http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, 2015; 38(3): 293–299 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2014.954047
RESEARCH ARTICLE
Protective effects of Scutellaria lindbergii root extract against oxidative-induced cell and DNA damage in mouse fibroblast-like cells
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
Melika Ehtesham-Gharaee1,2, Ameneh Eshaghi2, Susan Shojaee2, Javad Asili3, Seyed Ahmad Emami3, Javad Behravan1,2, and Fatemeh Mosaffa1,2 1
Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran, 2Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran, and 3Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Abstract
Keywords
Context: Scutellaria lindbergii Rech. f. (Lamiaceae) is an Iranian species of Scutellaria which has been shown to exert antimicrobial, antioxidant and cytotoxic effects. Objective: The protective properties of total methanol extract (TME) of S. lindbergii and its fractions (defatted and CH2Cl2) were investigated against cytotoxic and genotoxic effects of H2O2 in NIH 3T3 cell line as nonmalignant cells. Materials and methods: The cells were incubated with different concentrations of S. lindbergii root extracts [TME (15–250 mg ml1), defatted fraction (15–500 mg ml1) and CH2Cl2 fraction (5–40 mg ml1)] and toxic concentration of H2O2 (200 mM) at 37 C for 2 h concurrently and Cell viability was quantitated by MTT assay. The antigenotoxic effect of extracts was investigated using comet assay. The cells were incubated with extracts [TME (25–250 mg ml1), defatted fraction (25–500 mg ml1) and CH2Cl2 fraction (5–40 mg ml1)] and H2O2 (25 mM) at 4 C for 20 min, then the comet assay was performed. DNA damage was expressed as percentage tail DNA. Results: Total methanol extract of S. lindbergii and its fractions had a significant inhibitory effect on DNA damage. The IC50 values of TME, defatted fraction and CH2Cl2 fraction against DNA damage were determined as 48, 138 and 8 mg ml1, respectively. Conclusion: S. lindbergii extracts can prevent oxidative DNA damage, which is likely due to its flavonoids and phenolic compounds as antioxidant constituents.
Anticytotoxicity, antigenotoxicity, comet assay, hydrogen peroxide, Lamiaceae, MTT assay
Introduction Reactive oxygen species-mediated cell damage plays an important role in various human chronic diseases such as Crohn’s disease, certain cancers and a number of neurodegenerative diseases (Aherne et al., 2007). At the cellular level, oxidative stress can cause severe metabolic dysfunction, including lipid peroxidation, protein oxidation, membrane disruption and DNA damage, resulting in changes in signal transduction pathways, gene expression, cell mutagenesis and cell death (Aherne et al., 2007; Demirci et al., 2008). Natural products from plant sources have extensive past and present use in treatment of different kinds of ailments and serve as compounds of interest both in their natural form and as templates for synthetic modification (Chin et al., 2006). There is some evidence that some plant-derived natural compounds have protective effects towards DNA damage caused by oxidative stresses (Glei & Pool-Zobel, 2006; Plazar et al., 2008).
Address for correspondence: Dr. F. Mosaffa, Dept. of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, P. O. Box: 91775-1365. Iran. Tel: +98-5118823255-66, Fax: +98-511-8823251. E-mail: [email protected]
History Received 6 March 2014 Revised 25 June 2014 Accepted 8 August 2014 Published online 19 September 2014
Scutellaria L. (Lamiaceae) is a genus that contains about 300 species worldwide, excluding South Africa (Mabberley, 1993). This genus has 27 species and two hybrids in Iran, of which 10 species and two hybrids are endemic to the country (Emami & Aghazari, 2011; Jamzad, 2012). Scutellaria lindbergii Rech. f. is an Iranian species of the genus. General distribution of this species is limited to Iran and Afghanistan (Attar & Joharchi, 2002; Jamzad, 2012). In traditional medicine, this species is used as a source of flavonoids to treat a variety of diseases. The dried roots of S. baicalensis have been used for the treatment of suppurative dermatitis, diarrhea, inflammatory diseases, hyperlipidemia, and atherosclerosis in Chinese and Japanese traditional medicine (Middleton Jr et al., 2000). In traditional Chinese medicine, the Huangquin-tang formula which contains four medicinal herbs including Scutellariae Radix etc. is prescribed for the treatment of prostate cancer (Zuo et al., 2002) and it was also originally used to treat nausea, vomiting and diarrhea as far back as 1800 years ago (Lam et al., 2010). Several studies have been shown various biological and pharmacological activities of some Scutellaria species such as antibacterial (Hahm et al., 2001; Sato et al., 2000; Skaltsaa et al., 2005), antiviral (Nagai et al., 1990, 1995), antifungal (Cole et al., 1991), anti-inflammatory (Li et al., 2000),
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
294
M. Ehtesham-Gharaee et al.
antioxidant (Gao et al., 1999; Shao et al., 1999), Cytotoxicity and anticancer (Powell et al., 2003; Sonoda et al., 2004; Tayarani-Najaran et al., 2010; Yin et al., 2004). Experimental evidence suggests that most Lamiaceae spices possess a wide range of biological and pharmacological activities that may protect tissues against O2-induced damage and therefore lower the risk of human chronic diseases (Bozin et al., 2006; Craig, 1999) for example, Scutellaria baicalensis Georgi extract attenuates oxidative stress in cardiomyocytes and protect cells from lethal oxidant damage in an ischemia–reperfusion model (Shao et al., 1999). Therefore, the present work was carried out to investigate the cytoprotective and genoprotective effects of Scutellaria lindbergii extract and its fractions against cell damage and DNA damage induced by hydrogen peroxide in mouse embryo cell line (NIH 3T3) as non-malignant cells. Several studies have been carried out on some biological activities of S. lindbergii such as its antimicrobial, antioxidant (Fazly-Bazzaz et al., 2013) and cytotoxic effects (TayaraniNajaran et al., 2010), but to the authors knowledge this is the first study on cytoprotective and genoprotective activity of S. lindbergii.
Materials and methods Chemical and reagents
Drug Chem Toxicol, 2015; 38(3): 293–299
streptomycin and maintained at 37 C in a humidified atmosphere (90%) containing 5% CO2. Effect of S. lindbergii extracts on viability of NIH 3T3 cells The crude extracts of S. lindbergii were dissolved in the minimum amount of DMSO in a way that the final DMSO concentration was 0.5% when mixed with culture medium. The solutions of crude extracts in DMSO were added to the culture medium to obtain different concentrations of extracts [total methanol extract (TME) (10–1000 mg ml1), defatted fraction (10–1200 mg ml1) and CH2Cl2 fraction (5–160 mg ml1)] (Tayarani-Najaran et al., 2010). Cell viability was determined using a modified MTT assay (Mosmann, 1983; Tayarani-Najaran et al., 2010). Briefly, NIH 3T3 cells were seeded at 5000 cells per well in 96-well cell culture plates. After 24 h incubation, growth media was replaced with growth medium containing various concentrations of different extracts and then incubated for 24 and 48 h. Negative control wells (no extract) were prepared with 0.5% (v/v) DMSO in equal volume of medium. After removing the medium, MTT solution (5 mg ml1 in PBS) was added to the cells. The cells were incubated at 37 C for 4 h, the medium was aspirated, and the formazan product was solubilized with DMSO. The absorption was measured at 570 nm (620 nm as a reference) in an ELISA reader. The percentage cell viability for each concentration of plant extracts was calculated.
Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), trypsin-EDTA solution, penicillin/streptomycin 100 units, dimethylsulfoxide (DMSO) and all other fine chemicals were obtained from Merck, Germany. SYBR Green, 3-(4,5-dimethylthiazole-2-yl)-2,5-dimethyl tetrazolium bromide (MTT) and trypan blue were purchased from Sigma (St Louis, MO). Low melting point agarose (LMA) and normal melting point agarose (NMA) were purchased from Fermentas, Germany.
In order to determine the cytotoxic effect of H2O2, after 24 h incubation in 96-well plates (5000 cells/200 ml), the cells were treated with H2O2 (0–600 mM) for 0.5, 1 and 2 h and then cell viability was detected by the MTT method as described previously.
Plant material
Extract cytoprotective activity assay
Scutellaria lindbergii was collected from Kang valley (height 1800 m) near Mashhad (Razavi Khorasan Province, north-east of Iran) in July 2012 and identified by Mr. M. R. Joharchi, from Ferdowsi University of Mashhad Herbarium (FUMH). A voucher specimen of the species was deposited in the Herbarium of the Faculty of Pharmacy, Mashhad University of Medical Sciences (MUMS) under number: 11309.
In order to investigate of the protective effects of S. lindbergii extracts against H2O2 cytotoxicity either pre-treatment or co-treatment protocol was used.
Extraction and fractionation Dry powdered roots of the plant (100 g) were macerated separately in methanol and dichloromethane at room temperature for 48 h. Both extracts were concentrated at 50 C under reduced pressure to dryness. The concentrated methanol extract was then extracted with an equal volume of n-hexane, three times, to give a fraction containing polar compounds (defatted fraction) and then concentrated at 50 C under reduced pressure to dryness. Cell culture NIH 3T3 was obtained from Pasteur Institute (Tehran, Iran). The cell line was grown in DMEM medium containing 10% FCS, supplemented with 100 unit/ml penicillin and 100 mg/ml
Assay of H2O2-induced toxicity
Pre-treatment protocol Cells grown in DMEM containing 10% FBS in 96-well culture plates (5000 cells/well) were incubated for 24 h in the presence of different concentrations of extracts [TME (15, 31.5, 62.5, 125 and 250 mg ml1), defatted fraction (15, 31.5, 62.5, 125, 250 and 500 mg ml1) and CH2Cl2 fraction (5, 10, 20 and 40 mg ml1)]. After washing the cells with fresh DMEM without FBS twice, the cells were treated with H2O2 (200 mM) in the above FBS-free medium for 2 h. Then the medium was replaced with fresh DMEM containing 10% FBS and the cell viability was quantified by MTT (Chen et al., 2003). Co-treatment protocol Before treatment, the cells were seeded at 5000 cells per well in a 96-well plate and incubated for 24 h at 37 C. Then the cells were incubated with different concentrations of extracts and 200 mM H2O2 simultaneously, for 2 h and cell viability was determined by MTT assay.
DOI: 10.3109/01480545.2014.954047
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
Determination of DNA damage (comet assay) The alkaline single cell gel electrophoresis [SCGE (comet assay)] was performed under alkaline conditions according to the technique described by Singh et al. (1988) with slight modifications (Soltani et al., 2009). The cells were plated at 2.5 104 cells per well in a 24-well plate and incubated for 24 h at 37 C. Growing cells were treated with various concentrations of S. lindbergii extracts either TME (25, 125 and 250 mg ml1), defatted fraction (25, 250 and 500 mg ml1), CH2Cl2 fraction (5, 20 and 40 mg ml1) and H2O2 (25 mM) simultaneously, for 20 min in the dark at 4 C. Positive and negative control wells were treated with 25 mM H2O2 in PBS and 0.5% (v/v) DMSO-PBS, respectively. The cells were harvested and centrifuged at 3000 rpm for 10 min and then washed with PBS. The cell pellets were mixed with 40 mL of sterile PBS at room temperature. Then 140 mL of 1% (w/v) low melting point agarose (LMA) was added at 37 C and distributed onto microscopic slides coated with a layer of 1% (w/v) normal melting point agarose (NMA), covered with a coverslip and kept for 10 min at 4 C to solidify. After the coverslips were removed, the slides vertically 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) at 4 C for at least 2 h. The slides were presoaked in freshly prepared alkaline electrophoresis buffer (1 mM Na2EDTA, 0.3 N NaOH, pH 13.0) for 30 min. Electrophoresis was carried out for 45 min at 4 C (300 mA, 1 V/Cm). 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 washed thrice with a neutralizing solution (0.4 m tris HCl buffer, pH 7.5). Twenty microliters SYBR Green (1 x) was dispensed directly onto the slides and covered with a coverslip. The slides were studied by a fluorescent microscope (Nikon100, E 200, Japan) using a G-2 A filter, attached to a charge-coupled device camera connected to a personal computer. All experiments were carried out at least three times, each with three parallel slides per data point. For each slide, 50 selected cells were analyzed with TriTek Cometscore version 1.5 software (www.autocomet.com). The DNA damage was expressed as percentage tail DNA, where % tail DNA ¼ [tail DNA/(head DNA + tail DNA)] 100. A higher percentage tail DNA indicated a higher level of DNA damage.
Statistical analysis All of the experiments were performed in triplicate. Instat software and One-way analysis of variance (ANOVA) were used for data analysis. Differences between groups were evaluated by Tukey–Kramer test. All results were expressed as mean ± SEM of three independent experiments performed in triplicate. p50.05 was considered statistically significant.
Results Effect of S. lindbergii extracts on NIH 3T3 cell viability The cytotoxicity of total methanol extract of S. lindbergii and its fractions were examined on NIH 3T3 cell line. When NIH
S. lindbergii against cell and DNA damage
295
3T3 cells were incubated with different concentrations of S. lindbergii extracts for 24 and 48 h, TME showed a significant toxicity at concentration of 500 mg ml1 and higher (p50.001). A significant toxicity was observed for concentrations above 500 mg ml1 of defatted fraction (p50.001).While CH2Cl2 fraction showed a significant toxicity at concentration of 80 mg ml1 and higher (p50.01) (Table 1). The IC50 values of TME, defatted fraction and CH2Cl2 fraction against NIH 3T3 cells were determined as 421, 1139 and 150 mg ml1 after 24 h and 513, 578 and 104 mg ml1 after 48 h, respectively. After examination of 0.5% DMSO in culture medium, no significant toxicity was observed. Effect of H2O2 on NIH 3T3 cell viability MTT assay was carried out at different concentrations of H2O2 to determine the cytotoxic concentrations of H2O2 for NIH 3T3 cells. After 24 h incubation in 96-well plates (5000 cells/200 ml)and then incubation with different H2O2 concentrations (0–600 mM) for 0.5, 1 and 2 h, a significant cytotoxicity (450%) was observed at concentration of 200 mM and higher with 2 h incubation time compared to untreated control (p50.001) (Table 2). Thus, 200 mM H2O2 for 2 h were selected as appropriate concentration to induce toxicity to assess the effects of the S. lindbergii extracts in further experiments. Effect of S. lindbergii extracts on viability of NIH 3T3 cells against H2O2-induced cytotoxicity When both of pre-treatment and co-treatment protocol were used, no significant difference in cell viability was observed (results not shown) at different concentrations of all extracts (TME, defatted fraction and CH2Cl2 fraction) compared with the control (H2O2 alone). Effect of H2O2 or S. lindbergii extracts on NIH 3T3 cell DNA damage Induced effects of oxidative stress were measured by SCGE. After 20 min incubation of NIH 3T3 cells with H2O2 (25, 50 and 100 mM) at 4 C, a significant increase in DNA damage was induced with increasing concentration of H2O2 (p50.001 for all; Figure 1). The potential of TME (25, 125 and 250 mg ml1), defatted fraction (25, 250 and 500 mg ml1) and CH2Cl2 fraction (5, 20 and 40 mg ml1) of S. lindbergii to induce DNA damage was assessed using the comet assay. None of the plant extracts investigated significantly increased % tail DNA when compared with negative control cells (0.5% (v/v) DMSOPBS) (p40.05; Figure 2). Protective effect of S. lindbergii extracts against H2O2-induced DNA damage The genoprotective effects of different concentrations of TME (25, 125 and 250 mg ml1), defatted fraction (25, 250 and 500 mg ml1) and CH2Cl2 fraction (5, 20 and 40 mg ml1) of S. lindbergii in the presence of 25 mM H2O2, were examined for 20 min at 4 C. Comet assay was carried out and DNA damage was expressed as percentage tail DNA. As shown in
296
M. Ehtesham-Gharaee et al.
Drug Chem Toxicol, 2015; 38(3): 293–299
Table 1. Effect of different concentrations of S. lindbergii extracts [total methanol extract (TME), defatted fraction and CH2Cl2 fraction] on NIH 3T3 cells viability after 24 h and 48 h incubationa,b. 24 h
Concentration (mg/ml)
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
TME 0.0 (Control) 10.0 20.0 31.0 40.0 62.0 80.0 125.0 160.0 250.0 500.0 600.0 1000.0 1200.0
100.0 ± 0.03
48 h
Defatted fraction
CH2Cl2 fraction
100.0 ± 0.09
100.0 ± 0.03 100.0 ± 0.03 103.5 ± 0.07
97.0 ± 0.04
TME 100.0 ± 0.05
Defatted fraction
CH2Cl2 fraction
100.0 ± 0.06
100.0 ± 0.12 100.0 ± 0.05 102.0 ± 0.11
100.0 ± 0.05 110.0 ± 0.05
92.0 ± 0.03
100.0 ± 0.04
94.0 ± 0.05
100.0 ± 0.08
103.0 ± 0.11 100.0 ± 0.04
100.0 ± 0.01
100.0 ± 0.04
100.0 ± 0.10
66.6 ± 0.05**
50.6 ± 0.05**
45.0 ± 0.05*** 88.0 ± 0.01 22.0 ± 0.03***
98.5 ± 0.09 98.5 ± 0.04 37.9 ± 0.09*** 33.3 ± 0.08*** 28.8 ± 0.03***
20.0 ± 0.06***
31.3 ± 0.09*** 99.0 ± 0.02 22.0 ± 0.05*** 21.0 ± 0.04***
88.9 ± 0.11 88.9 ± 0.01 33.3 ± 0.06*** 11.1 ± 0.03*** 11.1 ± 0.03***
a
Viability was quantitated by MTT assay. Values are expressed as a percentage of negative control values. Data are mean ± SEM (n ¼ 3). **p50.01 and ***p50.001, significantly different from controls.
b
Table 2. Effect of different concentrations of H2O2 on NIH 3T3 cells viability after treatment for 0.5, 1 and 2 ha,b. Concentration (mM) 0.0 (Control) 25.0 50.0 100.0 200.0 400.0 600.0
0.5 h
1h
2h
100.0 ± 0.02 100.0 ± 0.02 98.3 ± 0.03 96.6 ± 0.04 95.0 ± 0.01 95.0 ± 0.02 91.7 ± 0.02
100.0 ± 0.03 100.0 ± 0.02 98.3 ± 0.03 99.5 ± 0.05 97.9 ± 0.02 93.7 ± 0.01 93.7 ± 0.02
100.0 ± 0.02 97.5 ± 0.04 87.5 ± 0.02 81.0 ± 0.02 8.2 ± 0.02*** 0.0 ± 0.02*** 0.0 ± 0.02***
a
Viability was quantitated by MTT assay. Values are expressed as a percentage of untreated control values. Data are mean ± SEM (n ¼ 3). ***p50.001 compared with untreated control.
b
Figure 3, TME, defatted fraction and CH2Cl2 fraction significantly decreased H2O2-induced DNA damage, when compared with cells treated only with H2O2 (25 mM). The total methanol extract of S. lindbergii at 125 and 250 mg ml1 and CH2Cl2 fraction at 20 and 40 mg ml1, exhibited a significant inhibitory effect (450%) on H2O2-induced DNA damage (p50.001), while a significant reduction (450%) in H2O2-induced DNA damage was observed at 500 mg ml1 of defatted fraction (p50.001). The IC50 values of TME, defatted fraction and CH2Cl2 fraction against DNA damage were determined as 48, 138 and 8 mg ml1, respectively.
Discussion Many herbs, spices, and their extracts have been reported as having high antioxidant capacity, such as some plants of the Lamiaceae family. The antioxidant activity of these plants is attributed to their phenolic compound content, such as phenolic acids, phenolic diterpenes and flavonoids (Velasco & Williams, 2011). Antioxidants are substances that at low concentrations delay or prevent the oxidation caused by reactive oxygen species (Saha et al., 2004). Antioxidants may act by decreasing oxygen concentration, preventing first chain
Figure 1. DNA damage of NIH 3T3 cells treated with H2O2 by the comet assay. NIH 3T3 cells were incubated for 20 min at 4 C with different concentrations of H2O2. DNA damage was expressed as percentage tail DNA. Results are mean ± SEM (n ¼ 4 slides 50 cells). ANOVA test, ***p50.001.
initiation by scavenging initial radicals, binding metal ion catalysts and decomposing primary products to non-radical compounds and may help the body to protect itself against various types of oxidative damage which are linked to a variety of diseases including cancer (Saha et al., 2004). Oxidative damage to DNA is thought to play a significant role in mutagenesis, aging and cancer (Starcevic et al., 2003). Antioxidant activity of some Scutellaria species has been shown in other previous studies (Gao et al., 1999; Shao et al., 1999). Free radical scavenging and antioxidant activities of baicalein, baicalin, wogonin and wogonoside, the four major flavonoids in the radix of Scutellaria baicalensis Georgi, were examined in different systems (Gao et al., 1999). The results showed that baicalein and baicalin scavenged hydroxyl radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and alkyl radical in a dose-dependent manner, while wogonin and wogonoside showed subtle or no effect on these radicals (Gao et al., 1999). In the other study, Baicalin inhibited lipid peroxidation in rat brain homogenate three times more than
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
DOI: 10.3109/01480545.2014.954047
Figure 2. DNA damage of NIH 3T3 cells treated with S. lindbergii extracts (total methanol extract, defatted fraction and CH2Cl2 fraction) by the comet assay. NIH 3T3 cells were incubated for 20 min at 4 C with different concentrations of all extracts. Results are mean ± SEM (n ¼ 4 slides 50 cells). ANOVA test, p40.05.
the flavonoid quercetin, and almost 375 times more than vitamin E, both known inhibitors of lipid peroxidation (Van Loon, 1997). Cytotoxicity and anticancer activity of some Scutellaria species have been shown in other previous studies (Yin et al., 2004) apigenin, 20 , 30 , 5, 7-tetrahydroxy flavone, viscidulin III, wogonin and luteolin, the flavonoids isolated from two Scutellaria species (Scutellaria baicalensis Georgi and Scutellaria rivularis Wall), inhibited the proliferation of HL-60 cells (Sonoda et al., 2004) and wogonin also induced apoptosis in HL-60 (Lee et al., 2002). Baicalin, isolated from Scutellaria baicalensis, has been reported to inhibit proliferation and induce apoptosis in human prostate cancer cells (Chan et al., 2000). In recent studies, S. lindbergii extracts exerted cytotoxic and apoptotic effects in different cancer cell lines (Tayarani-Najaran et al., 2010) and also were found to possess moderate antioxidant and antibacterial capacity (Fazly-Bazzaz et al., 2013). In a previously published work done by some of the authors of the present article, chemical composition of the CH2Cl2 fraction of Scutellaria litwinowii root extract, a much closed species to S. lindbergii, was investigated and wogonin, and neobaicalein were isolated. Wogonin and neobaicalein showed apoptotic effects in HeLa cells (Tayarani-Najaran et al., 2012). Therefore, in this study, we examined the cytoprotective and genoprotective effects of Scutellaria lindbergii methanolic extract and its fractions against cell and DNA damage induced by hydrogen peroxide in NIH 3T3 cells using MTT assay and comet assay, respectively. In this study, total methanol extract of S. lindbergii and its fractions were investigated because, the larger the variety of compounds that are extracted by the extractant, the better the chance that biologically active components will also be extracted if a specific class of chemical component is not targeted (Eloff, 1998). Alkaloids, sterols, triterpenes, indigo, flavanoids, carbohydrates and coumarins isolated in methanolic extract (Leite et al., 2006). Low polarity contaminants
S. lindbergii against cell and DNA damage
297
Figure 3. Protective effect of S. lindbergii extracts [total methanol extract (TME), defatted fraction and CH2Cl2 fraction] on NIH 3T3 cells DNA damaged induced by hydrogen peroxide by the comet assay. NIH 3T3 cells were incubated for 20 min at 4 C with a combination of 25 mM H2O2 with different concentrations of S. lindbergii extracts. VC, vehicle control (without H2O2 and extract). Results are mean ± SEM (n ¼ 4 slides 50 cells). ANOVA test, *p50.05, **p50.01 and ***p50.001, significantly different from sample treated with only H2O2 (25 mM).
such as fats, terpenes etc. cleared in defatted fraction and less polar compounds such as isoflavones, flavanones, methoxylated flavones and flavonols extracted in CH2Cl2 fraction (Markham, 1982). In this study, NIH 3T3 cells were used as an in vitro model to study the cytoprotective and genoprotective potential of Scutellaria lindbergii root extract and its fractions against H2O2 induced toxicity. Our data confirmed that S. lindbergii extract has cytotoxic activity against NIH 3T3 cells at high concentrations. The present study demonstrated that pretreatment and concurrent treatment of NIH 3T3 cells with total methanol extract of S. lindbergii and its fractions (at nontoxic concentrations) has not significantly protected the cells against H2O2 induced cytotoxicity. In the present study, we also examined the antigenotoxic effects of S. lindbergii extracts against DNA damage induced by hydrogen peroxide. H2O2, a well-defined oxidant agent, does not undergo any chemical reactions with DNA (Slamenova et al., 2002). It is envisaged that H2O2 can penetrate to the nucleus and react with ions of iron or copper to form OH radicals. Attack of HO radicals on DNA leads to fragmentation and strand breaks (Slamenova et al., 2002). The level of DNA damage (DNA strand breaks) was measured using the single cell gel electrophoresis, so-called comet assay. 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). Relative to other genotoxicity tests, the advantages of the SCGE assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its usage for any eukaryote cell, its flexibility, its low costs, its ease of application, and the short time needed to complete a study (Tice et al., 2000). Simultaneous treatment of cells with S. lindbergii extracts and
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
298
M. Ehtesham-Gharaee et al.
H2O2 displayed genoprotective activity of methanol extract and its fractions against H2O2-induced DNA damage. Total phenolic and flavonoid contents in each S. lindbergii extract have been expressed as tannic acid equivalent (TAE) and quercetin equivalents (QE), respectively. Dichloromethane extract contained higher flavonoid content (82.32 QE/g dried extract) while for the total phenolic content, methanol extract contained the highest amount (80.64 TAE/g dried extract) (Fazly-Bazzaz et al., 2013). A number of studies have focussed on the biological activities of phenolic compounds, which are potential antioxidants and free radical-scavengers for example, it has been reported a high correlation between DPPH (2, 2-diphenyl- 1-picrylhydrazyl) radical-scavenging activities and total polyphenolics (Kumar et al., 2008). In a study, using three different methods for testing antioxidant activity of oregano essential oil confirmed that the essential oil possess remarkable antioxidant properties which is due to the presence of phenolic monoterpenes: thymol and carvacrol (Kulisic et al., 2004). Three major polyphenolic components, namely wogonin, baicalin, and baicalein from S. baicalensis have been demonstrated as free radical scavengers of hydroxyl radical, DPPH radical and alkyl radical (Gao et al., 1999). Flavonoids are polyphenolic compounds which represent a large family of molecules including the flavones, flavonols, flavavones, anthocyanidins, and catechins. Flavonoids which share a basic structure with vitamin E are potent scavengers of free radicals such as hydroxyl and superoxide radicals, and also act as chelators of transient elements (Gate et al., 1999). Many flavonoids, such as quercetin, luteolin and catechins, are better antioxidants than the antioxidant nutrients vitamin C, vitamin E and L-carotene on a mole for mole basis (Gao et al., 1999). Therefore, it was speculated that genoprotective effect of S. lindbergii extracts is related to its flavonoids and phenolic compounds. It was found that the methanol extract and dichloromethane fraction significantly had the greater genoprotective activity compared with the defatted fraction. Since the methanol extract and dichloromethane fraction contained higher flavonoid and total phenolic contents, the difference in the genoprotective activity of extracts may be described by the difference in the total phenolic and flavonoid contents. Antioxidants can decrease the oxidative damage directly via reacting with free radicals or indirectly by inhibiting the activity or expression of free radical generating enzymes or enhancing the activity or expression of intracellular antioxidant enzymes (Lu et al., 2010). In a study antioxidant activities of S. linbergii Extracts were reported Using the thiobarbituric acid reactive species (TBARS) and DPPH radical scavenging assays (Fazly-Bazzaz et al., 2013). With respect to this report, genoprotective activities of S. linbergii extracts in simultaneous treatment of cells with extracts and H2O2 and also high amounts of free radical scavengers in S. lindbergii extracts such as flavonoids and phenolic compounds, radical scavenging can be suggested as the mechanism. However, the precise mechanism of the inhibition effects of the extracts against oxidative DNA damage needs further investigation. As a conclusion, this study confirms that S. linbergii root extract has protective effects against hydrogen peroxide-induced oxidative DNA damage.
Drug Chem Toxicol, 2015; 38(3): 293–299
Thus S. linbergii might have potential for the prevention and treatment of diseases or conditions resulting from oxidative stress such as cancer and aging.
Acknowledgements The authors are indebted to the Research Council of Mashhad University of Medical Sciences, Iran for approval and financial support of this project. Also this research work was part of Pharm. D. thesis of A. Eshaghi.
Declaration of interest The authors report no declarations of interest.
References Aherne SA, Kerry JP, O’Brien NM. (2007). Effects of plant extracts on antioxidant status and oxidant-induced stress in Caco-2 cells. Br J Nutr 97:321–328. Attar F, Joharchi MR. (2002). New plant records from Iran. Iran J Bot 9:223–228. Bozin B, Mimica-Dukic N, Simin N, Anackov G. (2006). Characterization of the volatile composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils. J Agric Food Chem 54:1822–1828. Chan FL, Choi HL, Chen ZY, et al. (2000). Induction of apoptosis in prostate cancer cell lines by a flavonoid, baicalin. Cancer Lett 160:219–228. Chen X, Nishida H, Konishi T. (2003). Baicalin promoted the repair of DNA single strand breakage caused by H2O2 in cultured NIH3T3 fibroblasts. Biol Pharm Bull 26:282–284. Chin YW, Balunas MJ, Chai HB, Kinghorn AD. (2006). Drug discovery from natural sources. AAPS J 8:E239–E253. Cole MD, Bridge PD, Dellar JE, et al. (1991). Antifungal activity of Neoclerodane diterpenoids from Scutellaria. Phytochemistry 30:1125–1127. Craig WJ. (1999). Health-promoting properties of common herbs. Am J Clin Nutr 70:491S–499S. Demirci M, Hiller KA, Bosl C, et al. (2008). The induction of oxidative stress, cytotoxicity, and genotoxicity by dental adhesives. Dent Mater 24:362–371. Eloff JN. (1998). Which extractant should be used for the screening and isolation of antimicrobial components from plants? J Ethnopharmacol 60:1–8 Emami SA, Aghazari F. (2011). Les Phanerogames Endemiques de la Flore d’Iran. Publications de I’Universite´ de Te´he´ran des Sciences Me´dicales. Fazly-Bazzaz BS, Atefeh Arab A, Emami SA, et al. (2013). Antimicrobial and antioxidant activities of methanol, dichloromethane and ethyl acetate extracts of Scutellaria lindbergii Rech.f. Chiang Mai J Sci 40:49–59. Gao Z, Huang K, Yang X, Xu H. (1999). Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi. Biochim Biophys Acta 1472:643–650. Gate L, Paul J, Ba GN, et al. (1999). Oxidative stress induced in pathologies: the role of antioxidants. Biomed Pharmacother 53:169–180. Glei M, Pool-Zobel BL. (2006). The main catechin of green tea, ()-epigallocatechin-3-gallate (EGCG), reduces bleomycin-induced DNA damage in human leucocytes. Toxicol In Vitro 20:295–300. Hahm DH, Yeom MJ, Lee EH, et al. (2001). Effect of Scutellariae Radix as a novel antibacterial herb on the ppk(polyphosphate kinase) mutant of Salmonella typhimurium. J Microbiol Biotechnol 11:1061–1065. Jamzad Z. (2012). Lamiaceae. In: Flora of Iran. Tehran: Research Institute of Forests & Rangelands. Kulisic T, Radonic A, Katalinic V, Milos M. (2004). Use of different methods for testing antioxidative activity of oregano essential oil. Food Chem 85:633–640. Kumar KS, Ganesan K, Rao PVS. (2008). Antioxidant potential of solvent extracts of Kappaphycus alvarezii (Doty) Doty – an edible seaweed. Food Chem 107:289–295.
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
DOI: 10.3109/01480545.2014.954047
Lam W, Bussom S, Guan F, et al. (2010). The four-herb Chinese medicine PHY906 reduces chemotherapy-induced gastrointestinal toxicity. Sci Transl Med 2:45ra59. Lee WR, Shen SC, Lin HY, et al. (2002). Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species, and activation of caspase 3 and Ca (2+)-dependent endonuclease. Biochem Pharmacol 63:225–236. Leite SP, Vieira JRC, De Medeiros PL, et al. (2006). Antimicrobial activity of Indigofera suffruticosa. Evid Based Complement Alternat Med 3:261–265. Li BQ, Fu T, Gong W-H, et al. (2000). The flavonoid baicalin exhibits anti-inflammatory activity by binding to chemokines. Immunopharmacology 49:295–306. Lu JM, Lin PH, Yao Q, Chen C. (2010). Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems. J Cell Mol Med 14:840–860. Mabberley DJ. (1993). The plant-book. Cambridge University Press. Markham KR. (1982). Techniques of flavonoid identification. London: Academic Press. Middleton Jr E, Kandaswami C, Theoharides TC. (2000). The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 52:673–751. Mosmann T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63. Nagai T, Miyaichi Y, Tomimori T, et al. (1990). Inhibition of influenza virus sialidase and anti-influenza virus activity by plant flavonoids. Chem Pharm Bull (Tokyo) 38:1329–1332. Nagai T, Moriguchi R, Suzuki Y, et al. (1995). Mode of action of the anti-influenza virus activity of plant flavonoid, 5,7,40 -trihydroxy-8methoxyflavone, from the roots of Scutellaria baicalensis. Antiviral Res 26:11–25. Plazar J, Filipic M, Groothuis GM. (2008). Antigenotoxic effect of Xanthohumol in rat liver slices. Toxicol In Vitro 22:318–327. Powell CB, Fung P, Jackson J, et al. (2003). Aqueous extract of herba Scutellaria barbatae, a chinese herb used for ovarian cancer, induces apoptosis of ovarian cancer cell lines. Gynecol Oncol 91:332–340. Rojas E, Lopez MC, Valverde M. (1999). Single cell gel electrophoresis assay: methodology and applications. J Chromatogr B Biomed Sci Appl 722:225–254. Saha K, Lajis NH, Israf DA, et al. (2004). Evaluation of antioxidant and nitric oxide inhibitory activities of selected Malaysian medicinal plants. J Ethnopharmacol 92:263–267. Sato Y, Suzaki S, Nishikawa T, et al. (2000). Phytochemical flavones isolated from Scutellaria barbata and antibacterial activity against
S. lindbergii against cell and DNA damage
299
methicillin-resistant Staphylococcus aureus. J Ethnopharmacol 72:483–488. Shao Z-H, Li C-Q, Vanden Hoek TL, et al. (1999). Extract from Scutellaria baicalensis Georgi attenuates oxidant stress in cardiomyocytes. J Mol Cell Cardiol 31:1885–1895. Singh NP, McCoy MT, Tice RR, Schneider EL. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191. Skaltsaa HD, Lazarib DM, Kyriazopoulosc P, et al. (2005). Composition and antimicrobial activity of the essential oils of Scutellaria sieberia Benth. and Scutellaria rupestris Boiss. et Heldr. ssp. adenotricha (Boiss. et Heldr.) Greuter et Burdet from Greece. Essent Oil Res 17:232–235. Slamenova D, Kuboskova K, Horvathova E, Robichova S. (2002). Rosemary-stimulated reduction of DNA strand breaks and FPGsensitive sites in mammalian cells treated with H2O2 or visible lightexcited methylene blue. Cancer Lett 177:145–153. Soltani F, Mosaffa F, Iranshahi M, et al. (2009). Auraptene from Ferula szowitsiana protects human peripheral lymphocytes against oxidative stress. Phytother Res 24:85–89. Sonoda M, Nishiyama T, Matsukawa Y, Moriyasu M. (2004). Cytotoxic activities of flavonoids from two Scutellaria plants in Chinese medicine. J Ethnopharmacol 91:65–68. Starcevic SL, Diotte NM, Zukowski KL, et al. (2003). Oxidative DNA damage and repair in a cell lineage model of human proliferative breast disease (PBD). Toxicol Sci 75:74–81. Tayarani-Najaran Z, Asili J, Parsaee H, et al. (2012). Wogonin and neobaicalein from Scutellaria litwinowii roots are apoptotic for HeLa cells. Rev Bras Farmacogn 22:268–276. Tayarani-Najaran Z, Mousavi SH, Asili J, Emami SA. (2010). Growthinhibitory effect of Scutellaria lindbergii in human cancer cell lines. Food Chem Toxicol 48:599–604. Tice RR, Agurell E, Anderson D, et al. (2000). Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221. Van Loon IM. (1997). The golden root: clinical applications of Scutellaria baicalensis Georgi flavonoids as modulators of the inflammatory response. Altern Med Rev 2:472–480. Velasco V, Williams P. (2011). Improving meat quality through natural antioxidants. Chilean J Agric Res 71:313–322. Yin X, Zhou J, Jie C, et al. (2004). Anticancer activity and mechanism of Scutellaria barbata extract on human lung cancer cell line A549. Life Sci 75:2233–2244. Zuo F, Zhou ZM, Yan MZ, et al. (2002). Metabolism of constituents in Huangqin-Tang, a prescription in traditional Chinese medicine, by human intestinal flora. Biol Pharm Bull 25:558–563.
RESEARCH ARTICLE
Protective effects of Scutellaria lindbergii root extract against oxidative-induced cell and DNA damage in mouse fibroblast-like cells
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
Melika Ehtesham-Gharaee1,2, Ameneh Eshaghi2, Susan Shojaee2, Javad Asili3, Seyed Ahmad Emami3, Javad Behravan1,2, and Fatemeh Mosaffa1,2 1
Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran, 2Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran, and 3Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Abstract
Keywords
Context: Scutellaria lindbergii Rech. f. (Lamiaceae) is an Iranian species of Scutellaria which has been shown to exert antimicrobial, antioxidant and cytotoxic effects. Objective: The protective properties of total methanol extract (TME) of S. lindbergii and its fractions (defatted and CH2Cl2) were investigated against cytotoxic and genotoxic effects of H2O2 in NIH 3T3 cell line as nonmalignant cells. Materials and methods: The cells were incubated with different concentrations of S. lindbergii root extracts [TME (15–250 mg ml1), defatted fraction (15–500 mg ml1) and CH2Cl2 fraction (5–40 mg ml1)] and toxic concentration of H2O2 (200 mM) at 37 C for 2 h concurrently and Cell viability was quantitated by MTT assay. The antigenotoxic effect of extracts was investigated using comet assay. The cells were incubated with extracts [TME (25–250 mg ml1), defatted fraction (25–500 mg ml1) and CH2Cl2 fraction (5–40 mg ml1)] and H2O2 (25 mM) at 4 C for 20 min, then the comet assay was performed. DNA damage was expressed as percentage tail DNA. Results: Total methanol extract of S. lindbergii and its fractions had a significant inhibitory effect on DNA damage. The IC50 values of TME, defatted fraction and CH2Cl2 fraction against DNA damage were determined as 48, 138 and 8 mg ml1, respectively. Conclusion: S. lindbergii extracts can prevent oxidative DNA damage, which is likely due to its flavonoids and phenolic compounds as antioxidant constituents.
Anticytotoxicity, antigenotoxicity, comet assay, hydrogen peroxide, Lamiaceae, MTT assay
Introduction Reactive oxygen species-mediated cell damage plays an important role in various human chronic diseases such as Crohn’s disease, certain cancers and a number of neurodegenerative diseases (Aherne et al., 2007). At the cellular level, oxidative stress can cause severe metabolic dysfunction, including lipid peroxidation, protein oxidation, membrane disruption and DNA damage, resulting in changes in signal transduction pathways, gene expression, cell mutagenesis and cell death (Aherne et al., 2007; Demirci et al., 2008). Natural products from plant sources have extensive past and present use in treatment of different kinds of ailments and serve as compounds of interest both in their natural form and as templates for synthetic modification (Chin et al., 2006). There is some evidence that some plant-derived natural compounds have protective effects towards DNA damage caused by oxidative stresses (Glei & Pool-Zobel, 2006; Plazar et al., 2008).
Address for correspondence: Dr. F. Mosaffa, Dept. of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, P. O. Box: 91775-1365. Iran. Tel: +98-5118823255-66, Fax: +98-511-8823251. E-mail: [email protected]
History Received 6 March 2014 Revised 25 June 2014 Accepted 8 August 2014 Published online 19 September 2014
Scutellaria L. (Lamiaceae) is a genus that contains about 300 species worldwide, excluding South Africa (Mabberley, 1993). This genus has 27 species and two hybrids in Iran, of which 10 species and two hybrids are endemic to the country (Emami & Aghazari, 2011; Jamzad, 2012). Scutellaria lindbergii Rech. f. is an Iranian species of the genus. General distribution of this species is limited to Iran and Afghanistan (Attar & Joharchi, 2002; Jamzad, 2012). In traditional medicine, this species is used as a source of flavonoids to treat a variety of diseases. The dried roots of S. baicalensis have been used for the treatment of suppurative dermatitis, diarrhea, inflammatory diseases, hyperlipidemia, and atherosclerosis in Chinese and Japanese traditional medicine (Middleton Jr et al., 2000). In traditional Chinese medicine, the Huangquin-tang formula which contains four medicinal herbs including Scutellariae Radix etc. is prescribed for the treatment of prostate cancer (Zuo et al., 2002) and it was also originally used to treat nausea, vomiting and diarrhea as far back as 1800 years ago (Lam et al., 2010). Several studies have been shown various biological and pharmacological activities of some Scutellaria species such as antibacterial (Hahm et al., 2001; Sato et al., 2000; Skaltsaa et al., 2005), antiviral (Nagai et al., 1990, 1995), antifungal (Cole et al., 1991), anti-inflammatory (Li et al., 2000),
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
294
M. Ehtesham-Gharaee et al.
antioxidant (Gao et al., 1999; Shao et al., 1999), Cytotoxicity and anticancer (Powell et al., 2003; Sonoda et al., 2004; Tayarani-Najaran et al., 2010; Yin et al., 2004). Experimental evidence suggests that most Lamiaceae spices possess a wide range of biological and pharmacological activities that may protect tissues against O2-induced damage and therefore lower the risk of human chronic diseases (Bozin et al., 2006; Craig, 1999) for example, Scutellaria baicalensis Georgi extract attenuates oxidative stress in cardiomyocytes and protect cells from lethal oxidant damage in an ischemia–reperfusion model (Shao et al., 1999). Therefore, the present work was carried out to investigate the cytoprotective and genoprotective effects of Scutellaria lindbergii extract and its fractions against cell damage and DNA damage induced by hydrogen peroxide in mouse embryo cell line (NIH 3T3) as non-malignant cells. Several studies have been carried out on some biological activities of S. lindbergii such as its antimicrobial, antioxidant (Fazly-Bazzaz et al., 2013) and cytotoxic effects (TayaraniNajaran et al., 2010), but to the authors knowledge this is the first study on cytoprotective and genoprotective activity of S. lindbergii.
Materials and methods Chemical and reagents
Drug Chem Toxicol, 2015; 38(3): 293–299
streptomycin and maintained at 37 C in a humidified atmosphere (90%) containing 5% CO2. Effect of S. lindbergii extracts on viability of NIH 3T3 cells The crude extracts of S. lindbergii were dissolved in the minimum amount of DMSO in a way that the final DMSO concentration was 0.5% when mixed with culture medium. The solutions of crude extracts in DMSO were added to the culture medium to obtain different concentrations of extracts [total methanol extract (TME) (10–1000 mg ml1), defatted fraction (10–1200 mg ml1) and CH2Cl2 fraction (5–160 mg ml1)] (Tayarani-Najaran et al., 2010). Cell viability was determined using a modified MTT assay (Mosmann, 1983; Tayarani-Najaran et al., 2010). Briefly, NIH 3T3 cells were seeded at 5000 cells per well in 96-well cell culture plates. After 24 h incubation, growth media was replaced with growth medium containing various concentrations of different extracts and then incubated for 24 and 48 h. Negative control wells (no extract) were prepared with 0.5% (v/v) DMSO in equal volume of medium. After removing the medium, MTT solution (5 mg ml1 in PBS) was added to the cells. The cells were incubated at 37 C for 4 h, the medium was aspirated, and the formazan product was solubilized with DMSO. The absorption was measured at 570 nm (620 nm as a reference) in an ELISA reader. The percentage cell viability for each concentration of plant extracts was calculated.
Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), trypsin-EDTA solution, penicillin/streptomycin 100 units, dimethylsulfoxide (DMSO) and all other fine chemicals were obtained from Merck, Germany. SYBR Green, 3-(4,5-dimethylthiazole-2-yl)-2,5-dimethyl tetrazolium bromide (MTT) and trypan blue were purchased from Sigma (St Louis, MO). Low melting point agarose (LMA) and normal melting point agarose (NMA) were purchased from Fermentas, Germany.
In order to determine the cytotoxic effect of H2O2, after 24 h incubation in 96-well plates (5000 cells/200 ml), the cells were treated with H2O2 (0–600 mM) for 0.5, 1 and 2 h and then cell viability was detected by the MTT method as described previously.
Plant material
Extract cytoprotective activity assay
Scutellaria lindbergii was collected from Kang valley (height 1800 m) near Mashhad (Razavi Khorasan Province, north-east of Iran) in July 2012 and identified by Mr. M. R. Joharchi, from Ferdowsi University of Mashhad Herbarium (FUMH). A voucher specimen of the species was deposited in the Herbarium of the Faculty of Pharmacy, Mashhad University of Medical Sciences (MUMS) under number: 11309.
In order to investigate of the protective effects of S. lindbergii extracts against H2O2 cytotoxicity either pre-treatment or co-treatment protocol was used.
Extraction and fractionation Dry powdered roots of the plant (100 g) were macerated separately in methanol and dichloromethane at room temperature for 48 h. Both extracts were concentrated at 50 C under reduced pressure to dryness. The concentrated methanol extract was then extracted with an equal volume of n-hexane, three times, to give a fraction containing polar compounds (defatted fraction) and then concentrated at 50 C under reduced pressure to dryness. Cell culture NIH 3T3 was obtained from Pasteur Institute (Tehran, Iran). The cell line was grown in DMEM medium containing 10% FCS, supplemented with 100 unit/ml penicillin and 100 mg/ml
Assay of H2O2-induced toxicity
Pre-treatment protocol Cells grown in DMEM containing 10% FBS in 96-well culture plates (5000 cells/well) were incubated for 24 h in the presence of different concentrations of extracts [TME (15, 31.5, 62.5, 125 and 250 mg ml1), defatted fraction (15, 31.5, 62.5, 125, 250 and 500 mg ml1) and CH2Cl2 fraction (5, 10, 20 and 40 mg ml1)]. After washing the cells with fresh DMEM without FBS twice, the cells were treated with H2O2 (200 mM) in the above FBS-free medium for 2 h. Then the medium was replaced with fresh DMEM containing 10% FBS and the cell viability was quantified by MTT (Chen et al., 2003). Co-treatment protocol Before treatment, the cells were seeded at 5000 cells per well in a 96-well plate and incubated for 24 h at 37 C. Then the cells were incubated with different concentrations of extracts and 200 mM H2O2 simultaneously, for 2 h and cell viability was determined by MTT assay.
DOI: 10.3109/01480545.2014.954047
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
Determination of DNA damage (comet assay) The alkaline single cell gel electrophoresis [SCGE (comet assay)] was performed under alkaline conditions according to the technique described by Singh et al. (1988) with slight modifications (Soltani et al., 2009). The cells were plated at 2.5 104 cells per well in a 24-well plate and incubated for 24 h at 37 C. Growing cells were treated with various concentrations of S. lindbergii extracts either TME (25, 125 and 250 mg ml1), defatted fraction (25, 250 and 500 mg ml1), CH2Cl2 fraction (5, 20 and 40 mg ml1) and H2O2 (25 mM) simultaneously, for 20 min in the dark at 4 C. Positive and negative control wells were treated with 25 mM H2O2 in PBS and 0.5% (v/v) DMSO-PBS, respectively. The cells were harvested and centrifuged at 3000 rpm for 10 min and then washed with PBS. The cell pellets were mixed with 40 mL of sterile PBS at room temperature. Then 140 mL of 1% (w/v) low melting point agarose (LMA) was added at 37 C and distributed onto microscopic slides coated with a layer of 1% (w/v) normal melting point agarose (NMA), covered with a coverslip and kept for 10 min at 4 C to solidify. After the coverslips were removed, the slides vertically 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) at 4 C for at least 2 h. The slides were presoaked in freshly prepared alkaline electrophoresis buffer (1 mM Na2EDTA, 0.3 N NaOH, pH 13.0) for 30 min. Electrophoresis was carried out for 45 min at 4 C (300 mA, 1 V/Cm). 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 washed thrice with a neutralizing solution (0.4 m tris HCl buffer, pH 7.5). Twenty microliters SYBR Green (1 x) was dispensed directly onto the slides and covered with a coverslip. The slides were studied by a fluorescent microscope (Nikon100, E 200, Japan) using a G-2 A filter, attached to a charge-coupled device camera connected to a personal computer. All experiments were carried out at least three times, each with three parallel slides per data point. For each slide, 50 selected cells were analyzed with TriTek Cometscore version 1.5 software (www.autocomet.com). The DNA damage was expressed as percentage tail DNA, where % tail DNA ¼ [tail DNA/(head DNA + tail DNA)] 100. A higher percentage tail DNA indicated a higher level of DNA damage.
Statistical analysis All of the experiments were performed in triplicate. Instat software and One-way analysis of variance (ANOVA) were used for data analysis. Differences between groups were evaluated by Tukey–Kramer test. All results were expressed as mean ± SEM of three independent experiments performed in triplicate. p50.05 was considered statistically significant.
Results Effect of S. lindbergii extracts on NIH 3T3 cell viability The cytotoxicity of total methanol extract of S. lindbergii and its fractions were examined on NIH 3T3 cell line. When NIH
S. lindbergii against cell and DNA damage
295
3T3 cells were incubated with different concentrations of S. lindbergii extracts for 24 and 48 h, TME showed a significant toxicity at concentration of 500 mg ml1 and higher (p50.001). A significant toxicity was observed for concentrations above 500 mg ml1 of defatted fraction (p50.001).While CH2Cl2 fraction showed a significant toxicity at concentration of 80 mg ml1 and higher (p50.01) (Table 1). The IC50 values of TME, defatted fraction and CH2Cl2 fraction against NIH 3T3 cells were determined as 421, 1139 and 150 mg ml1 after 24 h and 513, 578 and 104 mg ml1 after 48 h, respectively. After examination of 0.5% DMSO in culture medium, no significant toxicity was observed. Effect of H2O2 on NIH 3T3 cell viability MTT assay was carried out at different concentrations of H2O2 to determine the cytotoxic concentrations of H2O2 for NIH 3T3 cells. After 24 h incubation in 96-well plates (5000 cells/200 ml)and then incubation with different H2O2 concentrations (0–600 mM) for 0.5, 1 and 2 h, a significant cytotoxicity (450%) was observed at concentration of 200 mM and higher with 2 h incubation time compared to untreated control (p50.001) (Table 2). Thus, 200 mM H2O2 for 2 h were selected as appropriate concentration to induce toxicity to assess the effects of the S. lindbergii extracts in further experiments. Effect of S. lindbergii extracts on viability of NIH 3T3 cells against H2O2-induced cytotoxicity When both of pre-treatment and co-treatment protocol were used, no significant difference in cell viability was observed (results not shown) at different concentrations of all extracts (TME, defatted fraction and CH2Cl2 fraction) compared with the control (H2O2 alone). Effect of H2O2 or S. lindbergii extracts on NIH 3T3 cell DNA damage Induced effects of oxidative stress were measured by SCGE. After 20 min incubation of NIH 3T3 cells with H2O2 (25, 50 and 100 mM) at 4 C, a significant increase in DNA damage was induced with increasing concentration of H2O2 (p50.001 for all; Figure 1). The potential of TME (25, 125 and 250 mg ml1), defatted fraction (25, 250 and 500 mg ml1) and CH2Cl2 fraction (5, 20 and 40 mg ml1) of S. lindbergii to induce DNA damage was assessed using the comet assay. None of the plant extracts investigated significantly increased % tail DNA when compared with negative control cells (0.5% (v/v) DMSOPBS) (p40.05; Figure 2). Protective effect of S. lindbergii extracts against H2O2-induced DNA damage The genoprotective effects of different concentrations of TME (25, 125 and 250 mg ml1), defatted fraction (25, 250 and 500 mg ml1) and CH2Cl2 fraction (5, 20 and 40 mg ml1) of S. lindbergii in the presence of 25 mM H2O2, were examined for 20 min at 4 C. Comet assay was carried out and DNA damage was expressed as percentage tail DNA. As shown in
296
M. Ehtesham-Gharaee et al.
Drug Chem Toxicol, 2015; 38(3): 293–299
Table 1. Effect of different concentrations of S. lindbergii extracts [total methanol extract (TME), defatted fraction and CH2Cl2 fraction] on NIH 3T3 cells viability after 24 h and 48 h incubationa,b. 24 h
Concentration (mg/ml)
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
TME 0.0 (Control) 10.0 20.0 31.0 40.0 62.0 80.0 125.0 160.0 250.0 500.0 600.0 1000.0 1200.0
100.0 ± 0.03
48 h
Defatted fraction
CH2Cl2 fraction
100.0 ± 0.09
100.0 ± 0.03 100.0 ± 0.03 103.5 ± 0.07
97.0 ± 0.04
TME 100.0 ± 0.05
Defatted fraction
CH2Cl2 fraction
100.0 ± 0.06
100.0 ± 0.12 100.0 ± 0.05 102.0 ± 0.11
100.0 ± 0.05 110.0 ± 0.05
92.0 ± 0.03
100.0 ± 0.04
94.0 ± 0.05
100.0 ± 0.08
103.0 ± 0.11 100.0 ± 0.04
100.0 ± 0.01
100.0 ± 0.04
100.0 ± 0.10
66.6 ± 0.05**
50.6 ± 0.05**
45.0 ± 0.05*** 88.0 ± 0.01 22.0 ± 0.03***
98.5 ± 0.09 98.5 ± 0.04 37.9 ± 0.09*** 33.3 ± 0.08*** 28.8 ± 0.03***
20.0 ± 0.06***
31.3 ± 0.09*** 99.0 ± 0.02 22.0 ± 0.05*** 21.0 ± 0.04***
88.9 ± 0.11 88.9 ± 0.01 33.3 ± 0.06*** 11.1 ± 0.03*** 11.1 ± 0.03***
a
Viability was quantitated by MTT assay. Values are expressed as a percentage of negative control values. Data are mean ± SEM (n ¼ 3). **p50.01 and ***p50.001, significantly different from controls.
b
Table 2. Effect of different concentrations of H2O2 on NIH 3T3 cells viability after treatment for 0.5, 1 and 2 ha,b. Concentration (mM) 0.0 (Control) 25.0 50.0 100.0 200.0 400.0 600.0
0.5 h
1h
2h
100.0 ± 0.02 100.0 ± 0.02 98.3 ± 0.03 96.6 ± 0.04 95.0 ± 0.01 95.0 ± 0.02 91.7 ± 0.02
100.0 ± 0.03 100.0 ± 0.02 98.3 ± 0.03 99.5 ± 0.05 97.9 ± 0.02 93.7 ± 0.01 93.7 ± 0.02
100.0 ± 0.02 97.5 ± 0.04 87.5 ± 0.02 81.0 ± 0.02 8.2 ± 0.02*** 0.0 ± 0.02*** 0.0 ± 0.02***
a
Viability was quantitated by MTT assay. Values are expressed as a percentage of untreated control values. Data are mean ± SEM (n ¼ 3). ***p50.001 compared with untreated control.
b
Figure 3, TME, defatted fraction and CH2Cl2 fraction significantly decreased H2O2-induced DNA damage, when compared with cells treated only with H2O2 (25 mM). The total methanol extract of S. lindbergii at 125 and 250 mg ml1 and CH2Cl2 fraction at 20 and 40 mg ml1, exhibited a significant inhibitory effect (450%) on H2O2-induced DNA damage (p50.001), while a significant reduction (450%) in H2O2-induced DNA damage was observed at 500 mg ml1 of defatted fraction (p50.001). The IC50 values of TME, defatted fraction and CH2Cl2 fraction against DNA damage were determined as 48, 138 and 8 mg ml1, respectively.
Discussion Many herbs, spices, and their extracts have been reported as having high antioxidant capacity, such as some plants of the Lamiaceae family. The antioxidant activity of these plants is attributed to their phenolic compound content, such as phenolic acids, phenolic diterpenes and flavonoids (Velasco & Williams, 2011). Antioxidants are substances that at low concentrations delay or prevent the oxidation caused by reactive oxygen species (Saha et al., 2004). Antioxidants may act by decreasing oxygen concentration, preventing first chain
Figure 1. DNA damage of NIH 3T3 cells treated with H2O2 by the comet assay. NIH 3T3 cells were incubated for 20 min at 4 C with different concentrations of H2O2. DNA damage was expressed as percentage tail DNA. Results are mean ± SEM (n ¼ 4 slides 50 cells). ANOVA test, ***p50.001.
initiation by scavenging initial radicals, binding metal ion catalysts and decomposing primary products to non-radical compounds and may help the body to protect itself against various types of oxidative damage which are linked to a variety of diseases including cancer (Saha et al., 2004). Oxidative damage to DNA is thought to play a significant role in mutagenesis, aging and cancer (Starcevic et al., 2003). Antioxidant activity of some Scutellaria species has been shown in other previous studies (Gao et al., 1999; Shao et al., 1999). Free radical scavenging and antioxidant activities of baicalein, baicalin, wogonin and wogonoside, the four major flavonoids in the radix of Scutellaria baicalensis Georgi, were examined in different systems (Gao et al., 1999). The results showed that baicalein and baicalin scavenged hydroxyl radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and alkyl radical in a dose-dependent manner, while wogonin and wogonoside showed subtle or no effect on these radicals (Gao et al., 1999). In the other study, Baicalin inhibited lipid peroxidation in rat brain homogenate three times more than
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
DOI: 10.3109/01480545.2014.954047
Figure 2. DNA damage of NIH 3T3 cells treated with S. lindbergii extracts (total methanol extract, defatted fraction and CH2Cl2 fraction) by the comet assay. NIH 3T3 cells were incubated for 20 min at 4 C with different concentrations of all extracts. Results are mean ± SEM (n ¼ 4 slides 50 cells). ANOVA test, p40.05.
the flavonoid quercetin, and almost 375 times more than vitamin E, both known inhibitors of lipid peroxidation (Van Loon, 1997). Cytotoxicity and anticancer activity of some Scutellaria species have been shown in other previous studies (Yin et al., 2004) apigenin, 20 , 30 , 5, 7-tetrahydroxy flavone, viscidulin III, wogonin and luteolin, the flavonoids isolated from two Scutellaria species (Scutellaria baicalensis Georgi and Scutellaria rivularis Wall), inhibited the proliferation of HL-60 cells (Sonoda et al., 2004) and wogonin also induced apoptosis in HL-60 (Lee et al., 2002). Baicalin, isolated from Scutellaria baicalensis, has been reported to inhibit proliferation and induce apoptosis in human prostate cancer cells (Chan et al., 2000). In recent studies, S. lindbergii extracts exerted cytotoxic and apoptotic effects in different cancer cell lines (Tayarani-Najaran et al., 2010) and also were found to possess moderate antioxidant and antibacterial capacity (Fazly-Bazzaz et al., 2013). In a previously published work done by some of the authors of the present article, chemical composition of the CH2Cl2 fraction of Scutellaria litwinowii root extract, a much closed species to S. lindbergii, was investigated and wogonin, and neobaicalein were isolated. Wogonin and neobaicalein showed apoptotic effects in HeLa cells (Tayarani-Najaran et al., 2012). Therefore, in this study, we examined the cytoprotective and genoprotective effects of Scutellaria lindbergii methanolic extract and its fractions against cell and DNA damage induced by hydrogen peroxide in NIH 3T3 cells using MTT assay and comet assay, respectively. In this study, total methanol extract of S. lindbergii and its fractions were investigated because, the larger the variety of compounds that are extracted by the extractant, the better the chance that biologically active components will also be extracted if a specific class of chemical component is not targeted (Eloff, 1998). Alkaloids, sterols, triterpenes, indigo, flavanoids, carbohydrates and coumarins isolated in methanolic extract (Leite et al., 2006). Low polarity contaminants
S. lindbergii against cell and DNA damage
297
Figure 3. Protective effect of S. lindbergii extracts [total methanol extract (TME), defatted fraction and CH2Cl2 fraction] on NIH 3T3 cells DNA damaged induced by hydrogen peroxide by the comet assay. NIH 3T3 cells were incubated for 20 min at 4 C with a combination of 25 mM H2O2 with different concentrations of S. lindbergii extracts. VC, vehicle control (without H2O2 and extract). Results are mean ± SEM (n ¼ 4 slides 50 cells). ANOVA test, *p50.05, **p50.01 and ***p50.001, significantly different from sample treated with only H2O2 (25 mM).
such as fats, terpenes etc. cleared in defatted fraction and less polar compounds such as isoflavones, flavanones, methoxylated flavones and flavonols extracted in CH2Cl2 fraction (Markham, 1982). In this study, NIH 3T3 cells were used as an in vitro model to study the cytoprotective and genoprotective potential of Scutellaria lindbergii root extract and its fractions against H2O2 induced toxicity. Our data confirmed that S. lindbergii extract has cytotoxic activity against NIH 3T3 cells at high concentrations. The present study demonstrated that pretreatment and concurrent treatment of NIH 3T3 cells with total methanol extract of S. lindbergii and its fractions (at nontoxic concentrations) has not significantly protected the cells against H2O2 induced cytotoxicity. In the present study, we also examined the antigenotoxic effects of S. lindbergii extracts against DNA damage induced by hydrogen peroxide. H2O2, a well-defined oxidant agent, does not undergo any chemical reactions with DNA (Slamenova et al., 2002). It is envisaged that H2O2 can penetrate to the nucleus and react with ions of iron or copper to form OH radicals. Attack of HO radicals on DNA leads to fragmentation and strand breaks (Slamenova et al., 2002). The level of DNA damage (DNA strand breaks) was measured using the single cell gel electrophoresis, so-called comet assay. 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). Relative to other genotoxicity tests, the advantages of the SCGE assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its usage for any eukaryote cell, its flexibility, its low costs, its ease of application, and the short time needed to complete a study (Tice et al., 2000). Simultaneous treatment of cells with S. lindbergii extracts and
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
298
M. Ehtesham-Gharaee et al.
H2O2 displayed genoprotective activity of methanol extract and its fractions against H2O2-induced DNA damage. Total phenolic and flavonoid contents in each S. lindbergii extract have been expressed as tannic acid equivalent (TAE) and quercetin equivalents (QE), respectively. Dichloromethane extract contained higher flavonoid content (82.32 QE/g dried extract) while for the total phenolic content, methanol extract contained the highest amount (80.64 TAE/g dried extract) (Fazly-Bazzaz et al., 2013). A number of studies have focussed on the biological activities of phenolic compounds, which are potential antioxidants and free radical-scavengers for example, it has been reported a high correlation between DPPH (2, 2-diphenyl- 1-picrylhydrazyl) radical-scavenging activities and total polyphenolics (Kumar et al., 2008). In a study, using three different methods for testing antioxidant activity of oregano essential oil confirmed that the essential oil possess remarkable antioxidant properties which is due to the presence of phenolic monoterpenes: thymol and carvacrol (Kulisic et al., 2004). Three major polyphenolic components, namely wogonin, baicalin, and baicalein from S. baicalensis have been demonstrated as free radical scavengers of hydroxyl radical, DPPH radical and alkyl radical (Gao et al., 1999). Flavonoids are polyphenolic compounds which represent a large family of molecules including the flavones, flavonols, flavavones, anthocyanidins, and catechins. Flavonoids which share a basic structure with vitamin E are potent scavengers of free radicals such as hydroxyl and superoxide radicals, and also act as chelators of transient elements (Gate et al., 1999). Many flavonoids, such as quercetin, luteolin and catechins, are better antioxidants than the antioxidant nutrients vitamin C, vitamin E and L-carotene on a mole for mole basis (Gao et al., 1999). Therefore, it was speculated that genoprotective effect of S. lindbergii extracts is related to its flavonoids and phenolic compounds. It was found that the methanol extract and dichloromethane fraction significantly had the greater genoprotective activity compared with the defatted fraction. Since the methanol extract and dichloromethane fraction contained higher flavonoid and total phenolic contents, the difference in the genoprotective activity of extracts may be described by the difference in the total phenolic and flavonoid contents. Antioxidants can decrease the oxidative damage directly via reacting with free radicals or indirectly by inhibiting the activity or expression of free radical generating enzymes or enhancing the activity or expression of intracellular antioxidant enzymes (Lu et al., 2010). In a study antioxidant activities of S. linbergii Extracts were reported Using the thiobarbituric acid reactive species (TBARS) and DPPH radical scavenging assays (Fazly-Bazzaz et al., 2013). With respect to this report, genoprotective activities of S. linbergii extracts in simultaneous treatment of cells with extracts and H2O2 and also high amounts of free radical scavengers in S. lindbergii extracts such as flavonoids and phenolic compounds, radical scavenging can be suggested as the mechanism. However, the precise mechanism of the inhibition effects of the extracts against oxidative DNA damage needs further investigation. As a conclusion, this study confirms that S. linbergii root extract has protective effects against hydrogen peroxide-induced oxidative DNA damage.
Drug Chem Toxicol, 2015; 38(3): 293–299
Thus S. linbergii might have potential for the prevention and treatment of diseases or conditions resulting from oxidative stress such as cancer and aging.
Acknowledgements The authors are indebted to the Research Council of Mashhad University of Medical Sciences, Iran for approval and financial support of this project. Also this research work was part of Pharm. D. thesis of A. Eshaghi.
Declaration of interest The authors report no declarations of interest.
References Aherne SA, Kerry JP, O’Brien NM. (2007). Effects of plant extracts on antioxidant status and oxidant-induced stress in Caco-2 cells. Br J Nutr 97:321–328. Attar F, Joharchi MR. (2002). New plant records from Iran. Iran J Bot 9:223–228. Bozin B, Mimica-Dukic N, Simin N, Anackov G. (2006). Characterization of the volatile composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils. J Agric Food Chem 54:1822–1828. Chan FL, Choi HL, Chen ZY, et al. (2000). Induction of apoptosis in prostate cancer cell lines by a flavonoid, baicalin. Cancer Lett 160:219–228. Chen X, Nishida H, Konishi T. (2003). Baicalin promoted the repair of DNA single strand breakage caused by H2O2 in cultured NIH3T3 fibroblasts. Biol Pharm Bull 26:282–284. Chin YW, Balunas MJ, Chai HB, Kinghorn AD. (2006). Drug discovery from natural sources. AAPS J 8:E239–E253. Cole MD, Bridge PD, Dellar JE, et al. (1991). Antifungal activity of Neoclerodane diterpenoids from Scutellaria. Phytochemistry 30:1125–1127. Craig WJ. (1999). Health-promoting properties of common herbs. Am J Clin Nutr 70:491S–499S. Demirci M, Hiller KA, Bosl C, et al. (2008). The induction of oxidative stress, cytotoxicity, and genotoxicity by dental adhesives. Dent Mater 24:362–371. Eloff JN. (1998). Which extractant should be used for the screening and isolation of antimicrobial components from plants? J Ethnopharmacol 60:1–8 Emami SA, Aghazari F. (2011). Les Phanerogames Endemiques de la Flore d’Iran. Publications de I’Universite´ de Te´he´ran des Sciences Me´dicales. Fazly-Bazzaz BS, Atefeh Arab A, Emami SA, et al. (2013). Antimicrobial and antioxidant activities of methanol, dichloromethane and ethyl acetate extracts of Scutellaria lindbergii Rech.f. Chiang Mai J Sci 40:49–59. Gao Z, Huang K, Yang X, Xu H. (1999). Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi. Biochim Biophys Acta 1472:643–650. Gate L, Paul J, Ba GN, et al. (1999). Oxidative stress induced in pathologies: the role of antioxidants. Biomed Pharmacother 53:169–180. Glei M, Pool-Zobel BL. (2006). The main catechin of green tea, ()-epigallocatechin-3-gallate (EGCG), reduces bleomycin-induced DNA damage in human leucocytes. Toxicol In Vitro 20:295–300. Hahm DH, Yeom MJ, Lee EH, et al. (2001). Effect of Scutellariae Radix as a novel antibacterial herb on the ppk(polyphosphate kinase) mutant of Salmonella typhimurium. J Microbiol Biotechnol 11:1061–1065. Jamzad Z. (2012). Lamiaceae. In: Flora of Iran. Tehran: Research Institute of Forests & Rangelands. Kulisic T, Radonic A, Katalinic V, Milos M. (2004). Use of different methods for testing antioxidative activity of oregano essential oil. Food Chem 85:633–640. Kumar KS, Ganesan K, Rao PVS. (2008). Antioxidant potential of solvent extracts of Kappaphycus alvarezii (Doty) Doty – an edible seaweed. Food Chem 107:289–295.
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Victoria on 08/14/15 For personal use only.
DOI: 10.3109/01480545.2014.954047
Lam W, Bussom S, Guan F, et al. (2010). The four-herb Chinese medicine PHY906 reduces chemotherapy-induced gastrointestinal toxicity. Sci Transl Med 2:45ra59. Lee WR, Shen SC, Lin HY, et al. (2002). Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species, and activation of caspase 3 and Ca (2+)-dependent endonuclease. Biochem Pharmacol 63:225–236. Leite SP, Vieira JRC, De Medeiros PL, et al. (2006). Antimicrobial activity of Indigofera suffruticosa. Evid Based Complement Alternat Med 3:261–265. Li BQ, Fu T, Gong W-H, et al. (2000). The flavonoid baicalin exhibits anti-inflammatory activity by binding to chemokines. Immunopharmacology 49:295–306. Lu JM, Lin PH, Yao Q, Chen C. (2010). Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems. J Cell Mol Med 14:840–860. Mabberley DJ. (1993). The plant-book. Cambridge University Press. Markham KR. (1982). Techniques of flavonoid identification. London: Academic Press. Middleton Jr E, Kandaswami C, Theoharides TC. (2000). The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 52:673–751. Mosmann T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63. Nagai T, Miyaichi Y, Tomimori T, et al. (1990). Inhibition of influenza virus sialidase and anti-influenza virus activity by plant flavonoids. Chem Pharm Bull (Tokyo) 38:1329–1332. Nagai T, Moriguchi R, Suzuki Y, et al. (1995). Mode of action of the anti-influenza virus activity of plant flavonoid, 5,7,40 -trihydroxy-8methoxyflavone, from the roots of Scutellaria baicalensis. Antiviral Res 26:11–25. Plazar J, Filipic M, Groothuis GM. (2008). Antigenotoxic effect of Xanthohumol in rat liver slices. Toxicol In Vitro 22:318–327. Powell CB, Fung P, Jackson J, et al. (2003). Aqueous extract of herba Scutellaria barbatae, a chinese herb used for ovarian cancer, induces apoptosis of ovarian cancer cell lines. Gynecol Oncol 91:332–340. Rojas E, Lopez MC, Valverde M. (1999). Single cell gel electrophoresis assay: methodology and applications. J Chromatogr B Biomed Sci Appl 722:225–254. Saha K, Lajis NH, Israf DA, et al. (2004). Evaluation of antioxidant and nitric oxide inhibitory activities of selected Malaysian medicinal plants. J Ethnopharmacol 92:263–267. Sato Y, Suzaki S, Nishikawa T, et al. (2000). Phytochemical flavones isolated from Scutellaria barbata and antibacterial activity against
S. lindbergii against cell and DNA damage
299
methicillin-resistant Staphylococcus aureus. J Ethnopharmacol 72:483–488. Shao Z-H, Li C-Q, Vanden Hoek TL, et al. (1999). Extract from Scutellaria baicalensis Georgi attenuates oxidant stress in cardiomyocytes. J Mol Cell Cardiol 31:1885–1895. Singh NP, McCoy MT, Tice RR, Schneider EL. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191. Skaltsaa HD, Lazarib DM, Kyriazopoulosc P, et al. (2005). Composition and antimicrobial activity of the essential oils of Scutellaria sieberia Benth. and Scutellaria rupestris Boiss. et Heldr. ssp. adenotricha (Boiss. et Heldr.) Greuter et Burdet from Greece. Essent Oil Res 17:232–235. Slamenova D, Kuboskova K, Horvathova E, Robichova S. (2002). Rosemary-stimulated reduction of DNA strand breaks and FPGsensitive sites in mammalian cells treated with H2O2 or visible lightexcited methylene blue. Cancer Lett 177:145–153. Soltani F, Mosaffa F, Iranshahi M, et al. (2009). Auraptene from Ferula szowitsiana protects human peripheral lymphocytes against oxidative stress. Phytother Res 24:85–89. Sonoda M, Nishiyama T, Matsukawa Y, Moriyasu M. (2004). Cytotoxic activities of flavonoids from two Scutellaria plants in Chinese medicine. J Ethnopharmacol 91:65–68. Starcevic SL, Diotte NM, Zukowski KL, et al. (2003). Oxidative DNA damage and repair in a cell lineage model of human proliferative breast disease (PBD). Toxicol Sci 75:74–81. Tayarani-Najaran Z, Asili J, Parsaee H, et al. (2012). Wogonin and neobaicalein from Scutellaria litwinowii roots are apoptotic for HeLa cells. Rev Bras Farmacogn 22:268–276. Tayarani-Najaran Z, Mousavi SH, Asili J, Emami SA. (2010). Growthinhibitory effect of Scutellaria lindbergii in human cancer cell lines. Food Chem Toxicol 48:599–604. Tice RR, Agurell E, Anderson D, et al. (2000). Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221. Van Loon IM. (1997). The golden root: clinical applications of Scutellaria baicalensis Georgi flavonoids as modulators of the inflammatory response. Altern Med Rev 2:472–480. Velasco V, Williams P. (2011). Improving meat quality through natural antioxidants. Chilean J Agric Res 71:313–322. Yin X, Zhou J, Jie C, et al. (2004). Anticancer activity and mechanism of Scutellaria barbata extract on human lung cancer cell line A549. Life Sci 75:2233–2244. Zuo F, Zhou ZM, Yan MZ, et al. (2002). Metabolism of constituents in Huangqin-Tang, a prescription in traditional Chinese medicine, by human intestinal flora. Biol Pharm Bull 25:558–563.
