doi 10.1515/jcim-2013-0063
J Complement Integr Med. 2014; 11(1): 27–33
Kamal Razavi-Azarkhiavi, Javad Behravana, Fatemeh Mosaffa, Samira Sehatbakhsh, Kobra Shirani and Gholamreza Karimi*a
Protective effects of aqueous and ethanol extracts of rosemary on H2O2-induced oxidative DNA damage in human lymphocytes by comet assay Abstract Background: Rosemary (Rosmarinus officinalis) possesses various pharmacological properties such as antioxidant, anti-tumorigenesis and anti-mutagenesis activities. In this study, we investigated the possible protective effects of ethanol and aqueous extracts of rosemary on human lymphocyte DNA damage induced by H2O2. The extent of DNA lesions was measured using comet assay. Methods: Blood samples were taken from healthy volunteers and lymphocytes were isolated. The lymphocytes were then incubated in aqueous and ethanol extract of rosemary (0.05, 0.1, 0.5, 1 and 2.5 mg/mL) and H2O2 (50, 100 and 200 mM). Lymphocytes were also incubated with a combination of H2O2 (100 mM) with either 1 or 2.5 mg/mL of both extracts for 30 min at 4°C. Results: Our findings showed that H2O2 treatment led to a significant concentrate-dependent DNA damage in human lymphocyte when compared to respective controls (p < 0.001). The DNA damage which was initially occurred as the result of 100 µM H2O2 (Percentage tail DNA 55.1%) was inhibited due to the ethanol extract of rosemary at the doses tested (percentage tail DNA 4.7% and 4.03%). However, the aqueous extract has no effects on H2O2 genotoxicity. Conclusions: We suggest that antioxidant constituents in ethanol extract of rosemary can prevent human lymphocytes oxidative DNA damage which is due to its free radical scavenging activity.
Keywords: rosemary, DNA damage, human lymphocyte, comet assay, oxidative stress
a
Javad Behravan and Gholamreza Karimi contributed equally to this work. *Corresponding author: Gholamreza Karimi, Medical Toxicology Research Center and Pharmacy School, Mashhad University of Medical Sciences, Mashhad 1365-91775, Iran, E-mail: [email protected]
Kamal Razavi-Azarkhiavi, Department of Pharmacodynamy and Toxicology, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Javad Behravan, Fatemeh Mosaffa, Biotechnology Research Center and Pharmacy School, Mashhad University of Medical Sciences, Mashhad, Iran Samira Sehatbakhsh, Kobra Shirani, Department of Pharmacodynamy and Toxicology, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
Introduction Oxidative stress is associated with an overproduction of reactive oxygen species (ROS) together with attenuation in cellular antioxidant defenses [1–3]. The origin of ROS generation which are including hydroxyl radical (OH•), superoxide anion (O2•−), hydrogen peroxide (H2O2) and singlet oxygen can be intrinsically and/or extrinsically in the cell [4]. The antioxidant enzymes such as catalase, super oxide dismutase, glutathione reductase and glutathione peroxidase as well as non-enzymatic defense factors like glutathione play key roles in maintaining of oxidative–antioxidative balance [5, 6]. Free radical generation through the injury to cellular macromolecules such as membrane lipids, proteins and nucleic acids can lead to a various ranges of disorders including lung, kidney and liver diseases, atherosclerosis, diabetes mellitus and aging [7, 8]. DNA is one of the main targets of ROS which indicates the initial phase in carcinogenesis and mutagenesis. DNA protein cross-links, DNA strand breaks and alkali-labile sites are examples of oxidative DNA damage due to oxidative stress [9, 10]. The consumption of antioxidant dietary supplements can be a good strategy to verify the oxidative damages following ROS generation. Therefore, these days there is an increasing interest in using the natural antioxidant for therapeutic purposes. It has been demonstrated by various studies that natural compounds exhibit protective effects when used in oxidative stress–induced DNA damage models [11–13].
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Razavi-Azarkhiavi et al.: Effects of aqueous and ethanol extracts of rosemary
Rosemary (Rosmarinus officinalis), a native Mediterranean plant, is a member of Lamiaceae family [14]. The constituents of rosemary such as rosmarinic acid, caffeic acid, carnosol, rosmanol, 7-methyl-epirosmanol, isorosmanol, rosmadial, carnosic acid and other phenolic agents [15–17] gave it some protective properties including anti-inflammatory [18], anti-tumorigenesis, anti-mutagenesis [19, 20] and antioxidant activity [21]. The most effective antioxidant constituents of rosemary are carnosol and carnosic acid with more than 90% of the antioxidant activities of the rosemary extract [22]. Besides, it has been shown that rosemary play a protective role against genotoxic agents by suppression of DNA adducts formation [20, 23–26]. Alkaline single cell gel electrophoresis (SCGE) [Comet assay] as a sensitive, rapid and simple tool has been generally accepted for evaluating of DNA damage, repair studies, genotoxicity testing and human biomonitoring [27]. The aim of this study was to assess the anti-genotoxicity potential of ethanol and aqueous extracts of rosemary against H2O2-induced human lymphocytes DNA damage by using Comet assay.
Materials and methods Preparation of rosemary extract Aerial parts of rosemary were collected from the herbarium of the Pharmacy School, Mashhad University of Medical Sciences, Mashhad, Iran (Voucher no. 153-1815-06). After collection, the aerial parts of rosemary were cleaned, dried in the dark and powdered with a mechanical grinder.
Aqueous extract For the aqueous extract, 100 g aerial parts powder were mixed in 2 L distilled water, left to boil slowly for 15 min and filtered through cloth and qualitative Whatman filter. The extract was subsequently concentrated by heating in vacuo to the desired volume. Finally, the dry extract was stored at 4°C until used. The final product yielded 17% w/w.
Ethanol extract Powder was macerated in 500 mL ethanol (80%, v/v) for three consecutive days, then filtered and concentrated
under vacuo at 40°C to remove the ethanol content. The filtrate was re-suspended in saline normal with 1% Tween-20. The yield of dried ethanol extracts was 10%.
Isolation of human lymphocytes Blood samples were collected from healthy volunteers (25– 30 years) into tubes containing 10% ethylenediaminetetraacetic acid (EDTA) in PBS. About 5 mL of the blood was diluted 1:1 with PBS, and then carefully layered onto equivalent amounts of a lymphocyte separation medium (aqueous solution of Ficoll, 57 g/L; density of 1.077 g/mL) in a centrifugation tube. Samples were centrifuged at 447 g for 20 min, and then the gradient-separated lymphocytes were separated. Lymphocytes were diluted 1:1 with PBS, re-centrifuged at 252 g for 10 min and the final pellet was again suspended in 500 mL of PBS. Finally, the number of cells was counted in a Neubauer chamber. The cell concentration was adjusted to 5,000 cells/mL in preparation for the comet assay. Cell viability which was determined by trypan blue dye exclusion method [28] was greater than 97%.
Cellular treatment and DNA damage determination (comet assay) The comet assay was performed as described by Singh et al. in 1988 [29]. Human lymphocytes were incubated in different concentrations of H2O2 (50, 100 and 200 µM) as a positive control and different concentrations of aqueous or ethanol extract of rosemary (0.05, 0.1, 0.5, 1 and 2.5 mg/mL) alone. In the test groups, lymphocytes were exposed to H2O2 (100 µM) and 1 or 2.5 mg/mL of both extracts at 4°C for 30 min. In addition, we used the extract’s solvents without H2O2 as negative controls [30]. Samples were then centrifuged for 10 min at 1006 g and the cell pellets washed with PBS. In the next step, 100 mL of 0.75% (w/v) low melting point agarose (LMA) was add to cell pellets and mixed. The cell mixture was distributed onto microscope slides coated with 100 mL of 1% (w/v) normal melting agarose. The slides were then covered with a cover slip and let them to solidify for 10 min at 4°C. Afterward, the cover slips were removed and this step was again repeated. Then the slides were treated at least 2 h at 4°C (vertically and without a coverslip) in freshly prepared cold lysing solution containing 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% (v/v) triton
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Razavi-Azarkhiavi et al.: Effects of aqueous and ethanol extracts of rosemary
Results In this study, the human peripheral lymphocytes DNA damages following different treatments were assessed by using SCGE. As shown in Figure 1, H2O2 exposure (50, 100 and 200 µM) leads to a significant concentratedependent increase in DNA damage when compared to respective controls (p < 0.001). The genotoxic potential of aqueous and ethanol extracts of rosemary at the different doses (0.05, 0.1, 0.5, 1 and 2.5 mg/mL) were also evaluated on human peripheral lymphocytes (Figures 2 and 3). Our results
3
% DNA tail
X-100 and 10% DMSO [pH 10.0]. The slides were removed, washed with cold PBS and set into an electrophoresis tank side by side. DNA was unwinding in freshly prepared alkaline electrophoresis buffer for 30 min (1 mM Na2EDTA, 0.3 N NaOH, pH 13.0). Electrophoresis was run at 25 V for 45 min at 4°C. For minimizing additional DNA damage, all procedural steps were performed under yellow light conditions. The slides were then gently washed three times with neutralizing buffer (0.4 M Tris–HCl buffer, pH 7.5). A total of 20 µL ethidium bromide (20 mg/ mL) was dispensed directly onto slides and again covered with a cover slip. Finally, the slides were studied by a fluorescent microscope (Nikon 100, E 200, Japan) attached to a charge-coupled device camera. For calculating each analysis, 50 individual cells were selected. All tests were performed at least three times. Single cells were analyzed with TriTek Cometscore version 1.5 (www.autocomet.com). DNA damage was expressed as percentage tail DNA. A higher percentage tail DNA indicated a higher level of DNA damage. The percentage tail DNA was calculated by following formula: Percentage tail DNA ¼ [tail DNA/(head DNA þ tail DNA)] 100
2
1
Statistical analysis 0
The results were expressed as mean 6 standard error (SEM). Differences between groups were evaluated by one-way analysis of variance (ANOVA) followed by the Tukey’s test using SPSS analysis software (version 17.0). For the paired samples, comparison Student’s t test was used. p-Values less than 0.05 were considered significant.
0
0.05 0.1 0.5 1 Extract concentration (mg/mL)
2.5
Figure 2 The level of human lymphocytes DNA damage following treatment with different ethanol extract of rosemary compare to negative control. Lymphocytes were incubated for 30 min at 4°C with extract. Results are mean 6 SEM (n ¼ 4 slides 50 lymphocytes). ANOVA, p < 0.05.
7.5
100 * % DNA tail
% DNA tail
75 * *
50
5.0
2.5
25 0.0 0
0
50 100 H2O2 concentration (µM)
0
200
Figure 1 The level of DNA damage in H2O2-treated human lymphocytes. Lymphocytes were incubated for 30 min at 4°C with different concentrations of H2O2. Results are mean 6 SEM (n ¼ 4 slides 50 lymphocytes). ANOVA, p < 0.001.
0.05 0.1 0.5 1 Extract concentration (mg/mL)
2.5
Figure 3 The level of human lymphocytes DNA damage following treatment with different aqueous extract of rosemary compare to negative control. Lymphocytes were incubated for 30 min at 4°C with extract. Results are mean 6 SEM (n ¼ 4 slides 50 lymphocytes). ANOVA, p < 0.05.
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Razavi-Azarkhiavi et al.: Effects of aqueous and ethanol extracts of rosemary
75
60
% DNA tail
50
% DNA tail
40
30
50
25
20 g)
(1
*
Treatment Figure 4 Effect of ethanol extract of rosemary on lymphocyte DNA damage induced by H2O2. Human lymphocytes were incubated for 30 min at 4°C with a combination of H2O2 (100 µM) with different concentrations of ethanol extract of rosemary (1 and 2.5 mg/kg). Results are mean 6 SEM (n ¼ 4 slides 50 lymphocytes). ANOVA, *p < 0.001.
show that no significant DNA damage occurred due to treatment of cells with both extracts (p>0.05). As shown in Figure 4, the ethanol extract of rosemary at the doses of 1 and 2.5 mg/mL demonstrated a significant repressive effect on DNA damage induced by H2O2. (percentage tail DNA 4.7% 6 0.62 and 4.03% 6 0.63, respectively; p < 0.001). The data obtained from DNA damage in lymphocyte exposed to H2O2 (100 µM) and aqueous extract of rosemary (1, and 2.5 mg/mL) revealed that H2O2-induced DNA damage (percentage tail DNA 61.26% 6 1.65) was unaffected due to aqueous extract of rosemary treatment (Figure 5).
Discussion Natural antioxidants are considered to play an important role in enhancement of the endogenous antioxidant defenses against free radical generation and have a potential to create cellular oxidative–antioxidative balance by neutralizing the reactive species. Regarding, the oxidative DNA damage can be as a significant contributory cause of cancer, we investigated
2O 2+
H
g) m g/ k Ex t( 2. 5
2O 2+
H
H
2O 2+
Ex t
(1
H
m g/
2O 2
kg )
0
H
2O 2+
Ex t
*
m g/ k
H
10
Ex t( 2. 5
2O 2
m g/ kg )
0
Treatment Figure 5 Effect of aqueous extract of rosemary on lymphocyte DNA damage induced by H2O2. Human lymphocytes were incubated for 30 min at 4°C with a combination of H2O2 (100 µM) with different concentrations of aqueous extract of rosemary (1 and 2.5 mg/kg). Results are mean 6 SEM (n ¼ 4 slides 50 lymphocytes). ANOVA, p < 0.05.
the antigenotoxic activity of aqueous and ethanol extracts of rosemary toward oxidative DNA damage induced by hydrogen peroxide (H2O2) in human peripheral lymphocytes. H2O2 is an oxidant agent which does not direct any reaction with DNA. This free radical passes through the membranes and can enter to the nucleus. In presence of metal ions such as iron and copper, H2O2 over the Fenton reaction can generate highly reactive hydroxyl radical (•OH) [31]. Hydroxyl radicals attack to DNA and resulted in DNA strand breaks and fragmentation [32, 33]. The level of oxidative DNA damages following H2O2 exposure was detected by the single cell gel electrophoresis assay (comet assay), which is an inexpensive, sensitive, simple, versatile and rapid method for measurement of DNA strand breaks [34–37]. Because of the high sensitivity, studies at the individual cell level can be possible by using comet assay [34]. Our results indicated that treatment of human peripheral lymphocytes by different concentrations of H2O2 (50, 100 and 200 µM) leads to various levels of oxidative DNA damages which were determined by the comet assay (Figure 1). Besides, it was founded that the DNA damages due to H2O2 were dose-dependent. The genotoxicity potential of aqueous and ethanol extracts of rosemary were also investigated by SCGE. Our findings demonstrate that neither aqueous nor
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Razavi-Azarkhiavi et al.: Effects of aqueous and ethanol extracts of rosemary
ethanol extracts of rosemary in various concentrations resulted in human lymphocytes DNA damage. Slamenova et al. reported that long-term pre-incubation (24 h) of hamster V79 cells with 30 µg of rosemary/mL caused a significant increase of DNA damage, whereas no genotoxicity was observed due to other rosemary concentrations [38]. The difference in our findings may be due to the difference in cell line and time duration of exposure. It was also found that the ethanol extract of rosemary at the doses of 1 and 2.5 mg/mL displayed protective activities following chromosomal damage induced by H2O2 (Figure 6). Our results are similar to other studies which reported that long-term and short-term ethanol extract of rosemary treatment exhibits a protective effect against oxidative DNA damage in colon cancer cells CaCo-2 and hamster lung cells V79 [38]. Moreover, it has been reported that administration of rosemary essential oil to rats can increase resistance of hepatocytes against oxidative DNA damages. In this study, hepatocytes isolated from rats supplemented with rosemary oil were exposed to three genotoxins which attack to DNA includ-
A
B
31
ing H2O2, visible light-excited methylene blue and 2,3dimethoxy-1,4-naphthoquinone (DMNQ) [21]. However, no DNA protective activity was detected with the aqueous extracts of rosemary (p>0.05). The antioxidant properties of rosemary have been expressed by some studies [21, 39, 40]. It is also mentioned that antioxidant activity of rosemary is more potent than synthetic phenol antioxidant [41]. Carnosic acid and carnosol are responsible for more than 90% of rosemary antioxidant properties [15]. The antioxidant mechanism of rosemary extracts is attributed to its strong free radical scavenging activity as well as donation of electrons to free radicals to create less active species and inhibiting them from attacking to cellular biomolecules like proteins and DNA [42]. We can conclude that oxidative DNA damages in human peripheral lymphocytes, which were occurred following H2O2 treatment, were alleviated by ethanol extract of rosemary. This attenuation of DNA lesion can be attributing to antioxidative properties of the constituents present in the ethanol extract of rosemary. Acknowledgments: The authors are thankful to the Vice Chancellor of Research, Mashhad University of Medical Sciences for financial support. The results described in this paper are part of a Pharm. D. thesis.
Conflict of interest statement
C
D
Figure 6 DNA comet images of human lymphocytes. (A) Control, (B) Exposed to 100 μM H2O2 for 30 min. (C) and (D) Exposed to 1 and 2.5 mg ethanol extract of rosemary þ 100 μM H2O2
Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Received November 24, 2013; accepted January 12, 2014; previously published online February 4, 2014
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5. Brigelius-Flohe R. Tissue-specific functions of individual glutathione peroxidases. Free Radic Biol Med 1999;27:951–65. 6. Halliwell B. Drug antioxidant effects. A basis for drug selection? Drugs 1991;42:569–605. 7. Halliwell B, Gutteridge JM. Oxidative stress: adaptation, damage, repair and death. In free radicals in biology and medicine, 3rd ed. New York: Oxford Science Publications, 1999:246–350. 8. Elejalde JI. Oxidative stress, diseases and antioxidant treatment. An Med Interna 2001;18:326–35. 9. Kawanishi S, Hiraku Y, Murata M, Oikawa S. The role of metals in site-specific DNA damage with reference to carcinogenesis. Free Rad Biol Med 2002;32:822–32. 10. Shi H, Hudson LG, Liu KJ. Oxidative stress and apoptosis in metal ion-induced carcinogenesis. Free Rad Biol Med 2004;37:582–93. 11. Baratta GR. Antioxidant activity of selected essential oil components in two lipid model systems. Food Chem 2000;69:167–74. 12. Glei M, Pool-Zobel BL. The main catechin of green tea, (-) epigallocatechin-3-gallate (EGCG), reduces bleomycin induced DNA damage in human leucocytes. Toxicol Vitro 2006;20:295– 300. 13. Plazar J, Filipic M, Groothuis GM. Antigenotoxic effect of xanthohumol in rat liver slices. Toxicol Vitro 2008;22:318–27. 14. El-Din HM, Ghita EI, Badran SM, Gad AS, El-Said MM. Manufacture of low fat UF-soft cheese supplemented with rosemary extract (as natural antioxidant). J Am Sci 2010;6:570–9. 15. Aruoma OI, Halliwell B, Aeschbach R, Loligers J. Antioxidant and pro-oxidant properties of active rosemary constituents: carnosol and carnosic acid. Xenobiotica 1992;22:257–68. 16. Haraguchi H, Saito T, Okamura N, Yagi A. Inhibition of lipid peroxidation and superoxide generation by diterpenoids from Rosmarinus officinalis. Planta Med 1995;61:333–6. 17. Hernandez-Hernandez E, Ponce-Alquicira E, Jaramillo-Flores ME, Guerrero Legarreta I. Antioxidant effect rosemary (Rosmarinus officinalis L.) And oregano (Origanum vulgare L.) extracts on TBARS and colour of model raw pork batters. Meat Sci 2009;81:410–17. 18. Altinier G, Sosa S, Aquino RP, Mencherini T, Della Loggia R, Tubaro A. Characterization of topical anti-inflammatory compounds in Rosmarinus officinalis. L. J Agric Food Chem 1723;2007:1718–23. 19. Fahim F, Esmat A, Fadel H, Hassan K. Allied studies on the effect of Rosemarinus officinalis L. on experimental hepatotoxicity and mutagenesis. Int J Food Sci Nut 1999;50:413–27. 20. Singletary K, MacDonald C, Wallig M. Inhibition by rosemary and carosol of 7,12-dimethylbenz[α]anthracene (DMBA)induced rat mammary tumorigenesis and in vivo DMBA – DNA adduct formation. Cancer Lett 1996;104:43–8. 21. Horvathova E, Slamenova D, Navarova J. Administration of rosemary essential oil enhances resistance of rat hepatocytes against DNA-damaging oxidative agents. Food Chem 2010;123:151–6. 22. Posadas SJ, Caz V, Largo C, De la Gandara B, Matallanas B, Reglero G, et al. Protective effect of supercritical fluid rosem-
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Razavi-Azarkhiavi et al.: Effects of aqueous and ethanol extracts of rosemary
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J Complement Integr Med. 2014; 11(1): 27–33
Kamal Razavi-Azarkhiavi, Javad Behravana, Fatemeh Mosaffa, Samira Sehatbakhsh, Kobra Shirani and Gholamreza Karimi*a
Protective effects of aqueous and ethanol extracts of rosemary on H2O2-induced oxidative DNA damage in human lymphocytes by comet assay Abstract Background: Rosemary (Rosmarinus officinalis) possesses various pharmacological properties such as antioxidant, anti-tumorigenesis and anti-mutagenesis activities. In this study, we investigated the possible protective effects of ethanol and aqueous extracts of rosemary on human lymphocyte DNA damage induced by H2O2. The extent of DNA lesions was measured using comet assay. Methods: Blood samples were taken from healthy volunteers and lymphocytes were isolated. The lymphocytes were then incubated in aqueous and ethanol extract of rosemary (0.05, 0.1, 0.5, 1 and 2.5 mg/mL) and H2O2 (50, 100 and 200 mM). Lymphocytes were also incubated with a combination of H2O2 (100 mM) with either 1 or 2.5 mg/mL of both extracts for 30 min at 4°C. Results: Our findings showed that H2O2 treatment led to a significant concentrate-dependent DNA damage in human lymphocyte when compared to respective controls (p < 0.001). The DNA damage which was initially occurred as the result of 100 µM H2O2 (Percentage tail DNA 55.1%) was inhibited due to the ethanol extract of rosemary at the doses tested (percentage tail DNA 4.7% and 4.03%). However, the aqueous extract has no effects on H2O2 genotoxicity. Conclusions: We suggest that antioxidant constituents in ethanol extract of rosemary can prevent human lymphocytes oxidative DNA damage which is due to its free radical scavenging activity.
Keywords: rosemary, DNA damage, human lymphocyte, comet assay, oxidative stress
a
Javad Behravan and Gholamreza Karimi contributed equally to this work. *Corresponding author: Gholamreza Karimi, Medical Toxicology Research Center and Pharmacy School, Mashhad University of Medical Sciences, Mashhad 1365-91775, Iran, E-mail: [email protected]
Kamal Razavi-Azarkhiavi, Department of Pharmacodynamy and Toxicology, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Javad Behravan, Fatemeh Mosaffa, Biotechnology Research Center and Pharmacy School, Mashhad University of Medical Sciences, Mashhad, Iran Samira Sehatbakhsh, Kobra Shirani, Department of Pharmacodynamy and Toxicology, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
Introduction Oxidative stress is associated with an overproduction of reactive oxygen species (ROS) together with attenuation in cellular antioxidant defenses [1–3]. The origin of ROS generation which are including hydroxyl radical (OH•), superoxide anion (O2•−), hydrogen peroxide (H2O2) and singlet oxygen can be intrinsically and/or extrinsically in the cell [4]. The antioxidant enzymes such as catalase, super oxide dismutase, glutathione reductase and glutathione peroxidase as well as non-enzymatic defense factors like glutathione play key roles in maintaining of oxidative–antioxidative balance [5, 6]. Free radical generation through the injury to cellular macromolecules such as membrane lipids, proteins and nucleic acids can lead to a various ranges of disorders including lung, kidney and liver diseases, atherosclerosis, diabetes mellitus and aging [7, 8]. DNA is one of the main targets of ROS which indicates the initial phase in carcinogenesis and mutagenesis. DNA protein cross-links, DNA strand breaks and alkali-labile sites are examples of oxidative DNA damage due to oxidative stress [9, 10]. The consumption of antioxidant dietary supplements can be a good strategy to verify the oxidative damages following ROS generation. Therefore, these days there is an increasing interest in using the natural antioxidant for therapeutic purposes. It has been demonstrated by various studies that natural compounds exhibit protective effects when used in oxidative stress–induced DNA damage models [11–13].
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28
Razavi-Azarkhiavi et al.: Effects of aqueous and ethanol extracts of rosemary
Rosemary (Rosmarinus officinalis), a native Mediterranean plant, is a member of Lamiaceae family [14]. The constituents of rosemary such as rosmarinic acid, caffeic acid, carnosol, rosmanol, 7-methyl-epirosmanol, isorosmanol, rosmadial, carnosic acid and other phenolic agents [15–17] gave it some protective properties including anti-inflammatory [18], anti-tumorigenesis, anti-mutagenesis [19, 20] and antioxidant activity [21]. The most effective antioxidant constituents of rosemary are carnosol and carnosic acid with more than 90% of the antioxidant activities of the rosemary extract [22]. Besides, it has been shown that rosemary play a protective role against genotoxic agents by suppression of DNA adducts formation [20, 23–26]. Alkaline single cell gel electrophoresis (SCGE) [Comet assay] as a sensitive, rapid and simple tool has been generally accepted for evaluating of DNA damage, repair studies, genotoxicity testing and human biomonitoring [27]. The aim of this study was to assess the anti-genotoxicity potential of ethanol and aqueous extracts of rosemary against H2O2-induced human lymphocytes DNA damage by using Comet assay.
Materials and methods Preparation of rosemary extract Aerial parts of rosemary were collected from the herbarium of the Pharmacy School, Mashhad University of Medical Sciences, Mashhad, Iran (Voucher no. 153-1815-06). After collection, the aerial parts of rosemary were cleaned, dried in the dark and powdered with a mechanical grinder.
Aqueous extract For the aqueous extract, 100 g aerial parts powder were mixed in 2 L distilled water, left to boil slowly for 15 min and filtered through cloth and qualitative Whatman filter. The extract was subsequently concentrated by heating in vacuo to the desired volume. Finally, the dry extract was stored at 4°C until used. The final product yielded 17% w/w.
Ethanol extract Powder was macerated in 500 mL ethanol (80%, v/v) for three consecutive days, then filtered and concentrated
under vacuo at 40°C to remove the ethanol content. The filtrate was re-suspended in saline normal with 1% Tween-20. The yield of dried ethanol extracts was 10%.
Isolation of human lymphocytes Blood samples were collected from healthy volunteers (25– 30 years) into tubes containing 10% ethylenediaminetetraacetic acid (EDTA) in PBS. About 5 mL of the blood was diluted 1:1 with PBS, and then carefully layered onto equivalent amounts of a lymphocyte separation medium (aqueous solution of Ficoll, 57 g/L; density of 1.077 g/mL) in a centrifugation tube. Samples were centrifuged at 447 g for 20 min, and then the gradient-separated lymphocytes were separated. Lymphocytes were diluted 1:1 with PBS, re-centrifuged at 252 g for 10 min and the final pellet was again suspended in 500 mL of PBS. Finally, the number of cells was counted in a Neubauer chamber. The cell concentration was adjusted to 5,000 cells/mL in preparation for the comet assay. Cell viability which was determined by trypan blue dye exclusion method [28] was greater than 97%.
Cellular treatment and DNA damage determination (comet assay) The comet assay was performed as described by Singh et al. in 1988 [29]. Human lymphocytes were incubated in different concentrations of H2O2 (50, 100 and 200 µM) as a positive control and different concentrations of aqueous or ethanol extract of rosemary (0.05, 0.1, 0.5, 1 and 2.5 mg/mL) alone. In the test groups, lymphocytes were exposed to H2O2 (100 µM) and 1 or 2.5 mg/mL of both extracts at 4°C for 30 min. In addition, we used the extract’s solvents without H2O2 as negative controls [30]. Samples were then centrifuged for 10 min at 1006 g and the cell pellets washed with PBS. In the next step, 100 mL of 0.75% (w/v) low melting point agarose (LMA) was add to cell pellets and mixed. The cell mixture was distributed onto microscope slides coated with 100 mL of 1% (w/v) normal melting agarose. The slides were then covered with a cover slip and let them to solidify for 10 min at 4°C. Afterward, the cover slips were removed and this step was again repeated. Then the slides were treated at least 2 h at 4°C (vertically and without a coverslip) in freshly prepared cold lysing solution containing 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% (v/v) triton
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Razavi-Azarkhiavi et al.: Effects of aqueous and ethanol extracts of rosemary
Results In this study, the human peripheral lymphocytes DNA damages following different treatments were assessed by using SCGE. As shown in Figure 1, H2O2 exposure (50, 100 and 200 µM) leads to a significant concentratedependent increase in DNA damage when compared to respective controls (p < 0.001). The genotoxic potential of aqueous and ethanol extracts of rosemary at the different doses (0.05, 0.1, 0.5, 1 and 2.5 mg/mL) were also evaluated on human peripheral lymphocytes (Figures 2 and 3). Our results
3
% DNA tail
X-100 and 10% DMSO [pH 10.0]. The slides were removed, washed with cold PBS and set into an electrophoresis tank side by side. DNA was unwinding in freshly prepared alkaline electrophoresis buffer for 30 min (1 mM Na2EDTA, 0.3 N NaOH, pH 13.0). Electrophoresis was run at 25 V for 45 min at 4°C. For minimizing additional DNA damage, all procedural steps were performed under yellow light conditions. The slides were then gently washed three times with neutralizing buffer (0.4 M Tris–HCl buffer, pH 7.5). A total of 20 µL ethidium bromide (20 mg/ mL) was dispensed directly onto slides and again covered with a cover slip. Finally, the slides were studied by a fluorescent microscope (Nikon 100, E 200, Japan) attached to a charge-coupled device camera. For calculating each analysis, 50 individual cells were selected. All tests were performed at least three times. Single cells were analyzed with TriTek Cometscore version 1.5 (www.autocomet.com). DNA damage was expressed as percentage tail DNA. A higher percentage tail DNA indicated a higher level of DNA damage. The percentage tail DNA was calculated by following formula: Percentage tail DNA ¼ [tail DNA/(head DNA þ tail DNA)] 100
2
1
Statistical analysis 0
The results were expressed as mean 6 standard error (SEM). Differences between groups were evaluated by one-way analysis of variance (ANOVA) followed by the Tukey’s test using SPSS analysis software (version 17.0). For the paired samples, comparison Student’s t test was used. p-Values less than 0.05 were considered significant.
0
0.05 0.1 0.5 1 Extract concentration (mg/mL)
2.5
Figure 2 The level of human lymphocytes DNA damage following treatment with different ethanol extract of rosemary compare to negative control. Lymphocytes were incubated for 30 min at 4°C with extract. Results are mean 6 SEM (n ¼ 4 slides 50 lymphocytes). ANOVA, p < 0.05.
7.5
100 * % DNA tail
% DNA tail
75 * *
50
5.0
2.5
25 0.0 0
0
50 100 H2O2 concentration (µM)
0
200
Figure 1 The level of DNA damage in H2O2-treated human lymphocytes. Lymphocytes were incubated for 30 min at 4°C with different concentrations of H2O2. Results are mean 6 SEM (n ¼ 4 slides 50 lymphocytes). ANOVA, p < 0.001.
0.05 0.1 0.5 1 Extract concentration (mg/mL)
2.5
Figure 3 The level of human lymphocytes DNA damage following treatment with different aqueous extract of rosemary compare to negative control. Lymphocytes were incubated for 30 min at 4°C with extract. Results are mean 6 SEM (n ¼ 4 slides 50 lymphocytes). ANOVA, p < 0.05.
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Razavi-Azarkhiavi et al.: Effects of aqueous and ethanol extracts of rosemary
75
60
% DNA tail
50
% DNA tail
40
30
50
25
20 g)
(1
*
Treatment Figure 4 Effect of ethanol extract of rosemary on lymphocyte DNA damage induced by H2O2. Human lymphocytes were incubated for 30 min at 4°C with a combination of H2O2 (100 µM) with different concentrations of ethanol extract of rosemary (1 and 2.5 mg/kg). Results are mean 6 SEM (n ¼ 4 slides 50 lymphocytes). ANOVA, *p < 0.001.
show that no significant DNA damage occurred due to treatment of cells with both extracts (p>0.05). As shown in Figure 4, the ethanol extract of rosemary at the doses of 1 and 2.5 mg/mL demonstrated a significant repressive effect on DNA damage induced by H2O2. (percentage tail DNA 4.7% 6 0.62 and 4.03% 6 0.63, respectively; p < 0.001). The data obtained from DNA damage in lymphocyte exposed to H2O2 (100 µM) and aqueous extract of rosemary (1, and 2.5 mg/mL) revealed that H2O2-induced DNA damage (percentage tail DNA 61.26% 6 1.65) was unaffected due to aqueous extract of rosemary treatment (Figure 5).
Discussion Natural antioxidants are considered to play an important role in enhancement of the endogenous antioxidant defenses against free radical generation and have a potential to create cellular oxidative–antioxidative balance by neutralizing the reactive species. Regarding, the oxidative DNA damage can be as a significant contributory cause of cancer, we investigated
2O 2+
H
g) m g/ k Ex t( 2. 5
2O 2+
H
H
2O 2+
Ex t
(1
H
m g/
2O 2
kg )
0
H
2O 2+
Ex t
*
m g/ k
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Ex t( 2. 5
2O 2
m g/ kg )
0
Treatment Figure 5 Effect of aqueous extract of rosemary on lymphocyte DNA damage induced by H2O2. Human lymphocytes were incubated for 30 min at 4°C with a combination of H2O2 (100 µM) with different concentrations of aqueous extract of rosemary (1 and 2.5 mg/kg). Results are mean 6 SEM (n ¼ 4 slides 50 lymphocytes). ANOVA, p < 0.05.
the antigenotoxic activity of aqueous and ethanol extracts of rosemary toward oxidative DNA damage induced by hydrogen peroxide (H2O2) in human peripheral lymphocytes. H2O2 is an oxidant agent which does not direct any reaction with DNA. This free radical passes through the membranes and can enter to the nucleus. In presence of metal ions such as iron and copper, H2O2 over the Fenton reaction can generate highly reactive hydroxyl radical (•OH) [31]. Hydroxyl radicals attack to DNA and resulted in DNA strand breaks and fragmentation [32, 33]. The level of oxidative DNA damages following H2O2 exposure was detected by the single cell gel electrophoresis assay (comet assay), which is an inexpensive, sensitive, simple, versatile and rapid method for measurement of DNA strand breaks [34–37]. Because of the high sensitivity, studies at the individual cell level can be possible by using comet assay [34]. Our results indicated that treatment of human peripheral lymphocytes by different concentrations of H2O2 (50, 100 and 200 µM) leads to various levels of oxidative DNA damages which were determined by the comet assay (Figure 1). Besides, it was founded that the DNA damages due to H2O2 were dose-dependent. The genotoxicity potential of aqueous and ethanol extracts of rosemary were also investigated by SCGE. Our findings demonstrate that neither aqueous nor
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Razavi-Azarkhiavi et al.: Effects of aqueous and ethanol extracts of rosemary
ethanol extracts of rosemary in various concentrations resulted in human lymphocytes DNA damage. Slamenova et al. reported that long-term pre-incubation (24 h) of hamster V79 cells with 30 µg of rosemary/mL caused a significant increase of DNA damage, whereas no genotoxicity was observed due to other rosemary concentrations [38]. The difference in our findings may be due to the difference in cell line and time duration of exposure. It was also found that the ethanol extract of rosemary at the doses of 1 and 2.5 mg/mL displayed protective activities following chromosomal damage induced by H2O2 (Figure 6). Our results are similar to other studies which reported that long-term and short-term ethanol extract of rosemary treatment exhibits a protective effect against oxidative DNA damage in colon cancer cells CaCo-2 and hamster lung cells V79 [38]. Moreover, it has been reported that administration of rosemary essential oil to rats can increase resistance of hepatocytes against oxidative DNA damages. In this study, hepatocytes isolated from rats supplemented with rosemary oil were exposed to three genotoxins which attack to DNA includ-
A
B
31
ing H2O2, visible light-excited methylene blue and 2,3dimethoxy-1,4-naphthoquinone (DMNQ) [21]. However, no DNA protective activity was detected with the aqueous extracts of rosemary (p>0.05). The antioxidant properties of rosemary have been expressed by some studies [21, 39, 40]. It is also mentioned that antioxidant activity of rosemary is more potent than synthetic phenol antioxidant [41]. Carnosic acid and carnosol are responsible for more than 90% of rosemary antioxidant properties [15]. The antioxidant mechanism of rosemary extracts is attributed to its strong free radical scavenging activity as well as donation of electrons to free radicals to create less active species and inhibiting them from attacking to cellular biomolecules like proteins and DNA [42]. We can conclude that oxidative DNA damages in human peripheral lymphocytes, which were occurred following H2O2 treatment, were alleviated by ethanol extract of rosemary. This attenuation of DNA lesion can be attributing to antioxidative properties of the constituents present in the ethanol extract of rosemary. Acknowledgments: The authors are thankful to the Vice Chancellor of Research, Mashhad University of Medical Sciences for financial support. The results described in this paper are part of a Pharm. D. thesis.
Conflict of interest statement
C
D
Figure 6 DNA comet images of human lymphocytes. (A) Control, (B) Exposed to 100 μM H2O2 for 30 min. (C) and (D) Exposed to 1 and 2.5 mg ethanol extract of rosemary þ 100 μM H2O2
Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Received November 24, 2013; accepted January 12, 2014; previously published online February 4, 2014
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