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Effect of Calotropis procera latex on isoproterenol induced myocardial infarction in albino rats.

Summary

The alcoholic extract of the latex obtained from Calotropis procera (Asclepidaceae) was evaluated for protection against isoproterenol (20 mg/100g body wt., s.c.)-induced myocardial infarction in albino rats. The heart damage induced by isoproterenol was indicated by elevated levels of the marker enzymes such as Creatine Kinase-isoenzyme (CK-MB), Lactate dehydrogenase (LDH), Serum Glutamate Oxaloacetic Transaminase (SGOT) and Serum Glutamate Pyruvate Transaminase (SGPT) in serum with increased lipid peroxide and reduced glutathione content in heart homogenates. Microscopical examination (histopathology) was also performed on the myocardial tissue. Pretreatment with an ethanolic latex extract of Calotropis procera at a dose of 300 mg/kg body wt., administered orally thrice a day for 30 days, reduced significantly (p < 0.01) the elevated marker enzyme levels in serum and heart homogenates in isoproterenol-induced myocardial infarction. Histopathological observation revealed a marked protection by the extract in myocardial necrotic damage.

Key words: Calotropis procera, cardioprotective, isoproterenol, marker enzymes

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Introduction

Calotropis procera Ait. R.Br. (Asclepediacae) is a xerophytic shrub is distributed widely throughout Asia and Africa. It has a considerable reputation in India as potent medicine in the treatment of leucoderma, leprosy, ulcers, tumors, piles, diseases of the spleen, liver and abdomen (Kirthikar and Basu, 1933). The plant has been investigated phytochemically for cardenolides, anthocyanins, hydrocarbon and triterpenoids (Moscolo et al. 1988) and has been reported to show a number of pharmacological activities such as cardiotonic, hepatoprotective, antimicrobial and anticancer (Moscolo et al. 1988) as well as antifertility (Kamath and Rana, 2002). No reports exist, however as to anti-myocardial-infarction activity.

The present study was designed to determine the protective effect of an ethanolic extract of latex from Calotropis procera (CPLA) against isoproterenol-induced myocardial infarction.

Materials and Methods

Plant material

Latex from Calotropis procera was collected at midday from the forests in the Yelagiri hills, Tamil Nadu, India and the plant was authenticated by the Department of Botany, N.M.K R.V. College, Bangalore, India, where a voucher sample has been deposited.

Sun-dried latex was ground to a coarse powder (100 g) and macerated with 1000 ml of 70% ethanol for 72 h with occasional stirring. The extract (CPLA) obtained was concentrated to dryness (yield 8.4%, w/w). Different concentrations of CPLA were prepared by using 1% v/v tween 80 in distilled water.

Animals

Adult albino rats of either sex weighing 200-250 g each were used for the study. They were fed with commercial diet (Hindustan Lever Limited, India) and given water ad libitum, and maintained in clean, polypropylene cages at 25 [degrees]C with relative humidity.

Anti-myocardial-infarction activity

Rats of either sex were divided into 5 groups (n = 6). Group I received distilled water and served as normal, Group II received 1% v/v tween 80 solution and served as control; Groups III, IV, V and VI received 100 mg/kg, 200 mg/kg, 300 mg/kg and 400 mg/kg body wt., of CPLA, respectively, three times a day for 30 days. At the end of the treatment period, animals of all groups were administered with isoproterenol at a dose of 20mg/100g body wt., subcutaneously, twice, at an interval of 24 h (Sheela and Shyamaladevi, 2000).

Estimation of serum enzyme levels in rats

After experimental period blood was withdrawn from the retro orbital sinus, the serum was separated by centrifugation and used for the estimation of marker enzymes CK-MB (Okinaga et al. 1961), LDH (King, 1965), SGOT and SGPT (Reitman and Frankel, 1975).

Estimation of lipid peroxide and glutathione content in heart homogenates

Hearts of the animals of all the groups removed by dissection, and three hearts from each group were chosen for lipid peroxide content (Okhawa et al. 1979) and glutathione content (Moron et al. 1979) measurements. The protein content was determined via the method described by Lowry et al. (1951).

Histopathological studies

The remaining three myocardial tissues from all the groups were subjected to histopathological studies as described by Luna et al. (1960). The tissues were fixed using a 10% formalin solution in a phosphate buffer, and sections were prepared using paraffin blocks and stained with heamatoxylin and eosin after dewaxing. The sections were observed for histopathological changes.

Statistical tests

The student t test was used to determine significant intergroup difference.

Results

Effect of CPLA on serum enzyme levels in rats

Rats administered with isoproterenol showed a significant increase in the levels of CK-MB, LDH, SGOT and SGPT relative to serum of control group animals. Pretreatment with CPLA extract at a dose of 200 mg/kg body wt., p.o., significantly lowered (p < 0.01) isoproterenol-induced elevated levels, whereas the extract at a dose of 300 mg/kg body wt., decreased the serum levels of CK-MB and LDH enzymes (Table 1, Fig. 1, 2).

Effect of CPLA on enzyme levels in heart homogenates

In isoproterenol-induced myocardial damage, the level of lipid peroxide was increased and glutathione content was lowered. Rats pretreated with CPLA, at doses of 400 mg/kg and 300 mg/kg body wt. showed reduced lipid peroxide levels and steady glutathione content respectively (Table 2, Fig. 3).

[FIGURE 1 OMITTED]

Effect of CPLA on histopathology of heart

Microscopic examination of heart sections of rats treated with isoproterenol revealed extensive and diffuse vacuolar degeneration of the myocardial bundles and leucocytic infiltration. Rats pretreated with CPLA extract at doses of 200 mg/kg body wt. and further showed dose-dependent protection characterized by less leucocytic infiltration and degeneration of myofibrillar tissues.

Discussion

It is evident from the present study that the alcoholic extract of latex of Calotropis procera possesses anti-myocardial-infarction activity. Animals pretreated with extract at doses of 200 mg/kg and 300 mg/kg (body wt.) showed significantly (p < 0.01) lowered SGOT, SGPT, LDH and CK-MB enzyme levels in serum and these marker enzymes are useful in diagnosing myocardial infarction (Witteween et al. 1975). The induction of myocardial infarction in experimental animals by isoproterenol is probably be due to action on the sarcolemmal membrane, stimulation of adenylate cyclase, activation of [Na.sup.+] and [Ca.sup.+] channels, exaggerated [Ca.sup.+] inflow and energy consumption leading to cellular death (Milei et al. 1978). Furthermore, an increase in the level of marker enzymes in serum could be due to the leakage of enzymes from the heart as a result of isoproterenol-induced necrosis (Sheela and Shyamaladevi, 2000). The mechanism of the extract may operate via decreasing [Ca.sup.+] inflow and preventing the leakage of enzymes by reducing myocardial damage, but the exact mechanism of the protective action of this extract is not yet clearly known.

[FIGURE 2 OMITTED]

Free radicals are implicated in an increasing large number of diseases and are reported to have a deleterious effect on heart function (Ajitha and Rajnarayana, 2001). An increased level of lipid peroxides in the heart following isoproterenol administration indicates enhanced lipid peroxidation by free radicals (Sushmakumari et al. 1989). Due to this increased lipid peroxidation, glutathione levels are lowered (Flohe, 1989). CPLA pretreatment decreases lipid peroxide and maintained glutathione levels. In our earlier unpublished findings, CPLA extract showed in vitro free-radical scavenging activity. Hence, the possible mechanism may be due to an antioxidant property of extract.

[FIGURE 3 OMITTED]

It has been reported that the myocardial lesions in rats induced by isoproterenol is due to increase of calcium inflow (Milei et al. 1978). The CPLA extract enhances the competence of myocytolysis in rats to tolerate repeated challenges of isoproterenol Furthermore, histopathological observations revealed that the extract prevented the degeneration of myofibrillar tissue and leucocytic infiltration in myocardial necrosis.

The CPLA extract used in the present investigation was in crude form and likely to contain compounds such as anthocyanins, sterols, glycosides and alkaloids, which have been isolated from this plant (Moscolo et al. 1988). At this stage it is not possible to determine which of these compounds are responsible for protective action in the heart. But this action can be attributed to one of the above mentioned compounds. Experiments are underway to find the actual compounds responsible for the antimyocardial infarction activity. In conclusion, the results obtained from our study indicate that rats pretreated with CPLA extract are significantly protected from myocardial damage caused by isoproterenol.
Table 1. Effect of extract on SGOT, SGPT, LDH AND CK-MB enzyme levels in
serum of albino rats.

Groups Dose SGOT
 (body wt.) Mean [+ or -] SEM

Normal -- 68.76 [+ or -] 6.58
Control + isoproterenol -- 272.54 [+ or -] 2.35#
CPLA + isoproterenol 100 mg/kg 112.41 [+ or -] 4.81*
CPLA + isoproterenol 200 mg/kg 106.07 [+ or -] 4.56**
CPLA + isoproterenol 300 mg/kg 96.2 [+ or -] 2.45**
CPLA + isoproterenol 400 mg/kg 95.87 [+ or -] 3.2**

Groups SGPT LDH
 Mean [+ or -] SEM Mean [+ or -] SEM

Normal 32.15 [+ or -] 2.15 88.32 [+ or -] 2.41
Control + isoproterenol 212.19 [+ or -] 4.65# 180.52 [+ or -] 5.25#
CPLA + isoproterenol 62.57 [+ or -] 4.89* 165.51 [+ or -] 4.5
CPLA + isoproterenol 58.56 [+ or -] 6.61** 110.41 [+ or -] 2.58**
CPLA + isoproterenol 55.17 [+ or -] 2.29** 105.62 [+ or -] 1.21**
CPLA + isoproterenol 49.55 [+ or -] 2.51** 98.51 [+ or -] 2.51**

Groups CK-MB
 Mean [+ or -] SEM

Normal 545.6 [+ or -] 54.6
Control + isoproterenol 867.7 [+ or -] 42.5#
CPLA + isoproterenol 722.15 [+ or -] 41.65
CPLA + isoproterenol 601.5 [+ or -] 34.1**
CPLA + isoproterenol 598.6 [+ or -] 42.1**
CPLA + isoproterenol 585.4 [+ or -] 41.5**

Values are represented as IU/ml, except CK-MB, expressed as IU/L, and
values are expressed as Mean [+ or -] SEM.
** p < 0.01, * p < 0.05 significantly different from isoproterenol
group; # Significantly different from normal group.

Table 2. Effect of extract on lipid peroxide and glutathione content in
heart homogenates of albino rats.

 Lipid peroxide
 Dose content Glutathione Content
Groups (body wt.) Mean [+ or -] SEM Mean [+ or -] SEM

Normal -- 5.82 [+ or -] 3.2 2.66 [+ or -] 0.23
Control +
 isoproterenol -- 18.26 [+ or -] 2.20# 1.49 [+ or -] 0.11#
CPLA +
 isoproterenol 100 mg/kg 16.21 [+ or -] 1.81 1.52 [+ or -] 0.12
CPLA +
 isoproterenol 200 mg/kg 11.62 [+ or -] 2.12 1.85 [+ or -] 0.16
CPLA +
 isoproterenol 300 mg/kg 10.63 [+ or -] 1.90 1.89 [+ or -] 0.25**
CPLA +
 isoproterenol 400 mg/kg 9.82 [+ or -] 1.2* 1.92 [+ or -] 0.32**

Values are represented as nanomoles/gram of protein and expressed as
Mean [+ or -] SEM.
** p < 0.01, * p < 0.05 significantly different from isoproterenol
group; # Significantly different from normal group.


References

Ajitha M, Rajnaryana K (2001) Role of oxygen free radicals in Human Disease. Indian Drugs 38(11): 545-554

Flohe L (1989) Glutathione Chemical, Biochemical and Medical Aspects (eds Dolphin Poulson DR, Avramsvic O): 634-731. Wiley, New York

Kamath JV, Rana AC (2002) Preliminary study on antifertility activity of Calotropis procera roots in female rats. Fitoterap 73(2): 111-115

King J (1965) Practical Clinical enzymology. Van Nostrand Co. Ltd, London

Kirtikar KR, Basu BD (1935) Indian Medical Plants (Volume 3): 1606. Lalit Mohan, Allahabad

Lowry OH, Rosebrough NJ, Farr A, Ranwill R (1951) Protein determination with folin reagent. J Biol Chem 195: 133

Luna LC (1960) Manual of histological screening methods of armed forces institute of pathology. Ed. 125. Mc. Graw Hill book Co., New York

Mascolo N, Sharma R, Jain SC. Capassa F (1988) Ethnopharmacology of Calotropis procera. J Ethnopharmacol 22: 211-221

Milei J, Nunez RG, Rapaport M (1978) Pathogenesis of isoproterenol induced lesions in the rat myocardium. Cardiol 63(3): 139

Moron MS, Bepierre JW, Mannerwick B (1979) Levels of glutathione, glutathione reductase, glutathione-s-reductase in rat lung and liver. Biochem Biophys Acta 95: 351

Okhawa H, Ohishi N, Yogi K (1979) Assay for lipid Peroxides in animal tissue with thiobarbituric acid reaction. Anal Biochem 95: 351

Okinaga S, Kumugai HE, Sugaita M, Momo (1961) Serum creatine phosphokinase activity in progressive muscular dystrophy and neuromuscular disease. Arch Neurol Biol 4: 520-526

Reitman S, Frankel S (1975) In vitro determination of transaminase activity in serum. Am J Clin Path 28: 56

Sheela Sasikumar C, Shyamaladevi CS (2000) Protective effect of abana. A Polyherbal formulation on isoproterenol induced myocardial infarction in rats. Indian J Pharmacol 32: 198-201

Sushmakumari S, Jayadeep A, Sureshkumar S, Venugopal P (1989) Effect of carnitine of malionaldehyde, taurine and glutathione levels in hearts of rats subjected to myocardial stress by isoproterenol. Indian J Exp Biol 27: 234-237

Witteween Sagi AGJ, Hemker HC, Hollar L, Hermens W (1975) The quantitation of infarct size in man by means of plasma enzyme levels. British Heart Journal 37: 795

K. K. Mueen Ahmed (1), A. C. Rana (2) and V. K. Dixit (2)

(1) Dept. of Pharmacognosy, Govt. College of Pharmacy, Bangalore, India

(2) Dept. of Pharmaceutical Sciences, Dr. H.S. Gour Vishwavidyalaya, Sagar, India

Address

K. K. Mueen Ahmed, Dept. of Pharmacognosy, Hosur Road, Bangalore-560 027, India.

Phone: 91-984 5655 732; e-mail: makky@rediffmail.com
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Author:Ahmed, K.K. Mueen; Rana, A.C.; Dixit, V.K.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:9INDI
Date:Apr 1, 2004
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