential oils 88 Svetla Gateva et al. and their antigenotoxicity against bleomycin-induced DNA damage. Fitoterapia 81: 343–349. doi: 10.1016/j. fitote.2009.10.008. Wannes WA, Mhamdi B, Marzouk B. 2009. GC compara- tive analysis of leaf essential oils from two myrtle varieties at different phenological stages. Chroma- tographia, 69 (1/2): 145–150. doi: 10.1365/s10337- 008-0818-9.