9 : Efficacy of Solanum nigrum Linn. Extract on Myocardial Infarction in Rats
Abstract
Solanum nigrum Linn. has been suggested as useful traditional medicine for cardiovascular disorders. In this article an attempt has been made to evaluate the beneficial effect of S. nigrum extract (SNE) on isoproterenol induced myocardial infarction in rats. Myocardial infarction was determined by disturbances in serum and cardiac tissue marker enzymes such as lactate dehydrogenase (LDH), creatine kinase (CPK), aspartate transaminase (AST) and alanine transaminase (ALT). Myocardial infarction caused significant decrease in the activities of antioxidant defense enzymes with a concomitant increase in lipid peroxidation of heart tissue and these functional alterations were supported by severe modifications in cardiac tissue architecture. Pretreatment with SNE for 15 days prevented these biochemical alterations and restored the cytostructural integrity of heart tissue. Finally SNE shows better recovery from myocardial injury which can be attributed by its reducing effect on oxidative damage. These preliminary results could be useful to study and understand the biochemical events involved in this cardioprotective mechanism of S. nigrum.
Key words : Myocardial infarction, Solanum nigrum, Isoproterenol, Oxidative damage
1. Department of Biomedical Engineering, Sri Siva Subramaniya Nadar College of Engineering, SSN Nagar, Chennai – 603 110, India.
* Corresponding author : E-mail: malsleo80@yahoo.co.in
Introduction
Myocardial infarction (MI) is the acute condition of myocardial necrosis that occurs as a result of imbalance between coronary blood supply and myocardial demand (Boudina et al., 2002). Myocardial necrosis can be recognized by the appearance of different proteins released into the blood circulation due to the damaged myocytes which could be due to the induction of free radical mediated lipid peroxidation by isoproterenol (Suchalatha & Devi, 2004; Sushmakumari et al., 1989). Reactive oxygen species are generated from the leakage of electrons into oxygen from various systems in our body and the endogenous antioxidant enzymatic defense are very important source to neutralize the oxygen free radical mediated tissue injury (Karthikeyan & Rani, 2003). The use of available cardioprotective drugs is very much limited due to their various side effects. Therefore, recently most of the herbal drugs have been proved to be safe, clinically effective, better patient tolerance, relatively less expensive and globally competitive. Plant extracts, are some of the most attractive sources of new drugs and have been shown to produce promising results in the treatment of cardiovascular diseases.
Solanum nigrum Linn. (Family: Solanaceae) commonly known as ‘Black nightshade’ has been extensively used in traditional medicine in India for various diseases. The berries possess various medicinal properties such as sedative, diaphoretic, diuretic, hydragogue, expectorant and are useful in the diseases of liver, heart and eyes and is also effective against piles, fever and dysentery (Rastogi & Mehrotra, 1991). Phytochemical studies of Solanum nigrum revealed the presence of glycoalkaloids (solanine, solamargine, solanigrine and solasodine (0.09-0.65 %)), steroidal glycosides (b-solamargine, solasonine and a-b-solansodamine), steroidal saponins (diosogenin (0.4-1.2 %)), steroidal genin (gitogenin), tannin (7-10 %) and polyphenolic compounds (Kritikar & Basu, 1935; Saijo et al., 1982; Duke, 1985). Previous reports indicated that Solanum nigrum fruits possess beneficial activity as antiulcer, antioxidant antifungal and antitumor promoting agent in rats (Jainu & Devi, 2004; Prashanth et al., 2001; Moundipa & Domngang, 1991). The fruits of S. nigrum have been reported to play an adjuvant role in the hepatoprotective property (Sarwat et al., 1995; Raju et al., 2003). More recent reports revealed that the glycoprotein isolated from this plant exerted hypolipidemic activity (Lee et al., 2005). Sultana et al. demonstrated that Solanum nigrum protect DNA against oxidative damage and the results suggest that the observed hepatoprotective effect of Solanum nigrum might be due to the ability to suppress the oxidative degradation of DNA in the tissue debris (Sarwat et al., 1995). Dietary factors play a key role in the protection of various human diseases, including cardiovascular disease. Solanum nigrum have been used as a traditional food for congestive heart failure.
Experimental and clinical studies to determine cardioprotective properties of SNE are very limited, so it was thought worthwhile to undertake such investigation using methanolic extract of berries of Solanum nigrum. In addition, the beneficial efficacy of Solanum nigrum extract (SNE) was explained in terms of its effect on heart marker enzymes, antioxidant status, free radical production and it was also confirmed by histologic findings on myocardial integrity.
Materials and Methods Drugs and chemicals
Methanol (MeOH), isoproterenol, nicotinamide adenine dinucleotide phosphate (NADP), nicotinamide adenine dinucleotide reduced (NADPH), Sodium pyruvate were purchased from Sigma Chemical Co., St Louis, MO, USA. Bovine serum albumin was purchased from (Loba Chemie Co., Bombay, India). All substances were prepared immediately before use and the reagents used were of analytical grade.
Plant material
The mature fruits of Solanum nigrum used in this study were purchased from Native Care and Cure Center, India and identified with the standard sample by Dr. P. Brindha, Department of Pharmacognosy, Captain Srinivasa Murthy Drug Research Institute for Ayurveda, Chennai. A voucher specimen was deposited at the herbarium of the same institute.
Extract preparation
Solanum nigrum mature fruits were shade dried and coarsely powdered. 1 kg of this powdered plant material was soaked in 2 L of methanol for 5 days and then extracted in Soxhlet apparatus with methanol for 10 h. The last traces of the solvent were removed and concentrated to dryness under vacuo by using a rotary evaporator. The dried extract was weighed and then kept at –4 oC until ready for use. The yield of the extract was 16.7% w/w of powdered methanolic extract. In each experiment, the extract was diluted with water to desired concentration (Mallika et al., 2006).
Animals
Adult male albino rats of Wistar strain weighing about 120 – 150 g were used for the study. The animals were obtained from Tamil Nadu University of Veterinary and Animal Sciences (TANUVAS), Madhavaram, Chennai. The animal room was well ventilated with a 12 h light / dark cycle throughout the experimental period. They were maintained in clean, sterile, polypropylene cages and fed with commercial pelleted rat chow (M/S Hindustan Lever Limited, Bangalore, India) and water ad libitum. The study was approved by the institutional ethical committee, which follows the guidelines of CPSCEA (Committee for the purpose of control and supervision of experiment on animals), which complies with international norms of INSA.
Toxicity studies
SNE (100, 250, 500 and 1000 mg/kg body weight/day) dissolved in water and was administered orally to rats for 60 days for chronic toxicity study. Another group of animals, which received only vehicle alone (distilled water) through oral induction, were served as control. Morphological behavior and toxic symptoms of the animals were also checked for 24, 48 and 72 hrs and the animals were weighed biweekly for the whole treatment period for delayed toxicity.
Dosage schedule
Different doses of SNE (150, 300 and 500 mg/kg body weight) were dissolved in water and pretreated at different time intervals of 5, 15 and 20 days orally to assess the effective dose of SNE and duration of treatment against myocardial injury based on the activities of serum lactate dehydrogenase (LDH) (Nieland, 1955) and creatine kinase (CK) (Hall & Deluca, 1967) enzymes. Pretreatment with SNE at a dose of 300 mg/kg body weight for 15 days was found to be effective against myocardial infarction and hence this was fixed as optimum dosage for the subsequent biochemical analysis.
Experimental design
Animals were grouped into 4, each group consisting of six animals.
| Group 1: | Control rats received distilled water (1.0 ml/100g body weight) as a vehicle orally. |
| Group 2: | Rats were administered with ISPH (20 mg/100 g body weight suspended in 0.1 ml of 0.9% saline) subcutaneously twice at an interval of 24 h (Wexler & Greenberg, 1978). |
| Group 3: | Rats pretreated with SNE alone (300 mg/kg body weight dissolved in 1.0 ml of distilled water) orally for 15 days. |
| Group 4: | Rats pretreated with SNE (300 mg/kg body weight in 1.0 ml of distilled water) orally for 15 days and ISPH was administered as mentioned in Group II. |
After 15 days of SNE pretreatment, ISPH was induced i.p. for the next 2 days to induce MI. On 17th day the animals were anaesthetized with pentobarbital sodium (35 mg/kg, i.p.), the heart tissue were dissected out immediately and washed in ice-cold saline.
Biochemical estimations
The tissue (100 mg) was weighed accurately and homogenized in 5 ml of 0.1 M Tris-HCl buffer (pH 7.4) in ice-cold condition. The homogenate was centrifuged at 2500 x g and the clear supernatant solution was taken for the assay of marker enzymes such as aspartate transaminase (AST) (Mohur & Cooke, 1975), alanine transaminase (ALT) (18), lipid peroxides (LPO) (Okhawa et al., 1979) and protein (Lowry et al., 1951) in tissue and serum. Superoxide dismuatse (SOD), one of the principle antioxidant enzymes was estimated by following the method of Misra and Fridovich (1972) catalase (CAT) was measured by the method of Sinha (1972) and glutathione peroxidase (GPx) was estimated by the method of Roturck et al. (1979). Glutathione-S-transferase activity (GST) was measured using 1,chloro 2,4-dinitrobenzene as substrate according to Habig et al. (1974). The determination of total tissue sulphydryl (thiol) group (reduced glutathione level) was carried out according to the method of Ellman (1959). The nucleic acids such as DNA and RNA in tissue homogenate were estimated by Burton (1956) and Rawal et al. (1967) method.
Histological studies
Fresh heart tissue were excised and then fixed in 10% formalin for 24h. After dehydration through a graded series of alcohols, the tissue were cleaned in methyl benzoate, embedded in paraffin wax. Sections were cut into 5mm thickness and stained with hematoxylin and eosin. Again after dehydration and cleaning, the sections were mounted and observed under light microscope with magnification of 100x for histological changes.
Statistical analysis
The results are presented as mean ± S.D. The data’s were also analyzed by ANOVA (one-way analysis of variance) using SPSS package. The statistical analysis was performed using Dunnett’s T3 multiple comparison test for all parameters. The values were considered significant at the levels of p < 0.05, p < 0.01 and p < 0.001.
Results
The effective dosage was fixed by assessing the activities of serum marker enzymes such as LDH and CPK (Figs 1A and B). Dose-dependent protection was observed upon SNE pretreatment at different dose levels (150, 300 and 500 mg/kg body weight, daily) for 5, 15 and 20 days as compared with ISPH induced rats. A significant dose dependent protection was observed in 150, 300 and 500 mg/kg body weight of SNE treatment for 15 and 20 days and since it is safe to take minimum drug dose for short treatment period, a minimum dose of 300 mg/kg body weight of SNE daily for 15 days was chosen as optimum dosage and optimum duration to protect the myocardium effectively from the necrotic damage induced by ISPH and further biochemical analysis were carried out with this dosage alone.
The cardiac marker enzymes and antioxidants activities in the heart tissue of control and experimental animals are presented in Table 1. A significant decrease in ALT, AST, SOD, CAT, GSH, GST and GPX activities with a concomitant increase in LPO levels were observed in myocardial infarcted rats as compared with control. Rats pretreated with SNE significantly inhibited the alteration in these enzyme activities in tissue. SNE alone received animals registered near normal values when compared with control.
The ISPH induced rats showed a significant increase in heart tissue proteins and nucleic acids as compared to control animals (Table 2). SNE
Fig 1. Results are expressed as mean ± SD for 6 animals in each group. *, statistically significant from control and a, b, c, statistically significant from ISPH induced group respectively at p < 0.05, p < 0.01 and p < 0.001.
pretreated rats showed a significant decrease in protein and nucleic acids as compared with isoproterenol administered rats. Drug control rats showed a non-significant change in all these parameters as compared to control rats.
Microscopic examination of heart tissue of control rats (Fig 2a) showed normal myocardial fibers and muscle bundles with normal architecture. Heart tissue of myocardial infarcted rats (Fig 2b) showed separation of myocardial fibers with inflammatory mononuclear collections, edema and myocardial necrosis. SNE alone treated rats (Figs 2c) showed normal myocardial fibers with no pathological changes. Myocardial section of SNE pretreated rats showed slightly separated myocardial fibers with small focus of inflammatory mononuclear collections (Fig 2d) with absence of necrotic damage.
Table 1. Effect of SNE on the activities of antioxidant enzymes and transaminases in heart tissue of ISPH induced rats
Table 2. Effect of SNE on protein and nucleic acids in heart tissue of ISPH induced rats
Discussion
The present investigation suggested that heart tissue injury induced by ISPH was reduced dose-dependently by administration of SNE, suggesting that SNE have a protective action against myocardial infarction. The toxicity studies of SNE carried out in rats indicated no lethal effect at least upto an oral dose of 1.0 g/kg b.w for 60 days indicating that LD50 of SNE will be higher than that dose.
Fig 2. Histological examination of heart tissue section in control and experimental animals (Hematoxylin & Eosin, 100x). Section of heart tissue from control rat showing normal cardiac muscle architecture (Fig 2a). SNE treated rat showing apparently normal architecture with no pathologic changes (Fig 2c). ISPH-myocardial infarcted rat showing degenerative changes, hyalinization of muscle fibres and cellular infiltration (Fig 2b). SNE pretreated rat revealed less cellular infiltration, normal muscle fibres and the cardioprotective effect are evident from reduced myocardial damage even after ISPH administration (Fig 2d).
As a result of necrosis caused by toxic or diseased conditions many marker enzymes of myocardial infarction such as CPK, LDH and transaminases are found drained in blood stream (Suchalatha & Devi, 2004). The release of these myocardial marker enzymes into the serum could be due to the induction of free radical mediated lipid peroxidation by ISPH (Sushmakumari et al., 1989). Solanum nigrum acts as a potent scavenger of hydroxyl radicals and DPPH radicals (Son et al., 2003). This review suggests that SNE could have reduced the necrotic damage through anti-free radical action and prevented the leakage of enzymes from the tissue. Alkaloids have been reported to possess protective action against myocardial ischemia and diminish the release of CK and LDH enzymes (He et al., 1998). Similar observations have been recorded with SNE pretreatment in the present study, which could be attributed to the protective action of alkaloid present in the extract.
The principal finding of the present study is that myocardial infarction was associated with oxidative stress, as evidenced by increase in myocardial TBARS and depletion of tissue antioxidant status (SOD, CAT, GPx, GST, GSH and GR). Similar observations were made earlier by other studies (Tosaki et al., 1993; Seth et al., 1998). The imbalance in the activity of antioxidant enzymes like SOD, CAT and GPx causes accumulation of free radicals, which leads to tissue damage. Oral administration of SNE caused a significant decrease in the TBARS and marked rise in the activities of enzymic antioxidants and it shows better recovery profile along with histological improvement. SNE also exerted its antioxidant action by scavenging reactive oxygen species (Sultana et al., 1995) and enhancing the cellular antioxidants, like reduced glutathione superoxide dismutase, catalase and glutathione peroxidase of vascular endothelial cells (Jainu & Devi, 2004). Previous studies proved that anthocyanins present in Solanum nigrum possess significant antioxidant effect (Tsuda, 2000). The enhancement of antioxidant enzymes and reduced oxidative damage in SNE treated rats might be due to its free radical scavenging and cytoprotective action (Prashanth et al., 2001).
ISPH caused a significant increase in protein, DNA and RNA content in heart tissue when compared to control rats. The increased DNA content in isoproterenol treated rats has been reported to be probably attributable to fibroblast cells since, cardiac muscle cells do not undergo mitotic division (Kizer & Howell, 1970). Lochner et al. (1971) have reported that the increased protein synthesis following experimental myocardial infarction as a part of repair process may be stimulated after cellular necrosis. It has been reported that protein synthesis is preceded and accompanied by enhanced RNA synthesis. Venugopal et al. (2001) have reported that the adrenergic agents adrenaline and isoproterenol exert effects on cardiovascular cells and induces mRNA hybridization signals in the vascular cells of the heart and also in cardiocytes. Son et al. (2003) tested in vitro cytoprotection of Solanum nigrum and observed significant inhibition of cytotoxicity, reduced nucleic acid synthesis along with hydroxyl radical scavenging potential, which might be the mechanism of cytoprotection. It may be suggested that the antioxidant property of compounds present in extracts or due to the presence of polyphenolic compounds might be responsible for inhibiting the oxidative injury to DNA. Augmented endogenous antioxidants on heart and endothelial cells have important direct cytoprotective effects, especially in the event of oxidant stress induced injury. These biological evidences support the beneficial efficacy of the extract on proteins and nucleic acids levels.
In conclusion, the data suggest that Solanum nigrum, is beneficial for cardiovascular health and should be recommended as part of a healthy diet. Further research should also be carried out to identify specific compounds from Solanum nigrum that are responsible for most of its biological effects.
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