Experimental and Toxicologic Pathology 62 (2010) 61–74 Cardioprotective effect of ‘Khamira Abresham Hakim Arshad Wala’ a Unani formulation in isoproterenol-induced myocardial necrosis in rats Sameer Goyal a , Mohammed Khalid Siddiqui b , Khalid Mehmood Siddiqui b , Sachin Arora a , Rajan Mittal c , Sujata Joshi a , Dharamvir Singh Arya a, a Department of Pharmacology, All India Institute of Medical Sciences, New Delhi-110029, India b Central Council for Research in Unani Medicine, Ministry of Health and Family Welfare, Government of India, New Delhi-110058, India c Department of Pharmacology, Postgraduate Institute of Medical Education & Research, Chandigarh-160012, India Received 20 November 2008; accepted 7 February 2009 Abstract The present study was designed to investigate whether Khamira Abresham Hakim Arshad Wala (KAHAW), a preparation of Unani System of Medicine, is able to attenuate the isoproterenol (ISO)-induced myocardial necrosis on the basis of its effects on hemodynamic, antioxidant, histopathological and ultrastructural parameters. Male Wistar albino rats were administered KAHAW (200, 400 and 800 mg/kg/day, orally) or vehicle for 14 days with concurrent ISO administration (85 mg/kg, subcutaneously, 2 doses at 24 h interval) on 13th and 14th day. On the 15th day, vehicle+ISO-treated rats exhibit cardiac dysfunctions as indicated by decrease in systolic, diastolic, and mean arterial pressures, reduction in both maximum positive and maximum negative rates of developed left ventricular pressure (7LVdp/dt) and an increase in left ventricular end-diastolic pressure (LVEDP). Biochemical analysis of their heart homogenate presented reduced levels of enzymes viz., superoxide dismutase (SOD), catalase (CAT), lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB) isoenzyme. A marked reduction in reduced glutathione (GSH) levels along with increase in levels of thiobarbituric acid reactive substances (TBARS) was also observed in rat myocardium. Myocardial necrosis, edema and inflammation were evident from the light microscopic and ultrastructural changes. KAHAW at dose of 800 mg/kg/day significantly reversed majority of hemodynamic and antioxidant derangements. The protective role of KAHAW on ISO-induced myocardial necrosis was further confirmed by histopathological and ultrastructural examination. There was no significant change in heart rate in all experimental groups. KAHAW per se groups showed no significant change when compared with vehicle control group. The study results thus demonstrated the cardioprotective potential of KAHAW against ISO-induced myocardial necrosis and associated oxidative stress. r 2009 Elsevier GmbH. All rights reserved. Keywords: Abresham; Unani; Isoproterenol; Cardiotoxicity; Oxidative stress Introduction Myocardial infarction (MI) is an acute condition of necrosis of the myocardium that occurs as a result of ARTICLE IN PRESS www.elsevier.de/etp 0940-2993/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.02.115 Corresponding author. Tel.: +91 11 26594266; fax: +91 11 26584121. E-mail address: [email protected] (D.S. Arya).
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ARTICLE IN PRESS
0940-2993/$ - se
doi:10.1016/j.et
�Correspondfax: +9111 265
E-mail addr
Experimental and Toxicologic Pathology 62 (2010) 61–74
www.elsevier.de/etp
Cardioprotective effect of ‘Khamira Abresham Hakim Arshad Wala’ a
Unani formulation in isoproterenol-induced myocardial necrosis in rats
Sameer Goyala, Mohammed Khalid Siddiquib, Khalid Mehmood Siddiquib,Sachin Aroraa, Rajan Mittalc, Sujata Joshia, Dharamvir Singh Aryaa,�
aDepartment of Pharmacology, All India Institute of Medical Sciences, New Delhi-110029, IndiabCentral Council for Research in Unani Medicine, Ministry of Health and Family Welfare, Government of India,
New Delhi-110058, IndiacDepartment of Pharmacology, Postgraduate Institute of Medical Education & Research, Chandigarh-160012, India
Received 20 November 2008; accepted 7 February 2009
Abstract
The present study was designed to investigate whether Khamira Abresham Hakim Arshad Wala (KAHAW), apreparation of Unani System of Medicine, is able to attenuate the isoproterenol (ISO)-induced myocardial necrosis onthe basis of its effects on hemodynamic, antioxidant, histopathological and ultrastructural parameters. Male Wistaralbino rats were administered KAHAW (200, 400 and 800mg/kg/day, orally) or vehicle for 14 days with concurrentISO administration (85mg/kg, subcutaneously, 2 doses at 24 h interval) on 13th and 14th day. On the 15th day,vehicle+ISO-treated rats exhibit cardiac dysfunctions as indicated by decrease in systolic, diastolic, and mean arterialpressures, reduction in both maximum positive and maximum negative rates of developed left ventricular pressure(7LVdp/dt) and an increase in left ventricular end-diastolic pressure (LVEDP). Biochemical analysis of their hearthomogenate presented reduced levels of enzymes viz., superoxide dismutase (SOD), catalase (CAT), lactatedehydrogenase (LDH), creatine kinase-MB (CK-MB) isoenzyme. A marked reduction in reduced glutathione (GSH)levels along with increase in levels of thiobarbituric acid reactive substances (TBARS) was also observed in ratmyocardium. Myocardial necrosis, edema and inflammation were evident from the light microscopic andultrastructural changes. KAHAW at dose of 800mg/kg/day significantly reversed majority of hemodynamic andantioxidant derangements. The protective role of KAHAW on ISO-induced myocardial necrosis was further confirmedby histopathological and ultrastructural examination. There was no significant change in heart rate in all experimentalgroups. KAHAW per se groups showed no significant change when compared with vehicle control group. The studyresults thus demonstrated the cardioprotective potential of KAHAW against ISO-induced myocardial necrosis andassociated oxidative stress.r 2009 Elsevier GmbH. All rights reserved.
ARTICLE IN PRESSS. Goyal et al. / Experimental and Toxicologic Pathology 62 (2010) 61–7462
imbalance between coronary blood supply and myocar-dial demand (De Bono and Boon, 1992). Oxidativestress produced by free radicals or reactive oxygenspecies (ROS), as evidenced by marked increase inproduction of lipid peroxidative products and transientinhibition of endogenous antioxidant defense such assuperoxide dismutase (SOD), catalase (CAT) andreduced glutathione (GSH) has been shown to underliemyocardial damage during MI (Loper et al., 1961;Padmanabhan and Stanely Mainzen Prince, 2006a;Zhou et al., 2008). Therapeutic intervention via suppres-sion of free radical generation and/or augment endo-genous antioxidant enzymes may attenuate myocardialdysfunction.
Isoproterenol (ISO), a synthetic b-adrenoceptor ago-nist, has been found to induce myocardial injury in ratas a result of disturbance in physiological balancebetween production of free radicals and antioxidativedefense system (Rathore et al., 1998; Zhou et al., 2008).It is the acute condition of myocardial necrosiswhich caused cardiac dysfunctions, increased lipidperoxidation, altered activities of cardiac enzymesand antioxidants (Banerjee et al., 2003; Gupta et al.,2004; Rajadurai and Stanely Mainzen Prince, 2006).The pathophysiological and morphological changesobserved in ISO-treated rats have been similarto those observed in human MI (Nirmala andPuvanakrishnan, 1996; Devika and Stanely MainzenPrince, 2008).
Table 1. Formulation of Khamira Abresham Hakim Arshad Wala
Sr. no. Pharmaceutical names Source Botanic
1. Abresham (silk cocoon) Animal Bombax
2. Marjan (coral) Animal Coralliu
3. Mushk (musk) Animal Moschu
4. Shahed-ponay (honey) Animal Apis me
5. Marwareed (pearl) Animal Mytilus
6 Ambar (ambergris) Animal Physeter
7. Ood Gharqi (eaglewood) Plant Aquilari
8. Sumbal-Teeb (Indian spikenard) Plant Nordost
9. Turanj (citron) Plant Citrus m
10. Qaran fal (clove) Plant Syzygiu
11. Elaichi khurd (cardamom) Plant Elettaria
12. Sandal sufed Plant Santalum
13. Sajaz hindi (bay leaf) Plant Cinnamo
14. Anar (pomegranate) Plant Punica g
15. Bihi (quince) Plant Cydonia
16. Seb (apple) Plant Malus p
17. Zafraan (saffron) Plant Crocus s
18. Mastagi (mastich) Plant Pistacia
19. Keora (screwpine) Plant Pandanu
20. Powder sugar Plant Sacchar
21. Yashab (jade) Mineral Hydrate
22. Yaqoot (ruby, sapphire) Mineral Alumini
Words italicized are in Urdu/Persian and the parentheses include names in E
In Unani system of medicine diseases are treated withnatural resources like plants, animals and minerals.There are cumulative evidences suggesting the modula-tory role of polyherbal formulation in various humandiseases, including cardiovascular diseases (Prince et al.,2008). From ancient times Khamira Abresham HakimArshad Wala (KAHAW) (Table 1), a Unani formula-tion, has been used in the treatment of variouspathological conditions like arrhythmia (Kabeer,1951), atherosclerosis (Safiuddin, 1999), cerebral ische-mia (Yousuf et al., 2005) and cognitive impairment(Khan et al., 2006). To the best of our knowledge,efficacy of KAHAW in ISO-induced myocardial necro-sis and oxidative stress-related parameters has not beenscientifically explored till date. Therefore, the presentstudy was designed to investigate the effects of oraladministration of KAHAW on ISO-induced myocardialnecrosis in rats.
Materials and methods
Drugs and chemicals
Isoproterenol hemisulphate was dissolved in 0.9%saline and used within 10min of preparation. Creatinekinase-MB (CK-MB) isoenzyme detection kit waspurchased from Logotech India Pvt. Ltd. (Delhi, India)
.
al name and family Each 100 g contain (g)
mori L. (Bombycidae) 50.5
m rubrum L. (Gorgonidae) 0.6
s moschiferous L. (Moschidae) 0.5
llifera L. (Apiidae) 15
margaritiferus L. (Pterioidea) 0.9
catodon L. (Physeteridae) 0.6
a agallocha Roxb. (Thymelacaceae) 0.4
achy jatamansi (Valerianaceae) 0.5
edica L. (Rutacea) 0.5
m aromaticum L. (Myrtaceae) 0.5
cardamomum L. (Zingiberaceae) 0.5
album (Santalaceae) 0.6
mum tamala (Lauraceae) 0.5
ranatum L. (Punicacea) 1.4
oblonga (Rosaceae) 1.4
umila (Rosaceae) 1.4
ativus L. (Iridaceae) 0.5
lentiscus L. (Pistaciaceae) 0.5
s tectoriuss (pandanaceae) 0.9
arum officinarum L. (Gramineae) 20.5
d silica 0.9
um oxide 0.9
nglish.
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and KAHAW (Batch no. 230, 01/05) was purchasedfrom Hamdard (wakf) Laboratories (Ghaziabad, UP,India). KAHAW was dissolved in 0.9% saline andvortexed before use. All chemicals used in this studywere of analytical grade and purchased from SigmaChemicals (St. Louis, MO, USA).
Experimental animals
Male Wistar albino rats, weighing 150–200 g, wereobtained from the Central Animal House Facility of AllIndia Institute of Medical Sciences, New Delhi, India.The study protocol was reviewed and approved bythe Institutional Animal Ethics Committee (IAEC,No. 376/2007) and all study related activities conformedto the Indian National Science Academy Guidelines forthe use and care of experimental animals in research.Animals were kept in the departmental animal houseunder controlled conditions of temperature at 2572 1C,relative humidity of 6075% and light–dark cycle of12:12 h. They were fed food pellets (Ashirwad IndustriesLtd., Chandigarh, India) and water ad libitum. Animalswere maintained in polypropylene cages, each contain-ing a maximum of four animals.
Induction of experimental myocardial necrosis
Standardized dose of ISO (85mg/kg, s.c., for 2consecutive days at 24 h interval) was used for theinduction of myocardial necrosis (Mohanty et al., 2004;Loh et al., 2007).
Experimental groups
A total of 128 rats were used for the experimentalstudies. They were randomly divided into eight groups,with 16 rats in each group. Group 1 (vehicle control):rats were administered normal saline orally (1ml/kg/day)using intragastric tube for 14 days and on 13th and 14thday administered 0.3ml saline, s.c. at an interval of 24 h.Groups 2–4 (KAHAW alone): rats were treated orallywith KAHAW (200, 400 and 800mg/kg/day) for aperiod of 14 days and on 13th and 14th dayadministered 0.3ml saline, s.c. at an interval of 24 h.Group 5 (vehicle+ISO-control): rats were administerednormal saline orally (1ml/kg/day) for 14 days alongwith concurrent administration of ISO (85mg/kg, s.c. at24 h interval) on 13th and 14th day. Groups 6–8(KAHAW+ISO): rats were treated with KAHAW(200, 400 and 800mg/kg/day) orally for a period of 14days along with concurrent administration of ISO(85mg/kg, s.c. at 24 h interval) on 13th and 14th day.Changes in body weight, and food and water intakepatterns of animals in all groups were noted throughoutthe experimental period at regular intervals. All the
treatments were given between 10:00 and 12:00 h and therats once used were not reemployed in further experiments.
Surgery for recording hemodynamic parameters
The detailed surgical procedure for recording ofhemodynamic parameters has been described in ourprevious studies (Loh et al., 2007; Nandave et al., 2007).Briefly, rats were anesthetized with sodium pentobarbi-tone (60mg/kg, ip). Atropine (4mg/kg, ip) was adminis-tered along with the anesthetic to reducetracheobronchial secretions. Throughout the experimen-tal protocol body temperature of the animals wasmaintained at 37 1C. The neck was opened with aventral midline incision to perform tracheostomy andanimals were ventilated with room air from a positivepressure ventilator (Inco Pvt. Ltd., Ambala, India) usingcompressed air at a rate of 90 strokes/min and a tidalvolume of 10ml/kg. Ventilator setting and PO2 wereadjusted as needed to maintain the arterial blood gasparameters within the physiological range. The leftjugular vein was cannulated with polyethylene tube(internal diameter 0.28mm; outer diameter 0.35mm)(R. K Ltd., Delhi, India) attached to a three-waycannula (Meditop Corporation (M) Sdn. Bhd, Malay-sia) for continuous infusion of 0.9% saline. The rightcarotid artery was cannulated with polyethylene tube(internal diameter 0.30mm; outer diameter 0.40mm)(R. K Ltd., Delhi, India) attached to a three-waycannula. The cannula was heparinized (Heparin300 IU/ml) and connected to CARDIOSYS CO-101(Experimentria, Budapest, Hungary) system using apressure transducer for the measurement of systolic,diastolic, mean arterial pressures and heart rate. Theheart was exposed through the left fifth intercostal spacewith the help of cervical cautery (Delta MedicalAppliances, Mumbai, India). For the measurement ofleft ventricular hemodynamic variables, a wide bore(1.5mm) sterile metal cannula connected to a pressuretransducer (Gould Statham P23ID, Gould Electronics,Oxnard, CA, USA) was inserted into the left ventricularcavity through the apex of the heart. The left ventricularpressure dynamics were recorded on a polygraph(Model 7D, Grass Instrument Co., Quincy, MA, USA)after 10min of stabilization of blood pressure. Afterrecording of hemodynamic parameters, animals of allgroups were sacrificed with an overdose of anesthesia(sodium pentobarbitone 100mg/kg, intravenously),their hearts were excised and processed for biochemical,histopathological and ultrastructural studies. For bio-chemical analysis, hearts were removed and stored inliquid nitrogen, whereas for light microscopic studieshearts were fixed in 10% buffered formalin. Forultrastructural studies, small pieces of myocardial tissue(approximately 1–2mm in thickness) were immediatelyfixed in ice-cold Karnovsky’s fixative for 10–12 h.
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Biochemical estimation
Processing of heart tissue
The hearts were taken out from liquid nitrogen,weighed and a 10% homogenate was prepared in ice-chilled phosphate buffer (50mM, pH 7.4). Aliquots ofhomogenate were stored to be used for the estimation ofthiobarbituric acid reactive substances (TBARS) andGSH. The remaining homogenate was then centrifugedat 5000 rpm for 20min at 4 1C and supernatant was usedfor the estimation of lactate dehydrogenase (LDH),CK-MB isoenzyme, SOD, CAT and protein.
Reduced glutathione estimation
Myocardial GSH was estimated by the method ofMoron et al. (1979). Briefly, 100 ml tissue homogenatewas mixed with 100 ml of 10% trichloro acetic acid(TCA) and centrifuged at 5000 rpm for 20min. Subse-quently, 0.05ml of supernatant was incubated with areaction mixture containing 3.0ml 0.3M phosphatebuffer (pH 8.4) and 0.5ml DTNB [5,5-dithiobis-(2-nitrobenzoic acid)]. After 10min of incubation theabsorbance was measured at 412 nm using spectro-photometer. GSH content was determined from astandard curve obtained using commercially availablestandard GSH (Sigma Chemicals, USA). Levels of GSHare expressed as mmol/g tissue.
Lipid peroxidation in hearts was determined bymeasuring malondialdehyde (MDA) content (Okhawaet al., 1979). Briefly, 100 ml tissue homogenate wasfurther centrifuged with 100 ml of 10% TCA at5000 rpm, for 20min at 4 1C. Subsequently, 0.2mlhomogenate was mixed with 0.2ml of 8.1% sodiumdodecyl sulfate, 1.5ml of 30% acetic acid (pH 3.5) and1.5ml of 0.8% thiobarbituric acid. The reaction mixturewas heated for 60min at 95 1C and then cooled.After cooling, 1.0ml of distilled water and 5.0ml ofn-butanol:pyridine (15:1 v/v) were added and centri-fuged at 5000 rpm for 20min. The absorbance of thepink colored organic layer was measured at 532 nm.1,1,3,3-tetraethoxypropane (Sigma Chemicals, USA)was used as the standard. MDA contents are expressedas nmol/g tissue.
Catalase estimation
Catalase activity was estimated by the methoddescribed by Aebi (1974). To 50 ml tissue supernatant,1.0ml of 50mM phosphate buffer (pH 7) and 0.1ml of30mM hydrogen peroxide were added and a decrease inabsorbance at 240 nm was measured every 5 s for 30 s.Catalase content is expressed as U/mg protein.
Superoxide dismutase estimation
SOD activity was determined by the method ofMarklund and Marklund (1974). To 100 ml of tissuesupernatant, 2.85ml of 0.1M phosphate buffer (pH 8.4)and 50 ml of 7.5mM pyrogallol were added andabsorbance was measured at 420 nm for 3min at 30 sintervals. SOD levels are expressed as U/mg protein.
Lactate dehydrogenase estimation
Myocardial LDH was estimated by the method ofCabaud and Wroblewski (1958). The reaction mixturecontained 2.90ml of 0.2M Tris-buffer, 10 ml tissuesupernatant, 100 ml of 30mM sodium pyruvate and100 ml of NADH. The rate of change in absorbance wasmeasured at 340 nm for 2min at 30 s intervals. Theoxidation of NADH in the above-mentioned reactionwas proportional to LDH present in the sample. LDHwas determined from a standard curve obtained usingcommercially available LDH (Sigma Chemicals, USA).LDH levels are expressed as IU/mg protein.
Creatine kinase-MB isoenzyme estimation
CK-MB isoenzyme was estimated using a commercialkit from Logotech Kit (Delhi, India). The absorbance ofreaction mixture was measured at 340 nm for 3min at60 s intervals. CK-MB levels are expressed as IU/mgprotein.
Protein estimation
The method of Bradford (1976) was used for thedetermination of protein. To 10 ml of tissue’s super-natant, 100 ml of 1N NaOH and 1ml of Bradfordreagent were added, vortexed and absorbance weremeasured at 595 nm. Protein level was determined usingstandard curve obtained by using known concentrationsof commercially available Bovine Serum Albumin(Sigma Chemicals, USA).
Histopathology
Light microscopic study
The 10% buffer formalin fixed tissues were embeddedin paraffin and serial sections (3 mm thick) were cut usingmicrotome (Leica RM 2125, Germany). Each sectionwas stained with hematoxylin and eosin (H&E). Thesections were examined under the light microscope(Nikon, Tokyo, Japan) and photomicrographs weretaken. Representative area images were captured in animage analysis system. The Image Analyzer consisted ofBX-50 Research Microscope (Olympus, Japan), Coolsnap 10 bit digital camera (Media Cybernetics, USA)and Pentium 4 computer (Compaq, USA) with an imageanalysis software Image plus Pro (Media Cybernetius,USA). At least four hearts from each group wereassessed for light microscopic studies. A minimum of
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10 fields per slide were examined and graded for severityof changes using scores on a scale of severe (+++),moderate (++), mild (+) and nil (�). The pathologistperforming histopathological evaluation was blinded tothe treatment assignment of different study groups.
Transmission electron microscopic study
The Karnovsky’s fixed tissues were washed inphosphate buffer (0.1M, pH 7.4, 6 1C) and post-fixedfor 2 h in 1% osmium tetroxide in the same buffer at4 1C. The specimens were then washed in phosphatebuffer, dehydrated with graded acetone and thenembedded in araldite CY212 to make tissue blocks.Semi-thin (1 mm) as well as ultrathin sections (70–80 nm)were cut by ultramicrotome (Ultracut E, Reichert,Austria). The sections were stained with uranyl acetateand lead acetate and examined under transmissionelectron microscope (Morgagni 268D, Fei Co., TheNetherlands) operated at 60 kV by a morphologistblinded to the groups studied. At least four hearts fromeach group were examined for ultrastructural changes.
Data analysis
The quantitative variables are presented asmean7S.D. Kruskal–Wallis test followed by post-hocNewman–Keul range test was applied to comparehemodynamic and biochemical variables among differ-ent groups. A value of Po0.05 was consideredsignificant.
Results
Mortality
The mortality rate in surgery for recording hemody-namic parameter was 5% due to bleeding or impropercannulation in right carotid artery.
Table 2. Effect of KAHAW on arterial pressure.
Treatment groups SAP (mmHg) DA
Vehicle control 127.50716.35 100
KAHAW (200mg/kg) 126.3373.72 113
KAHAW (400mg/kg) 127.0072.89 113
KAHAW (800mg/kg) 125.0074.2 115
ISO-control 91.00715.96� 76
KAHAW (200mg/kg)+ISO 93.6773.50 78
KAHAW (400mg/kg)+ISO 100.5076.8 85
KAHAW (800mg/kg)+ISO 122.8379.2] 94
SAP: systolic arterial pressure; DAP: diastolic arterial pressure; MAP: mean a
for each group (n ¼ 8/group).�Po0.0001 ISO-control vs. vehicle control.]Po0.001 KAHAW+ISO vs. ISO-control.
Effect of KAHAW on arterial pressure
Table 2 shows the effect of KAHAW on arterialpressure. ISO-control rats showed significantly decreasedsystolic (from 127.50716.35 to 91.00715.96mmHg),diastolic (from 100.6775.9 to 76.00717.69mmHg) andmean (from 109.5975.94 to 80.99716.14mmHg) arterialblood pressures (Po0.0001) as compared to vehicle controlgroup. Although ISO treatment led to increase in heart rate(from 388.67716.58 to 432.83749.55 per min), but theincrease was not found to be statistically significant(P40.05). In the KAHAW+ISO-treated rats, KAHAW(200, 400 and 800mg/kg/day) increased SAP, MAP andDAP dose-dependently in comparison to ISO-controlgroup. KAHAW (800mg/kg/day) significantly increasedsystolic (from 91.00715.96 to 122.8379.2mmHg), dia-stolic (from 76.00717.69 to 94.3373.72mmHg) and mean(from 80.99716.14 to 103.6574.65mmHg) arterial bloodpressures (Po0.001) as compared to ISO-control rats,whereas there was no significant change in heart rate ofKAHAW-treated groups. Administration of KAHAWalone (200, 400 and 800mg/kg/day) did not evoke anysignificant changes in hemodynamic parameters (P40.05).
Effect of KAHAW on left ventricular
contractile functions
Figs. 1–3 show the deleterious effect of ISO on leftventricular end-diastolic pressure (LVEDP), +LVdp/dtand �LVdp/dt, respectively, and their restoration towardsthe control values by KAHAW. ISO administrationresulted in myocardial dysfunction as indicated byincreased LVEDP (from 2.6270.84 to 6.0470.45mmHg),decreased +LVdp/dt (from 2437.57204.78 to 2004.177183.31mmHg/s) and �LVdp/dt (from 2366.67776.92 to1813.33786.12mmHg/s) as compared to vehicle controlrats. The effects were found to be statistically significant(Po0.0001). In the KAHAW+ISO-treated rats, KA-HAW (200, 400 and 800mg/kg/day) increased +LVdp/dtand �LVdp/dt and decreased LVEDP dose-dependently
P (mmHg) MAP (mmHg) HR (per min)
.6775.9 109.5975.94 388.67716.58
.41713.93 117.7179.91 387.3377.2
.66714.93 118.10712.23 395.0076.5
.41711.02 118.60712.78 393.0079.7
.00717.69� 80.99716.14� 432.83749.55
.1779.8 83.1676.9 424.0073.3
.3377.6 90.1677.2 401.6774.3
.3373.72] 103.6574.65] 396.33717.95
rterial pressure; HR: heart rate. All values are expressed as mean7S.D.
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Fig. 1. Effect of KAHAW on left ventricular end-diastolic pressure (LVEDP) in ISO-induced myocardial necrosis in rats. All values
are expressed as mean7S.D. for each group (n ¼ 8/group). *Po0.0001 ISO-control vs. vehicle control, ]Po0.001 KAHAW+ISO
vs. ISO-control.
0
500
1000
1500
2000
2500
3000
mm
Hg/
s
Vehicle Control KAHAW 200 mg/kgKAHAW 400 mg/kg KAHAW 800 mg/kgISO-control KAHAW 200 mg/kg + ISOKAHAW 400 mg/kg + ISO KAHAW 800 mg/kg + ISO
*#
Fig. 2. Effect of KAHAW on left ventricular maximum rate of
positive pressure development (+LVdp/dt) in ISO-induced
myocardial necrosis in rats. All values are expressed as
mean7S.D. for each group (n ¼ 8/group). *Po0.0001 ISO-
control vs. vehicle control, ]Po0.001 KAHAW+ISO vs. ISO-
control.
0
500
1000
1500
2000
2500
3000
mm
Hg/
s
Vehicle Control KAHAW 200 mg/kg
*
#
KAHAW 400 mg/kg KAHAW 800 mg/kgISO-control KAHAW 200 mg/kg + ISOKAHAW 400 mg/kg + ISO KAHAW 800 mg/kg + ISO
Fig. 3. Effect of KAHAW on left ventricular maximum rate of
negative pressure development (�LVdp/dt) in ISO-induced
myocardial necrosis in rats. All values are expressed as
mean7S.D. for each group (n ¼ 8/group). *Po0.0001 ISO-
control vs. vehicle control, ]Po0.001 KAHAW+ISO vs. ISO-
control.
S. Goyal et al. / Experimental and Toxicologic Pathology 62 (2010) 61–7466
in comparison to ISO-control group. KAHAW (800mg/kg/day) increased +LVdp/dt (from 2004.177183.31 to2409.177154.12mmHg/s) and �LVdp/dt (from 1813.33786.12 to 23107164.77mmHg/s) as compared to ISO-control rats (Po0.001). Similarly, it also prevented the risein LVEDP (from 6.0470.45 to 370.5mmHg) caused byISO treatment (Po0.001). Administration of KAHAWalone (200, 400, 800mg/kg/day) did not produce anysignificant changes in left ventricular functions ascompared to vehicle control rats.
Effect of KAHAW on the activities of CAT, SOD
and GSH content
Table 3 shows the detrimental effect of ISO onactivities of CAT, SOD and GSH content and the
protective effect of KAHAW. Cardiotoxicity-inducedwith ISO exhibited a significant (Po0.0001) decreasein activities of CAT (from 39.9272.77 to 28.6274.71U/mg protein), SOD (from 10.3871.48 to6.5570.83U/mg protein) and GSH level (from2.9070.12 to 1.1270.22 mM/gram tissue) as com-pared to vehicle control rats. Although KAHAW(200, 400 and 800mg/kg/day) dose-dependentlycounteracted the deleterious effect of ISO by increasingthe content of these antioxidants, significance couldbe achieved with 800mg/kg/day dose of KAHAWonly (Po0.001). Administration of KAHAW alone(200, 400 and 800mg/kg/day) did not show significantchanges in antioxidants as compared to vehicle controlrats.
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Table 3. Effect of KAHAW on the activities of CAT, SOD and GSH content.
CAT: catalase; SOD: superoxide dismutase; GSH: reduced glutathione. All values are expressed as mean7S.D. for each group (n ¼ 8/group).�Po0.0001 ISO-control vs. vehicle control.]Po0.001 KAHAW+ISO vs. ISO-control.
0
10
20
30
40
50
nmol
/g ti
ssue
Vehicle Control KAHAW 200 mg/kgKAHAW 400 mg/kg KAHAW 800 mg/kgISO-control KAHAW 200 mg/kg + ISOKAHAW 400 mg/kg + ISO KAHAW 800 mg/kg + ISO
*#
Fig. 4. Effect of KAHAW on the level of thiobarbituric acid
reactive substances (TBARS) in ISO-induced myocardial
necrosis in rats. All values are expressed as mean7S.D. for
each group (n ¼ 8/group). *Po0.0001 ISO-control vs. vehicle
control, ]Po0.001 KAHAW+ISO vs. ISO-control.
Fig. 5. Effect of KAHAW on the level of CK-MB isoenzyme
in ISO-induced myocardial necrosis in rats. All values are
expressed as mean7S.D. for each group (n ¼ 8/group).
*Po0.0001 ISO-control vs. vehicle control, ]Po0.001 KA-
HAW+ISO vs. ISO-control.
Fig. 6. Effect of KAHAW on the level of lactate dehydrogen-
ase (LDH) in ISO-induced myocardial necrosis in rats.
All values are expressed as mean7S.D. for each group
(n ¼ 8/group). *Po0.0001 ISO-control vs. vehicle control,]Po0.001 KAHAW+ISO vs. ISO-control.
S. Goyal et al. / Experimental and Toxicologic Pathology 62 (2010) 61–74 67
Effects of KAHAW on the level of TBARS, CK-MB
isoenzyme and LDH
Figs. 4–6 show the level of TBARS, and activitiesof CK-MB isoenzyme and LDH in myocardium ofcontrol and drug treated rats. In ISO-control rats,level of lipid peroxidation marker, TBARS was sig-nificantly increased (from 25.1871.93 to 39.5170.96 nmol/g tissue, Po0.0001) whereas cardiac injurymarkers, CK-MB isoenzyme (from 142.1775.1 to101.3979.5 IU/mg protein) and LDH (from86.5673.3 to 49.8579.5 IU/mg protein) were signifi-cantly (Po0.0001) decreased when compared to vehiclecontrol group. Pretreatment with KAHAW (200, 400and 800mg/kg/day) dose-dependently decreasedTBARS levels and increased activities of CK-MBisoenzyme and LDH. KAHAW in 800mg/kg/daydose significantly (Po0.001) decreased TBARSlevel (from 39.5170.96 to 28.6573.60 nmol/g tissue)while it increased the activities of CK-MB isoenzyme(from 101.3979.5 to 137.6774.7 IU/mg protein)and LDH (from 49.8579.5 to 81.7971.79 IU/mg
protein) as compared to ISO-control rats. Per se,KAHAW did not have any significant effect on theabove parameters.
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Table 4. Effect of KAHAW on histopathological evaluation of rat myocardium in different experimental groups.
Treatment groups Myonecrosis Inflammatory cells Edema
Vehicle control – – –
KAHAW (200mg/kg) – – –
KAHAW (400mg/kg) – – –
KAHAW (800mg/kg) – – –
ISO-control +++ +++ +++
KAHAW (200mg/kg)+ISO ++ +++ ++
KAHAW (400mg/kg)+ISO + ++ +
KAHAW (800mg/kg)+ISO – – +
+++ severe, ++ moderate, + mild, � nil.
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Effects of KAHAW on histopathological changes of
rat myocardium
Table 4 shows the effect of KAHAW on the extent ofhistopathological changes in myocardial tissues innormal and ISO-treated rats. Fig. 7A shows the lightmicrograph of vehicle control heart showing normalarchitecture. Light micrograph of ISO-control groupshows focal confluent necrosis of muscle fibers withinflammatory cell infiltration, edema with fibroblasticproliferation and myophagocytosis along with extra-vasation of red blood cells (Fig. 7B). The degree ofmyocardial damage in KAHAW 200mg/kg/day+ISOgroup was similar to that of ISO-control group withsimilar morphological changes (Fig. 7F). In KAHAW400mg/kg/day+ISO group, there was less edema andmyonecrosis with less inflammatory cells (Fig. 7G).Light micrograph of KAHAW 800mg/kg/day+ISO-treated heart tissue shows mild edema but no infarction.The myocardial fibers are normal in architecture(Fig. 7H). KAHAW (200–800mg/kg) administrationalone did not lead to any histopathological changes inthe myocardium (Figs. 7C–E).
Effect of KAHAW on ultrastructural changes
(transmission electron microscopy)
Normal cardiac architecture was seen in electronmi-crographs of animals in vehicle control and KAHAWalone groups (Figs. 8A–D). However, electronmicro-graphs of ISO-control rat hearts show extensive loss oflipid droplets, myonecrosis along with significantdisruption of myofilaments and Z-band architecture(Fig. 9A). Similar ultrastructural changes were seen inKAHAW 200 and 400mg/kg/day+ISO groups,although the changes were milder in the 400mg/kggroup (Figs. 9B and C). On the other hand, treatmentwith KAHAW at 800mg/kg/day in ISO-treated ratsshowed occasional disruption of myofilaments anddisarrangement of cristae. Almost normal ultrastructurewas seen in this group (Fig. 9D).
Discussion
Present study demonstrates the cardioprotectivepotential of Khamira Abresham Hakim Arshad Wala,a Unani formulation, in ISO-induced model of myo-cardial necrosis, as evidenced by amelioration of cardiacdysfunction, improvement in endogenous antioxidantdefense system and decreased lipid peroxidation.Further, in light microscopic and ultrastructural studies,KAHAW pretreated groups showed normal architec-ture of cells when compared with their respectivecontrols. The protective role of this formulation invarious disorders like cardiovascular, neurological andother diseases has already been suggested (Kabeer, 1951;Safiuddin, 1999; Yousuf et al., 2005; Khan et al., 2006).However, there is no report of this formulation in theprevention of myocardial injury-induced by ISO.
ISO-induced myocardial necrosis is the most authen-ticated model of MI in rats (Padmanabhan and StanelyMainzen Prince, 2006b; Loh et al., 2007; Zhou et al.,2008). ISO mediates its positive inotropic and chrono-tropic effects through b1 adrenoceptor (Brodde, 1991;Rajadurai and Stanely Mainzen Prince, 2006). Subcu-taneous injection of ISO has been shown to causemyocardial necrosis, particularly in subendocardialregions of left ventricles and interventricular septum.The lesions resemble those produced by myocardialischemia in human being (Rona et al., 1959; Dhallaet al., 1978). Several mechanisms of ISO-inducedmyocardial necrosis have been reported. Imbalancebetween oxygen supply and demand to myocyte aftercoronary hypotension and myocardial hyperactivity(due to increased heart rate and contractility) has beensuggested to underlie the myocardial damage (Yeagerand Whitehurst, 1982). Other probable mechanismsassociated with myocardial necrosis are increased cyclicadenosine monophosphate (Bhagat et al., 1978), in-creased intracellular Ca2+ overload (Bloom and Davis,1972), depletion of high-energy phosphate stores(Fleckenstein et al., 1974) and oxidative stress (Mohantyet al., 2004; Rajadurai and Stanely Mainzen Prince,2006). One of the putative mechanisms among these isincrease in oxidative stress due to metabolic products of
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Fig. 7. (A) Light micrograph of vehicle control rat heart showing normal architecture of myocytes (H&E, 200� ). (B) Light
micrograph of ISO-control group heart showing focal confluent necrosis of muscle fiber with inflammatory cell infiltration, edema
with fibroblastic proliferation and phagocytosis along with extravasation of red blood cells (H&E, 200� ). (C–E) Light micrographs
of KAHAW (200–800mg/kg/day) alone treated rat heart showing normal cardiac fibers without any fraying or infarction (H&E,
200� ). (F) Light micrograph of KAHAW 200mg/kg/day+ISO showing marked myocardial necrosis, edema with inflammatory
cells infiltration (H&E, 200� ). (G) Light micrograph of KAHAW 400mg/kg/day+ISO showing less myocardial injury, less edema
and inflammatory cells (H&E, 200� ). (H) Light micrograph of KAHAW 800mg/kg/day+ISO-treated heart tissue showing mild
edema but no infarction. The myocardial fibers are normal in architecture (H&E, 200� ).
S. Goyal et al. / Experimental and Toxicologic Pathology 62 (2010) 61–74 69
ISO and generation of redundant free radicals (Singalet al., 1982). Oxidation of catecholamines causesincreased production of ROS (Reichenbach and Benditt,1970; Bors et al., 1978; Singal et al., 1983) resulting inmyocardial dysfunction (Yates and Dhalla, 1975).
A growing body of evidence has shown that followingISO-induced myocardial necrosis, produces systolic anddiastolic dysfunction (Loh et al., 2007; Nandave et al.,2007), increased end-diastolic volume, end-diastolicpressure, left ventricular wall thickness (Zhou et al.,
2008) and ST-segment elevation (Peacock et al., 2007;Rajadurai and Stanely Mainzen Prince, 2007a). Tobetter understand the protective effect of KAHAW oncardiac function, hemodynamic parameters were in-cluded in the present study. KAHAW (200, 400 and800mg/kg/day) dose-dependently prevented ISO-in-duced left ventricular systolic and diastolic dysfunctionas evidenced by improvement in SAP, DAP and MAP.Rona (1985) and Zhou et al. (2008) have demonstratedthat an increase in heart rate is responsible for increased
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Fig. 8. (A) Electronmicrograph (magnification 4800� ) of normal myocardial tissue, showing normal architecture of myofibrils and
mitochondria. (B–D) Electronmicrographs (magnification 4800� ) of KAHAW (200–800mg/kg/day) alone treated groups showing
normal myocardial tissue, myofibrils and mitochondria.
Fig. 9. (A) Electronmicrograph of myocardium of ISO-control group (magnification 3500� ) showing lipid droplets, myocardial
necrosis with marked disruption of Z-bands and myofilaments along with changes in mitochondria and vacuolization of myofibrils
and mitochondrial damage. (B) Electronmicrograph of rat heart of KAHAW 200mg/kg/day+ISO group showing severe
myonecrosis, swollen nucleus and vacuoles. In mitochondria, loss of double membrane, disarrangement of cristae and accumulation
of electron dense material are seen (3500� ). (C) Electronmicrograph of rat heart of KAHAW 400mg/kg/day+ISO group showing
swelling of nucleus, less myocardial necrosis, edema along with vacuolization and mitochondrial changes (3500� ). (D)
Electronmicrograph of heart of KAHAW 800mg/kg/day+ISO showing irregular disruption of myofilaments along with focal loss
of mitochondrial double membrane and disarrangement of cristae. Almost normal structure is seen in most areas (3500� ).
S. Goyal et al. / Experimental and Toxicologic Pathology 62 (2010) 61–7470
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oxygen consumption that leads to accelerated myocar-dial necrosis. However, in our study, there was nosignificant difference in heart rate among all experimentgroups when compared with their respective controlgroups. KAHAW also improved left ventricular end-diastolic function by increasing inotropic (+LVdp/dt,marker of myocardial contraction) and lusitropic(�LVdp/dt, marker of myocardial relaxation) states ofthe heart. It also ameliorated ISO-induced increase inLVEDP, a marker of pre-load that again reflects animprovement of left ventricular function. Our resultssuggest that KAHAW (800mg/kg/day) offers protectionto the myocardium by attenuating the ventriculardysfunction through maintaining the hemodynamicparameters to near normal status in ISO-treated rats.
It has been well documented that ISO causesincreased oxidative stress in rat heart as evidenced byreduction in myocardial SOD and CAT activities,reduced GSH level, and rise in plasma TBARS(Banerjee et al., 2003; Devika and Stanely MainzenPrince, 2008; Panda and Naik, 2008). Myocyte injury isalso shown by increase in plasma CK-MB isoenzymeand LDH activities (Kurian et al., 2005; Prabhu et al.,2006; Rajadurai and Stanely Mainzen Prince, 2007b;Zhou et al., 2008). In the present study, we found thatKAHAW dose-dependently increased activities of SOD,CAT and GSH level in ISO-injected rats. However,KAHAW at 800mg/kg showed a significant effect ascompared to ISO-control group. Abresham (silk co-coon), an important constituent of KAHAW, isreported to contain a protein ‘sericin’ that resistsoxidation and acts as free radical scavenger (Zhang,2002; Yousuf et al., 2005; Khan et al., 2006). Anotherconstituent of KAHAW is ‘bihi (quince)’ which containsvitamins A, C, B1 and B2. It is well known that B groupvitamins (Wang et al., 1999; Hagar, 2002) and vitamin C(Carlson et al., 2006; Rajadurai and Stanely MainzenPrince, 2007b; Devika and Stanely Mainzen Prince,2008) have antioxidant and cardioprotective effects inanimal models of MI. Several studies have reported themodulation of antioxidants (SOD, CAT and GSH) bytraditional herbal formulations or plant extracts invarious pathological conditions (Banerjee et al., 2003;Mohanty et al., 2004; Yousuf et al., 2005; Khan et al.,2006; Karthikeyan et al., 2007; Mohanty et al., 2008).Khamira Abresham Uood Mastagiwala, another Unaniformulation, contains abresham as a major activeconstituent and has been shown to increase the activitiesof antioxidant enzymes in focal cerebral ischemia in rats(Yousuf et al., 2005). Similarly, KAHAW has beenshown to decrease the generation of free radicals in ananimal model of cognitive impairment (Khan et al.,2006). Our results are in consonance with the abovefinding, wherein KAHAW showed increase in activitiesof antioxidants in rat hearts. Therefore possibility existsthat the protection offer by KAHAWmight be due to its
antioxidant activity, which could exert a beneficial effectagainst pathological alterations caused by free radicalsin ISO-induced myocardial necrosis.
ROS are highly toxic byproducts of aerobic metabo-lism. They react unfavorably with surrounding macro-molecules resulting in severe cell and tissue damage(Halliwell and Chirico, 1993). Lipid peroxidation is animportant pathogenic event in myocardial necrosisand accumulation of lipid hydroperoxides reflectsdamage of the cardiac constituents (Hamberg et al.,1974; Gutteridge, 1982). The increased levels of lipidperoxides in ISO-induced myocardial necrosis might bedue to free radical-mediated membrane damage (Zhouet al., 2006; Karthikeyan et al., 2007). The resultspresented in this study indicate that KAHAW (200, 400and 800mg/kg/day) pretreatment can decrease ISO-induced TBARS (malondialdehyde) elevation. Thedecreased level of malondialdehyde in heart tissuesmight be due to the enhanced activities in antioxidantenzymes (SOD, CAT and GSH). Our results are inagreement with previous reports (Kato et al., 1998;Sathish et al., 2003; Yousuf et al., 2005; Khan et al.,2006) that abresham has ability to decrease lipidperoxidation end products (TBARS).
Furthermore, we observed a decrease in the activitiesof myocyte injury markers like CK-MB isoenzyme andLDH in hearts of ISO-control rats. When myocardialcells, containing CK-MB isoenzyme and LDH aredamaged or destroyed due to deficient oxygen supplyor glucose, the integrity of cell membrane gets disturbedand it might become more porous and permeable ormay rupture that results in the leakage of these enzymes.This accounts for the decreased activities of CK-MBisoenzyme and LDH in heart tissue of ISO-treated rats.This might be due to the damage caused to thesarcolemma by the b-agonist, that has rendered it leaky.Our results showed significant decrease in the levels ofLDH and CK-MB isoenzyme in heart tissue in ISO-injected rats, which are in line with the previous reports(Sabeena Farvin et al., 2004; Gurgun et al., 2008; Zhouet al., 2008) and indicate ISO-induced necrotic damageof the myocardial membrane. Oral administration ofKAHAW in 800mg/kg/day dose significantly increasedthe activities of CK-MB isoenzyme and LDH ascompared to the ISO-control group. We suggest thatthis could be due to protective effect of KAHAW on themyocardium, reducing the myocardial damage andthereby restricting the leakage of CK-MB isoenzymeand LDH.
To further characterize the modulatory action ofKAHAW on ISO-induced myocardial necrosis, lightmicroscopic study was performed. In histopathologicalstudy, ISO-induced myocardium showed severe in-fracted area with edema and inflammatory cells andseparation of cardiac muscle fibers (Fig. 7B). Althoughmyocardium of KAHAW 200 and 400mg/kg/day
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pretreated groups also showed myocardial damage(Figs. 7F and G), only mild edema and few inflamma-tory cells were observed in the myocardium of KAHAW800mg/kg/day pretreated group. Also the cardiacfibers were normal in architecture (Table 4, Fig. 7H).Myocardium of KAHAW alone treated groupshowed normal cardiac fibers without any pathologicalchanges. This indicates that KAHAW does not possessany adverse effects under normal conditions. Intransmission electron microscopic studies also KAHAW(800mg/kg/day) showed almost normal ultrastructure inmost areas of myocardium except irregular disruption ofmyofilaments along with focal loss of mitochondrialdouble membrane and disarrangement of cristae in somearea as compared to ISO-control group. These datafurther confirmed the cardioprotective action of oraladministration of KAHAW in ISO-induced myocardialnecrosis.
Conclusion
It is concluded that KAHAW can mitigate thecardiotoxic effect of ISO in rat heart. The present studyprovides experimental evidence that KAHAW augmen-ted the myocardial antioxidant enzymes level, preservedhisto-architecture and improved cardiac performancefollowing ISO administration. These findings suggest thebeneficial effects of KAHAW on rat heart againstexperimental myocardial necrosis.
Acknowledgments
Authors are thankful to the Central Council forResearch in Unani Medicine, Ministry of Health andFamily Welfare, Government of India, New Delhi, forfinancial assistance. The authors are also thankful toMr. Brij Mohan Sharma for his expert technicalassistance during the course of study.
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