doi 10.1515/jcim-2014-0009
J Complement Integr Med. 2014; 11(2): 121–127
Sousan Shojaee, Hamideh Parhiz, Ameneh Eshaghi, Seyed Ahmad Emami, Javad Asili, Javad Behravan and Fatemeh Mosaffa*
In vitro protective effects of Scutellaria litwinowii root extract against H2O2-induced DNA damage and cytotoxicity
Abstract Background: Despite the wide range of investigations on the therapeutic potentials of herbal extracts in cancer cell lines, there is not usually enough data on their probable effects on normal cells. Recently, both antitumoral and anti-oxidant properties of Scutellaria litwinowii extracts have been reported on different cancer cell lines. In the present study, the possible protective effects of S. litwinowii root extracts against cytotoxicity and DNA damage induced by hydrogen peroxide (H2O2) on normal cells were investigated using MTT and Comet assays, respectively. Methods: A method of sequential extraction with solvents of different polarities was used to generate methanolic, de-fatted, and dichloromethane fractions. Both MTT and Comet assays were performed here to assess, respectively, changes in cell viability and levels of DNA damage from H2O2. In a pre-treatment regimen, test materials were applied to the cells prior to H2O2 treatment, while in a co-treatment protocol, cells were treated simultaneously with H2O2 and extracts. Results: In the co-treatment studies, only methanolic extract at 1,000 μg/mL (p < 0.001) imparted a significant protective effect as shown in the MTT assay. Pre-treatment of cells for 24 h with different concentrations of the test materials did not lead to any significant protection. Results from the Comet assays in the co-treatment studies suggested a significant (p < 0.01) protective effect of the test materials against DNA damage caused by H2O2. However, in the pre-treatment studies, only methanolic extract at ≥500 μg/mL showed a protective effect (p < 0.01). Conclusions: Considering the probable high levels of phenolic and flavonoid compounds in the methanolic extract, these compounds may impart the noted protective effects of the S. litwinowii root through the scavenging of free radicals.
Keywords: Comet assay, cytotoxicity, DNA damage, NIH 3T3 cell line, Scutellaria litwinowii
*Corresponding author: Fatemeh Mosaffa, Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran, E-mail: [email protected] Sousan Shojaee, Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Hamideh Parhiz, Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Ameneh Eshaghi, Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Seyed Ahmad Emami, Javad Asili, Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Javad Behravan, Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
Introduction The Lamiaceae family comprises a large group of plants that contain significant amounts of polyphenolic compounds. The dried root of Scutellaria from this family – and one of the most commonly used herbs in China and several Far East countries – contains a large amount of flavonoids. Plants in the Lamiaceae family have a wide range of biological and pharmacological activities that appear to protect tissues against damage induced by oxidants. Oxidative stress and cellular damage mediated by oxidants like reactive oxygen species are involved in
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Shojaee et al.: Cell and DNA protective effects of S. litwinowii
development of many chronic diseases, including inflammatory\autoimmune diseases, certain types of cancer, and some neurodegenerative diseases. Recently, anti-tumor and anti-oxidant effects of extracts prepared from Scutellaria litwinowii have been reported in different cancer cell lines [1–3]. TayaraniNajaran et al. [1] showed the material led to an inhibition of the proliferation of a variety of malignant cell lines that involved apoptotic cell death. The observed cytotoxic and apoptogenic effects were thought to be attributed to three major plant flavonoids, that are, baicalin, baicalein, and wogonin [1, 4]. The extract – using anti-oxidant mechanisms – also effectively protected PC12 cells against serum/glucose-deprivation-induced cell death [3]. Despite a range of investigations concerning the therapeutic potentials of S. litwinowii, there is not enough data available about probable effects\anti-oxidant mechanisms as they pertain to normal cells. The present study, therefore, sought to assess potential protective effects of S. litwinowii root extract against cell and DNA damage induced by hydrogen peroxide in normal cells.
Materials and methods Cells NIH 3T3 cells were obtained from the Pasteur Institute of Iran (Tehran, Iran). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS; each from Biosera, Tehran) and were stored at 37°C under a humidified atmosphere containing 5% CO2.
S. litwinowii root extract and its fractions S. litwinowii samples were collected from the Hosseinabad valley in northeast Iran. The plant identity was confirmed by M.R. Joharchi at the Ferdowsi University Mashhad Herbarium and a voucher specimen was placed in the herbarium of the Faculty of Pharmacy at Mashhad University of Medical Sciences. Dried powdered S. litwinowii root was used for preparing extract and fractions [1]. Powdered root (approx. 400 g) was first extracted with methanol and then dried (and concentrated) at 50°C under low pressure (i.e. 5 psi). The concentrated methanolic extract was then extracted three times with n-hexane to obtain a fraction containing polar compounds (deemed de-fatted fraction). The de-fatted fraction was, in turn, extracted
with dichloromethane and dried (deemed CH2Cl2 fraction). In each case, the final materials were weighed to assess yield; for the methanolic extract, yield was 62.5 mg/g starting material, while for the de-fatted and CH2Cl2 fractions, the yields were calculated to be 0.18 g and approx. 0.15 g/g starting material, respectively.
Assessment of cell viability using MTT assay The dried extract and fractions of S. litwinowii were each dissolved in a minimum amount of DMSO such that the final DMSO concentration was approx. 1% when mixed and ultimately introduced into DMEM/FBS culture medium. Each prepared solution (in DMSO; Merck, Munich, Germany) was added to the medium to obtain the concentrations of the methanolic extract (10–1,000 µg/ mL), de-fatted fraction (10–1,200 µg/mL), and CH2Cl2 fraction (5–160 µg/mL) to be used in the assays. Cell viability studies were performed in triplicate using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay. In brief, NIH 3T3 cells were seeded at 5 103 cells/well in 96-well cell plates and incubated for 24 h. Thereafter, the medium was replaced with one containing various concentrations of different extracts, and the cells were incubated further for 24 or 48 h. In all experiments, medium containing 1% DMSO was used as a negative control. At the end of each incubation period, the medium was removed and replaced with medium containing MTT (5 mg/mL in PBS; Sigma, Munich) and the plates incubated for 4 h at 37°C. The medium was then aspirated and 100 μL of DMSO was added to each well to solubilize the formazan that had formed in the cells. Absorbance was then measured at 545 nm (620 nm as reference) in a STAT FAX-2100 microplate reader (Awareness Technology, Palm City, FL, USA). Cell viability was calculated as percent absorbance of cells treated with different concentrations of test material relative to that in untreated cells.
Cytoprotection assay against H2O2-induced toxicity To quantify the cytotoxic effect of H2O2, after 24 h incubation of the cells in 96-well plates (5,000 cells/200 µL/ well), the cells were treated with increasing concentrations of H2O2 (0–800 µM) for 0.5, 1, and 2 h and then cell viability was assessed as above. The least toxic concentration of H2O2 (2 h incubation) was subsequently
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Shojaee et al.: Cell and DNA protective effects of S. litwinowii
used in studies of H2O2 exposure for 24 (100 μM) and 48 h (600 μM) post-seeding.
Pre-treatment procedure To determine any protective effect of the test materials against toxic effects from H2O2, cells grown in 96-well plates were incubated for 24 h in the presence of different concentrations of total methanolic extract (10, 32.5, 62.5, 125, 250, 500, or 1,000 μg/mL), de-fatted fraction (10, 32.5, 62.5, 125, 250, or 500 μg/mL), and CH2Cl2 fraction (5, 10, 20, 40, or 80 μg/mL). After washing the cells with fresh DMEM (without FBS) twice, the cells were treated with H2O2 (600 µM) in FBS-free medium for 2 h. The medium was then replaced with fresh DMEM containing 10% FBS and cell viability quantified using MTT.
Co-treatment procedure For a co-treatment procedure, cells were cultured at 5,000 cells/well in 96-well plates and incubated for 24 h at 37°C. The cells were then treated with different concentrations of extracts and 100 µM H2O2 simultaneously, for 2 h, and cell viability was then determined using MTT.
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normal-melting point agarose. Each sample was then covered with a coverslip and placed at 4°C to solidify. After 10 min, the coverslips were removed and the slides were immersed in freshly prepared cold lysis buffer (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% [v/v] Triton X100, 10% DMSO [pH 10.0]) at 4°C for at least 2 h. The slides were then pre-soaked in freshly prepared alkaline electrophoresis buffer (1 mM Na2EDTA, 0.3 N NaOH [pH 13.0]) for 30 min. Electrophoresis was then conducted for 45 min at 4°C (300 mA, 1 V/C m) in a UV-deprived environment to minimize additional DNA damage. The slides were then placed in a neutralizing tank and washed twice with cold neutralizing buffer (0.4 M Tris HCl buffer [pH 7.5]) and once with distilled water. SYBR Green (1 ; 20 µL) was then added directly onto the slides and the sample was then coverslipped. The stained slides were then assessed by fluorescent microscopy (Nikon100, E 200; Tokyo, Japan), and photos were recorded. All experiments were done at least three times, each with three slides/datapoint. For each slide, 50 selected cells were analyzed using TriTek Cometscore v.1.5 software (www.autocomet.com). DNA damage was calculated as percentage tail DNA ¼ 100 [tail DNA/(head DNA þ tail DNA)]. A greater percentage tail DNA indicated a higher level of DNA damage.
Statistics Comet assay A Comet assay was carried out under alkaline conditions according to the method of Singh et al. [5]. The cells were cultured at 2.5 104 cells/well in 24-well plates and incubated for 24 h at 37°C. For a co-treatment regimen, growing cells were treated simultaneously for 30 min in the dark at 2–8°C with H2O2 (25 µM) and various concentrations of S. litwinowii total methanolic extract (25, 250, or 1,000 μg/mL), de-fatted fraction (25, 250, or 500 μg/ mL), and CH2Cl2 fraction (10, 40, or 80 μg/mL). For a pretreatment regimen, the same concentrations of the different test materials were used, but the cells were incubated with H2O2 after the 24 h. Positive and negative control wells were treated with 25 µM H2O2 in PBS and 0.5% (v/v) DMSO/PBS, respectively. At the end of the respective regimens, cells were harvested and centrifuged at 3,000 rpm for 10 min, and the pellets were washed with PBS. A volume of 140 µL of 1% (w/v) low-melting point agarose (at 37°C) was added to each cell suspension and the material was placed on to microscope slides coated with a layer of 1% (w/v)
The results were expressed as mean±SD of three independent experiments performed in triplicate. All data were analyzed by Instat software (La Jolla, CA, USA) using a one-way analysis of variance (ANOVA). Differences between groups were also evaluated using a Tukey–Kramer post hoc test. A p-value≤0.05 was deemed statistically significant.
Results Effects of S. litwinowii test materials on the NIH 3T3 cell viability NIH 3T3 cells were treated with increasing concentrations of methanolic extract, de-fatted, and dichloromethane fractions of S. litwinowii root for 24 and 48 h, and then cell viability was compared to the control group. Based on MTT assay results, statistically there was no significant difference between the viability of cells treated with the methanolic extract at 24 and 48 h and the negative control group (Figure 1A). The de-fatted fraction at 24 h had no
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Figure 1 Effects of different concentrations of S. litwinowii materials on NIH 3T3 cell viability after 24 and 48 h incubations. (A) Total methanolic extract. (B) De-fatted fraction. (C) CH2Cl2 fraction. Viability was quantified by MTT assay. Values are expressed as percentage of negative control values. Data are presented as mean (SD) of three independent experiments, each performed in triplicate. **Value significantly different vs negative control (p < 0.001; ANOVA).
cytotoxic effects at ≤ 500 μg/mL, but higher concentrations caused significant cytotoxicity (p ≤ 0.001) (Figure 1B). At 48 h, concentrations of ≥ 500 μg/mL resulted in cytotoxicity (Figure 1B). For the CH2Cl2 fraction, cytotoxicity appeared at a concentration of ≥160 μg/mL after 24 h, and at ≥80 μg/mL after 48 h (p ≤ 0.001) (Figure 1C).
Protective effects of root extract and its fractions against H2O2-induced cytotoxicity Treatment of the NIH 3T3 cell with 600 µM (pre-treatment regimen) and 100 µM (co-treatment regimen) H2O2 for 2 h resulted in mean viability levels of 32.0 and 19.2%, respectively. Using the co-treatment protocol, methanolic extract at 1,000 μg/mL was able to protect the cells against the toxic effects of H2O2 (control ¼ H2O2 alone) (p ≤ 0.001) (Figure 2). However, no significant changes in viability were observed with the other test materials.
In the pre-treatment protocol, no significant changes in viability were found using any of the different concentrations of test materials (Figure 3).
Protective effects of extracts against H2O2induced DNA damage H2O2-induced DNA damage in the NIH 3T3 cells with and without treatment with different doses of the test materials (pre-treatment or co-treatment) was measured using a Comet assay and expressed as percentage tail DNA. Treatment of the cells with 25 µM H2O2 for 2 h resulted in a 36.3% mean tail DNA level. With the co-treatment regimen, all doses of extract and each fraction significantly reduced the level of H2O2-induced DNA damage compared to that seen in control cells (cells treated only with H2O2) (Figure 4A and B). With the pre-treatment protocol, methanolic extract at ≥500 μg/mL significantly
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Shojaee et al.: Cell and DNA protective effects of S. litwinowii
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Figure 2 Effects of S. litwinowii materials against H2O2 toxicity in NIH 3T3 cells. Effects of simultaneous treatment of the cells with the total methanolic extract (Methanol Ext), de-fatted fraction (Defatted Fr), and CH2Cl2 fraction (CH2Cl2 Fr) against 100 μM H2O2 for 2 h were assessed using an MTT assay. Data are presented as mean (SD) of three independent experiments, each performed in triplicate. **Value significantly different vs untreated control (**p ≤ 0.001).
Figure 3 Effects of pre-treatment with S. litwinowii materials against H2O2 toxicity in NIH 3T3 cells. Effects of a 24-h pre-treatment of the cells with the total methanolic extract (Methanol Ext), de-fatted fraction (Defatted Fr), and CH2Cl2 fraction (CH2Cl2 Fr) against 600 μM H2O2 (2 h) were assessed using an MTT assay. Data shown are means (SD) of three independent experiments, each performed in triplicate.
(p ≤ 0.01) decreased the level of induced DNA damage. In contrast, there were no significant effects from the de-fatted and CH2Cl2 fractions tested at any dose tested when compared to control cell levels of damage (Figure 4C and D).
Discussion In recent years, extensive studies on plants, their essences, and their extracts have been carried out in order to find novel and natural anti-oxidant compounds. Herbal polyphenols, flavonoids, and phenolic compounds can be considered as major groups of these
natural anti-oxidants. Anti-oxidants are compounds which in low concentrations are capable of preventing or delaying the oxidation caused by active oxygen species [6–8]. They act through reducing oxygen concentration and preventing chain reactions by sweeping free radicals, metallic chelate ions, and conversion of radicals to their non-radical species which will finally result in less tissue damage [7]. Previous studies on Scutellaria have shown that the different species of this plant impart anti-oxidant effects [9–11]. For example, a methanolic extract of Scutellaria baicalensis imparted significant anti-inflammatory effects on gingival fibroblasts [12]. Wogonin was believed to be the main flavone in this extract responsible for the
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Figure 4 Effects of S. litwinowii materials against H2O2-induced DNA damage in NIH 3T3 cells. Total methanolic extract (Methanol Ext), de-fatted fraction (Defatted Fr), and CH2Cl2 fraction (CH2Cl2 Fr) were assessed at the indicated doses. NIH 3T3 cells were treated with 25 µM H2O2 for 30 min at 4°C in the presence (A and B) and 24 h after exposure (C and D) to the different concentrations of S. litwinowii materials. Results shown are mean % tail DNA (SD) of three independent experiments (n ¼ 4 slides 50 cells/experiment). Value significantly different vs samples treated with H2O2 only (*p ≤ 0.01, **p ≤ 0.001; ANOVA).
anti-inflammatory effect [13]. In earlier investigations, S. baicalensis flavonoids such as baicalin, baicalein, and wogonin were also found to exhibit promising radical scavenging and anti-oxidant activities in various test systems [10, 14]. Regarding anti-oxidant and anti-cancer effects of different Scutellaria species, studies of effects from the Iranian species S. litwinowii are important. In a previous study by Tayarani-Najaran et al. [2], cytotoxic and apoptotic effects from S. litwinowii on different cancer cell lines were reported. In another study using a methanolic extract, or de-fatted or dichloromethane fractions of S. litwinowii, cytotoxic and apoptogenic effects in cancer cell lines were found to likely have been caused by baicalin, baicalein, and wogonin flavonoids present in these materials [1]. Since S. litwinowii root was assumed to possess the majority of these anti-oxidant compounds, the present study evaluated any protective effects of S. litwinowii root against oxidative damage induced in normal cells by hydrogen peroxide. For this study, sequential extraction with different solvents was used to increase the efficiency of extraction of various metabolites with different polarities [15].
In the current study, protective effects of the extract materials against DNA damage were seen to be more significant during co-treatment rather than pre-treatment regimens. Since the mechanisms involved in simultaneous treatment of extract and hydrogen peroxide are essentially those related to free-radical sweeping, a protective mechanism by the S. litwinowii root extract(s) could be one based on free-radical scavenging. Further, a comparison of the results obtained with the methanolic extract and the dichloromethane and de-fatted fractions indicated to us that the protective effect of the methanolic extract was significantly greater than that of either other fraction. It has been shown that phenolic compounds are responsible for many of the anti-oxidant effects associated with plant materials [16]. Based on their chemistries, in the current extraction protocol, it would be expected that the highest amount of total phenols and flavonoids from the test root materials would be found in methanolic extracts; thus, the higher protective effect of the methanolic extract compared to the other materials. Regarding the other non-polar, phenolic, and/or flavonoid compounds, it would then be expected that the
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Shojaee et al.: Cell and DNA protective effects of S. litwinowii
de-fatted extract would have the second highest amounts of the materials, followed by the dichloromethane extract. Similar results were also noted in studies in various systems by Gao et al. [10] and Kulisic et al. [16] using materials derived from, respectively, S. baicalensis and Origanum vulgare essence.
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Acknowledgments: This work was supported financially by a research grant from the Vice Chancellor for Research of Mashhad University of Medical Sciences, Mashhad, Iran. The results described in this paper were part of the S. Shojaee’s PharmD thesis.
Conflict of interest statement
Conclusions The data from this study illustrated that S. litwinowii root extracts could help to protect NIH 3T3 cells against H2O2induced DNA damage; these effects we surmise are likely due to phenolic constituents that are acting as antioxidants. Free-radical sweeping can be one assumed mechanism for this observed effect. Still, more in-depth mechanistic studies – as well as in vivo experiments – are needed before any conclusions can be reached as to any greater utility of these natural products/extracts for benefiting human health.
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: Vice Chancellor for Research, Mashhad University of Medical Sciences. Employment or leadership: None declared. Honorarium: None declared. Received February 18, 2014; accepted March 1, 2014; previously published online April 12, 2014
References 1. Tayarani-Najaran Z, Emami SA, Asili J, Mirzaei A, Mousavi SH. Analyzing cytotoxic and apoptogenic properties of Scutellaria litwinowii root extract on cancer cell lines. Evidence Based Complement Alternat Med 2011;2011:160682. 2. Tayarani-Najaran Z, Mousavi SH, Vahdati-Mashhadian N, Emami SA, Parsaee H. Scutellaria litwinowii induces apoptosis through both extrinsic and intrinsic apoptotic pathways in human promyelocytic leukemia cells. Nutr Cancer 2012;64:80–8. 3. Afsharzadeh M, Tayarani-Najaran Z, Zare A, Mousavi SH. Protective effect of Scutellaria litwinowii extract on serum/ glucose-deprived cultured PC12 cells and determining the role of reactive oxygen species. J Toxicol 2012;2012:413279. 4. Tayarani-Najarani Z, Asili J, Parsaee H, Mousavi SH, Mashhadian NV, Mirzaee A, et al. Wogonin and neobaicalein from Scutellaria litwinowii roots are apoptotic for HeLA cells. Rev Bras Farmacogn 2012;22:268–76. 5. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184–91. 6. Halliwell B, Aeschbach R, Löliger J, Aruoma O. The characterization of antioxidants. Food Chem Toxicol 1995;33:601–17. 7. Saha K, Lajis N, Israf D, Hamzah A, Khozirah S, Khamis S, et al. Evaluation of antioxidant and nitric oxide inhibitory activities of selected Malaysian medicinal plants. J Ethnopharmacol 2004;92:263–7. 8. Gate L, Paul J, Ba GN, Tew K, Tapiero H. Oxidative stress induced in pathologies: the role of antioxidants. Biomed Pharmacother 1999;53:169–80.
9. Nagai T, Moriguchi R, Suzuki Y, Tomimori T, Yamada H. Mode of action of the anti-influenza virus activity of plant flavonoid, 5, 7, 4′-trihydroxy-8-methoxyflavone, from the roots of Scutellaria baicalensis. Antiviral Res 1995;26:11–25. 10. Gao Z, Huang K, Yang X, Xu H. Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis georgi. BBA-Gen Subjects 1999;1472:643–50. 11. Sato Y, Suzaki S, Nishikawa T, Kihara M, Shibata H, Higuti T. Phytochemical flavones isolated from Scutellaria barbata and antibacterial activity against methicillin-resistant Staphylococcus aureus. J Ethnopharmacol 2000;72:483–8. 12. Chung CP, Park JB, Bae KH. Pharmacological effects of methanolic extract from the root of Scutellaria baicalensis and its flavonoids on human gingival fibroblast. Planta Med 1995;61:150–3. 13. Chi YS, Lim H, Park H, Kim HP. Effects of wogonin, a plant flavone from Scutellaria radix, on skin inflammation: in vivo regulation of inflammation-associated gene expression. Biochem Pharm 2003;66:1271–8. 14. Van Loon IM. The golden root: clinical applications of Scutellaria baicalensis georgi flavonoids as modulators of the inflammatory response. Altern Med Rev 1997;2:472–80. 15. Eloff J. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med 1998;64:711–13. 16. Kulisic T, Radonic A, Katalinic V, Milos M. Use of different methods for testing antioxidative activity of oregano essential oil. Food Chem 2004;85:633–40.
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J Complement Integr Med. 2014; 11(2): 121–127
Sousan Shojaee, Hamideh Parhiz, Ameneh Eshaghi, Seyed Ahmad Emami, Javad Asili, Javad Behravan and Fatemeh Mosaffa*
In vitro protective effects of Scutellaria litwinowii root extract against H2O2-induced DNA damage and cytotoxicity
Abstract Background: Despite the wide range of investigations on the therapeutic potentials of herbal extracts in cancer cell lines, there is not usually enough data on their probable effects on normal cells. Recently, both antitumoral and anti-oxidant properties of Scutellaria litwinowii extracts have been reported on different cancer cell lines. In the present study, the possible protective effects of S. litwinowii root extracts against cytotoxicity and DNA damage induced by hydrogen peroxide (H2O2) on normal cells were investigated using MTT and Comet assays, respectively. Methods: A method of sequential extraction with solvents of different polarities was used to generate methanolic, de-fatted, and dichloromethane fractions. Both MTT and Comet assays were performed here to assess, respectively, changes in cell viability and levels of DNA damage from H2O2. In a pre-treatment regimen, test materials were applied to the cells prior to H2O2 treatment, while in a co-treatment protocol, cells were treated simultaneously with H2O2 and extracts. Results: In the co-treatment studies, only methanolic extract at 1,000 μg/mL (p < 0.001) imparted a significant protective effect as shown in the MTT assay. Pre-treatment of cells for 24 h with different concentrations of the test materials did not lead to any significant protection. Results from the Comet assays in the co-treatment studies suggested a significant (p < 0.01) protective effect of the test materials against DNA damage caused by H2O2. However, in the pre-treatment studies, only methanolic extract at ≥500 μg/mL showed a protective effect (p < 0.01). Conclusions: Considering the probable high levels of phenolic and flavonoid compounds in the methanolic extract, these compounds may impart the noted protective effects of the S. litwinowii root through the scavenging of free radicals.
Keywords: Comet assay, cytotoxicity, DNA damage, NIH 3T3 cell line, Scutellaria litwinowii
*Corresponding author: Fatemeh Mosaffa, Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran, E-mail: [email protected] Sousan Shojaee, Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Hamideh Parhiz, Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Ameneh Eshaghi, Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Seyed Ahmad Emami, Javad Asili, Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Javad Behravan, Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
Introduction The Lamiaceae family comprises a large group of plants that contain significant amounts of polyphenolic compounds. The dried root of Scutellaria from this family – and one of the most commonly used herbs in China and several Far East countries – contains a large amount of flavonoids. Plants in the Lamiaceae family have a wide range of biological and pharmacological activities that appear to protect tissues against damage induced by oxidants. Oxidative stress and cellular damage mediated by oxidants like reactive oxygen species are involved in
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122
Shojaee et al.: Cell and DNA protective effects of S. litwinowii
development of many chronic diseases, including inflammatory\autoimmune diseases, certain types of cancer, and some neurodegenerative diseases. Recently, anti-tumor and anti-oxidant effects of extracts prepared from Scutellaria litwinowii have been reported in different cancer cell lines [1–3]. TayaraniNajaran et al. [1] showed the material led to an inhibition of the proliferation of a variety of malignant cell lines that involved apoptotic cell death. The observed cytotoxic and apoptogenic effects were thought to be attributed to three major plant flavonoids, that are, baicalin, baicalein, and wogonin [1, 4]. The extract – using anti-oxidant mechanisms – also effectively protected PC12 cells against serum/glucose-deprivation-induced cell death [3]. Despite a range of investigations concerning the therapeutic potentials of S. litwinowii, there is not enough data available about probable effects\anti-oxidant mechanisms as they pertain to normal cells. The present study, therefore, sought to assess potential protective effects of S. litwinowii root extract against cell and DNA damage induced by hydrogen peroxide in normal cells.
Materials and methods Cells NIH 3T3 cells were obtained from the Pasteur Institute of Iran (Tehran, Iran). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS; each from Biosera, Tehran) and were stored at 37°C under a humidified atmosphere containing 5% CO2.
S. litwinowii root extract and its fractions S. litwinowii samples were collected from the Hosseinabad valley in northeast Iran. The plant identity was confirmed by M.R. Joharchi at the Ferdowsi University Mashhad Herbarium and a voucher specimen was placed in the herbarium of the Faculty of Pharmacy at Mashhad University of Medical Sciences. Dried powdered S. litwinowii root was used for preparing extract and fractions [1]. Powdered root (approx. 400 g) was first extracted with methanol and then dried (and concentrated) at 50°C under low pressure (i.e. 5 psi). The concentrated methanolic extract was then extracted three times with n-hexane to obtain a fraction containing polar compounds (deemed de-fatted fraction). The de-fatted fraction was, in turn, extracted
with dichloromethane and dried (deemed CH2Cl2 fraction). In each case, the final materials were weighed to assess yield; for the methanolic extract, yield was 62.5 mg/g starting material, while for the de-fatted and CH2Cl2 fractions, the yields were calculated to be 0.18 g and approx. 0.15 g/g starting material, respectively.
Assessment of cell viability using MTT assay The dried extract and fractions of S. litwinowii were each dissolved in a minimum amount of DMSO such that the final DMSO concentration was approx. 1% when mixed and ultimately introduced into DMEM/FBS culture medium. Each prepared solution (in DMSO; Merck, Munich, Germany) was added to the medium to obtain the concentrations of the methanolic extract (10–1,000 µg/ mL), de-fatted fraction (10–1,200 µg/mL), and CH2Cl2 fraction (5–160 µg/mL) to be used in the assays. Cell viability studies were performed in triplicate using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay. In brief, NIH 3T3 cells were seeded at 5 103 cells/well in 96-well cell plates and incubated for 24 h. Thereafter, the medium was replaced with one containing various concentrations of different extracts, and the cells were incubated further for 24 or 48 h. In all experiments, medium containing 1% DMSO was used as a negative control. At the end of each incubation period, the medium was removed and replaced with medium containing MTT (5 mg/mL in PBS; Sigma, Munich) and the plates incubated for 4 h at 37°C. The medium was then aspirated and 100 μL of DMSO was added to each well to solubilize the formazan that had formed in the cells. Absorbance was then measured at 545 nm (620 nm as reference) in a STAT FAX-2100 microplate reader (Awareness Technology, Palm City, FL, USA). Cell viability was calculated as percent absorbance of cells treated with different concentrations of test material relative to that in untreated cells.
Cytoprotection assay against H2O2-induced toxicity To quantify the cytotoxic effect of H2O2, after 24 h incubation of the cells in 96-well plates (5,000 cells/200 µL/ well), the cells were treated with increasing concentrations of H2O2 (0–800 µM) for 0.5, 1, and 2 h and then cell viability was assessed as above. The least toxic concentration of H2O2 (2 h incubation) was subsequently
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Shojaee et al.: Cell and DNA protective effects of S. litwinowii
used in studies of H2O2 exposure for 24 (100 μM) and 48 h (600 μM) post-seeding.
Pre-treatment procedure To determine any protective effect of the test materials against toxic effects from H2O2, cells grown in 96-well plates were incubated for 24 h in the presence of different concentrations of total methanolic extract (10, 32.5, 62.5, 125, 250, 500, or 1,000 μg/mL), de-fatted fraction (10, 32.5, 62.5, 125, 250, or 500 μg/mL), and CH2Cl2 fraction (5, 10, 20, 40, or 80 μg/mL). After washing the cells with fresh DMEM (without FBS) twice, the cells were treated with H2O2 (600 µM) in FBS-free medium for 2 h. The medium was then replaced with fresh DMEM containing 10% FBS and cell viability quantified using MTT.
Co-treatment procedure For a co-treatment procedure, cells were cultured at 5,000 cells/well in 96-well plates and incubated for 24 h at 37°C. The cells were then treated with different concentrations of extracts and 100 µM H2O2 simultaneously, for 2 h, and cell viability was then determined using MTT.
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normal-melting point agarose. Each sample was then covered with a coverslip and placed at 4°C to solidify. After 10 min, the coverslips were removed and the slides were immersed in freshly prepared cold lysis buffer (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% [v/v] Triton X100, 10% DMSO [pH 10.0]) at 4°C for at least 2 h. The slides were then pre-soaked in freshly prepared alkaline electrophoresis buffer (1 mM Na2EDTA, 0.3 N NaOH [pH 13.0]) for 30 min. Electrophoresis was then conducted for 45 min at 4°C (300 mA, 1 V/C m) in a UV-deprived environment to minimize additional DNA damage. The slides were then placed in a neutralizing tank and washed twice with cold neutralizing buffer (0.4 M Tris HCl buffer [pH 7.5]) and once with distilled water. SYBR Green (1 ; 20 µL) was then added directly onto the slides and the sample was then coverslipped. The stained slides were then assessed by fluorescent microscopy (Nikon100, E 200; Tokyo, Japan), and photos were recorded. All experiments were done at least three times, each with three slides/datapoint. For each slide, 50 selected cells were analyzed using TriTek Cometscore v.1.5 software (www.autocomet.com). DNA damage was calculated as percentage tail DNA ¼ 100 [tail DNA/(head DNA þ tail DNA)]. A greater percentage tail DNA indicated a higher level of DNA damage.
Statistics Comet assay A Comet assay was carried out under alkaline conditions according to the method of Singh et al. [5]. The cells were cultured at 2.5 104 cells/well in 24-well plates and incubated for 24 h at 37°C. For a co-treatment regimen, growing cells were treated simultaneously for 30 min in the dark at 2–8°C with H2O2 (25 µM) and various concentrations of S. litwinowii total methanolic extract (25, 250, or 1,000 μg/mL), de-fatted fraction (25, 250, or 500 μg/ mL), and CH2Cl2 fraction (10, 40, or 80 μg/mL). For a pretreatment regimen, the same concentrations of the different test materials were used, but the cells were incubated with H2O2 after the 24 h. Positive and negative control wells were treated with 25 µM H2O2 in PBS and 0.5% (v/v) DMSO/PBS, respectively. At the end of the respective regimens, cells were harvested and centrifuged at 3,000 rpm for 10 min, and the pellets were washed with PBS. A volume of 140 µL of 1% (w/v) low-melting point agarose (at 37°C) was added to each cell suspension and the material was placed on to microscope slides coated with a layer of 1% (w/v)
The results were expressed as mean±SD of three independent experiments performed in triplicate. All data were analyzed by Instat software (La Jolla, CA, USA) using a one-way analysis of variance (ANOVA). Differences between groups were also evaluated using a Tukey–Kramer post hoc test. A p-value≤0.05 was deemed statistically significant.
Results Effects of S. litwinowii test materials on the NIH 3T3 cell viability NIH 3T3 cells were treated with increasing concentrations of methanolic extract, de-fatted, and dichloromethane fractions of S. litwinowii root for 24 and 48 h, and then cell viability was compared to the control group. Based on MTT assay results, statistically there was no significant difference between the viability of cells treated with the methanolic extract at 24 and 48 h and the negative control group (Figure 1A). The de-fatted fraction at 24 h had no
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Shojaee et al.: Cell and DNA protective effects of S. litwinowii
Figure 1 Effects of different concentrations of S. litwinowii materials on NIH 3T3 cell viability after 24 and 48 h incubations. (A) Total methanolic extract. (B) De-fatted fraction. (C) CH2Cl2 fraction. Viability was quantified by MTT assay. Values are expressed as percentage of negative control values. Data are presented as mean (SD) of three independent experiments, each performed in triplicate. **Value significantly different vs negative control (p < 0.001; ANOVA).
cytotoxic effects at ≤ 500 μg/mL, but higher concentrations caused significant cytotoxicity (p ≤ 0.001) (Figure 1B). At 48 h, concentrations of ≥ 500 μg/mL resulted in cytotoxicity (Figure 1B). For the CH2Cl2 fraction, cytotoxicity appeared at a concentration of ≥160 μg/mL after 24 h, and at ≥80 μg/mL after 48 h (p ≤ 0.001) (Figure 1C).
Protective effects of root extract and its fractions against H2O2-induced cytotoxicity Treatment of the NIH 3T3 cell with 600 µM (pre-treatment regimen) and 100 µM (co-treatment regimen) H2O2 for 2 h resulted in mean viability levels of 32.0 and 19.2%, respectively. Using the co-treatment protocol, methanolic extract at 1,000 μg/mL was able to protect the cells against the toxic effects of H2O2 (control ¼ H2O2 alone) (p ≤ 0.001) (Figure 2). However, no significant changes in viability were observed with the other test materials.
In the pre-treatment protocol, no significant changes in viability were found using any of the different concentrations of test materials (Figure 3).
Protective effects of extracts against H2O2induced DNA damage H2O2-induced DNA damage in the NIH 3T3 cells with and without treatment with different doses of the test materials (pre-treatment or co-treatment) was measured using a Comet assay and expressed as percentage tail DNA. Treatment of the cells with 25 µM H2O2 for 2 h resulted in a 36.3% mean tail DNA level. With the co-treatment regimen, all doses of extract and each fraction significantly reduced the level of H2O2-induced DNA damage compared to that seen in control cells (cells treated only with H2O2) (Figure 4A and B). With the pre-treatment protocol, methanolic extract at ≥500 μg/mL significantly
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Shojaee et al.: Cell and DNA protective effects of S. litwinowii
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Figure 2 Effects of S. litwinowii materials against H2O2 toxicity in NIH 3T3 cells. Effects of simultaneous treatment of the cells with the total methanolic extract (Methanol Ext), de-fatted fraction (Defatted Fr), and CH2Cl2 fraction (CH2Cl2 Fr) against 100 μM H2O2 for 2 h were assessed using an MTT assay. Data are presented as mean (SD) of three independent experiments, each performed in triplicate. **Value significantly different vs untreated control (**p ≤ 0.001).
Figure 3 Effects of pre-treatment with S. litwinowii materials against H2O2 toxicity in NIH 3T3 cells. Effects of a 24-h pre-treatment of the cells with the total methanolic extract (Methanol Ext), de-fatted fraction (Defatted Fr), and CH2Cl2 fraction (CH2Cl2 Fr) against 600 μM H2O2 (2 h) were assessed using an MTT assay. Data shown are means (SD) of three independent experiments, each performed in triplicate.
(p ≤ 0.01) decreased the level of induced DNA damage. In contrast, there were no significant effects from the de-fatted and CH2Cl2 fractions tested at any dose tested when compared to control cell levels of damage (Figure 4C and D).
Discussion In recent years, extensive studies on plants, their essences, and their extracts have been carried out in order to find novel and natural anti-oxidant compounds. Herbal polyphenols, flavonoids, and phenolic compounds can be considered as major groups of these
natural anti-oxidants. Anti-oxidants are compounds which in low concentrations are capable of preventing or delaying the oxidation caused by active oxygen species [6–8]. They act through reducing oxygen concentration and preventing chain reactions by sweeping free radicals, metallic chelate ions, and conversion of radicals to their non-radical species which will finally result in less tissue damage [7]. Previous studies on Scutellaria have shown that the different species of this plant impart anti-oxidant effects [9–11]. For example, a methanolic extract of Scutellaria baicalensis imparted significant anti-inflammatory effects on gingival fibroblasts [12]. Wogonin was believed to be the main flavone in this extract responsible for the
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Shojaee et al.: Cell and DNA protective effects of S. litwinowii
Figure 4 Effects of S. litwinowii materials against H2O2-induced DNA damage in NIH 3T3 cells. Total methanolic extract (Methanol Ext), de-fatted fraction (Defatted Fr), and CH2Cl2 fraction (CH2Cl2 Fr) were assessed at the indicated doses. NIH 3T3 cells were treated with 25 µM H2O2 for 30 min at 4°C in the presence (A and B) and 24 h after exposure (C and D) to the different concentrations of S. litwinowii materials. Results shown are mean % tail DNA (SD) of three independent experiments (n ¼ 4 slides 50 cells/experiment). Value significantly different vs samples treated with H2O2 only (*p ≤ 0.01, **p ≤ 0.001; ANOVA).
anti-inflammatory effect [13]. In earlier investigations, S. baicalensis flavonoids such as baicalin, baicalein, and wogonin were also found to exhibit promising radical scavenging and anti-oxidant activities in various test systems [10, 14]. Regarding anti-oxidant and anti-cancer effects of different Scutellaria species, studies of effects from the Iranian species S. litwinowii are important. In a previous study by Tayarani-Najaran et al. [2], cytotoxic and apoptotic effects from S. litwinowii on different cancer cell lines were reported. In another study using a methanolic extract, or de-fatted or dichloromethane fractions of S. litwinowii, cytotoxic and apoptogenic effects in cancer cell lines were found to likely have been caused by baicalin, baicalein, and wogonin flavonoids present in these materials [1]. Since S. litwinowii root was assumed to possess the majority of these anti-oxidant compounds, the present study evaluated any protective effects of S. litwinowii root against oxidative damage induced in normal cells by hydrogen peroxide. For this study, sequential extraction with different solvents was used to increase the efficiency of extraction of various metabolites with different polarities [15].
In the current study, protective effects of the extract materials against DNA damage were seen to be more significant during co-treatment rather than pre-treatment regimens. Since the mechanisms involved in simultaneous treatment of extract and hydrogen peroxide are essentially those related to free-radical sweeping, a protective mechanism by the S. litwinowii root extract(s) could be one based on free-radical scavenging. Further, a comparison of the results obtained with the methanolic extract and the dichloromethane and de-fatted fractions indicated to us that the protective effect of the methanolic extract was significantly greater than that of either other fraction. It has been shown that phenolic compounds are responsible for many of the anti-oxidant effects associated with plant materials [16]. Based on their chemistries, in the current extraction protocol, it would be expected that the highest amount of total phenols and flavonoids from the test root materials would be found in methanolic extracts; thus, the higher protective effect of the methanolic extract compared to the other materials. Regarding the other non-polar, phenolic, and/or flavonoid compounds, it would then be expected that the
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Shojaee et al.: Cell and DNA protective effects of S. litwinowii
de-fatted extract would have the second highest amounts of the materials, followed by the dichloromethane extract. Similar results were also noted in studies in various systems by Gao et al. [10] and Kulisic et al. [16] using materials derived from, respectively, S. baicalensis and Origanum vulgare essence.
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Acknowledgments: This work was supported financially by a research grant from the Vice Chancellor for Research of Mashhad University of Medical Sciences, Mashhad, Iran. The results described in this paper were part of the S. Shojaee’s PharmD thesis.
Conflict of interest statement
Conclusions The data from this study illustrated that S. litwinowii root extracts could help to protect NIH 3T3 cells against H2O2induced DNA damage; these effects we surmise are likely due to phenolic constituents that are acting as antioxidants. Free-radical sweeping can be one assumed mechanism for this observed effect. Still, more in-depth mechanistic studies – as well as in vivo experiments – are needed before any conclusions can be reached as to any greater utility of these natural products/extracts for benefiting human health.
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: Vice Chancellor for Research, Mashhad University of Medical Sciences. Employment or leadership: None declared. Honorarium: None declared. Received February 18, 2014; accepted March 1, 2014; previously published online April 12, 2014
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