Tribulus Terrestris (l) Protects Heart and Liver from Beta Adrenergic-Stimulated Cardiotoxicity: Biochemical and Histological study in Wistar Rats

S. B Mahammad Rahmathulla1, K. V Sailaja2,Prof. Kodidhela Lakshmi Devi*
  1. Department of Biochemistry, Sri Krishnadevaraya University, Anantapur, India
  2. Department of Biochemistry, Sri Krishnadevaraya University, Anantapur, India
Corresponding Author:K. Lakshmi Devi. Sri Krishnadevaraya University, Anantapur, India E-mail address: [email protected], [email protected]
Received:12 January 2011 Accepted: 28 January2011
Citation: S B Mahammad Rahmathulla, K. V Sailaja, Prof Kodidhela Lakshmi Devi* “Tribulus Terrestris (l) Protects Heart and Liver from Beta Adrenergic-Stimulated Cardiotoxicity: Biochemical and Histological study in Wistar Rats” Int. J. Drug Dev. & Res., January-March 2013, 5(1): 264-270.
Copyright: © 2013 IJDDR, S B Mahammad Rahmathulla et al. This is an open access paper distributed under the copyright agreement with Serials Publication, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Acute myocardial infarction is a wearisome risk inherent in all major surgeries. Preoperative evaluation has been directed mainly to assess its risk because it carries with it a very high mortality. Living donor liver transplantation has been subject to inquiry not only because of the morbidity risk but also because of the mortality risk it carries to the live donor. The present study investigates the protective effects of Tribulus terrestris Fruit aqueous Extract (TTFAEt) in myocardially infarcted rats. The oral administration of TTFAEt to rats for 40 days afforded good protection against isoproterenol-induced alterations in cardiac levels of lipid peroxidation (LPO) as well as the activities of antioxidant enzymes like Superoxide dismutase (SOD), Catalase (CAT), glutathione peroxidase (GPx), Glutathione-S-transferase (GST). Antioxidants can prevent reactive oxygen species-mediated damage and thus may have potential application in the prevention and cure of chronic diseases. Myocardial infarction produces a significant abnormal liver functioning. Liver tissue marker enzymes injury like alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). The protective effect of TTFAEt was further supported by the reversal of isoproterenol-induced histological changes in the liver. The results suggest that TTFAEt protect the heart and circulatory system and also hepatoprotective and thereby maintain the near normal architecture of liver tissues.


Key words

TTFAEt, isoproterenol, oxidative stress, myocardial infarction, Lipid peroxidation, Anti-oxidant enzymes and liver histopathology.


Worldwide, myocardial infarction (MI) is one of the leading causes of decease1. Oxidative stress is the major etiopathological factor in ISO-induced myocardial necrosis. A relatively low amount of endogenous antioxidant makes the heart vulnerable to oxidative stress-induced damage 2. Considerable efforts have been made in the exploration of the potential for exogenous antioxidants and free radical scavengers to supplement endogenous antioxidant system and limit free radical injury, with mixed success and failure 3. One reason why exogenous antioxidants have limited success in the prevention of myocardial injury may be due to the inaccessibility of large molecules to the key intracellular sites of oxidative damage. Under such circumstances, other options need to be explored which will help in circumventing this problem whether, by any means, it is possible to stimulate or augment the endogenous antioxidant defense system of the heart 4. One of the proposed mechanisms of action of such drugs is by enhancing basal level of cellular endogenous antioxidant enzymes (SOD, CAT, GSHPx), and nucleic acid biosynthesis 5.
The liver is the largest organ in the body and it is involved in various metabolic functions. Liver disease has serious secondary effects on the functions of many other organs in the body. Conversely, diseases of other organs can interfere with liver function, either directly or indirectly 6. In cardiovascular diseases, hepatic damage always occurs 7. Liver cells contain many enzymes that may be released into the blood in various pathological processes 8.
Tribulus terrestris has long been a constituent in tonics in Indian ayurveda practice, where it is known by its Sanskrit name, gokshura. It is also used as an aphrodisiac, diuretic and nervine in Ayurveda, and in Unani, another medical system of India. The saponins (furostanol) and flavanoids present in the leaves and fruits are the active principles responsible for its vasodilatory and diuretic properties 9. Effects of Tribulus saponins on ventricular remodeling after myocardial infarction in hyperlipidemic rats 10. Flavonoids are reported to have antioxidant and hepatoprotective properties 11. The focus of the present study was to evaluate the effect of TTFAEt on heart and liver against isoproterenol-induced myocardial infarction in albino rats.


Tribulus terrestris was received as a gift from Chemiloids manufacturers and exporters of chemicals, alkaloids and herbal extracts, based in Vijayawada, Andhra Pradesh, India.
Adult male albino rats of Wistar strain, weighing 120- 150g, were procured from the National Institute of Nutrition, Hyderabad, India. The study was approved by Animal Ethics Committee of S. K University, Anantapur (REGD. No. 470/01/a/ CPCSEA, DT. 24th Aug 2001.). The rats were fed with commercial pellet rat chow (M/s. Sai Durga Feeds and Foods, Bangalore., India) and water ad libitum and maintained under standard laboratory conditions with 12 :12 h light : dark cycle. The rats were divided into four groups of six animals each. Group I rats served as positive control received 1ml of physiological saline subcutaneously (sc) for two days while Group II rats were pretreated with TTFAEt orally (85 mg/kg bw/day) for a period of 40 days group III rats were administered ISO sc (85 mg/kg body weight/day) dissolved in physiological saline twice at an interval of 24 h for two consecutive days. while group IV rats were pretreated with TTFAEt orally (85mg /kg bw for a period of 40 days) and then received ISO 85 mg/kg bw/ day dissolved in physiological saline sc twice at an interval of 24 h for two consecutive days. At the end of 40 days, the animals were fasted for 12 hours to minimize dietary effects and anaesthetized with thiopentone sodium (35 mg/kg BW, ip). Blood was drawn from the external jugular vein of the rats into heparinised tubes and plasma was separated from the cells by centrifugation immediately used for the estimation of proteins, lipid peroxidation. Liver was excised immediately for analysis.


Immediately after the sacrifice, the heart and liver were excised and washed in ice-cold isotonic saline and blotted with a filter paper.
Subsequently, the hearts were weighed and a portion of the tissue was homogenized in 0.1M Tris–HCl buffer (pH 7.4). The homogenate was centrifuged at 7000rpm for 15 min and the resulting supernatant was used for the estimation of the following parameters: lipid peroxidation (Okhawa et al. 1979), enzymatic antioxidants: GST (Habig et al.,1981), GPX (Rotruck 1973), SOD (Misra and Fridovich 1972), CAT (Sinha 1972) and nonenzymatic antioxidants: Reduced glutathione (Maron et al.,1979).

Liver marker enzymes

The liver samples were homogenized in 0.1 M Tris HCl buffer solution (pH 7.5, 4°C) to give a homogenate, the tissue marker enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were assayed using commercial kits purchased from Qualigens Diagnostics (Mumbai, India).


Small pieces of liver tissue were flushed with normal saline, fixed in 10% formalin solution, and processed. Sections 5 m thick were cut, stained with hematoxylin and eosin, and observed under the light microscope for cell necrosis, vacuolar degenerative changes, inflammation, and fibrosis.


Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT). Results were expressed as mean ±S.D. for six rats in each group. P values < 0.05 were considered significant.


In Table 1, Rats induced with ISO showed a considerable (P < 0.05) increase in the levels of TBARS and LOOH in the plasma and heart compared to normal control rats. Oral pretreatment with TTFAEt to ISO-induced rats showed considerable (P < 0.05) decrease in the levels of TBARS and LOOH in plasma and the heart compared with ISO alone induced rats.
Isoproterenol-induced myocardial infarction in rats was used as an experimental model. The activities of enzymic antioxidants such as SOD, catalase, GPx, GRx and GST in the heart of normal and ISO-induced rats are shown in Table 2. ISO-induced rats exhibited a significant (P < 0.05) decrease in the activities of these enzymic antioxidants in the heart compared to normal control rats. Pretreatment with TTFAEt to ISO-induced rats significantly increased the activities of these enzymes compared with ISO alone induced rats.
Each value is mean ±S.D, for six rats in each group. (P < 0.05, DMRT). SOD Units: one unit is defined as the enzyme concentration required to inhibit the optical density at 560nm of chromogen production by 50% in 1min. CDNB: 1-chloro-2, 4-dinitrobenzene.
The results of the activities of Liver marker enzymes in normal and experimental group of rat liver are shown in Figure 1. Significant decrease in levels of the liver tissue marker enzymes AST, ALT and LDH were noted in isoproterenol-induced rats when compared with control rats. A slight increase in levels of the marker enzymes was noted in drug-treated rats when compared with isoproterenol- induced rats. No significant changes were noticed for drug control rats when compared with control rats. Near normal levels were maintained in drug-treated rats when compared with control rats.
Each column is mean ±S.D for six rats in each group. The histological observations of the liver sections Fig. II are in agreement with the biochemical changes. Group I showed normal architecture, Group II showed near normal architecture with slight sinusoidal dilatation, Group III showed inflammatory necrosis and fibrosis, and in Group IV near normal hepatic architecture was restored.


Isoproterenol-induced myocardial oxidative stress has also been reported earlier 12. Its metabolic effects are seen in adipose tissue, skeletal muscle, and liver. Decreased levels of tissue marker enzyme in myocardially infarcted rats can be attributed to the damaged structural integrity of the liver because these are cytoplasmic in location and are released into circulation after cellular damage13.
Antioxidants constitute the foremost defense system that limits the toxicity associated with free radicals. The equilibrium between antioxidants and free radicals is an important process for the effective removal of oxidative stress in intracellular organelles. However, in pathological conditions like MI, the generation of ROS can dramatically upset this balance with an increased demand on the antioxidant defense system. Free radical scavenging enzymes such as SOD, catalase, GPx, GRx and GST are the first line of cellular defense against oxidative injury. These enzymes are lowered due to enhanced lipid peroxidation. Superoxide radicals generated at the site of damage in MI modulates SOD and catalase resulting in the lowered activities of these enzymes and accumulation of superoxide anion, which also damages the myocardium 14. Rats pretreated with TTFAEt showed increased activities of these enzymes which suggest that TTFAEt may have the ability to prevent the deleterious effects induced by free radicals in ISO-induced rats. GPx and GST activities are significantly depressed in ISO induced rats, which may be due to the reduced availability of GSH. Inactivation of GRx in the heart leads to accumulation of oxidized glutathione (GSSG) 15. GSSG inactivates the enzymes containing SH-group and inhibits protein synthesis 16. Increase in the activities of GPx, GRx and GST on pretreatment with TTFAEt in ISO-induced MI group shows the antioxidant potential of TTFAEt against injury caused by free radicals. GSH is important in protecting the myocardium against free radical mediated injury and thus reduction in cellular GSH content could impair recovery after short period of ischemia. Depressed GSH levels may be associated with an enhanced protective mechanism to oxidative stress in MI. ISO administration was found to reduce the levels of GSH in plasma and cardiac tissue and these observations concur with earlier finding.
Liver is an imperative organ play a major role in metabolism and excretion of xenobiotics from the body. Liver injury or liver dysfunction is a major health problem that encounters not only health care professionals but also the pharmaceutical industry and drug regulatory agencies. Leakage of cellular enzymes into plasma indicates a hallmark sign of hepatic injury or damage 17-18. Measurements of aminotransferases and LDH assess hepatocyte integrity. Even mild changes in their levels may be due to the presence of potentially significant liver disease 19-20. Damage due to isoproterenol might have led to the leakage of these enzymes from liver tissue into the blood stream, but TTFAEt administration might have minimized the effect of isoproterenol and would have prevented the leakage, thereby maintaining the values at near normalcy in drug-treated rats when compared with control rats. ALT (in cytoplasm) and AST (in cell cytoplasm and mitochondria) occur in much higher concentration in liver than elsewhere and consequently their decrease reflects hepatic damage more specifically21.
The hepatic histology generally correlates with the clinical or biochemical severity of cardiovascular disease. Liver dysfunction leading to the associated histological changes has long been recognized as a complication of severe heart failure as reported earlier 22. Central hepatic necrosis associated with left-sided heart failure is recognized mostly 23. Group I showed normal architecture. Group II showed near normal architecture with slight sinusoidal dilatation. In Group III sinusoids adjacent to the terminal hepatic veins are dilated, and hepatocytes show nuclear vacuolation. Hyaline thickening of the central vein is seen. In this form of liver damage only a small area of normal-appearing hepatocytes remains in the perivenular area. Necrosis and fibrosis are common occurrences, which is similar to earlier reports 24-25. In the case of Group IV rat’s near-normal hepatic architecture is seen.
Saponins, the active constituent of TTFAEt inhibit the metabolism of arachidonic acid via the Cyclooxygenase and lipooxygenase pathways that generates reactive oxygen species; resulting in a decrease in the levels of lipid peroxides 26.
T. terrestris Flavonoids have been shown to possess hepatoprotective activities 27-28. The hepatoprotective effect of T. terrestris may therefore be due to the presence of flavonoids (Figs. 1). The studied plant extract contains antioxidants and hepatoprotective activity through a regulatory action on cellular permeability, stability and suppressing oxidative stress. A number of scientific reports indicated that certain flavonoids, triterpenoids and steroids have protective effects on the liver 29-30.


The current study demonstrates that TTFAEt offered significant protection against ISO induced myocardial necrosis and hepatic damage through a unique property of enhancement of basal endogenous antioxidants, hepatoprotective activity through a regulatory action on cellular permeability, stability and antioxidant property without producing any cytotoxic effects. The mechanism of action of the active compounds present in TTFAEt to attenuate cardiac tissue damage requires further extensive study.


Authors would like to thank Department of Biochemistry S.K. University, Anantapur, A.P, India, M/S Chemiloids, Vijayawada, Andhra pradesh, India and we grateful to Prof Md. Akthar, Department of Statistics, S.K. University for assisting with data analysis.

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