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Cytotoxic and Genotoxic Studies of Essential Oil from Rosa Damascene Mill., Kashan, Iran

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Cytotoxic and Genotoxic Studies of Essential Oil from Rosa Damascene Mill., Kashan, Iran ORIGINAL ARTICLE Cytotoxic and genotoxic studies of essential oil from Rosa damascene Mill., Kashan, Iran Mohammad Shokrzadeh1, Emran Habibi2, Mona Modanloo3 1Department of Toxicology-Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, 2Department of Phar- macognosy, Faculty of Pharmacy, Mazandaran University of Medical Sciences; Sari, Iran , 3Pharmaceutical Sciences Research Centre, Student research committe Mazandaran University of Medical Sciences, Sari, Iran ABSTRACT Aim Rosa damascene Mill. belongs to the family of Roseaceae and its essential oil is produced in large amounts in Iran. The wide application of rose oil has raised questions about potential adverse health effects. We have investigated cytotoxic activity and geno- toxic effects of Rosa oil from Kashan, Iran. Methods The cytotoxic effect and IC50 of the essential oil on the cell lines was studied followed by MTT assay. In this assay mi- Corresponding author: tochondrial oxidoreductase enzymes with reducing the tetrazolium Mona Modanloo dye MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) reflect the number of viable cells. Genotoxic effect of Pharmaceutical Sciences Research the oil was evaluated by micronucleus assay by evaluating produ- Center, Student research committee, ced micronuclei due to cytogenetic damage in binucleated lymp- Mazandaran University of Medical hocytes. Sciences, Results The results showed that essential oil significantly had Khazarabad Road, 48175-861 Sari, Iran; cytotoxic and genotoxic effects at doses over 10µg/mL (p<0.05). Phone: +98 911 1520 410; Also, essential oil of Rose showed lower IC50 in cancer cell line Fax: +981133543084; (A549) in comparison with the normal cell line (NIH3T3). Email: [email protected] Conclusion Cytotoxic and genotoxic properties of essential oil of Mohammad Shokrzadeh ORCID ID: http:// Rose in Kashan, Iran, are safe at a dose of 10µg/mL. Also, a good www.orcid.org/0000-0002-0071-6530 cytotoxic effect was shown and could be introduced as an antican- cer compound. Further studies are needed with regard to anti-can- cer effects of Rose essential oil. Original submission: 28 February 2017; Key words: micronucleus assay, MTT, Rose oil Revised submission: 15 March 2017; Accepted: 09 May 2017. doi: 10.17392/901-17 Med Glas (Zenica) 2017; 14(2): 152-157 152 Shokrzadeh et al. Rose oil cytotoxic and genotoxic effect INTRODUCTION MATERIALS AND METHODS Iran has a long history in cultivation and consump- Essential oil distillation tion of Rosa damascena, and it is known as an im- portant producer of rose oil in the world (1). Rosa Flowers were picked by hand before sunrise in damascena Mill. belongs to the family of Rose- May 2015 in the city of Kashan (Vidorj region), aceae (2). This ornamental is not only known as Iran. Botanical identification was confirmed by one of the most valuable sources of flavours and morphologic characteristics at the Department of fragrances in the world, but also it has some appli- Pharmacognosy, Sari Faculty of Pharmacy. The cations in medicine and food industry (3). flowers were subjected to steam distillation wit- Some evidence showed that Rose oil has some hin the same day (400gr fresh flowers in 2.0 L beneficial effects in the treatment of various di- water). After 3h hydrodistillation the obtained oil seases like premenstrual breast tenderness, in- was dried using anhydrous sodium sulphate. Pure o flammatory reactions, gall care and spasms (4). essential oil was stored at 4 C in a dark place (1). It also seems to have antidepressant and relaxing Dimethyl sulphate (DMSO) was used to dissolve effects. It is helpful for long-lasting cough, wo- essential oil (the final concentration of DMSO und healing, allergies and severe headache (3). was not over 1%) (14). Some evidence reported various potential adver- Cell culture se effects of Rosa damascene (5-7). On the other hand, a new study showed that while it is cytotoxic Experiments were carried out with cell lines in high doses, it did not show genotoxic effects (8). NIH3T3 (non-tumour fibroblast) and A549 (hu- man NSCLC cell line) (Pasteur Institute of Iran, The MTT test is an appropriate assay that has Tehran, Iran). often been used to investigate cytotoxicity cau- sed by medical plants (6). It is a rapid, low-cost Cells were cultured in Dulbecco’s Modified Ea- method based on the reduction of yellow te- gle Medium (DMEM) (Gibco-BRL, Germany) trazolium salt, MTT (3-(4,5-dimethylthiazol- with 10% fetal bovine serum (Gibco-BRL, Ger- 2-yl)-2,5-diphenyltetrazolium bromide), to dark many) and 100µg/mL streptomycin (Gibco-BRL, blue formazan in mitochondria of active cells (9). Germany) and 100IU/mL penicillin (Gibco-BRL, Thus, the amount of produced formazan has a di- Germany). Cell cultures were adjusted to allow rect relation with viable cells (10). for exponential growth. The micronucleus assay in binucleate human MTT assay lymphocytes is an effective tool to measure cyto- genetic damage of agents with different mechani- The protocol was adapted from the method des- sms of genotoxicity in vitro (11). Recent studies cribed by Shokrzadeh et al. (15). have shown that genomic instability is an early Cells (104 cells) were cultured with 200 µL event that occurs in some malignancies and it can DMEM/F12 medium containing bovine serum in be detected by examination of the peripheral blood 96 wells plate and incubated in 37 °C for 24 hours. lymphocytes as a sample of precursor cells (12). Stock solutions of Rose oil and cisplatin (a plati- Different studies reported some differences in num coordination complex with potent anti-neo- the composition of Rose oil in various regions plastic activity induces apoptosis in cancer cells, (8, 13, 14). possibly via caspase-3 activation) (16) were pre- Due to high application of Rose essential oil and pared in DMSO (1%) and phosphate buffered sa- with regard to the fact that Kashan is one of the line (PBS), respectively. biggest producers of Rose oil in Iran, this study After 48 hours of cell incubation with different aimed at assessing cytotoxicity of Rosa damasce- doses of essential oil (1, 10, 50, 100, 150 and 200 ne Mill.’s essential oil on both normal and cancer µl), 20µL MTT solution (5 mg/mL) was added to cell lines using MTT assay and also evaluating its each well. After 4 hours incubation at 37oC, the genotoxicity on human blood lymphocytes using formazan was dissolved in DMSO. Finally the micronuclei assay. optical density (OD) of wells was measured on a micro plate ELISA reader at 570 nm. All expe- 153 Medicinski Glasnik, Volume 14, Number 2, August 2017 riments were performed twice and each experi- less than 0.05 was considered as significant. The ment was run in triplicate, and mean values were IC50 (half maximal inhibitory concentration) va- recorded. lues were calculated by PRISM software using A linear relation between cell viability and OD of nonlinear regression. Standard deviations repre- each well is an exact determination of cell proli- sent average results of double experiments. The feration (17). The percentage of cell viability was IC50 values were compared using the student’s calculated using the equation (18): [Mean (OD) T-test measuring the effectiveness of a substance of treated cells/mean OD of control cells (1% to cause cell death or inhibit cell growth. So the DMSO)]×100 lower amount of IC50 represents a higher toxi- city of a compound, which leads to death or inhi- Micronucleus assay bition of cell growth (23). Fresh blood was collected from 10 healthy, no RESULTS smoking, no alcoholic, male donors aged betwe- en 25-35 years by venepuncture in heparini- During this study 0.2 mL essential oil of 1200 gr zed falcons; 0.5 mL of whole blood was added Rose flowers was produced. Comparing the results to 4.5mL of Roswell Park Memorial Institute in both MTT and micronucleus tests, data showed (RPMI) culture medium 1640 supplemented no significant difference between DMSO control with fetal bovine serum containing L-glutamin, group and the control without DMSO (Figures 1-4). antibiotics and phytohemagglutinin (PHA), and different doses of Rose oil (1, 10, 50, 100, 150 and 200 µL) were added. Cytochalasin B (Cyt-B) (Sigma, MO, USA) at the final concentration of 6µg/mL was added at 44h post PHA stimulation. Cyt B prevents complete cytokinesis in mito- sis, thus causing an appearance of multi-nucle- ar cells (19). The binucleated lymphocytes were harvested 28 hour after adding Cyt-B, they were treated by hypotonic KCl (0.075M) to red blood cell (RBC) Figure 1. Effect of Rosa damascene essential oil on A549 cell lysis. Then fixative solution (methanol: acetic viability *significant difference compared to the control group acid= 6:1) was added to the cells prior to slide (p<0.05) preparation and staining. For slide preparation 2-3 drops of cell suspension were thrown on a clean slide. The slides were stained with Giemsa solution (4%) for 7-10 mins. They were obser- ved at 40× and 100× magnifications using a light microscope to estimate mitotic index (the cells with 2 or more nuclei per 1000 observed cells) and micronuclei frequency (the number of micro- nuclei in at least 1000 binucleated cells) (8,20) binucleated cells are lymphocytes that were once divided by mitosis (21). The experiment was per- formed twice. Mitotic Index has a direct relation Figure 2. Effect of Rosa damascene essential oil on NIH3T3 with cells’ proliferative activity (8,22). cell viability *significant difference compared to the control group (p<0.05) Statistical analysis The MTT test with increasing doses of oil showed One way analysis of variance and tukey’s a decrease in viability in both normal and cancer honestly significant differences (HSD) test were cells, A549 and NIH3T3, respectively (Figures 1, used for multiple comparisons of data.
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