http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–8 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1035795
ORIGINAL ARTICLE
Protective effect of Lepidium sativum seed extract against hydrogen peroxide-induced cytotoxicity and oxidative stress in human liver cells (HepG2) Ebtesam S. Al-Sheddi1, Nida N. Farshori1, Mai M. Al-Oqail1, Javed Musarrat2,3, Abdulaziz A. Al-Khedhairy2,3, and Maqsood A. Siddiqui2,3
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1
Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, 2Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia, and 3Al-Jeraisy Chair for DNA Research, King Saud University, Riyadh, Saudi Arabia
Abstract
Keywords
Context: Garden cress [Lepidium sativum (Brassicaceae)] has been widely used to treat a number of ailments in traditional medicine. The pharmacological and preventive potential of Lepidium sativum, such as anti-inflammatory, antipyretic, antihypertensive, anti-ashthamatic, anticancer, and anti-oxidant, are well known. Objective: The present investigation was designed to study the protective effects of chloroform extract of Lepidium sativum seed (LSE) against oxidative stress and cytotoxicity induced by hydrogen peroxide (H2O2) in human liver cells (HepG2). Materials and methods: Cytotoxicity of LSE and H2O2 was identified by (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), neutral red uptake (NRU) assays, and morphological changes in HepG2. The cells were pre-exposed to biologically safe concentrations (5–25 mg/ml) of LSE for 24 h, and then cytotoxic (0.25 mM) concentration of H2O2 was added. After 24 h of the exposures, cell viability by MTT, NRU assays, and morphological changes in HepG2 were evaluated. Further, protective effects of LSE on reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), and reduced glutathione (GSH) levels induced by H2O2 were studied. Results: Pre-exposure of LSE significantly attenuated the loss of cell viability up to 48% at 25 mg/ ml concentration against H2O2 (LD50 value ¼ 2.5 mM). Results also showed that LSE at 25 mg/ml concentration significantly inhibited the induction of ROS generation (45%) and LPO (56%), and increases the MMP (55%) and GSH levels (46%). Discussion and conclusion: The study suggests the cytoprotective effects of LSE against H2O2induced toxicity in HepG2. The results also demonstrate the anti-oxidative nature of LSE.
Glutathione, lipid peroxidation, MMP, morphological changes, ROS generation
Introduction Oxidative stress plays a significant role in the etiology of variety of human diseases (Dhalla et al., 2000; Rahman, 2005). The role of oxidative stress in liver cells induced by various toxins is also well known (Rodeiro et al., 2008). Reports showed that overproduction of reactive oxygen species (ROS) plays a major role in the hepatocarcinoma (Lima et al., 2006; Zhang et al., 2011), which leads to cellular damage (Lin et al., 2007; Zhang et al., 2012). A number of in vitro studies have demonstrated that oxidative stress induced by chemical oxidants, such as hydrogen peroxide (H2O2), leads to cell death (Cai et al., 2008; Hwang & Yen, 2008; Kim et al., 2009; Siddiqui et al., 2011). H2O2 has also been reported to induce apoptotic changes, which subsequently lead to death in a Correspondence: Dr. Maqsood A. Siddiqui, Assistant Professor, Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia. Tel: +966 542967835. E-mail: maqsoodahmads @gmail.com
History Received 4 June 2014 Revised 8 February 2015 Accepted 23 March 2015 Published online 17 April 2015
variety of cell systems (Jung et al., 2006; Kanno et al., 2003; Sattayasai et al., 2013), including human liver cells (HepG2) (Alia et al., 2005; Chen et al., 2011). Therefore, we have selected H2O2 to induce the oxidative stress-mediated cytotoxicity in the HepG2. Garden cress [Lepidium sativum (Brassicaceae)] has been widely used to treat a number of ailments in traditional system of medicine. Lepidium sativum is a fast-growing edible herb belonging to the family Brassicaceae (Cruciferae) or mustard family, and is being cultivated as culinary vegetable in North America, Europe, and all over Asia including India (Al-Sheddi et al., 2013; Nadkarni, 1976). The pharmacological and preventive properties of L. sativum, such as antioxidant (Yadav et al., 2010), anti-inflammatory, anticoagulant (Al-Yahya et al., 1994), antidiabetic (Eddoaks et al, 2005), antidiarrheal (Manohar et al., 2009), antihypertensive, diuretic (Mohamed et al., 2003; Umang et al., 2009), antirheumatic (Ahsan et al., 1989), anti-asthmatic (Paranjape & Mehta, 2006), chemoprotective (Fekadu et al,
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2
E. S. Al-Sheddi et al.
2002), hypercholesterolemic (Al Hamedan, 2010), laxative (Najeeb et al., 2011), and fracture healing (Abdullah & Abdullah, 2007) are well documented. The seeds of L. sativum are also known to be useful in leprosy, skin, dysentery, diarrhea, splenomegal, and asthma diseases (Kirtikar & Basu, 2006). The phytoconstituent of L. sativum include carbohydrate, protein, fatty acid, riboflavin, niacin, flavonoids, isothiocynates glycoside, essential aromatic oils, and fatty oils (Al-Sheddi et al., 2013). In view of the significant lack of the mechanism(s) defining the protective effects of L. sativum on oxidative stress and ROS generation in HepG2, the present study was designed to develop a model system to investigate the protective effects of chloroform extract of L. sativum seed (LSE) against H2O2induced cytotoxicity in HepG2. The HepG2 are involved in the metabolism (Kim et al., 2011), and compose a good model to assess the toxicity or detoxification of various compounds against oxidative stress inducers (Farshori et al., 2013; Lima et al., 2006; Zhang et al., 2012).
Materials and methods Chemicals and consumables DMEM culture medium, antibiotics-atimycotic solution, fetal bovine serum (FBS), and trypsin were purchased from Invitrogen, Life Sciences, Grand Island, NY. Consumables and culture wares used in the study were procured from Nunc, Roskilde, Denmark. H2O2 and all other specified chemicals and reagents were purchased from Sigma Chemical Company Pvt. Ltd. St. Louis, MO. Preparation of extract The L. sativum seeds used in this study were obtained from the local market of Riyadh, Saudi Arabia in October 2013. The seeds were identified by Dr. Mohammad Atiqur Rahman, taxonomist of Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University, Saudi Arabia and a specimen (#15967) was submitted in the herbarium of the King Saud University. The seeds were screened manually to remove bad ones. They were then dried to constant weight in an oven at 70 C, ground using mechanical grinder, put in air-tight containers, and stored in a desiccator. For the preparation of chloroform extract, the seeds were macerated in chloroform and then filtered. The procedure was repeated several times. The solvent was then evaporated using a rotary evaporator and the residue so obtained was formed as the chloroform extract.
Pharm Biol, Early Online: 1–8
Experimental design To determine the biologically safe concentrations of LSE, HepG2 were exposed to 5–500 lg/ml of LSE and to determine the LD50 value of H2O2, the cells were exposed to 0.01–2 mM of H2O2 for 24 h. Further, to evaluate the protective effects of LSE on HepG2, the cells were pre-exposed to biologically safe concentrations (5, 10, and 25 mg/ml) of LSE. Then H2O2 (0.25 mM) was added and cells were incubated at 37 C for 24 h. After the exposures, percentage cell viability, morphological changes, ROS generation, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), and reduced glutathione (GSH) were evaluated. Drug solutions The LSE was not completely soluble in culture medium; therefore, the stock solutions of extract were prepared in dimethylsulphoxide (DMSO) and diluted in culture medium to reach the desired concentrations. The concentration of DMSO in culture medium was not more than 0.1% and this medium was used as control. Hydrogen peroxide (H2O2) was freshly diluted in culture medium before adding to the cells. Cytotoxicity screening of LSE extract and H2O2 The biologically safe or non-cytotoxic concentration of LSE and cytotoxicity of H2O2 was evaluated using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), neutral red uptake (NRU), and cellular morphology assays. For the cytotoxicity assessment, HepG2 were exposed to various concentrations (5–500 mg/ml) of LSE and H2O2 (0.01–2 mM) for 24 h. The cellular morphological analysis was performed by phase contrast-inverted microscope. MTT assay Percentage cell viability was assessed using the MTT assay as described (Siddiqui et al., 2008). Briefly, HepG2 (1 104) were allowed to adhere for 24 h CO2 incubator at 37 C in 96well culture plates. After 24 h exposure of HepG2 with increasing concentrations (5–500 mg/ml) of LSE and H2O2 (0.01–2 mM) for 24 h, MTT (5 mg/ml of stock in PBS) was added (10 ll/well in 100 ll of cell suspension), and plates were incubated for 4 h. The supernatant was discarded and 200 ml of DMSO was added to each well and mixed gently. The developed color was read at 550 nm using multiwell microplate reader (Thermo Scientific, Waltham, MA). Untreated sets were also run under identical conditions and served as control.
Cell culture
NRU assay
HepG2, sourced from the American Type Culture Collection (Accession HB-8065, Rockville, MD) were cultured in DMEM, supplemented with 10% fetal bovine serum, 0.2% sodium bicarbonate, and antibiotic/antimycotic solution (100, 1 ml/100 ml of medium). Cells were grown in 5% CO2 at 37 C in high humid atmosphere. Before the experiments, the viability of cells was assessed following the protocol of Siddiqui et al. (2008). HepG2 showing more than 98% cell viability and passage number between 20 and 22 were used in this study.
NRU assay was carried out following the protocol described by Siddiqui et al. (2010). Briefly, after 24 h exposure of HepG2 with increasing concentrations (5–500 mg/ml) of LSE and H2O2 (0.01–2 mM) for 24 h, the medium was aspirated and cells were washed twice with PBS, and incubated for 3 h in a medium supplemented with neutral red (50 lg/ml). The medium was washed off rapidly with a solution containing 0.5% formaldehyde and 1% calcium chloride. Cells were subjected to further incubation of 20 min at 37 C in a mixture of acetic acid (1%) and ethanol (50%) to extract the dye.
Effect of Lepidium sativum extract against H2O2
DOI: 10.3109/13880209.2015.1035795
The plates were read at 540 nm using multiwell microplate reader (Thermo Scientific, Waltham, MA). The values were compared with the control sets run under identical conditions. Morphological analysis Morphological changes in HepG2 exposed to increasing concentrations of LSE (5–500 mg/ml) and H2O2 (0.01–2 mM) for 24 h were observed by phase contrast inverted microscope (Olympus, Center Valley, PA) attached with automatic image analysis software. Further, to observe the protective effects of LSE on cellular morphology, HepG2 were treated with biologically safe concentrations (5, 10, and 25 mg/ml) of LSE before exposure to cytotoxic concentration of H2O2 (0.25 mM) for 24 h.
3
(0.4 M Tris and 0.02 M EDTA; pH 8.9) and 0.01 M of 5,50 -dithionitrobenzoic acid (DTNB) to reach a final volume of 3 ml. The tubes were incubated for 10 min at 37 C in a shaking water bath. The absorbance of yellow color developed was read at 412 nm. Statistical analysis Results were expressed as mean ± SE of at least three independent experiments (each in triplicate). Statistical analysis was performed using one-way ANOVA using Dunnett’s post hoc test employed to compare the values between control and treated groups. The values showing p50.05 were considered as statistically significant.
Results The production of intracellular ROS was measured using 2,7-dichlorofluorescin diacetate (DCFH-DA) as described by Wang and Joseph (1999) with slight modifications. In brief, after the exposure of HepG2 with LSE and H2O2, the cells were washed twice with PBS. The cells were then exposed to 20 mM of DCFH-DA fluorescent dye for 1 h at 37 C in dark. The fluorescence intensity of specific dye was measured at 485 nm excitation and 530 nm emission wavelengths using spectrofluorometer (Fluoroskan, Thermo Scientific, Waltham, MA).
Cytotoxicity assessments of LSE and H2O2 The cytotoxicity of LSE was assessed using MTT assay, NRU assay, and cellular morphology, after exposing the HepG2 to various concentrations of LSE and H2O2. Our results showed that LSE at 5, 10, and 25 mg/ml concentrations did not cause any significant effect on cell viability of HepG2 (Figures 1(A, B) and 2). Therefore, the concentrations 5, 10, and 25 mg/ml of LSE were chosen to study the protective effects against H2O2-induced toxicity in HepG2. Further, based on the LD50 value obtained, 0.25 mM of H2O2 was used to induce the toxicity in further experiments (Figure 3A and B).
MMP was measured following the protocol of Zhang et al. (2011). In brief, control and treated cells were washed twice with PBS. The cells were further treated with 10 mg/ml of Rhodamine-123 fluorescent dye for 1 h at 37 C in dark. The fluorescence intensity of Rhodamine-123 was measured at 485 nm excitation and 530 nm emission wavelengths using spectrofluorometer. Lipid peroxidation LPO was performed using thiobarbituric acid-reactive substances (TBARS) protocol (Buege & Aust, 1978). Briefly, after the exposure of HepG2 with LSE and H2O2, HepG2 were collected by centrifugation and sonicated in ice cold potassium chloride (1.15%) and centrifuged again for 10 min at 3000 g. The resulting supernatant (1 ml) was collected and 2 ml of thiobarbituric acid (TBA) reagent (15% TCA, 0.7% TBA, and 0.25N HCl) was added. The solution was heated at a temperature of 100 C for 15 min in a boiling bath. The sample was then placed at a cold temperature and centrifuged at 1000 g for 10 min. The absorbance of the supernatant was measured at 535 nm. GSH level Intracellular reduced GSH level was estimated as described by Chandra et al. (2002) with some modifications. Briefly, HepG2 exposed to LSE and H2O2 were collected by centrifugation and the cellular proteins were precipitated by incubating 1 ml sonicated cell suspension with 10% TCA (1 ml) on ice for 1 h followed by centrifugation at 3000 rpm for 10 min. The supernatant was then added to 2 ml buffer
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Figure 1. Cytotoxicity assessment by (A) MTT assay and (B) NRU assay in HepG2 following the exposure of various concentrations of LSE for 24 h. Values are mean ± SE of three independent experiments. *p50.05, **p50.01 versus control.
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Pharm Biol, Early Online: 1–8
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An increase of 45% in the cell viability of HepG2 was recorded at 25 mg/ml of LSE (Figure 4A). A similar kind of concentration-dependent increase in the cell viability was also observed by NRU assay in LSE pre-exposed HepG2. An increase of 48% in cell viability of HepG2 was recorded at 25 mg/ml of LSE (Figure 4B).
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Morphological changes The alterations in the morphology of HepG2 on exposure of LSE and H2O2 are shown in Figure 5(A–E). Cells exposed to 0.25 mM of H2O2 reduced the normal morphology of HepG2, and cell adhesion capacity as compared with control. Most of the cells exposed to H2O2 lost their typical morphology and appeared smaller in size (Figure 5B). HepG2 exposed to increasing concentrations of LSE for 24 h prior to H2O2 exposure significantly restore their original morphology in a concentration-dependent manner (Figure 5C–E).
Concentrations (H2O2) Cell Viability (percent control)
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Figure 2. Alterations in the morphology of HepG2 exposed for 24 h. Various concentrations of LSE for 24 h. Image were taken using phase contrastinverted microscope at 20 magnification.
Protective effects of LSE on ROS generation induced by H2O2 **
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Concentrations (H2O2)
Figure 3. Cytotoxicity assessments by (A) MTT and (B) NRU assays in HepG2 following the exposure of various concentrations of hydrogen peroxide (H2O2) for 24 h. Values are mean ± SE of three independent experiments. *p50.05, **p50.01 versus control.
Protective effects of LSE against H2O2-induced cell death MTT and NRU assays The protective potential of LSE in HepG2 observed by MTT and NRU assays is presented in Figure 4(A) and (B). A significant (p50.01) reduction in percentage cell viability was observed in HepG2 following the exposure of H2O2 (0.25 mM) for 24 h by MTT assay (Figure 4A), and NRU assay (Figure 4B). HepG2 pre-treated with LSE at 5, 10, and 25 mg/ml for 24 h significantly attenuated the H2O2 induced loss of cell viability in a concentration-dependent manner.
The results of ROS generation in HepG2 exposed to H2O2 and various concentrations of LSE are presented in Figure 6(A). Exposure of HepG2 with H2O2 at 0.25 mM for 24 h resulted in an increase of 154 ± 5.6% of increase (p50.01) in the production of ROS. Pre-treatment of cells with LSE at 5, 10, and 25 mg/ml concentration significantly reduced the levels of H2O2-induced ROS production in HepG2 (Figure 6A). Protective effects of LSE on MMP The intensity of mitochondrial membrane potential (MMP) in HepG2 exposed to H2O2 and various concentrations of LSE is presented in Figure 6(B). Results showed that HepG2 exposed to H2O2 (0.25 mM) for 24 h resulted in 46 ± 2.6% reduction (p50.01) in the level of MMP. Pre-treatment of cells with LSE at 5, 10, and 25 mg/ml concentrations prior to H2O2 treatment significantly increased the intensity of MMP in HepG2 (Figure 6B). Protective effects of LSE on LPO Protective potential of various concentrations of LSE on H2O2 induced lipid peroxidation in HepG2 is summarized in Figure 6(C). As shown in figure, exposure of H2O2 resulted
Effect of Lepidium sativum extract against H2O2
DOI: 10.3109/13880209.2015.1035795
(B)
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ORIGINAL ARTICLE
Protective effect of Lepidium sativum seed extract against hydrogen peroxide-induced cytotoxicity and oxidative stress in human liver cells (HepG2) Ebtesam S. Al-Sheddi1, Nida N. Farshori1, Mai M. Al-Oqail1, Javed Musarrat2,3, Abdulaziz A. Al-Khedhairy2,3, and Maqsood A. Siddiqui2,3
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1
Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, 2Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia, and 3Al-Jeraisy Chair for DNA Research, King Saud University, Riyadh, Saudi Arabia
Abstract
Keywords
Context: Garden cress [Lepidium sativum (Brassicaceae)] has been widely used to treat a number of ailments in traditional medicine. The pharmacological and preventive potential of Lepidium sativum, such as anti-inflammatory, antipyretic, antihypertensive, anti-ashthamatic, anticancer, and anti-oxidant, are well known. Objective: The present investigation was designed to study the protective effects of chloroform extract of Lepidium sativum seed (LSE) against oxidative stress and cytotoxicity induced by hydrogen peroxide (H2O2) in human liver cells (HepG2). Materials and methods: Cytotoxicity of LSE and H2O2 was identified by (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), neutral red uptake (NRU) assays, and morphological changes in HepG2. The cells were pre-exposed to biologically safe concentrations (5–25 mg/ml) of LSE for 24 h, and then cytotoxic (0.25 mM) concentration of H2O2 was added. After 24 h of the exposures, cell viability by MTT, NRU assays, and morphological changes in HepG2 were evaluated. Further, protective effects of LSE on reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), and reduced glutathione (GSH) levels induced by H2O2 were studied. Results: Pre-exposure of LSE significantly attenuated the loss of cell viability up to 48% at 25 mg/ ml concentration against H2O2 (LD50 value ¼ 2.5 mM). Results also showed that LSE at 25 mg/ml concentration significantly inhibited the induction of ROS generation (45%) and LPO (56%), and increases the MMP (55%) and GSH levels (46%). Discussion and conclusion: The study suggests the cytoprotective effects of LSE against H2O2induced toxicity in HepG2. The results also demonstrate the anti-oxidative nature of LSE.
Glutathione, lipid peroxidation, MMP, morphological changes, ROS generation
Introduction Oxidative stress plays a significant role in the etiology of variety of human diseases (Dhalla et al., 2000; Rahman, 2005). The role of oxidative stress in liver cells induced by various toxins is also well known (Rodeiro et al., 2008). Reports showed that overproduction of reactive oxygen species (ROS) plays a major role in the hepatocarcinoma (Lima et al., 2006; Zhang et al., 2011), which leads to cellular damage (Lin et al., 2007; Zhang et al., 2012). A number of in vitro studies have demonstrated that oxidative stress induced by chemical oxidants, such as hydrogen peroxide (H2O2), leads to cell death (Cai et al., 2008; Hwang & Yen, 2008; Kim et al., 2009; Siddiqui et al., 2011). H2O2 has also been reported to induce apoptotic changes, which subsequently lead to death in a Correspondence: Dr. Maqsood A. Siddiqui, Assistant Professor, Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia. Tel: +966 542967835. E-mail: maqsoodahmads @gmail.com
History Received 4 June 2014 Revised 8 February 2015 Accepted 23 March 2015 Published online 17 April 2015
variety of cell systems (Jung et al., 2006; Kanno et al., 2003; Sattayasai et al., 2013), including human liver cells (HepG2) (Alia et al., 2005; Chen et al., 2011). Therefore, we have selected H2O2 to induce the oxidative stress-mediated cytotoxicity in the HepG2. Garden cress [Lepidium sativum (Brassicaceae)] has been widely used to treat a number of ailments in traditional system of medicine. Lepidium sativum is a fast-growing edible herb belonging to the family Brassicaceae (Cruciferae) or mustard family, and is being cultivated as culinary vegetable in North America, Europe, and all over Asia including India (Al-Sheddi et al., 2013; Nadkarni, 1976). The pharmacological and preventive properties of L. sativum, such as antioxidant (Yadav et al., 2010), anti-inflammatory, anticoagulant (Al-Yahya et al., 1994), antidiabetic (Eddoaks et al, 2005), antidiarrheal (Manohar et al., 2009), antihypertensive, diuretic (Mohamed et al., 2003; Umang et al., 2009), antirheumatic (Ahsan et al., 1989), anti-asthmatic (Paranjape & Mehta, 2006), chemoprotective (Fekadu et al,
Pharmaceutical Biology Downloaded from informahealthcare.com by Gazi Univ. on 05/03/15 For personal use only.
2
E. S. Al-Sheddi et al.
2002), hypercholesterolemic (Al Hamedan, 2010), laxative (Najeeb et al., 2011), and fracture healing (Abdullah & Abdullah, 2007) are well documented. The seeds of L. sativum are also known to be useful in leprosy, skin, dysentery, diarrhea, splenomegal, and asthma diseases (Kirtikar & Basu, 2006). The phytoconstituent of L. sativum include carbohydrate, protein, fatty acid, riboflavin, niacin, flavonoids, isothiocynates glycoside, essential aromatic oils, and fatty oils (Al-Sheddi et al., 2013). In view of the significant lack of the mechanism(s) defining the protective effects of L. sativum on oxidative stress and ROS generation in HepG2, the present study was designed to develop a model system to investigate the protective effects of chloroform extract of L. sativum seed (LSE) against H2O2induced cytotoxicity in HepG2. The HepG2 are involved in the metabolism (Kim et al., 2011), and compose a good model to assess the toxicity or detoxification of various compounds against oxidative stress inducers (Farshori et al., 2013; Lima et al., 2006; Zhang et al., 2012).
Materials and methods Chemicals and consumables DMEM culture medium, antibiotics-atimycotic solution, fetal bovine serum (FBS), and trypsin were purchased from Invitrogen, Life Sciences, Grand Island, NY. Consumables and culture wares used in the study were procured from Nunc, Roskilde, Denmark. H2O2 and all other specified chemicals and reagents were purchased from Sigma Chemical Company Pvt. Ltd. St. Louis, MO. Preparation of extract The L. sativum seeds used in this study were obtained from the local market of Riyadh, Saudi Arabia in October 2013. The seeds were identified by Dr. Mohammad Atiqur Rahman, taxonomist of Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University, Saudi Arabia and a specimen (#15967) was submitted in the herbarium of the King Saud University. The seeds were screened manually to remove bad ones. They were then dried to constant weight in an oven at 70 C, ground using mechanical grinder, put in air-tight containers, and stored in a desiccator. For the preparation of chloroform extract, the seeds were macerated in chloroform and then filtered. The procedure was repeated several times. The solvent was then evaporated using a rotary evaporator and the residue so obtained was formed as the chloroform extract.
Pharm Biol, Early Online: 1–8
Experimental design To determine the biologically safe concentrations of LSE, HepG2 were exposed to 5–500 lg/ml of LSE and to determine the LD50 value of H2O2, the cells were exposed to 0.01–2 mM of H2O2 for 24 h. Further, to evaluate the protective effects of LSE on HepG2, the cells were pre-exposed to biologically safe concentrations (5, 10, and 25 mg/ml) of LSE. Then H2O2 (0.25 mM) was added and cells were incubated at 37 C for 24 h. After the exposures, percentage cell viability, morphological changes, ROS generation, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), and reduced glutathione (GSH) were evaluated. Drug solutions The LSE was not completely soluble in culture medium; therefore, the stock solutions of extract were prepared in dimethylsulphoxide (DMSO) and diluted in culture medium to reach the desired concentrations. The concentration of DMSO in culture medium was not more than 0.1% and this medium was used as control. Hydrogen peroxide (H2O2) was freshly diluted in culture medium before adding to the cells. Cytotoxicity screening of LSE extract and H2O2 The biologically safe or non-cytotoxic concentration of LSE and cytotoxicity of H2O2 was evaluated using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), neutral red uptake (NRU), and cellular morphology assays. For the cytotoxicity assessment, HepG2 were exposed to various concentrations (5–500 mg/ml) of LSE and H2O2 (0.01–2 mM) for 24 h. The cellular morphological analysis was performed by phase contrast-inverted microscope. MTT assay Percentage cell viability was assessed using the MTT assay as described (Siddiqui et al., 2008). Briefly, HepG2 (1 104) were allowed to adhere for 24 h CO2 incubator at 37 C in 96well culture plates. After 24 h exposure of HepG2 with increasing concentrations (5–500 mg/ml) of LSE and H2O2 (0.01–2 mM) for 24 h, MTT (5 mg/ml of stock in PBS) was added (10 ll/well in 100 ll of cell suspension), and plates were incubated for 4 h. The supernatant was discarded and 200 ml of DMSO was added to each well and mixed gently. The developed color was read at 550 nm using multiwell microplate reader (Thermo Scientific, Waltham, MA). Untreated sets were also run under identical conditions and served as control.
Cell culture
NRU assay
HepG2, sourced from the American Type Culture Collection (Accession HB-8065, Rockville, MD) were cultured in DMEM, supplemented with 10% fetal bovine serum, 0.2% sodium bicarbonate, and antibiotic/antimycotic solution (100, 1 ml/100 ml of medium). Cells were grown in 5% CO2 at 37 C in high humid atmosphere. Before the experiments, the viability of cells was assessed following the protocol of Siddiqui et al. (2008). HepG2 showing more than 98% cell viability and passage number between 20 and 22 were used in this study.
NRU assay was carried out following the protocol described by Siddiqui et al. (2010). Briefly, after 24 h exposure of HepG2 with increasing concentrations (5–500 mg/ml) of LSE and H2O2 (0.01–2 mM) for 24 h, the medium was aspirated and cells were washed twice with PBS, and incubated for 3 h in a medium supplemented with neutral red (50 lg/ml). The medium was washed off rapidly with a solution containing 0.5% formaldehyde and 1% calcium chloride. Cells were subjected to further incubation of 20 min at 37 C in a mixture of acetic acid (1%) and ethanol (50%) to extract the dye.
Effect of Lepidium sativum extract against H2O2
DOI: 10.3109/13880209.2015.1035795
The plates were read at 540 nm using multiwell microplate reader (Thermo Scientific, Waltham, MA). The values were compared with the control sets run under identical conditions. Morphological analysis Morphological changes in HepG2 exposed to increasing concentrations of LSE (5–500 mg/ml) and H2O2 (0.01–2 mM) for 24 h were observed by phase contrast inverted microscope (Olympus, Center Valley, PA) attached with automatic image analysis software. Further, to observe the protective effects of LSE on cellular morphology, HepG2 were treated with biologically safe concentrations (5, 10, and 25 mg/ml) of LSE before exposure to cytotoxic concentration of H2O2 (0.25 mM) for 24 h.
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(0.4 M Tris and 0.02 M EDTA; pH 8.9) and 0.01 M of 5,50 -dithionitrobenzoic acid (DTNB) to reach a final volume of 3 ml. The tubes were incubated for 10 min at 37 C in a shaking water bath. The absorbance of yellow color developed was read at 412 nm. Statistical analysis Results were expressed as mean ± SE of at least three independent experiments (each in triplicate). Statistical analysis was performed using one-way ANOVA using Dunnett’s post hoc test employed to compare the values between control and treated groups. The values showing p50.05 were considered as statistically significant.
Results The production of intracellular ROS was measured using 2,7-dichlorofluorescin diacetate (DCFH-DA) as described by Wang and Joseph (1999) with slight modifications. In brief, after the exposure of HepG2 with LSE and H2O2, the cells were washed twice with PBS. The cells were then exposed to 20 mM of DCFH-DA fluorescent dye for 1 h at 37 C in dark. The fluorescence intensity of specific dye was measured at 485 nm excitation and 530 nm emission wavelengths using spectrofluorometer (Fluoroskan, Thermo Scientific, Waltham, MA).
Cytotoxicity assessments of LSE and H2O2 The cytotoxicity of LSE was assessed using MTT assay, NRU assay, and cellular morphology, after exposing the HepG2 to various concentrations of LSE and H2O2. Our results showed that LSE at 5, 10, and 25 mg/ml concentrations did not cause any significant effect on cell viability of HepG2 (Figures 1(A, B) and 2). Therefore, the concentrations 5, 10, and 25 mg/ml of LSE were chosen to study the protective effects against H2O2-induced toxicity in HepG2. Further, based on the LD50 value obtained, 0.25 mM of H2O2 was used to induce the toxicity in further experiments (Figure 3A and B).
MMP was measured following the protocol of Zhang et al. (2011). In brief, control and treated cells were washed twice with PBS. The cells were further treated with 10 mg/ml of Rhodamine-123 fluorescent dye for 1 h at 37 C in dark. The fluorescence intensity of Rhodamine-123 was measured at 485 nm excitation and 530 nm emission wavelengths using spectrofluorometer. Lipid peroxidation LPO was performed using thiobarbituric acid-reactive substances (TBARS) protocol (Buege & Aust, 1978). Briefly, after the exposure of HepG2 with LSE and H2O2, HepG2 were collected by centrifugation and sonicated in ice cold potassium chloride (1.15%) and centrifuged again for 10 min at 3000 g. The resulting supernatant (1 ml) was collected and 2 ml of thiobarbituric acid (TBA) reagent (15% TCA, 0.7% TBA, and 0.25N HCl) was added. The solution was heated at a temperature of 100 C for 15 min in a boiling bath. The sample was then placed at a cold temperature and centrifuged at 1000 g for 10 min. The absorbance of the supernatant was measured at 535 nm. GSH level Intracellular reduced GSH level was estimated as described by Chandra et al. (2002) with some modifications. Briefly, HepG2 exposed to LSE and H2O2 were collected by centrifugation and the cellular proteins were precipitated by incubating 1 ml sonicated cell suspension with 10% TCA (1 ml) on ice for 1 h followed by centrifugation at 3000 rpm for 10 min. The supernatant was then added to 2 ml buffer
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Figure 1. Cytotoxicity assessment by (A) MTT assay and (B) NRU assay in HepG2 following the exposure of various concentrations of LSE for 24 h. Values are mean ± SE of three independent experiments. *p50.05, **p50.01 versus control.
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An increase of 45% in the cell viability of HepG2 was recorded at 25 mg/ml of LSE (Figure 4A). A similar kind of concentration-dependent increase in the cell viability was also observed by NRU assay in LSE pre-exposed HepG2. An increase of 48% in cell viability of HepG2 was recorded at 25 mg/ml of LSE (Figure 4B).
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Morphological changes The alterations in the morphology of HepG2 on exposure of LSE and H2O2 are shown in Figure 5(A–E). Cells exposed to 0.25 mM of H2O2 reduced the normal morphology of HepG2, and cell adhesion capacity as compared with control. Most of the cells exposed to H2O2 lost their typical morphology and appeared smaller in size (Figure 5B). HepG2 exposed to increasing concentrations of LSE for 24 h prior to H2O2 exposure significantly restore their original morphology in a concentration-dependent manner (Figure 5C–E).
Concentrations (H2O2) Cell Viability (percent control)
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Figure 2. Alterations in the morphology of HepG2 exposed for 24 h. Various concentrations of LSE for 24 h. Image were taken using phase contrastinverted microscope at 20 magnification.
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Figure 3. Cytotoxicity assessments by (A) MTT and (B) NRU assays in HepG2 following the exposure of various concentrations of hydrogen peroxide (H2O2) for 24 h. Values are mean ± SE of three independent experiments. *p50.05, **p50.01 versus control.
Protective effects of LSE against H2O2-induced cell death MTT and NRU assays The protective potential of LSE in HepG2 observed by MTT and NRU assays is presented in Figure 4(A) and (B). A significant (p50.01) reduction in percentage cell viability was observed in HepG2 following the exposure of H2O2 (0.25 mM) for 24 h by MTT assay (Figure 4A), and NRU assay (Figure 4B). HepG2 pre-treated with LSE at 5, 10, and 25 mg/ml for 24 h significantly attenuated the H2O2 induced loss of cell viability in a concentration-dependent manner.
The results of ROS generation in HepG2 exposed to H2O2 and various concentrations of LSE are presented in Figure 6(A). Exposure of HepG2 with H2O2 at 0.25 mM for 24 h resulted in an increase of 154 ± 5.6% of increase (p50.01) in the production of ROS. Pre-treatment of cells with LSE at 5, 10, and 25 mg/ml concentration significantly reduced the levels of H2O2-induced ROS production in HepG2 (Figure 6A). Protective effects of LSE on MMP The intensity of mitochondrial membrane potential (MMP) in HepG2 exposed to H2O2 and various concentrations of LSE is presented in Figure 6(B). Results showed that HepG2 exposed to H2O2 (0.25 mM) for 24 h resulted in 46 ± 2.6% reduction (p50.01) in the level of MMP. Pre-treatment of cells with LSE at 5, 10, and 25 mg/ml concentrations prior to H2O2 treatment significantly increased the intensity of MMP in HepG2 (Figure 6B). Protective effects of LSE on LPO Protective potential of various concentrations of LSE on H2O2 induced lipid peroxidation in HepG2 is summarized in Figure 6(C). As shown in figure, exposure of H2O2 resulted
Effect of Lepidium sativum extract against H2O2
DOI: 10.3109/13880209.2015.1035795
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