INTRODUCTION:

Myocardial infarction (MI) is the most lethal manifestation of cardiovascular diseases and has been the object of intense investigation by clinicians and basic medical Scientists. It is the necrotic condition that occurs due to imbalance between coronary blood supply and demand¹. Currently, there is 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^2-3.

Use of herbs for the treatment of cardiovascular diseases in Ayurveda, Chinese and Unani systems of medicine has given a new lead to understand the pathophysiology of these diseases. Therefore, it is rational to use the formulations which have been prepared by using natural resources for identifying and selecting inexpensive and safer approaches for the management of cardiovascular diseases along with the current therapy.

Draksharishta is a polyherbal hydroalcoholic ayurvedic preparation and is used as blood purifier, in the treatment of anaemia and advised as a choice of remedy in respiratory problems. The chief ingredient of Draksharishta is draksha, fruits of Vitis vinifera⁴. The composition and properties of fruits of Vitis vinifera, have been extensively investigated and it was reported that they contain large amount of phenolic compounds as catechins, epicatechin, quercetin, gallic acid, dimeric, trimeric and tetrameric procyanidins⁵. These compounds have many favourable effects on human health such as lowering of human low density lipoproteins, reduction of heart disease and cancer etc^6-9.

Therefore, we undertook the present investigation to evaluate the cardio protective effect of Draksharishta-T and Draksharishta-M prepared by traditional and modern methods respectively on isoproterenol (ISO) induced myocardial infarction (MI) in albino rats.

MATERIALS AND METHODS:

Preparation of Draksharishta-T

This was prepared by the method as given in The Ayurvedic Formulary of India, Part-I⁴. All the ingredients of Draksharishta were procured from local market, Jamnagar while jaggery was procured from local market, Mehsana. Authentication of all the ingredients of Draksharishta was done by Dr. G. D. Bagchi, Scientist, Department of Taxonomy and Pharmacognosy, Central Institute of Medicinal and Aromatic Plants, Lucknow. Prepared herbarium has been deposited in the Central Institute of Medicinal and Aromatic Plants, Lucknow for future reference. Identification of all the individual plant material was done as per The Ayurvedic Pharmacopoeia of India. Quantity of ingredients taken for the preparation of batch size 3.25 l of Draksharishta has been calculated according to the formula as given in The Ayurvedic Formulary of India, Part-I, 2000.

According to this method, dried fruits of Vitis vinifera after proper crushing were placed in polished vessel of brass along with prescribed quantity of water (13 l), and allowed to steep overnight. After overnight steeping, this material was warmed at medium flame until the water for decoction reduced to one fourth of the prescribed quantity (3.25 l), then the heating was stopped and it was filtered through unstarched muslin cloth in cleaned and fumigated vessel and after that jaggery was added and mixed properly. Then the prescribed quantity of coarsely powdered prakshepa dravyas as Cinnamomum zeylanicum (stem bark), Eletteria cardamomum (seeds), Cinnamomum tamala (leaves), Mesua ferrea (stamens), Callicarpa macrophylla (flowers), Piper nigrum (fruits), Piper longum (fruits), Embelia ribes (fruits) were added and then dhataki flowers (Woodfordia floribunda) were added for inducing fermentation and after that this sweet filtered fluid was placed for fermentation in incubator for fifteen days at 33ºC±1 ºC. After fifteen days completion of fermentation was confirmed by standard tests¹⁰. The fermented preparation was filtered with unstarched muslin cloth and kept in cleaned covered vessel for further next seven days. Then, it was poured in clean amber coloured glass bottles previously rinsed with ethyl alcohol, packed and labelled properly.

Preparation of Draksharishta-M

Method of preparation of Draksharishta-M was same as followed for Draksharishta-T only dhataki flowers were replaced by yeast for inducing fermentation¹¹.

Animals

Adult wistar albino rats, weighing between 200-220g of either sex were acclimatized to normal environmental conditions in the animal house for one week. The animals were housed in standard polypropylene cages and maintained under controlled room temperature (22^oC±2^oC) and humidity (55±5%) with 12:12 hour light and dark cycle. All the animals were given a standard chow diet (Hindustan Lever Limited), and water ad libitum. The guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) of the Government of India were followed and prior permission was granted from the Institutional Animal Ethics Committee (CPCSEA No. 07/09).

Experimental Procedure

The cardio protective effect of Draksharishta-T, Draksharishta-M and marketed Draksharishta was determined on ISO-induced MI in albino rats¹². All the animals were randomly divided into six groups comprising six animals in each group. Animals of normal control and positive control group received normal saline as vehicle and positive control animals received ISO (85 mg/kg) intraperitoneally (i.p.). Remaining groups were pretreated with Inderal*10 (Piramal Healthcare Limited, Baddi, India) which contains propranolol hydrochloride 10 mg at the dose of 10 mg/kg per os per day¹³ and with Draksharishta-T, Draksharishta-M and marketed Draksharishta at the dose of 2 ml/kg per os per day for thirty days to all the ISO treated animals. MI was induced in all the groups except normal control by administering ISO (85 mg/kg) intraperitoneally on 29^th and 30^th day, at an interval of 24 h. At the end of the experimental period, i.e. 24 h after the last injection of ISO, on 31^st day, the blood samples were withdrawn by retro orbital bleeding under mild ether anaesthesia and were centrifuged at 2000 rpm for 10 minutes for the separation of serum. The animals were subsequently sacrificed with an over dose of ether anaesthesia, hearts were removed, weighed and immediately processed for biochemical studies. The ratio of heart weight to body weight (mg/g) was also measured.

Biochemical analysis of serum

The separated serum was analysed for various serum marker enzymes as lactate dehydrogenase¹⁴, creatine kinase¹⁵, alanine aminotransferase and aspartate aminotransferase¹⁶. Serum was also assessed for lipid profile as serum cholesterol¹⁷, serum HDL and LDL¹⁸ and triglycerides¹⁹. Span and Erba diagnostic kits were used for the measurement of all these serum marker enzymes.

Biochemical analysis of myocardial tissue

A 10% homogenate of myocardial tissue was prepared in 50 mM phosphate buffer of pH 7.4. This homogenate was centrifuged at 2000 rpm for 10 min and an aliquot of the supernatant was used for the estimation of malonyldialdehyde²⁰ and glutathione²¹.

Statistical analysis

The results are expressed as mean ± SEM. Statistical analysis of data among the various groups was performed by using one way analysis of variance (ANOVA) followed by Tukey’s test using Graph Pad Prism software of statistics.

RESULTS:

The effects of pretreatment of Draksharishta-T, Draksharishta-M and its marketed preparation on serum lactate dehydrogenase (LDH), creatine kinase (CK-MB), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in ISO-induced MI in albino rats have been shown in Table 1. Results showed that in ISO-control group significant (P<0.001) increase was observed in the level of serum marker enzymes as serum LDH, CK-MB, AST and ALT as compared to normal control group. Pretreatment with Draksharishta-T, M at the dose of 2 ml/kg orally for thirty days significantly (P<0.001) reduced serum LDH, CK-MB, AST and ALT in ISO-induced MI in albino rats as compared to ISO- control group. Pre-treatment with marketed Draksharishta also showed similar effects on serum LDH, CK-MB, AST and ALT nearby same as produced by Draksharishta-T and M in ISO- induced MI in albino rats.

Pretreatment with all the test preparations of Draksharishta significantly improved serum lipid profile in ISO- induced MI in albino rats as compared to ISO-control group as shown in Table 2. Pretreatment with Draksharishta-T, M and its marketed preparation significantly (P<0.001) reduced serum cholesterol, triglycerides (TG), serum low density lipoproteins (LDL) while showed significant (P<0.001) increase in serum HDL as compared to ISO- control group.

Draksharishta-T, M and its marketed preparation pretreated groups significantly (P<0.001) reduced the increased heart weight and heart to body weight ratio as compared to ISO-control group as shown in Table 3.

It was observed that ISO-control group showed significant (P<0.001) rise in the basal level of myocardial lipid per-oxidation marker malonyldialdehyde (MDA) in myocardial tissue and caused significant (P<0.001) decrease in glutathione (GSH) content in cardiac tissue. Pretreatment with Draksharishta-T, M and its marketed preparation significantly (P<0.001) reduced MDA content and showed significant (P<0.001) rise in GSH content in cardiac tissue as compared to ISO – control group as shown in Table 4.

Table 1. Effect of Draksharishta-T, M and marketed Draksharishta on serum LDH, CK-MB, ALT and AST in ISO-induced MI in albino rats

Group	Dose( ml/kg/day p.o.)	LDH( U/L)	CK-MB( U/L)	ALT(IU/L)	AST(IU/L)
Normal control	2ml normal saline	192.51± 2.48	107.35±1.96	64.21±4.72	118.54±4.61
ISO control	2 ml normal saline	506.12±6.25^a	278.50±3.24^a	176.15±6.48^a	304.48±3.82^a
Inderal*10+ISO	10 mg	212.42±2.92^b	123.56±4.28^b	85.42±3.17^b	167.24±4.26^b
Drst-T+ISO	2ml	270.52±3.71^b	161.56±1.82^b	102.54±4.18^b	190.21±3.79^b
Drst-M+ISO	2ml	273.16±2.94^b	163.14±4.17^b	104.26±3.62^b	192.58±2.78^b
Marketed Drst+ISO	2ml	272.41± 2.43^b	164.28±1.46^b	103.81±2.97^b	192.96±4.12^b

All values are expressed as mean ± standard error mean (n = 6).

a P<0.001 significant as compared to normal control

b P<0.001 significant as compared to ISO control

ISO, isoproterenol; MI, myocardial infarction; Drst, Draksharishta

Table 2. Effect of Draksharishta-T, M and marketed Draksharishta on serum lipid profile in ISO-induced MI in albino rats

Group	Dose ( ml/kg p.o./day)	Serum cholesterol ( mg/dl)	Serum HDL ( mg/dl)	Serum LDL ( mg/dl)	Serum triglycerides ( mg/dl)
Normal control	2 ml normal saline	148.52±5.81	53.24±3.72	76.92±6.46	89.74±4.67
ISO control	2 ml normal saline	320.29±6.24^a	29.30±1.72^a	252.41±9.26^a	206.15±5.92^a
Inderal*10 +ISO	10 mg	161.48±12.21^b	50.12±4.26^b	90.25±1.48^b	102.41±2.73^b
Drst-T+ISO	2 ml	178.53±1.87^b	47.79±2.81^b	105.43±2.36^b	126.55±1.93^b
Drst-M+ISO	2 ml	180.84±2.98^b	47.21±3.18^b	107.14±4.92^b	132.45±2.49^b
marketed Drst+ISO	2 ml	180.16±4.16^b	47.32±1.68^b	106.84±3.58^b	130.0±3.71^b

All values are expressed as mean ± standard error mean (n = 6).

a P<0.001 significant as compared to normal control

b P<0.001 significant as compared to ISO control

ISO, isoproterenol; MI, myocardial infarction; Drst, Draksharishta

Table 3. Effect of Draksharishta-T, M and marketed Draksharishta on heart weight and heart to body weight ratio in ISO-induced MI in albino rats

Group	Dose ( ml/kg p.o./day)	Heart weight (mg)	Body Weight ( g)		Heart to body weight ratio ( mg/g)
Group	Dose ( ml/kg p.o./day)	Heart weight (mg)	On 1^st Day	After 14 days	Heart to body weight ratio ( mg/g)
Normal control	2 ml normal saline	972±46	208.6±3.8	209.2±2.7	4.646±0.42
ISO control	2 ml normal saline	1215±37^a	208.2±4.6	208.4±2.2	5.830±0.38^a
ISO + Inderal*10	10 mg	994±42^b	208.1±2.4	207.9±4.1	4.781±0.51^b
ISO+Drst-T	2 ml	1029±51^b	210.8±5.4	210.6±2.9	4.886±0.24^b*
ISO+Drst-M	2 ml	1033±34^b	211.2±4.9	211.0±3.6	4.895±0.47^b*
ISO + marketed Drst	2 ml	1031±41^b	211.0±3.8	210.8±5.6	4.890±0.37^b*

All values are expressed as mean ± standard error mean (n = 6).

a P<0.001 significant as compared to normal control

b P<0.001; b*P<0.01 significant as compared to ISO control

ISO, isoproterenol; MI, myocardial infarction; Drst, Draksharishta

Table 4. Effect of Draksharishta-T, M and marketed Draksharishta on heart MDA and GSH concentration in ISO-induced MI in albino rats

Group	Dose ( ml/kg p.o. /Day)	MDA( nmol/g tissue)	GSH ( µmol/g tissue)
Normal control	2 ml normal saline	110.12±4.28	1.48±0.081
ISO control	2 ml normal saline	246.23±7.43^a	0.89±0.043^a
Inderal*10+ISO	10 mg	125.27±3.72^b	1.21±0.036^b
Drst-T+ISO	2 ml	147.28±2.89^b	1.12±0.048^b
Drst-M+ISO	2 ml	149.57±3.54^b	1.10±0.037^b
marketed Drst+ISO	2 ml	149.13±4.27^b	1.11±0.059^b

All values are expressed as mean ± standard error mean (n = 6).

a P<0.001 significant as compared to normal control

b P<0.001 significant as compared to ISO control

ISO, isoproterenol; MI, myocardial infarction; Drst, Draksharishta

DISCUSSION:

Isoproterenol (ISO), a synthetic catecholamine in higher dose produces cardiotoxic effects on the myocardium. Amongst the various mechanisms proposed to explain ISO-induced cardiac damage, generation of highly cytotoxic free radicals through the auto-oxidation of catecholamines has been implicated as one of the important causative factor²². This free radical mediated lipid per-oxidation of membrane phospholipids and consequent changes in membrane permeability is the primary target responsible for cardio toxicity induced by ISO.

Studies have shown that oxidative stress results in the reduction of the efficacy of the β-adrenoceptor agonists probably due to reduction in c AMP formation. 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 hyper stimulation leads to cardio toxicity²³. Oxidative stress may also depress the sarcolemmal Ca²⁺ transport and result in the development of intracellular Ca²⁺overload and ventricular dysfunction²⁴. Hence, therapeutic intervention with therapeutic activity may be useful in preventing these deleterious changes.

Changes in serum LDH and CK-MB activities have been considered some of the important biomarkers of MI. A significant increase in serum LDH, CK-MB, AST and ALT was observed in ISO control group. Pre-treatment with Draksharishta-T, Draksharishta-M and marketed Draksharishta in ISO-induced MI in albino rats significantly restored serum LDH, CK-MB, AST and ALT activity as compared to the ISO control group was suggestive of their cardio-protective effect.

In ISO control group significant rise in serum lipid profile was also observed. Pre-treatment with Draksharishta-T, Draksharishta-M and marketed Draksharishta for thirty days significantly reduced serum cholesterol, LDL and TG level while showed significant rise in serum HDL level in ISO-induced MI in albino rats. A rise in LDL may cause deposition of cholesterol in the arteries and aorta and hence it is a direct risk factor for coronary heart disease. LDL carries cholesterol from liver to the peripheral cells and smooth muscles and cells of the arteries²⁵. HDL promotes the removal of cholesterol from peripheral cells and facilitates its delivery back to the liver. Therefore, increased levels of HDL are desirable²⁶.

In the ISO control group, a significant increase in heart weight and heart weight to body weight ratio was observed which was reversed by Draksharishta-T, Draksharishta-M and marketed Draksharishta treatment in ISO-induced MI in albino rats. It suggests the cardio-protective property of all these test formulations.

In the current investigation, ISO-induced MI produced oxidative stress as indicated by increased heart lipid peroxides as MDA and decreased heart GSH content. Pre-treatment with Draksharishta-T, Draksharishta-M and marketed Draksharishta significantly reduced heart lipid peroxides level as MDA and showed significant rise in GSH content in ISO-induced MI in albino rats. Thus, all the test formulations as Draksharishta-T, M and marketed Draksharishta maintained membrane integrity as evidenced by decline in cardiac MDA levels.

In summary, the present study strongly suggests that multiple mechanisms may be responsible for the cardio-protective effect of Draksharishta-T, Draksharishta-M and marketed Draksharishta. All these test formulations as Draksharishta-T, Draksharishta-M and marketed Draksharishta produced myocardial adaptive changes (augmentation of endogenous antioxidants as GSH) on chronic administration. In addition, they restored the integrity of the myocardium, subsequent to ISO-induced oxidative stress. Draksharishta contains rich concentration of polyphenolic compounds which possess good antioxidant activity. Thus, the obtained result suggests that presence of self generated alcohol could be beneficial in the faster absorption of polyphenolic compounds present in Draksharishta which might be responsible for showing scavenging of ISO-induced free radicals.

Thus, the present study provides scientific basis for the cardio protective potential of Draksharishta validating their usage in Ayurveda. Considering its safety, efficacy and traditional acceptability, clinical trials should be conducted to support its therapeutic use in ischemic heart diseases.

REFERENCES:

1. Bolli R. Myocardial ischemic metabolic disorder leading to cell death. Reviews of Postgraduate Cardiology 1994; 13:649-53.

2. Dhar ML, Dhar MM, Dhawan BN, Ray C. Screening of Indian plants for biological activity. Journal of Experimental Biology 1968; 6:232-47.

3. Hertog MGL, Feskens EJM, Hollam PCH, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart diseases. Lancet 1993;342:1007-20.

4. The Ayurvedic Formulary of India, Part-I. 2000, 1^st edition, The Controller of Publications, Delhi, 15-16.

5. Baydar NG, Ozkan G and Sagdic O. Total phenolic contents and antibacterial activities of grapes (Vitis vinifera L.) extracts. Food Control 2004; 15: 335-339.

6. Frankel EN, Kanner J, German JB, Parks E and Kinsella JE. Inhibition of oxidation of human low density lipoprotein by phenolic substances in red wine. The Lancet 1993; 341(20): 454-457.

7. Mayer AS, Yi OS, Person DA, Waterhouse DL and Frankel EN. Inhibition of human low density lipoprotein oxidation in relation to composition of phenolic antioxidants in grapes (Vitis vinifera). Journal of Agriculture and Food Chemistry 1997; 45: 1638-1643.

8. Teissedre PL, Frankel EN, Waterhouse AL, Peleg H and German GB. Inhibition of in vitro human LDL oxidation by phenolic antioxidants from grapes and wines. Journal of Science, Food and Agriculture 1996; 70: 55-61.

9. Waterhouse AL. Wine antioxidants may reduce heart disease and cancer. Presentation of American Chemical Society, Washington; 1994.

10. Mishra S. Bhaisazya Kalpana Vigyan. Varanasi, India: Chaukambha Surbharati Prakashan; 2005.p. 253-254.

11. Alam M, Radhamani S, Ali U and Purushottam KK. Microbiological Screening of Dhataki flowers. Journal of Research in Ayurveda and Siddha 1984; 2(4):371-375.

12. Rona G, Chapel CI, Balazs T, Gaudry R. An infarct like myocardial lesion and other toxic manifestations produced by isoproterenol in the rat. Archives of Pathology 1959;76:443-55.

13. Tripathi KD. Essentials of Medical Pharmacology. 6th ed. New Delhi (India): Jaypee Brothers Medical Publishers Limited; 2008. p. 137-8, 537.

14. Varley H. Practical Clinical Biochemistry. 4th ed. NY: William Heinemann; 1967.p. 161-2.

15. Lamprecht W, Stan F, Weisser H, Heinz F. Determination of creatine phosphate and adenosine triphosphate with creatine kinase. In: Methods of Enzymatic analysis. Ed. HU Vergmeyer. NY: Academic Press; 1974. 1776-8.

16. Mohun AF, Cook IGY. Simple methods for measuring serum levels of Glutamic oxaloacetic and Glutamic pyruvic transaminases in routine laboratories. Journal of Clinical Pathology 1957;10 (4):394-9.

17. Allain CC, Pool LS, Chan CS, Richmond W. Enzymatic determination of serum cholesterol. Clinical Chemistry 1974;20:447-75.

18. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the Concentration of Low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clinical Chemistry 1972;18 :499-502.

19. Muller PH, Schmulling RM, Liebich HM, Eggstein M. A fully enzymatic triglyceride determination. Journal of Clinical Chemistry 1977;15:457-64.

20. Ohkawa H, Ohisi N, Yagi K. Assay for lipid peroxides in animal tissue by thiobarbituric acid reaction. Analytical Biochemistry 1979;95:351-8.

21. Ellman GL. Tissue Sulphydril groups. Archives of Biochemistry & Biophysics 1959;82:72-7.

22. Nirmala C, Puvanakrishnan R. Isoproterenol induced myocardial infarction in rats; functional and biochemical alterations. Medicine Science and Research 1994;22:575-7.

23. Haenen GR, Veerman M, Bast A. Reduction of beta adrenoceptor functions by oxidative stress in heart. Free Radical Biology and Medicine 1990;9:279-88.

24. Tappia PS, Heta T, Dhalla NS. Role of oxidative stress in catecholamine induced changes in cardiac sarcolemmal Ca²⁺ transport. Archives of Biochemistry and Biophysics 2001;377:85-92.

25. Pederson TR. Low density lipoprotein cholesterol lowering is and will be the key to the future of lipid management. American Journal of Cardiology 2001;87(5A):8B-12B.

26. Bolden WE, Pearson TA. Raising low levels of High density lipoprotein cholesterol is an important target of therapy. American Journal of Cardiology 2000;85(5):645-50.

Received on 18.05.2014 Modified on 12.06.2014

Research J. Science and Tech. 6(3): July- Sept., 2014; Page 151-155