Cardioprotective Activity of Amritarishta on Isoproterenol Induced Myocardial Infarction

Preeti Tiwari*

Head of Department of Pharmacognosy, Dr K. N. Modi Institute of Pharmaceutical Education and Research, Modinagar (U. P.)

ABSTRACT:

The present study was designed to evaluate the cardio protective activity of Amritarishta-T, Amritarishta-M prepared by traditional and modern methods respectively and its marketed preparation on isoproterenol (ISO) induced myocardial infarction (MI) in albino rats. Wistar albino rats of either sex were randomly divided into 06 groups comprising 06 animals in each group as normal control, ISO control, pretreatment with Inderal10 (10 mg/kg) per os, pretreatment with Amritarishta-T, M and its marketed preparation at the dose of 2 ml/kg per os per day for 30 days. MI was induced in all the groups except normal control, by administering ISO (85 mg/kg) intraperitoneally, on 29th and 30th day. On 31st day, level of serum marker enzymes was determined and serum lipid profile was also measured. Then, animals were subsequently sacrificed, hearts were removed, weighed and immediately processed for biochemical studies. Pretreatment with Inderal10 and all the test preparations of Amritarishta significantly prevented the ISO-induced adverse changes in the levels of serum marker enzymes as creatine kinase (CK-MB), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and also improved serum lipid profile. All the test formulations pretreated groups showed significant increase in glutathione (GSH) content and significantly reduced malonyldialdehyde (MDA). Thus, experimental finding suggests that the cardio protective activity of Amritarishta-T, M and its marketed preparation may be due to an augmentation of endogenous antioxidants as GSH and inhibition of lipid peroxidation of cardiac membrane.

KEYWORDS: Myocardial infarction, Isoproterenol, Amritarishta

1. 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.

Amritarishta is a polyherbal hydroalcoholic Ayurvedic preparation and is used as antioxidant and advised as a choice of remedy in mostly all types of fevers⁴. The chief ingredient of Amritarishta is guduchi, dried stem of Tinospora cordifolia.

The chemical constituents reported from stems of Tinospora cordifolia belong to different classes such as alkaloids as tinosporin^5-6, glycosides as cordifoliosides-A and cordifolioside-B^7-8, steroids as β- sitosterol⁹, sesquiterpenoid as tinocordifolin¹⁰ and a large amount of phenolic compounds as gallic aciod, ellagic acid, catechin and epicatechin¹¹. These compounds have many notable medicinal properties as antidiabetic¹², hepatoprotective¹³, antioxidant¹⁴, antimalarial¹⁵, immunomodulatory¹⁶ and antineoplastic properties¹⁷.

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

2. MATERIALS AND METHODS:

2.1 Preparation of Amritarishta-T:

This was prepared by the method as given in The Ayurvedic Formulary of India, Part-I⁴. All the ingredients of Amritarishta were procured from local market, Jamnagar while jaggery was procured from local market, Mehsana. Authentication of all the ingredients of Amritarishta 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 3.072 l of Amritarishta has been calculated according to the formula as given in The Ayurvedic Formulary of India, Part-I, 2000.

According to this method, coarsely powdered stems of guduchi (Tinospora cordifolia) with prescribed ingredients as Aegle marmelos (stem bark), Oroxylum indicum (roots), Gmelina arborea (stem bark), Stereospermum suaveolns (stem bark), Premna integrifolia (stem bark), Hedysarum gangeticum (entire plant), whole plant of Paederia foetida, entire plant of Solanum indicum, entire plant of Solanum xanthocarpum and Tribulus terrestris were placed in polished vessel of brass along with prescribed quantity of water (12.288l) and allowed to steep. After 12 h of steeping, this material was warmed at medium flame until the water for decoction reduced to one fourth of the prescribed quantity(3.072 l) , then the heating was stopped and it was filtered in cleaned vessel and after that jaggery was added and mixed properly. Then, prakshepa dravyas as svet jiraka, raktapuspaka, saptaparni, sunthi, marica, pippali, nagakesara, mustaka, katuka, ativisa and indravaruni in fine powdered form were added and this sweet filtered material was placed for fermentation in incubator for fifteen days at 33±1°C. After 15 days completion of fermentation was confirmed by standard tests¹⁸. The fermented preparation was filtered with cotton cloth and kept in clean covered vessel for further next seven days. Then, when the fine suspended particles settled down, it is strained again and poured in amber colored glass bottles previously rinsed with ethyl alcohol, packed and properly labelled.

2.2 Preparation of Amritarishta-M:

Method of preparation of Amritarishta-M was same as followed for Amritarishta-T only in addition to jaggery, yeast was also added for inducing fermentation¹⁹.

2.3 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 (22ºC±2ºC) 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 Experimentals on Animals (CPCSEA) of the Government of India were followed and prior permission was granted from the Institutional Animals Ethics Committee (CPCSEA No. 07/09).

2.4 Experimental procedure

The cardio protective effect of Amritarishta-T, Amritarishta-M and marketed Amritarishta was determined on ISO-induced MI in albino rats²⁰. 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 day²¹ and with Amritarishta-T, Amritarishta-M and marketed Amritarishta 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.

2.5 Biochemical analysis of serum

The separated serum was analysed for various serum marker enzymes as lactate dehydrogenase²², creatine kinase²³, alanine aminotransferase and aspartate aminotransferase²⁴. Serum was also assessed for lipid profile as serum cholesterol²⁵, serum HDL and LDL²⁶ and triglycerides²⁷. Span and Erba diagnostic kits were used for the measurement of all these serum marker enzymes.

2.6 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 malonyldialdehyde²⁸ and glutathione²⁹.

2.7 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.

3. RESULTS:

The effects of pretreatment of Amritarishta-T, Amritarishta-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 Fig.1(A) and Fig. (B). 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 Amritarishta-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 Amritarishta also showed similar effects on serum LDH, CK-MB, AST and ALT nearby same as produced by Amritarishta-T and M in ISO- induced MI in albino rats.

Pretreatment with all the test preparations of Amritarishta significantly improved serum lipid profile in ISO- induced MI in albino rats as compared to ISO-control group as shown in Fig. 2. Pretreatment with Amritarishta-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.

Amritarishta-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 Fig.3 (A) and Fig.3 (B) respectively.

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 Amritarishta-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 Fig. 4(A) and Fig. 4(B) respectively.

Groups

Fig. 1(A) Effect of Amritarishta-T, M and marketed preparation on serum LDH and CK-MB

All values are expressed as mean ±SEM (n = 6); ISO, isoproterenol

a P<0.001 significant as compared to normal

b P<0.001 significant as compared to ISO control

Groups

Fig. 1(B) ) Effect of Amritarishta-T, M and marketed preparation on serum ALT and AST

All values are expressed as mean ±SEM (n = 6); ISO, isoproterenol

a P<0.001 significant as compared to normal

b P<0.001 significant as compared to ISO control

Groups

Fig.4(A) ) Effect of Amritarishta-T, M and marketed preparation cardiac MDA of ISO induced MI in albino rats

All values are expressed as mean ±SEM (n = 6); ISO, isoproterenol

a P<0.001 significant as compared to normal

b P<0.001 significant as compared to ISO control

Groups

Fig.4(B) Effect of Amritarishta-T, M and marketed preparation cardiac GSH content of ISO induced MI in albino rats

All values are expressed as mean ±SEM (n = 6); ISO, isoproterenol

a P<0.001 significant as compared to normal

b P<0.001 significant as compared to ISO control

4. 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 factor³⁰. 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 toxicity³¹. Oxidative stress may also depress the sarcolemmal Ca²⁺ transport and result in the development of intracellular Ca²⁺overload and ventricular dysfunction³². 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 Amritarishta-T, Amritarishta-M and marketed Amritarishta 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 Amritarishta-T, Amritarishta-M and marketed Amritarishta 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 arteries³³. HDL promotes the removal of cholesterol from peripheral cells and facilitates its delivery back to the liver. Therefore, increased levels of HDL are desirable³⁴.

In the ISO control group, a significant increase in heart weight and heart weight to body weight ratio was observed which was reversed by Amritarishta-T, Amritarishta-M and marketed Amritarishta 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 Amritarishta-T, Amritarishta-M and marketed Amritarishta 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 Amritarishta-T, M and marketed Amritarishta 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 Amritarishta-T, Amritarishta-M and marketed Amritarishta. All these test formulations as Amritarishta-T, Amritarishta-M and marketed Amritarishta 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. Amritarishta mainly contains withanolides and the 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 Amritarishta which might be responsible for showing scavenging of ISO-induced free radicals.

The present study provides scientific basis for the cardio protective potential of Amritarishta 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.

5. REFERENCES:

1. Bolli R. Myocardial ischemic metabolic disorder leading to cell death. Rev Post Cardiol 1994; 13:649-53.

2. Dhar ML, Dhar MM, Dhawan BN, Ray C. Screening of Indian plants for biological activity. J Exp Biol 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, 6.

5. Kumar S, Verma NS, Pande D and Srivastava PS. In vitro regeneration and screening of berberinein Tinospora cordifolia. Journal of Medicinal and Aromatic Plant Science 2000;22:61.

6. Biset NG and Nwaiwu J.Quaternary alkaloids of Tinospora species. Planta Medica 1983;48:275-9.

7. Maurya R, Wazir V, Tyagi A and Kapil RS. Cordifoliosides A and B, two new phenylpropene disaccharides from Tinospora cordifolia possessing immunostimulant activity. Natural Product Letter 1996;8:7-10.

8. Gangan VD, Pradhan P, Sipahimalani AT and Banerji A. Cordifoliosides A, B,C:Norditerpene furan glycosides from Tinospora cordifolia. Phytochemistry 1994; 37:781-6.

9. Dixit SN and Khosa RL. Chemical investigation of Tinospora cordifolia. Indian Journal of Applied Chemistry 1971;34:46-7.

10. Maurya R and Handa SS. Tinocordifolin, a sesquiterpene from Tinospora cirdifolia. Phytochemistry 1998;49:1343-6.

11. Kidwai AR, Salooja KC, Sharma VN, Siddiqui S. Chemical examination of Tinospora cordifolia. Journal of Science and Indian Research 1949; 8:115-8.

12. Stanely M, Prince P and Menon VP. Antioxidant action of Tinospora cordifolia root extract in alloxan diabetic rats.Phytotherapy Research 2001; 15:213-8.

13. Mehrotra R, Katiyar CK and Gupta AP. Hepatoprotective compositions and composition for treatment of conditions related to hepatitis-B and E infection. US Patent 749296. 2000.

14. Prince PS and Menon VP. Antioxidant activity of Tinospora cordifolia roots in experimental diabetes. Journal of Ethnopharmacology 1999; 65:277-81.

15. Ikram M, Khattak SG and Gilani SN. Antipyretic studieson some indigenous Pakistani medicinal plants. Journal of Ethnopharmacology 1987; 19:185-92.

16. Manjrekar PN, Jolly CI and Narayanan S. Comparative studies of immunomodulatory activity of Tinospora cordifolia and Tinospora sinensis. Fitoterapia 2000; 71:254-7.

17. Jagetia GC, Nayak V and Vidyasagar MS. Evaluation of the antineoplastic activity of guduchi (Tinospora cordifolia) in cultured HeLa cells. Cancer Letter 1998;127:71-82.

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

19. 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.

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

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

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

23. 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. p. 1776-8.

24. Mohun AF, Cook IGY. Simple methods for measuring serum levels of Glutamic oxaloacetic and Glutamic pyruvic transaminases in routine laboratories. J Clin Pathol 1957;10 (4):394-9.

25. Allain CC, Pool LS, Chan CS, Richmond W. Enzymatic determination of serum cholesterol. Clin Chem 1974;20:447-75.

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

27. Muller PH, Schmulling RM, Liebich HM, Eggstein M. A fully enzymatic triglyceride determination. J Clin Chem 1977;15:457-64.

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

29. Ellman GL. Tissue Sulphydril groups. Arch Biochem Biophys 1959;82:72-7.

30. Nirmala C, Puvanakrishnan R. Isoproterenol induced myocardial infarction in rats; functional and biochemical alterations. Med Sci Res 1994;22:575-7.

31. Haenen GR, Veerman M, Bast A. Reduction of beta adrenoceptor functions by oxidative stress in heart. Free Radic Biol Med 1990;9:279-88.

32. Tappia PS, Heta T, Dhalla NS. Role of oxidative stress in catecholamine induced changes in cardiac sarcolemmal Ca²⁺ transport. Arch Biochem Biophys 2001; 377:85-92.

33. Pederson TR. Low density lipoprotein cholesterol lowering is and will be the key to the future of lipid management. Am J cardiol 2001; 87(5A):8B-12B.

34. Bolden WE, Pearson TA. Raising low levels of High density lipoprotein cholesterol is an important target of therapy. Am J cardiol 2000;85(5):645-50.

Received on 01.12.2013

Modified on 12.12.2013

Accepted on 14.12.2013

Research J. Pharmacology and Pharmacodynamics. 5(6): November –December 2013, 356-361