Author for correspondence: S. K. Gupta, Head, Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India (fax 91-11-26588663, 91-11-26588641, e-mail firstname.lastname@example.org).
Abstract: The present study was designed to evaluate the cardioprotective potential of hydro-alcoholic extract of Withania somnifera on the basis of haemodynamic, histopathological and biochemical parameters in the isoprenaline-(isoproterenol) induced myocardial necrosis in rats and to compare with Vitamin E, a known cardioprotective antioxidant. Wistar albino male rats (150–200 g) were divided into six main groups: sham, isoprenaline control, Withania somnifera/Vitamin E control and Withania somnifera/Vitamin E treatment groups. Withania somnifera was administered at doses 25, 50 and 100 mg/kg and Vitamin E at a dose of 100 mg/kg, orally for 4 weeks. On days 29 and 30, the rats in the isoprenaline control and Withania somnifera/Vitamin E treatment groups were given isoprenaline (85 mg/kg), subcutaneously at an interval of 24 hr. On day 31, haemodynamic parameters were recorded and the hearts were subsequently removed and processed for histopathological and biochemical studies. A significant decrease in glutathione (P<0.05), activities of superoxide dismutase, catalase, creatinine phosphokinase and lactate dehydrogenase (P<0.01) as well as increase in lipid peroxidation marker malonyldialdehyde level (P<0.01) was observed in the hearts of isoproterenol control group rats as compared to sham control. However, we have not observed any significant changes in activity of glutathione peroxidase and protein levels. Left ventricular dysfunction was seen as a decrease in heart rate, left ventricular rate of peak positive and negative pressure change and elevated left ventricular end-diastolic pressure in the control group was recorded. On histopathological examination, myocardial damage was further confirmed. Our data show that Withania somnifera (25, 50 and 100 mg/kg) exerts a strong cardioprotective effect in the experimental model of isoprenaline-induced myonecrosis in rats. Augmentation of endogenous antioxidants, maintenance of the myocardial antioxidant status and significant restoration of most of the altered haemodynamic parameters may contribute to its cardioprotective effect. Among the different doses studied, Withania somnifera at 50 mg/kg dose produced maximum cardioprotective effect.
Myocardial infarction is the most lethal manifestation of cardiovascular diseases and has been the object of intense investigation by clinicians and basic medical scientists (Bolli 1994). Currently, there is an increasing realization that herbs can influence the course of heart diseases and its treatment by providing an integrated structure of nutritional substances which aid in restoring and maintaining balanced body systems (Dhar et al. 1968; Hertog et al. 1993). Use of herbs for the treatment of cardiovascular diseases in Ayurveda, Chinese and Unani systems of medicine has given a new lead to understanding the pathophysiology of these diseases. Therefore, it is rational to use our natural resources for identifying and selecting inexpensive and safer approaches for the management of cardiovascular diseases along with the current therapy.
Withania somnifera most commonly known as Ashwagandha belongs to the natural order Solanaceae. The roots of Withania somnifera have been extensively used as a valuable drug in Ayurveda, the Indian System of Medicine. Although its therapeutic potential as immunomodulatory, adaptogenic, antioxidant, hypoglycaemic and anticancer activities have been reported (Malhotra et al. 1961; Lavie et al. 1965). Very few studies assessing its cardioprotective potential are presently available (Dhuley et al. 2000).
The present study is designed to evaluate the efficacy of Withania somnifera in the experimental model of isoprenaline-induced myonecrosis. The effect of Withania somnifera on several molecular parameters for myocardial injury has been explored to elucidate its mechanism of action. The effect of Withania somnifera on modulation of biochemical parameters such as lipid peroxidation product malonyldialdehyde, endogenous antioxidants such as glutathione, antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase and myocardial enzymes creatinine phosphokinase and lactate dehydrogenase have been studied. For a better correlation between biochemical and functional changes in the myocardium, alterations in mean arterial pressure, heart rate, left ventricular peak positive pressure change (rate of pressure development), left ventricular rate of peak negative pressure change (rate of pressure decline) and elevated left ventricular end-diastolic pressure were also included in the study. Protective actions of Withania somnifera on heart have also been confirmed histopathologically.
Materials and Methods
All chemicals including vitamin E used in the study were obtained from Sigma Chemicals, USA and were of analytical grade. Hydro-alcoholic root extract of Withania somnifera was a generous gift from Dabur Research Foundation, India.
Wistar albino male rats, weighing 150–200 g, were used in the study. The study protocol was approved by the institutional Ethics Committee and conducted according to the Indian National Science Academy Guidelines for the Use and Care of Experimental Animals. Rats were obtained from the Central Animal House facility of All India Institute of Medical Sciences, New Delhi, India. They were kept in standard laboratory conditions under natural light and dark cycle. The animals were fed normal diet (Hindustan Lever Ltd, Chandigarh, India) and water ad libitum.
Treatment protocol. The animals were divided into six main groups. Withania somnifera control and treated groups were divided further into three subgroups each. All the groups/subgroups consisted of eight animals each.
In all the animals haemodynamic parameters were recorded on day 31. The animals were subsequently sacrificed with an overdose of anaesthesia, hearts were removed and immediately processed for histopathological studies. For performing biochemical estimations, hearts were stored in liquid nitrogen until further analysis.
Haemodynamic studies. Rats of all the experimental groups were anaesthetized intraperitoneally with pentobarbitone sodium (60 mg/kg). Atropine was co-administered along with the anaesthetic to maintain heart rate during the surgical procedure and reduce broncho-tracheal secretions. The body temperature was monitored and maintained at 37 ° throughout the experimental protocol. The neck was opened with a ventral midline incision, tracheostomy was performed and the rats ventilated with room air from a positive pressure ventilator (Inco, India) at a rate of 70 strokes/min. and a tidal volume of 10 ml/kg. Ventilator setting and PO2 were adjusted as needed to maintain the arterial blood gas parameters within the physiological range. The left jugular vein was cannulated with polyethylene tube for continuos infusion of 0.9% saline solution. The right carotid artery was cannulated and the cannula filled with heparinized saline and connected with CARDIOSYS CO-101 (Experimentria, Hungary) by a pressure transducer for the measurement of blood pressure and heart rate. A left thoractomy was preformed at the fifth intercostal space and the heart was exposed. A wide bore (1.5 mm) sterile metal cannula connected to a pressure transducer (Gould Statham P23ID, USA) was inserted into the cavity of the left ventricle from the posterior apical region of the heart for recording left ventricular pressure dynamics on Polygraph (Grass 7D, USA). After the completion of the surgical procedure, the thoracic cavity was covered with saline-soaked gauze to prevent the heart from drying. The animals were then allowed to stabilize for 15 minutes before recording the basal haemodynamic variables.
Histopathological studies. At the end of the experiment, myocardial tissue was immediately fixed in 10% buffered neutral formalin solution. The fixed tissues were embedded in paraffin and serial sections were cut. The sections were examined under light microscope (Nikon. Tokyo, Japan) after haematoxylin and eosin staining and photomicrographs were taken. Representation area images were captured in an image analysis system. The Image Analyzer consists of BX-50 Research Microscope (Olympus, Japan), Coolsnap 10 bit Digital Camera (Media Cyberneticus, USA) and Pentium 4 computer (compaq, India) with an image analysis software Image Plus Pro (Media Cyberneticus, USA). The area of myofiber loss was calculated by subtracting from an area of interest (AOI), the area occupied by muscle fibers and it was expressed as % of total area (Dinda et al. 2001).
Statistical analysis. Descriptive statistics mean and standard deviation were calculated for all variables of each group. In the isoprenaline group, one-way Analysis of Variance (ANOVA) was applied for statistical analysis with post-hoc analysis (Bonferroni Multiple Range Test) and P value <0.05 has been considered as statistical significance level.
Biochemical assessment of injury.
Effect of Withania somnifera on antioxidant parameters. The basal myocardial glutathione content was found to be 1.86±0.69 μmol/g tissue. On chronic feeding of Withania somnifera for 30 days, no significant change in basal glutathione level was observed in Withania somnifera (25 mg/kg) control group as compared to the sham. However, a marked increase in the glutathione contents (P<0.05) was observed in Withania somnifera (50 and 100 mg/kg) control groups as compared to sham (table 2). Subsequent to isoprenaline challenge, a significant decrease in myocardial glutathione (P<0.05) was observed in the isoprenaline control group in comparison to sham. However, a statistically significant restoration in glutathione levels (P<0.05) was seen when rats were pre-and co-treated with Withania somnifera at 50 and 100 mg/kg doses as compared to the isoprenaline control (table 2).
Table 2. Biochemical parameters in the different experimental groups. #P<0.05, ##P<0.01 versus sham; *P<0.05, **P<0.01, ***P<0.001 versus isoprenaline (ISP) control. Values are mean±S.D. of eight experiments. One unit of creatinine phosphokinase (CPK) will transfer 1 umol of phosphate from phosphocreatine to adenosine diphosphate (ADP) per min. at pH 7.4 at 30 °. One unit of lactate dehydrogenase (LDH) will reduce 1 μmol of pyruvate to D-lactate per min. at pH 7 at 25 °.
The basal myocardial catalase, glutathione peroxidase and superoxide dismutase activities were found to be 21.1±3.1, 0.33±0.2 and 7.94±2.9 units/mg protein respectively. Withania somnifera (25, 50 and 100 mg/kg) treatment per se significantly augmented endogenous antioxidant enzymes superoxide dismutase (P<0.05, fig. 1) and glutathione peroxidase (P<0.05, fig. 2). In addition, a significant increase in basal myocardial catalase activity in Withania somnifera (50 and 100 mg/kg) (P<0.05) control groups was also observed (fig. 3). Isoprenaline-induced myocardial necrosis resulted in a significant depletion of antioxidant enzymes: catalase (P<0.01, fig. 3), superoxide dismutase (P<0.05, fig. 1) compared to sham control. In the Withania somnifera 25, 50, 100 mg/kg treated groups, a significant increase in the activities of antioxidant enzymes glutathione peroxidase (P<0.05, fig. 2) and superoxide dismutase (P<0.05, fig. 1) was observed compared to isoprenaline control group. However, the myocardial catalase activity was not significantly altered in all the three Withania somnifera-treated groups when compared to the isoprenaline control (fig. 3).
The basal levels of myocardial lipid peroxidation marker, malonyldialdehyde was found to be 66.5± 8.9 nmol/g tissue. We observed that the malonyldialdehyde levels were significantly elevated (P<0.05) in the isoprenaline control group (table 2). Withania somnifera (25, 50 and 100 mg/kg) treatment significantly inhibited lipid peroxidation (P<0.05) and preserved membrane integrity.
Basal myocardial enzyme activities, lactate dehydrogenase and creatinine phosphokinase were found to be 210±36.9 and 162±27.3 units/mg protein. A fall in myocardial enzymes creatinine phosphokinase (P<0.01, table 2), lactate dehydrogenase (P<0.05, table 2) was observed in the isoprenaline control group compared to sham control. Withania somnifera treatment at the above doses elucidated a significant restoration in lactate dehydrogenase (P<0.01) and creatinine phosphokinase (P<0.05) enzymes (table 2).
Effect of Vitamin E on biochemical parameters. No significant effect on basal glutathione (table 2), superoxide dismutase (fig. 1), glutathione peroxidase (fig. 2), and catalase (fig. 3) activities was observed in the vitamin E control group compared to sham control. However, vitamin E treatment resulted in a significant protection in the biochemical markers compared to the isoprenaline challenge groups. A significant restoration in glutathione content (P<0.05, table 2), antioxidant enzymes (glutathione peroxidase (P<0.05, fig. 2) and superoxide dismutase (P<0.05, fig. 1)) and myocardial enzymes (lactate dehydrogenase (P<0.05, table 2) and creatinine phosphokinase (P<0.05, table 2)) was observed in the vitamin E-treated group as compared to isoprenaline control. Vitamin E also markedly reduced lipid peroxidation as evidenced by reduction (P<0.01) in malonyldialdehyde levels (table 2).
Ventricular function assessments. A slight fall in diastolic arterial pressure and a significant fall in systolic arterial pressure, mean arterial pressure (P<0.05, table 3) and heart rate (P<0.05, table 3) was observed in the isoprenaline group as compared to sham control. In addition, isoprenaline administration resulted in left ventricular dysfunction, as indicated by a significant fall in left ventricular rate of pressure development and decline (P<0.05, table 4) and a rise in left ventricular end-diastolic pressure (P<0.05, Table 4) as compared to sham control.
Table 3. Haemodynamic parameters in the different experimental groups. #P<0.05, ##P<0.01 versus sham; *P<0.05, **P<0.01, ***P<0.001 versus isoprenaline (ISP) control. Values are mean±S.D. of eight experiments.
Table 4. Haemodynamic parameters in the different experimental groups. #P<0.05, ##P<0.01 versus sham; *P<0.05, **P<0.01, ***P<0.001 versus isoprenaline control. Values are mean±S.D. of eight experiments.
Withania somnifera or vitamin E treatment in the present study failed to improve blood pressure recordings significantly (table 4) as compared to the isoprenaline control group. Nonetheless, a significant improvement in heart rate (P<0.05) with Withania somnifera 25, 50 and 100 mg/kg and vitamin E was observed (table 3). Moreover, both vitamin E (P<0.01) and Withania somnifera (25, 50 and 100 mg/kg, P<0.05) markedly reduced left ventricular end-diastolic pressure as compared to the isoprenaline control (table 4).
The lusitropic state, myocardial relaxation (left ventricular pressure decline) was significantly restored by Withania somnifera 25 mg/kg dose (P<0.05), Withania somnifera 50 mg/kg dose (P<0.001) and Withania somnifera 100 mg/kg dose (P<0.01) and vitamin E (P<0.01) doses as compared to the isoprenaline control (table 4). On the other hand, 50 mg/kg Withania somnifera as well as vitamin E slightly improved contractility as compared to the isoprenaline control (table 4).
Histopathological assessment of injury.
In the sham group the % area of fiber loss was found to be 0.6±0.3 (mainly representing interstitial space observed in the area of interest). On histopathological examination, in the isoprenaline control group, significant myocardial membrane damage and infiltration of inflammatory cells as compared to sham group was observed. Also extensive myonecrosis with fibroblastic proliferation and presence of chronic inflammatory cells were observed in the isoprenaline control group compared to that of sham. The myofiber loss due to necrosis in the isoprenaline control group was (18.4±4.2%, (P<0.001)) as compared to sham. In the present study, Withania somnifera (50 and 100 mg/kg,) and vitamin E (100 mg/kg,) treatment significantly prevented myonecrosis as indicated by significant reduction in the infiltration of inflammatory cells, vacuolar changes as well as oedema as compared to the isoprenaline control group. The % fiber loss in the vitamin E-treated group was 12.9±3.2% (P<0.05) and in the Withania somnifera (50 and 100 mg/kg) groups were 3.5±1.6% (P<0.001) and 4.3±1.2% (P<0.001) respectively as compared to isoprenaline control. However, Withania somnifera (25 mg/kg) treatment did not significantly prevent myofiber loss (15.9±3.8%) as compared to the isoprenaline control.
Isoprenaline, a synthetic catecholamine has cardiotoxic effects on the myocardium. Amongst the various mechanisms proposed to explain isoprenaline-induced cardiac damage, generation of highly cytotoxic free radicals through auto-oxidation of catecholamines has been implicated as one of the important causative factor (Handforth et al. 1962; Csapo et al. 1972; Singal et al. 1983; Nirmala & Puvanakrishnan 1994). This free radical-mediated peroxidation of membrane phospholipids and consequent changes in membrane permeability is the primary target responsible for cardiotoxicity induced by isoprenaline.
Studies have shown that oxidative stress results in the reduction of the efficacy of the β-adrenoceptor agonists probably due to reduction in cAMP formation, caused by an impaired coupling between the receptor and adenylate cylase. The reduction in of maximal β-adrenoceptor-mediated response might be the result of cytotoxic aldehydes that are produced during the oxidative stress. This β-adrenoceptor hyperstimulation leads to cardiotoxicity (Haenen et al. 1990). Oxidative stress may also depress the sarcolemmal Ca2+ transport and result in the development of intracellular Ca2+ overload and ventricular dyfunction (Tappia et al. 2001). Hence, therapeutic intervention with antioxidant activity may be useful in preventing these deleterious changes (Noronha-Dutra et al. 1984).
As described earlier, increase in malonyldialdehyde levels was observed in the heart tissue after isoprenaline administration. In addition, isoprenaline administration reduced the glutathione content as well as the antioxidant enzyme activity (superoxide dismutase, catalase and glutathione peroxidase) in cardiac tissue and this observation concurs with earlier reports (Nirmala & Purvanakrishnan 1994). The fall in the activity of glutathione peroxidase in the isoprenaline group might be co-related to decrease availability of its substrate, reduced glutathione. Moreover, due to impairment in both enzymatic and non-enzymatic antioxidant defense mechanism, it is quite possible that the free radicals are not effectively neutralized and therefore the myocardium shows enhanced susceptibility to lipid peroxidation.
Besides alterations in the antioxidant parameters, changes in lactate dehydrogenase and creatine phosphokinase activities have been considered as some of the important markers of myocardial infarction (Jennings et al. 1990). A significant depletion of lactate dehydrogenase and cretanine phosphokinase in the isoprenaline group was observed. Furthermore, on histopathological examination, the presence of focal myonecrosis with myophagocytosis and lymphocytic infiltration (myocarditis) in the subendocardial region was observed. Biochemical and histopathological confirmation of cardiotoxic effect produced by isoprenaline (85 mg/kg), in the present investigation has established the suitability of this model for studying the cardioprotective effect of Withania somnifera.
The observation that vitamin E and Withania somnifera treatment significantly restored lactate dehydrogenase and creatine phosphokinase activity compared to the isoprenaline control group, was suggestive of their cardioprotective effect. Both these drug treatments restored the myocardial antioxidant status and maintained membrane integrity as evidenced by a decline in malonyldialdehyde levels. Furthermore, histopathological examination confirmed the cardioprotective effects of Withania somnifera and vitamin E.
As described earlier, haemodynamic parameters were also incorporated into the experimental design for a better understanding of the co-relation between biochemical and functional changes in the myocardium subjected to isoprenaline-induced damage. Previous studies have reported that exposure of the heart to oxidation stress depresses the ventricular functions and these changes are significantly prevented by antioxidants (Dormandy 1978). The results of the present study also support these observations. In the isoprenaline control group, myocardial dysfunction was clearly evident by a significant fall in mean arterial pressure, heart rate, left ventricular rate of peak positive and negative pressure change and a rise in left ventricular end-diastolic pressure, which might be due to isoprenaline-induced myocardial necrosis. The left ventricular rate of negative pressure change was more markedly depressed indicating a more diastolic dysfunction per se which may result in the persistence of elevated left ventricular end-diastolic pressure. It is speculated that deteriorating myocardial contractile status following isoprenaline-induced necrosis might be responsible for the significant fall in mean arterial pressure. Normally a fall in mean arterial pressure increases heart rate and myocardial contractility due to reflex sympathetic action. However, none of these effects were observed in the present study, suggesting impairment in the conducting system of the heart following isoprenaline-induced myocardial necrosis. Although Withania somnifera and vitamin E treatment did not significantly increase mean arterial pressure, an increase in heart rate was observed as compared to the isoprenaline control group. Moreover, both drugs appeared to preserve left ventricular function as evidenced by significant restoration of left ventricular rate of peak positive and negative pressure change and correction of elevated left ventricular end-diastolic pressure. Preservation of cardiac reflexes resulting in increased heart rate and ventricular function to maintain cardiac output, may be on account of myocardial salvage, produced by Withania somnifera and vitamin E.
A concept is now emerging of ‘adaptogenic drugs’, drugs that increase non-specific resistance of the users to a variety of stresses, first time reported by Brehman and associates in Eleuthrococcus and Panax geniseng (Lei & Chiou 1986). Adaptogenic property of various herbs like Ocimum sanctum, Bacopa monniera and Withania somnifera has already been reported in various experimental studies (Devi & Ganasoundari 1999; Bhattacharya et al. 2000). The major active constituents of Ashwagandha from which its primary medicinal properties emanate, are based upon the actions of certain steroidal alkaloids and lactones as a class of constituents called withanolides (Lavie et al. 1965). The root contains the steroid lactone (withaferin A) and related withanolides, along with various alkaloids. It is reported that Sitoindosides VII, VIII, IX and X are likely adaptogenically active substances present in Withania somnifera. The exact mechanism of such myocardial adaptation is not known. However, it has been proposed that it works through the induction of a number of antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase and antioxidant such as glutathione and proteins like heat shock protein (Archana & Namasivayam 1999; Bhattacharya et al. 2000). There was a concomitant increase in glutathione, catalase and glutathione peroxidase along with superoxide dismutase activity in the Withania somnifera (50 and 100 mg/kg) control groups. The lowest dose of Withania somnifera only stimulated the synthesis of antioxidants glutathione and catalase. The observed increase in glutathione levels might be due to its enhanced synthesis. It is speculated that increase in glutathione reductase activity may account for maintaining glutathione in its reduced state. Vitamin E, in the present study, however did not exhibit any such adaptogenic property as no significant increase in the levels of the endogenous antioxidants with vitamin E group was observed.
In summary, the present study strongly suggests that multiple mechanisms may be responsible for the cardioprotective effect of Withania somnifera. It produced myocardial adaptive changes (augmentation of endogenous antioxidants) on chronic administration. In addition, it restored the antioxidant status of the myocardium, subsequent to isoprenaline-induced oxidative stress. These beneficial effects also translated into functional recovery of the myocardium as evidenced by favourable modulation of haemodynamic variables. Histopathological assessment further confirmed the protective effect of Withania somnifera on the myocardium. Furthermore, the results of this study on the whole, demonstrate that although cardioprotective effect was also provided by Withania somnifera (25 and 100 mg/kg), maximum protective effects was observed by pre and co-treatment with Withania somnifera at 50 mg/kg. Withania somnifera (50 mg/kg) dose was found to be the most effective in functional recovery of the heart and favourable restoration of biochemical and histopathological alterations.
Clinical studies have demonstrated the efficacy of Ashwagandha in the treatment of osteoarthritis and hypercholesterolemia (Kulkarni et al. 1991; Andallu & Radhika 2000). The present study provides scientific basis for the cardioprotective potential of Ashwagandha validating its usage in Ayurveda. Considering its safety, efficacy and traditional acceptability, clinical trials should be conducted to support its therapeutic use in ischaemic heart diseases.
The authors gratefully acknowledge the gift of Withania somnifera from Dabur Research Foundation, India. The authors also thank Mr. B.M. Sharma for his valuable technical assistance.