Keywords
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| Scoparia dulcis Linn, Hepatotoxicity, Antioxidants, Lipid peroxidation, N?Nitrosodiethylamine (DEN) |
INTRODUCTION
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| Hepatotoxicity is an injury to the liver associated with its dysfunction caused by exposure to various drugs and toxicants. The hepatotoxicant may be divided into genotoxic and non-genotoxic. Genotoxicants directly interact with DNA, forming covalent adducts and induces genetic changes upon cell replication. Non-genotoxic chemicals stimulate tumour formation by altering kinetics of cell proliferation, cell death and cell differentiation through a variety of epigenetic pathways.[1] Exposure to an agent that causes induction of an enzyme important in the biotransformation of a drug, may led to increased formation of its reactive metabolites and greater risk of toxicity. |
| N-nitosodiethylamine (DEN) is a potent hepatotoxin and if co-administered with promoters like phenol-barbital, it also causes hepatocarcinogenesis. [2, 3, 4] DEN, a polycyclicaromatic hydrocarbon is a common environmental pollutant which is produced by the incomplete combustion of organic materials, fossil fuels, cigarette smoke, residential heating units, power plants and in certain occupational settings. [5, 6] It is reported that generation of reactive oxygen species (ROS) by DEN causes by-products of cellular metabolism that have direct effect on cell development, growth and survival. [7] |
| Medicinal plants, since time immemorial have been in use for treatment of various diseases all over the world. [8] Scoparia dulcis L. (SD) belongs to the Family: Scrophulariaceae is a glabrous under shrub commonly found in the tropical and subtropical regions. Phytochemical screening has shown that Scoparia dulcis contains diterpenoids, flavonoids, [9] tannins, alkaloids, triterpenes, hexacosonol, G-sitosterol, ketone, dulcitone and amellin, an antidiabetic compound. [10] The plant of S. dulcis is known to possess antidiabetic activity, [11] antiulcer activities. [12] An aqueous extract of S. dulcis showed marked in vitro antioxidant activity, which supports the therapeutic effects claimed by traditional practitioners. [13] An active and safe drug is needed for the treatment of liver toxicity and hepatitis. [14] The active principles present in Scoparia dulcis have been shown to exhibit cytotoxic and antitumor activity. [15] The present study was undertaken to conclude the hepatoprotective effect of the ethanolic extract of S. dulcis in DEN induced experimental rats. |
MATERIALS AND METHODS
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| Preparation of Plant Extract |
| Scoparia dulcis L. whole plants were collected during the month of January from Chidambaram, Tamilnadu, India. The plant was authenticated by Chief Botanist, Captain Srinivasa Multi Drug Research Institute for Ayurveda and Siddha (CCRAS). A voucher specimen has been deposited at the department of pharmacology and Environmental Toxicology, Dr.ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai, Tamilnadu, India. The whole fresh plant was dried under shade at room temperature for seven days and then reduced to a coarse powder. This powder was used for the preparation of aqueous extracts. The extract obtained was filtered and concentrated to a viscous consistency at 55ºC (the yield was 12.6 % w/w for aqueous extracts). |
| Animals |
| Healthy adult Wistar albino rats of both sexes weighing between 110±20g were used for the present study. They were obtained from the Central Animal House Facility, Dr.ALMPGIBMS, Taramani, University of Madras, Chennai (IAEC No: 07/012/08). The animals were kept in polypropylene cages and received standardized rat pellet and water ad libitum. All the procedures were done in compliance with the guidelines issued by the Institutional Animal Ethics Committee. |
| Chemicals |
| DEN was purchased from Sigma Chemical Company, St Louis, MO, U.S.A. All other Chemical including solvents used were of high purity and of analytical grade marketed by Glaxo Laboratories, Mumbai and Sisco Research Laboratories Pvt. Ltd, Mumbai, India. |
| DEN induced Hepatotoxicity in animal models |
| Laboratory animals treated with low and intermediate doses of DEN (50 and 100 mg/kg), light and moderate liver necrosis has been documented with or without associated inflammatory process. In the animals treated with the highest dose of DEN (200mg/kg) extensive liver necrosis has been observed with associated inflammatory process. [16] Therefore it was decided to use DEN for inducing hepatotoxicity in the present investigation. In the present investigation rats were divided into four groups of six animals each. Group I: Control animals were administered with normal saline (0.9%). Group II:Animals received a single intra-peritoneal injection of DEN at a dose of 200mg/kg in normal saline to induce hepatotoxicity. Group III: After inducing toxicity, animals were administered with the aqueous extract of Scoparia dulcis L. orally at a concentration of 500mg/kg body weight for a period of 28 successive days. Group IV: Animals received only plant extract orally at a concentration of 500mg/kg body weight for a period of 28 successive days. At the end of the experiment, all the animals were sacrificed by cervical decapitation. Blood and liver samples were collected to for the estimate of various biochemical parameters. |
| Biochemical Estimations |
| Estimation of Nucleic Acids Estimation of Deoxyribonucleic acid (DNA) DNA was estimated by the method of [17]. Estimation of Ribonucleic acid (RNA) RNA was estimated by the method of [18]. |
| Estimation of Marker Enzymes |
| AST and ALT by the method of [19], ALP and ACP was assayed by the method of [20] as described by Balasubramanian et al., [21], 5’- Nucleotidase was assayed by the method of [22], Gamma glutamyl transpeptidase was estimated by the method of [23], LDH was assayed by the method of [24]. |
| Estimation of Macromolecular Damages |
| Assay of lipid peroxidation (LPO) The level of lipid peroxides was assayed by the method of [25], Peroxide induced tissue lipid peroxidation, Ascorbate and ferrous sulphate induced tissue lipid peroxidation was assayed by the method of [26]. |
| Estimation of Enzymic Antioxidants |
| The activity of superoxide dismutase (SOD) and catalase (CAT) was determined by the method of [28], The activity of glutathione peroxidase (GPx) was assayed by the method of [29]. |
| Estimation of Non-Enzymic Antioxidants |
| Glutathione was estimated by the method of [30], the level of ascorbic acid was estimated by the method of [31], and the level of vitamin E was estimated by the method of [32]. |
| Statistical Analysis |
| Data are presented as the mean ± standard deviation (SD). One way analysis of variance (ANOVA) followed by Tukey’s multiple comparison method was used to compare the means of different groups of by using SPSS 12.5 student’s versions. Comparisons were made between group II and IV with group I and III for animal studies. P<0.01 was considerable statistically significant in all cases. |
RESULTS
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| Table 1show the levels of nucleic acids (DNA and RNA) in liver of control and experimental animals. In group II animals, the levels of nucleic acids were significantly decreased due to DEN induction (p<0.01). These were significantly increased in drug treated group III animals (p<0.01). However, no significant changes were observed in group IV drug control animals when compared to the control group I animals. |
| Table 2 shows the activities of marker enzymes such as AST, ALT, ACP, ALP, LDH, 5´-ND and R-GT levels in serum of control and experimental animals. Significantly increased levels of marker enzymes were noticed in serum of DEN induced hepatotoxicity animals, when compared with group I control animals (p<0.01). The levels of marker enzymes were found to be decreased in Scoparia dulcis L. treated group III animals when compared with group II animals (p<0.01). No significant changes in marker enzymes activity were observed in group IV only extract administrated animals when compared to group I control animals. |
| Values are expressed as mean + SD for six animals in each group a - Group I Vs Group II, III and IV, b - Group II Vs Group III and IV, c - Group III Vs Group IV The significance at the level of p<0.01 |
| Table 3 represents the activities of marker enzymes such as AST, ALT, ACP, ALP, LDH, 5´-ND and R-GT levels in liver of control and experimental animals. The activities of marker enzymes were decreased in Group II toxicity bearing animals (p<0.01). On the contrary, in Scoparia dulcis L. treated Group III animals, the levels of marker enzymes were reversed to near normal when compared to group II animals (p<0.01). However, there were no significant changes were observed in Group IV aqueous extract of Scoparia dulcis L. administrated animals when compared with Group I control animals. |
| Values are expressed as mean + SD for six animals in each group a - Group I Vs Group II, III and IV, b - Group II Vs Group III and IV, c - Group III Vs Group IV The significance at the level of p<0.01 |
| Table 4 & 5 shows the effect of Scoparia dulcis L. on Lipid peroxidation in the serum & liver of control and experimental animals respectively. In serum and liver, the basal, hydrogen peroxide, ascorbate and ferrous sulphate induced LPO were significantly increased in group II toxicity induced animals (p<0.01) when compared with control group I animals. Conversely, the administration of Scoparia dulcis L. significantly reduces the peroxidation reaction in group III drug treated animals (p<0.01). However, no marked alterations of lipid peroxidation were observed in group IV drug control animals when compared to the group I control animals. |
| Values are expressed as mean + SD for six animals in each group a - Group I Vs Group II, III and IV, b - Group II Vs Group III and IV, c - Group III Vs Group IV The significance at the level of p<0.01 |
| Values are expressed as mean + SD for six animals in each group a - Group I Vs Group II, III and IV, b - Group II Vs Group III and IV, c - Group III Vs Group IV The significance at the level of p<0.01 |
| Table 6 shows the activities of Enzymic and Nonenzymic antioxidants in the serum of control and experimental animals. In group II DEN induced animals, the activities of enzymic antioxidants SOD, CAT, GPx and non-enzymic antioxidants GSH, Vit-C and Vit-E were significantly decreased as compared to that of group I control animals (p<0.01). These enzymes levels were increased significantly reverted back to near normal in drug treated group III animals when compared with group II animals (p<0.01). No remarkable changes were observed in group IV drug control animals when compared with group I control animals. |
| Table 7 shows the activities of superoxide dismutase, catalase, glutathione peroxidise, glutathione, ascorbic acid and S-tocopherol in the liver of control and experimental animals. A significant decrease in the level of these enzymes were observed in the DEN induced group II animals (p<0.01) when compared to group I control animals. Interestingly, a significant increase in the level of these antioxidant enzymes was observed in the Scoparia dulcis L. treated group III animals (p<0.01). No remarkable changes were observed in group IV drug control animals when compared with group I control animals. |
| Values are expressed as mean + SD for six animals in each group a - Group I Vs Group II, III and IV, b - Group II Vs Group III and IV, c - Group III Vs Group IV The significance at the level of p<0.01 |
DISCUSSION
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| Hepatotoxicity refers to liver dysfunction as a result of an overload of toxic chemicals or drugs in the body. DEN is reported to generate reactive oxygen species (ROS) that leads to an oxidative stress. It is well established that protein, lipids and DNA are the prime targets of oxidative injury. [33] The interruption in uracil nucleotide dependent biosynthesis of nucleic acid results in DNA damage. The decrease in the levels of DNA and RNA due to chemical injury to liver has been reported earlier by [34] and [35]. The observed decrease in both DNA and RNA levels in DEN induced animals may be due to its inhibitory effects on the DNA synthesis and RNA transcription in hepatocytes and lead to the damage of the plasma membrane. Treatment with Scoparia dulcis L. aqueous extract probably prevented DEN induced UTP depletion and a subsequent suppression of nucleic acids by restricting the formation of toxic metabolites from DEN. |
| The liver functions as a filter that separates out harmful substances from the bloodstream. When there is an excess of chemicals filtering through the liver, the overload is more than the liver can process and leads to hepatotoxicity. The levels of certain enzymes are drastically altered in any kind of liver damage of which transaminases are regarded as the important markers of liver function. These marker enzymes sensitively reflect the status of the liver damage. [36] Along with transaminases ACP, ALP, LDH, 5´-ND and R-GT are also sensitive markers of hepatic damage. [37, 38] In the present investigation, the increased levels of marker enzyme in serum and decreased level in the liver are indicators of cellular damage and loss of functional integrity of the cell membrane due to DEN administration. Treatment with aqueous extract Scoparia dulcis L. brought back these enzymes to near normal level by probably preserving the functional integrity of the hepatocytes, showing its hepatoprotective action against DEN induced liver damage. |
| Lipid peroxidation mediated by free radical is considered to be primarily responsible for cell membrane destruction and cell damage. LPO has been implicated in a number of deleterious effects on cells, such as increased membrane rigidity, osmotic fragility, decreased cellular deformation and reduced membrane fluidity. Increase in the levels of TBARS indicates enhanced LPO, leading to tissue injury and failure of the antioxidant defence mechanism. [39] DEN administration resulted in the formation of LPO products and leads to cellular damage in the group II animals. In the present study, Scoparia dulcis L. extract administration decreased lipid peroxides level which may be due to the free radical scavenging activity of the plant extract. |
| Antioxidants acts as radical scavenger and inhibit LPO and other free radical-mediated processes, thereby protecting the human body from various diseases. [40] There is a natural dynamic balance between the output of free radicals generated in the body and the antioxidant defense system that quenches or scavenges them and thereby protecting the body against pathogenesis. [41] The first lines of defence against free radicals are endogenous enzymic antioxidants such as SOD, Cat, GPx and the Second lines of defence are the non-enzymic antioxidants such as glutathione, Vitamin-C and Vitamin-E. [42] DEN administration resulted to uncompromised free radical production overwhelming the cellular defence. In the present investigation, Scoparia dulcis L. extract stabilized the normal fluctuations in enzymic and non-enzymic antioxidant levels due to its antioxidant efficacy. |
| The biochemical alterations observed in hepatotoxicity status seems to be mainly due to an oxy radical mediated mechanism, involving LPO, under conditions of reduced antioxidant defences. Scoparia dulcis L. protects against LPO by mechanisms that may be dependent on changes in the antioxidant defence system. These observations indicate that, Scoparia dulcis L. administration may be protective against DEN induced hepatotoxicity. |
CONCLUSION
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| From our investigation, we concluded that aqueous extracts of Scoparia dulcis L. possessed important hepatoprotective and antioxidant activity against DEN induced liver toxicity in Wistar albino rats may be due to its antioxidant and free radical scavenging properties. Therefore, number of scientific reports indicated certain flavonoids, alkaloids, triterpenoids and steroids have protective effect on liver due to its antioxidant properties. Presence of those phytochemicals in Scoparia dulcis L. may be responsible for the protective effect in DEN induced liver damage in experimental rats. |
Tables at a glance
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