PHYTOTHERAPY RESEARCH Phytother. Res. 24: 85–89 (2010) Published online 14 May 2009 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ptr.2874
Auraptene from Ferula szowitsiana Protects Human Peripheral Lymphocytes Against Oxidative Stress Fatemeh Soltani,1,2 Fatemeh Mosaffa,1,2 Mehrdad Iranshahi,1,2* Gholamreza Karimi,3,4 Mohammad Malekaneh,5 Fatemeh Haghighi6 and Javad Behravan1,2 1
Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran 3 Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran 4 Medical Toxicology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran 5 Department of Clinical Biochemistry, Birjand University of Medical Sciences, Birjand, Iran 6 Department of Pathology, Birjand University of Medical Sciences, Birjand, Iran 2
The antigenotoxicity effects of auraptene on DNA damage in human peripheral lymphocytes were studied using alkaline single cell gel electrophoresis. Auraptene at concentrations of 5, 10, 25, 50, 100, 200 and 400 mM was tested under simultaneous treatment with 25 mM H2O2. The data are expressed as % tail DNA and compared with ascorbic acid at concentrations of 25, 50, 100, 200 and 400 mM. Auraptene significantly reduced the genotoxicity of H2O2 at concentrations higher than 25 mM (p < 0.001). Interestingly, the antigenotoxicity activity of auraptene was higher than ascorbic acid (p < 0.01), however, at some concentrations (25, 50 and 200 mM) there was no significant difference between auraptene and ascorbic acid (p > 0.05). It seems that the significant antigenotoxicity effects of auraptene may be due to the prenyl moiety and also the suppression of superoxide anion (O2-) generation. This study suggests that the antigenotoxic property of auraptene is of great pharmacological importance and might be beneficial for cancer prevention. Copyright ” 2009 John Wiley & Sons, Ltd. Keywords: antigenotoxicity; comet assay; auraptene.
INTRODUCTION Oxidative stress is known to play an important role in the etiology of several human diseases such as cancer, atherosclerosis, arthritis and aging (Bonomini et al., 2008; Ishii, 2007; Laviano et al., 2007; Seven et al., 2008). Oxidative stress can occur through the overproduction of reactive oxygen species (ROS). Reactive oxygen species are formed during normal cell aerobic respiration (Barzilai and Yamamoto, 2004). They may not only have an impact on DNA damage but also influence DNA messengers to modulate DNA replication and the cell cycle (Hu et al., 1995). Thus, to eliminate oxidative stress such as reactive oxygen species in the body, many researchers have been attempting to develop antioxidant agents (Baratta and Ruberto, 2000; Kogure et al., 2004; Torres et al., 2006; Yen et al., 2002). There is some evidence that some plant-derived chemicals have protective effects towards DNA damage due to oxidative stresses (Glei and Pool-Zobel, 2006; Plazar et al., 2008). Coumarins are a large class of natural derivatives mainly found in the families Umbelliferae and Rutaceae (Curini et al., 2006). Auraptene, 7-geranloxycoumarin (Fig. 1), is synthesized by various plant species * Correspondence to: Dr Mehrdad Iranshahi, Department of Pharmacognosy, Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences (MUMS), P.O. Box: 91775-1365, Mashhad, Iran. E-mail:
[email protected]
Copyright © 2009 John Wiley & Sons, Ltd.
such as Ferula and Citrus (Iranshahi et al., 2007; Ju-Ichi, 2005). It is endowed with interesting medicinal properties including antileishmanial, antihelicobacter and antioxidant activities (Iranshahi et al., 2007; Murakami and Ohigashi, 2006; Takeda et al., 2007). Auraptene has been reported to be an effective inhibitor of chemical carcinogenesis in some rodent models (Kohno et al., 2006). In addition to an array of biological effects, a significant cancer chemoprevention activity of auraptene was reported (Iranshahi et al., 2008; Ju-Ichi, 2005). Also, it can suppress superoxide anion (O2−) generation from inflammatory leucocytes in in vitro experiments (Murakami et al., 2000). Despite the interesting biological activities of auraptene, there is no published report about the antigenotoxicity effect of this compound. Recently, the antigenotoxicity effect of umbelliprenin, 7-farnesyloxycoumarin (Fig. 1) which has a structure close to auraptene, was reported (Soltani et al., 2008). In view of the fact that the only difference between umbelliprenin and auraptene structures is the higher length of the prenyl chain in umbelliprenin and due to the lack of information about its antigenotoxicity, this paper was addressed to evaluate the antigenotoxicity effects of auraptene against DNA damage induced by hydrogen peroxide in human lymphocytes. The study used the most common technique, the comet assay (Rojas et al., 1999) which is a rapid, simple, sensitive and reliable biochemical method for evaluating DNA damage in cells or in tissues. Furthermore, the antigenotoxicity effect of auraptene was compared with those reported for umbelliprenin. Received 27 January 2009 Revised 11 March 2009 Accepted 24 March 2009
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Umbelliprenin Figure 1. Chemical structures of auraptene and umbelliprenin.
EXPERIMENTAL Auraptene isolation. Auraptene (C19H22O3, MW 298.4 g/mol) was purified (>95%) as described previously from the dried roots of Ferula szowitsiana D.C. collected from the mountains of Golestan forest (Golestan province, Iran) (Iranshahi et al., 2007). For the comet assay, the stock concentrations of auraptene were prepared in 2% (v/v) DMSO-PBS. Immediately before use, they were diluted in the PBS or H2O2 to obtain the final concentrations of treat-ments for screening the genotoxicity or antigenotoxicity effects. Isolation of human lymphocytes. The blood was obtained from ten healthy volunteers (aged 25–30). Fasting blood was collected into cell preparation tubes containing 10% EDTA in PBS as an anticoagulant agent. Five mL of the whole blood was diluted 1 : 1 with PBS and carefully layered on the top of a lymphocyte separation medium having a density of 1.077 g/mL (aqueous solution of Ficoll, 57 g/L) in a centrifugation tube and in a ratio of 1 : 1. After centrifugation for 20 min at 2000 rpm, the gradient-separated lymphocytes were recovered, diluted 1 : 1 with PBS and centrifuged a second time at 1500 rpm for 10 min. The cell pellets were resuspended in 500 μL of PBS and the cells counted in a Neubauer chamber. The cell concentration was adjusted to 5000 cells/μL in preparation for the comet assay. The cell viability was determined using the trypan blue dye exclusion technique (Philips, 1973) and was seen to be more than 98%. Determination of DNA damage (comet assay). The comet assay was performed under alkaline conditions according to the method described by Singh et al. (1988) with slight modifications as described recently (Mosaffa et al., 2006; Soltani et al., 2008). Lymphocytes were simultaneously treated with auraptene (5,10, 25, 50,100, 200 or 400 μm) or ascorbic acid (25, 50, 100, 200 and 400 μm) with H2O2 (25 μm) for 15 min at 4 °C to avoid repair of the induced oxidative DNA damage (Collins et al., 1995) The cells treated with 2% (v/v) DMSO-PBS or auraptene (5,10, 25, 50,100, 200 or 400 μm) without hydrogen peroxide were used as negative controls. The Copyright © 2009 John Wiley & Sons, Ltd.
samples were then centrifuged at 3000 rpm for 10 min and the cells washed with PBS. The cell pellets were mixed with 100 μL of 0.75% (w/v) low melting point agarose (LMA) and distributed onto microscope slides coated with 100 μL of 1% (w/v) normal melting agarose (NMA), covered with a coverslip and kept for 10 min at 4 °C to solidify. After the coverslips were removed, the slides were covered with another 100 μL of (0.75% w/v) low melting point agarose, covered with a coverslip and kept for 10 min at 4 °C. Then the slides were immersed in freshly prepared cold lysing solution (2.5 m NaCl, 100 mm Na2EDTA, 10 mm Tris, 1% (v/v) triton X-100, 10% dimethyl sulfoxide, pH 10.0). The slides were treated at 4 C for at least 2 h (vertically without a coverslip) with lysing solution. Then the slides were washed with cold PBS and placed in an electrophoresis tank horizontally side by side. The DNA was allowed to unwind for 30 min in freshly prepared alkaline electrophoresis buffer (1 mm Na2EDTA, 0.3 n NaOH, pH 13.0). Electrophoresis was run at 25 V for 45 min at 4 °C. All procedural steps were performed under yellow light conditions to minimize additional DNA damage. The slides were then placed vertically in a neutralizing tank and gently washed three times for 5 min with neutralizing buffer (0.4 m Tris–HCl buffer, pH 7.5). Twenty microliters of 20 μg/mL ethidium bromide was dispensed directly onto the slides and covered with a coverslip. The slides were studied using a fluorescent microscope (Nikon100) attached to a CCD camera connected to a personal computer. Fifty individual cells were selected for calculations for each analysis; all experiments were carried out at least three times, each with two parallel slides per data point. Single cells were analysed with TriTek Cometscore version 1.5 software. The DNA damage was expressed as % tail DNA, where % tail DNA = [tail DNA/(head DNA + tail DNA)] × 100. A higher % tail DNA indicated a higher level of DNA damage. Statistical analysis. Differences between groups were evaluated by means of one-way analysis of variance (ANOVA) followed by the Dunnett’s test. The protective effect of auraptene and ascorbic acid or umbelliprenin was compared using Student’s t-test for paired samples. Values are expressed as mean ± standard error (SE). Statistical significance was accepted at the p < 0.05 level.
RESULTS AND DISCUSSION Among short-term toxicity assays, the comet assay, also known as the single-cell gel-electrophoresis (SCGE) assay, was used. It is a very sensitive test for the quantification of DNA damage and provides direct determination of DNA single- and double-strand breaks in individual cells (Rojas et al., 1999). H2O2, a known genotoxic agent (Andreoli et al., 1999), is also used to induce oxidative DNA damage. Figure 2 represents photomicrographs of DNA damage of lymphocytes exposed to 2% (v/v) DMSOPBS (negative control), 25 μm H2O2 (positive control) and a combination of H2O2 and 400 μm auraptene. Figure 3 shows % tail DNA in undamaged lymphocytes by negative control and different concentrations of Phytother. Res. 24: 85–89 (2010) DOI: 10.1002/ptr
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Figure 2. Representative comet images from untreated control lymphocytes (a) H2O2-treated lymphocytes (positive control) (b); and simultaneous treatment with auraptene at 400 μM and H2O2 (c,d); (DNA was stained with ethidium bromide; 40× magnification).
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Concentration(mM) Figure 3. DNA damage of human lymphocytes treated with auraptene (AUR). Human lymphocytes were incubated for 15 min at 4 °C with different concentrations of auraptene. Results are mean ± SEM (n = 6 slides × 50 lymphocytes) (ANOVA test).
auraptene without H2O2. The level of DNA damage in lymphocytes exposed to H2O2 and auraptene or ascorbic acid is shown in Fig. 4, and comparison between the protection effects of umbelliprenin and auraptene is illustrated in Fig. 5. The positive control (25 μm H2O2) induced approximately 64% DNA damage. Experiments using negative control cell groups resulted in very Copyright © 2009 John Wiley & Sons, Ltd.
slight DNA damage with % tail DNA ranging from 3.4 to 6.5 (p > 0.05) (Fig. 3). The results indicated that the DNA damage induced by H2O2 was significantly decreased by auraptene at concentrations of 25, 50, 100, 200 and 400 μm (p < 0.001) and the % tail DNA ranged from 48.72 to 36.31) (Fig. 4). Interestingly, the antigenotoxicity effect of auraptene at the tested concentrations was higher than that of ascorbic acid (p < 0.01, t-test), however, at some concentrations (25, 50 and 200 μm) the protective activity was similar to ascorbic acid activity (p > 0.05, t-test). The findings also revealed that auraptene, under the experimental conditions tested, is not genotoxic (Fig. 3) and that auraptene, possesses a high degree protective activity against DNA damage induced by H2O2 (Fig. 4). Since H2O2 acts via reactive oxygen species (ROS), the results confirmed the antioxidant ability of auraptene. In the past two decades, prenyloxycoumarins have been recognized as interesting and valuable biologically active natural products. Auraptene, 7geranyloxycoumarin, is probably the most interesting bioactive member of this family. It possesses a variety of biological activities (Kawabata et al., 2006; Tanaka, 1998; Tanaka et al., 1999). Many investigations have shown that auraptene is one of the most promising natural chemopreventive agents against cancer of the liver, skin, tongue, oesophagus and colon in rodents (Curini et al., 2006). In addition to its chemoprotective activity, auraptene can suppress the generation of superoxide anion (O2−) by inflammatory leucocytes in Phytother. Res. 24: 85–89 (2010) DOI: 10.1002/ptr
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Figure 4. Effect of auraptene (AUR) and ascorbic acid (AA) on lymphocyte DNA damaged by 25 μM H2O2. Human lymphocytes were incubated for 15 min at 4 °C with a combination of 25 μM H2O2 with different concentrations of auraptene or ascorbic acid. Results are mean ± SEM (n = 6 slides × 50 lymphocytes). (ANOVA test) ***p < 0.001, significantly different to value for samples treated with H2O2 only.
Figure 5. Effect of auraptene (AUR) and umbelliprenin (UMB) on lymphocyte DNA damaged by 25 μM H2O2. Human lymphocytes were incubated for 15 min at 4 °C with a combination of 25 μM H2O2 with different concentrations of auraptene or umbelliprenin. Results are mean ± SEM (n = 6 slides × 50 lymphocytes). (ANOVA test) **p < 0.01, ***p < 0.001, significantly different to value for samples treated with H2O2 only.
vitro (Murakami et al., 2000). No report on the antigenotoxicity effects of auraptene has been published in literature. Recently, a significant antigenotoxic effect of umbelliprenin, a structural analogue of auraptene on H2O2-induced primary DNA damage in human peripheral lymphocytes, was reported (Soltani et al., 2008). Since auraptene and umbelliprenin are similar in their structure, the only difference being the higher length prenyl moiety in the umbelliprenin structure, it is logical to compare the antigenotoxic results of these two compounds. Auraptene at concentrations lower than 50 μm was more potent than umbelliprenin (p < 0.001), however no significant difference was observed at other concentrations (p > 0.05). The slight difference between the results of two mentioned compounds may be attributed to the difference in water solubility. In a previous work, it was also revealed that auraptene possessed slightly more cancer chemoprevention activity compared with umbelliprenin, probably confirming our results (Iranshahi et al., 2008). According to the studies regarding the structure– activity relationships of granyloxycoumarins, the prenyl moiety is essential for the activity of these compounds (Curini et al., 2006). The protective activity of auraptene
against oxidative DNA damage induced by H2O2 in lymphocytes might be due to the improvement in the activities of intracellular enzymes involved in the antioxidant mechanism, which can be further studied. It is suggested that the inhibitory effect of auraptene on H2O2 induced oxidative DNA damage might be also related to the activity of the geranyl group in the structure of this compound. In conclusion, the results demonstrated that auraptene could be a suitable agent for preventing chemically induced DNA damage in vitro. Although an antigenotoxic effect found in a plant-derived compound does not necessarily mean that it is an anticarcinogen, it hints at the possibility of acting as one. However, the precise mechanism of the inhibition effect of auraptene against oxidative DNA damage in human lymphocytes needs further investigation.
Acknowledgements The authors are indebted to the Research Council of Mashhad University of Medical Sciences, Iran for approval and financial support of this project.
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Phytother. Res. 24: 85–89 (2010) DOI: 10.1002/ptr
