Appl Biochem Biotechnol DOI 10.1007/s12010-014-1276-5
Protective Effect of Punica granatum Peel and Vitis vinifera Seeds on DEN-Induced Oxidative Stress and Hepatocellular Damage in Rats Ashok K. Kumar & Vijayalakshmi K
Received: 6 May 2014 / Accepted: 23 September 2014 # Springer Science+Business Media New York 2014
Abstract This study was designed to find out the efficacy of ethanol extracts of Punica granatum peel and Vitis vinifera seeds on diethylnitrosamine (DEN)-induced oxidative stress and hepatocellular damage in Wistar rats. Rats were divided into four groups. The first group served as normal control, and the second group received DEN at a dose of 200 mg/kg body weight by single intraperitoneal administration. The third one received DEN as in DEN-treated group and co-treated with 400 mg/kg P. granatum peel extract. The final group also received DEN and co-treated with 400 mg/kg V. vinifera seed extract. DEN administration to rats resulted in significantly elevated levels of serum SGPT, SGOT, ALP, and GGT which is indicative of hepatocellular damage. DEN-induced oxidative stress was confirmed by elevated levels of lipid peroxides and decreased activities of superoxide dismutase, catalase, and glutathione peroxidase in the serum and liver tissues. The status of non-enzymatic antioxidants like vitamin C, vitamin E, and reduced glutathione were also found to be decreased in serum and tissues of DEN-administered rats. Co-treatment with the P. granatum peel and V. vinifera seed extracts orally for 12 weeks significantly reversed the DEN-induced alterations in the serum and liver tissues. Keywords Punica granatum . Vitis vinifera . Antioxidant . Diethylnitrosamine . Hepatocellular damage
Introduction Hepatocellular carcinoma (HCC) is one of the most common and frequent malignancy throughout the world and is the fifth most common cancer and the third leading cause of cancer mortality in the world [1] with an estimated 560,000 newly diagnosed cases per year [2, 3]. HCC is the malignant transformation of hepatic cells and is a common complication of A. K. Kumar (*) Department of Biotechnology, Arulmigu Meenakshi Amman College of Engineering, Vadamavandal, 604 410 Thiruvanamalai District, Tamil Nadu, India e-mail: [email protected] V. K Department of Biochemistry, Bharathi Women’s College, 600 108 Chennai, Tamil Nadu, India
Appl Biochem Biotechnol
chronic hepatitis mainly caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) infection in China [4]. The factor that induces the formation of HCC includes exposure to environmental carcinogens, iron overload, fatty liver diseases, and alcohol abuse [5]. Diethylnitrosamine is an N-nitroso alkyl compound, categorized as a potent hepatotoxin and hepatocarcinogen in experimental animals, intiating reproducible tumors on administration [6–8]. As oxidative stress plays a central role in diethylnitrosamine-induced hepatotoxicity, the use of antioxidants would offer better protection to counteract liver damage [9]. Since the increase in the use of synthetic chemicals in cancer therapy has led to many side effects and undesirable hazards, there is a worldwide trend to use natural resource which is therapeutically effective, culturally acceptable, and economically within the reach of the poor people [10]. The reduced cancer risk and lack of toxicity associated with high intake of natural products suggest that specific concentrations of phytochemicals from these plant sources may produce cancer chemopreventive effects without causing significant levels of toxicity. Diets rich in fruits and vegetables have been associated with reduced risk of developing chronic diseases such as cardiovascular disease, cancer, and diabetes [11]. The Punica granatum and Vitis vinifera are common fruits consumed throughout the world. The peel and seeds of the mentioned fruits respectively are the waste materials of food processing industries. Both the fruit wastes are rich source of polyphenols, potent antioxidants that include phenols, flavonoids, and tannins. Flavonoids and phenolic compounds widely distributed in plants which have been reported to exert multiple biological effect, including antioxidant, free radical scavenging abilities, and anti-inflammatory and anticarcinogenic effects [12]. The present study aims to carry out a systematic investigation on the protective effect of P. granatum peel and V. vinifera seeds on diethylnitrosamine (DEN)-induced oxidative stress and hepatocellular damage by analyzing serum glutamate pyruvate transaminase (SGPT), serum glutamic-oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), and gammaglutamyl transferase (GGT) levels along with the non-enzymatic and enzymatic antioxidants.
Materials and Methods Plant Material The P. granatum and V. vinifera belongs to families Lythraceae and Vitaceae, respectively. These fruits were collected from the Koyambedu Market, Chennai, India from the same cultivar. The plants were identified and authenticated by Dr. P. Jayaraman, Director of National Institute of Herbal Science, Plant Anatomy Research Centre, Chennai. These voucher specimens are maintained in Plant Anatomy Research Centre, Chennai (PARC/2009/360 and PARC/2009/361). Drugs and Chemicals Diethylnitrosoamine, phenobarbital, hydrogen peroxide, ascorbic acid, α-tocopherol acetate, and glutathione were obtained from Sigma Chemicals. All other chemicals used for this study were of analytical grade. Preparation of Extract P. granatum peel and V. vinifera seeds were removed from the fruits and washed thoroughly using sterilized water. The selected plant materials were shade-dried and pulverized to fine
Appl Biochem Biotechnol
powder individually in a mechanical grinder. One hundred grams of coarse powder was weighed and extracted with ethanol by keeping it for 24 h in the orbital shaker at room temperature [13]. These extracts were collected after filtration using Whatman No. 1 filter paper. The solvent was concentrated under reduced pressure in a rotary evaporator until all the solvent is completely evaporated from the extract. The alcoholic extracts obtained were used for further studies. Selection of Animal Male Wistar rats (150–200 g) were obtained from Kings Institute, Chennai, India, and used for the studies. The animals were housed in cages and maintained under standard conditions of humidity, temperature (25.2 °C), and light (12-h light/12-h dark) at BRULAC, Saveetha University, Chennai, India. They were fed with standard pelleted diet (M/s Pranav Agro Industries Ltd., India) and had free access to water. Experimental animals were handled according to the university and institutional legislation, regulated by the committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India (IAEC No. Biochem BWC.004/2009). Experimental Design Hepatic cancer was induced in rats using DEN (200 mg/kg body weight by i.p.), and after 2 weeks, the carcinogenic effect was promoted by 0.05 % phenobarbital, which was supplemented to the experimental animals through drinking water for 20 successive weeks. The experimental animals were divided in to four groups of seven each. Group I Group II
Normal vehicle control Hepatic cancer induced in rats using DEN (200 mg/kg body weight by single i.p. injection). Phenobarbital, which was supplemented to the experimental animals through drinking water for 20 successive weeks Group III Cotreatment with P.granatum peel extract (EPGP) (400 mg/Kg body weight) by oral gavage for 12 weeks. Group IV Cotreatment with V. vinifera seed extract (EVVS) (400 mg/kg body weight) by oral gavage for 12 weeks
Collection of Samples Collection of Blood and Preparation of Liver Homogenate All the experimental animals were sacrificed by cervical decapitation after the experimental period. Blood was collected, and serum was separated by centrifugation before the sacrifice of animal and used for further studies. Liver homogenate was prepared according to the method described by El-Demerdash et al. [14]. The abdominal cavity of rats was dissected immediately after sacrifice, and the liver was rapidly removed. This liver were excised, washed with ice-cold 0.9 % sodium chloride (w/v) to remove the blood, cut in to small pieces by fine scissors, and then homogenized (10 % w/v) separately in ice-cold 1.15 % potassium chloride, 0.01 M sodium phosphate buffer, pH 7.4 with a Potter–Elvehjem glass homogenizer. The
Appl Biochem Biotechnol
homogenate was centrifuged at 10,000×g 20 min at 4 °C. Supernatant of the liver homogenate was collected into sterilized tubes and stored at −20 °C for further studies.
Biochemical Studies Determination of Serum Markers of Liver Damage SGOT and SGPT activities were determined in rats using the method described by Mohun and Cook [15]. Serum alkaline phosphatase activity was assayed by the method of King and Armstrong [16]. GGT level in serum was estimated using kit supplied from Agappe Diagnostics, Kerala, India, and the assay was carried out following standard procedure provided in the kit manual. Lipid Perioxides and Antioxidant Enzyme Determination Lipid peroxides content was measured using a thiobarbituric acid assay as reported and described earlier by Ohkawa et al. [17] and expressed as level of malondialdehyde (MDA). The activity of superoxide dismutase was determined by the method of Marklund and Marklund [18]. The catalase activity was assayed by the method of Sinha [19]. The activity of GPx was assayed by the method of Rotruck et al. [20]. The concentration of reduced glutathione was measured by the method of Moron et al. [21]. The concentration of ascorbic acid and Vitamin E were estimated by the methods of Omaye et al. [22] and Desai [23]. Statistical Analysis Data were analyzed with SPSS version 20 (IBM, USA). The test values were analyzed using one-way analysis of variance (ANOVA), followed by least significant difference (LSD) test. Statistical significance was defined as p
Protective Effect of Punica granatum Peel and Vitis vinifera Seeds on DEN-Induced Oxidative Stress and Hepatocellular Damage in Rats Ashok K. Kumar & Vijayalakshmi K
Received: 6 May 2014 / Accepted: 23 September 2014 # Springer Science+Business Media New York 2014
Abstract This study was designed to find out the efficacy of ethanol extracts of Punica granatum peel and Vitis vinifera seeds on diethylnitrosamine (DEN)-induced oxidative stress and hepatocellular damage in Wistar rats. Rats were divided into four groups. The first group served as normal control, and the second group received DEN at a dose of 200 mg/kg body weight by single intraperitoneal administration. The third one received DEN as in DEN-treated group and co-treated with 400 mg/kg P. granatum peel extract. The final group also received DEN and co-treated with 400 mg/kg V. vinifera seed extract. DEN administration to rats resulted in significantly elevated levels of serum SGPT, SGOT, ALP, and GGT which is indicative of hepatocellular damage. DEN-induced oxidative stress was confirmed by elevated levels of lipid peroxides and decreased activities of superoxide dismutase, catalase, and glutathione peroxidase in the serum and liver tissues. The status of non-enzymatic antioxidants like vitamin C, vitamin E, and reduced glutathione were also found to be decreased in serum and tissues of DEN-administered rats. Co-treatment with the P. granatum peel and V. vinifera seed extracts orally for 12 weeks significantly reversed the DEN-induced alterations in the serum and liver tissues. Keywords Punica granatum . Vitis vinifera . Antioxidant . Diethylnitrosamine . Hepatocellular damage
Introduction Hepatocellular carcinoma (HCC) is one of the most common and frequent malignancy throughout the world and is the fifth most common cancer and the third leading cause of cancer mortality in the world [1] with an estimated 560,000 newly diagnosed cases per year [2, 3]. HCC is the malignant transformation of hepatic cells and is a common complication of A. K. Kumar (*) Department of Biotechnology, Arulmigu Meenakshi Amman College of Engineering, Vadamavandal, 604 410 Thiruvanamalai District, Tamil Nadu, India e-mail: [email protected] V. K Department of Biochemistry, Bharathi Women’s College, 600 108 Chennai, Tamil Nadu, India
Appl Biochem Biotechnol
chronic hepatitis mainly caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) infection in China [4]. The factor that induces the formation of HCC includes exposure to environmental carcinogens, iron overload, fatty liver diseases, and alcohol abuse [5]. Diethylnitrosamine is an N-nitroso alkyl compound, categorized as a potent hepatotoxin and hepatocarcinogen in experimental animals, intiating reproducible tumors on administration [6–8]. As oxidative stress plays a central role in diethylnitrosamine-induced hepatotoxicity, the use of antioxidants would offer better protection to counteract liver damage [9]. Since the increase in the use of synthetic chemicals in cancer therapy has led to many side effects and undesirable hazards, there is a worldwide trend to use natural resource which is therapeutically effective, culturally acceptable, and economically within the reach of the poor people [10]. The reduced cancer risk and lack of toxicity associated with high intake of natural products suggest that specific concentrations of phytochemicals from these plant sources may produce cancer chemopreventive effects without causing significant levels of toxicity. Diets rich in fruits and vegetables have been associated with reduced risk of developing chronic diseases such as cardiovascular disease, cancer, and diabetes [11]. The Punica granatum and Vitis vinifera are common fruits consumed throughout the world. The peel and seeds of the mentioned fruits respectively are the waste materials of food processing industries. Both the fruit wastes are rich source of polyphenols, potent antioxidants that include phenols, flavonoids, and tannins. Flavonoids and phenolic compounds widely distributed in plants which have been reported to exert multiple biological effect, including antioxidant, free radical scavenging abilities, and anti-inflammatory and anticarcinogenic effects [12]. The present study aims to carry out a systematic investigation on the protective effect of P. granatum peel and V. vinifera seeds on diethylnitrosamine (DEN)-induced oxidative stress and hepatocellular damage by analyzing serum glutamate pyruvate transaminase (SGPT), serum glutamic-oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), and gammaglutamyl transferase (GGT) levels along with the non-enzymatic and enzymatic antioxidants.
Materials and Methods Plant Material The P. granatum and V. vinifera belongs to families Lythraceae and Vitaceae, respectively. These fruits were collected from the Koyambedu Market, Chennai, India from the same cultivar. The plants were identified and authenticated by Dr. P. Jayaraman, Director of National Institute of Herbal Science, Plant Anatomy Research Centre, Chennai. These voucher specimens are maintained in Plant Anatomy Research Centre, Chennai (PARC/2009/360 and PARC/2009/361). Drugs and Chemicals Diethylnitrosoamine, phenobarbital, hydrogen peroxide, ascorbic acid, α-tocopherol acetate, and glutathione were obtained from Sigma Chemicals. All other chemicals used for this study were of analytical grade. Preparation of Extract P. granatum peel and V. vinifera seeds were removed from the fruits and washed thoroughly using sterilized water. The selected plant materials were shade-dried and pulverized to fine
Appl Biochem Biotechnol
powder individually in a mechanical grinder. One hundred grams of coarse powder was weighed and extracted with ethanol by keeping it for 24 h in the orbital shaker at room temperature [13]. These extracts were collected after filtration using Whatman No. 1 filter paper. The solvent was concentrated under reduced pressure in a rotary evaporator until all the solvent is completely evaporated from the extract. The alcoholic extracts obtained were used for further studies. Selection of Animal Male Wistar rats (150–200 g) were obtained from Kings Institute, Chennai, India, and used for the studies. The animals were housed in cages and maintained under standard conditions of humidity, temperature (25.2 °C), and light (12-h light/12-h dark) at BRULAC, Saveetha University, Chennai, India. They were fed with standard pelleted diet (M/s Pranav Agro Industries Ltd., India) and had free access to water. Experimental animals were handled according to the university and institutional legislation, regulated by the committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India (IAEC No. Biochem BWC.004/2009). Experimental Design Hepatic cancer was induced in rats using DEN (200 mg/kg body weight by i.p.), and after 2 weeks, the carcinogenic effect was promoted by 0.05 % phenobarbital, which was supplemented to the experimental animals through drinking water for 20 successive weeks. The experimental animals were divided in to four groups of seven each. Group I Group II
Normal vehicle control Hepatic cancer induced in rats using DEN (200 mg/kg body weight by single i.p. injection). Phenobarbital, which was supplemented to the experimental animals through drinking water for 20 successive weeks Group III Cotreatment with P.granatum peel extract (EPGP) (400 mg/Kg body weight) by oral gavage for 12 weeks. Group IV Cotreatment with V. vinifera seed extract (EVVS) (400 mg/kg body weight) by oral gavage for 12 weeks
Collection of Samples Collection of Blood and Preparation of Liver Homogenate All the experimental animals were sacrificed by cervical decapitation after the experimental period. Blood was collected, and serum was separated by centrifugation before the sacrifice of animal and used for further studies. Liver homogenate was prepared according to the method described by El-Demerdash et al. [14]. The abdominal cavity of rats was dissected immediately after sacrifice, and the liver was rapidly removed. This liver were excised, washed with ice-cold 0.9 % sodium chloride (w/v) to remove the blood, cut in to small pieces by fine scissors, and then homogenized (10 % w/v) separately in ice-cold 1.15 % potassium chloride, 0.01 M sodium phosphate buffer, pH 7.4 with a Potter–Elvehjem glass homogenizer. The
Appl Biochem Biotechnol
homogenate was centrifuged at 10,000×g 20 min at 4 °C. Supernatant of the liver homogenate was collected into sterilized tubes and stored at −20 °C for further studies.
Biochemical Studies Determination of Serum Markers of Liver Damage SGOT and SGPT activities were determined in rats using the method described by Mohun and Cook [15]. Serum alkaline phosphatase activity was assayed by the method of King and Armstrong [16]. GGT level in serum was estimated using kit supplied from Agappe Diagnostics, Kerala, India, and the assay was carried out following standard procedure provided in the kit manual. Lipid Perioxides and Antioxidant Enzyme Determination Lipid peroxides content was measured using a thiobarbituric acid assay as reported and described earlier by Ohkawa et al. [17] and expressed as level of malondialdehyde (MDA). The activity of superoxide dismutase was determined by the method of Marklund and Marklund [18]. The catalase activity was assayed by the method of Sinha [19]. The activity of GPx was assayed by the method of Rotruck et al. [20]. The concentration of reduced glutathione was measured by the method of Moron et al. [21]. The concentration of ascorbic acid and Vitamin E were estimated by the methods of Omaye et al. [22] and Desai [23]. Statistical Analysis Data were analyzed with SPSS version 20 (IBM, USA). The test values were analyzed using one-way analysis of variance (ANOVA), followed by least significant difference (LSD) test. Statistical significance was defined as p
