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Preventive effect of crocin of Crocus sativus on hemodynamic, biochemical, histopathological and ultrastuctural alterations in isoproterenol-induced cardiotoxicity in rats.

ABSTRACT

We investigated the effects of crocin, a pharmacologically active constituent of Crocus sativus L, in isoproterenol (ISO)-induced cardiotoxicity with reference to hemodynamic, antioxidant, histopathological and ultrastructural parameters. Rats were administered crocin (5, 10 and 20 mg/kg/day) or vehicle orally for 21 days along with ISO (85 mg/kg, subcutaneously, at 24h interval) on 20th and 21st day. On 22nd day ISO-control rats showed cardiac dysfunction as indicated by lowering of systolic, diastolic and mean arterial blood pressures. In addition, a significant decrease in maximum positive and negative rate of developed left ventricular pressure ([+ or -]LVdp/[dt.sub.max]) and an increase in left ventricular end-diastolic pressure (LVEDP) were observed. Furthermore, a marked reduction in the activities of myocardial creatine kinase-MB (CK-MB) isoenzyme, lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) levels along with an increase in content of malondialdehyde (MDA) were observed. Myocardial necrosis, edema and inflammation were evident from the light microscopic and ultrastructural changes. Crocin at the dose of 20 mg/kg/day significantly modulated hemodynamic and antioxidant derangements. The preventive role of crocin on ISO-induced MI was reconfirmed by histopathological and ultrastructural examinations. The effect at the dose of 20 mg/kg/day of crocin was more pronounced than that of other two doses (5 and 10 mg/kg/day). The results suggest that crocin may have cardioprotective effect in ISO-induced cardiac toxicity through modulation of oxidative stress in such a way that maintains the redox status of the cell.

[C] 2009 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords: Cardiotoxicity Saffron Crocus sativus Myocardial infarction Oxidative stress Antioxidant

Introduction

Myocardial infarction (MI) is an acute condition of necrosis of the myocardium that occurs as a result of imbalance between myocardial demand and coronary blood supply (De Bono and Boon 1992). It is well established that reactive oxygen species (ROS) have been implicated in the pathophysiology of MI (Rajadurai and Stanely Mainzen Prince 2006a). Isoproterenol (ISO), a synthetic [beta]-adrenoceptor agonist induces cardiotoxicity in the form of MI in rats as a result of disturbance in physiological balance between production of free radicals and antioxidative defense system (Zhou et al. 2008). Several recent studies demonstrate the protective action of antioxidants from variety of sources on incidence of MI (Rajadurai and Stanely Mainzen Prince 2006b). Antioxidants not only suppress the formation of ROS but also have a modulatory effect on the survival and death signaling of ROS (Ulrich-Merzenich et al. 2009). Efforts are now being focused on to look for newer antioxidants that can prevent ISO-induced MI and maintain redox homeostasis of the cell.

Crocus sativus L. commonly known as saffron belongs to Iridaceae family. The extract of Crocus sativus L contains many compounds such as crocin, picrocrocin and safranal. Among the pharmacologically active constituents of Crocus sativus L., crocin (water soluble carotenoids) is the most important and abundant antioxidant (Abdullaev 1993). Studies have shown that crocin has antioxidant effects in ischemia-reperfusion models of stroke (Zheng et al. 2007) and renal injury (Hosseinzadeh et al. 2005).

Keeping in mind the documented protective role of antioxidants in MI and the potential antioxidant property of crocin, we hypothesized that crocin may have cardioprotective effect. Thus, we conducted the present experiments to examine the effects of crocin on hemodynamic, biochemical, histopathological and ultrastructural changes in ISO-induced model of Ml in order to understand the molecular mechanisms of its potential cardioprotective effects.

Materials and methods

Drugs and Chemicals

Isoproterenol hemisulphate was dissolved in 0.9% saline and used within 10 minutes of preparation. CK-MB isoenzyme detection kit was purchased from Logotech India Pvt Ltd (Delhi, India). Crocin was purchased from Fluka Riedel-deHaen (Buchs SG, Schweiz). All chemicals used in this study were of analytical grade and purchased from Sigma Chemicals (St. Louis, MO, USA).

Experimental animals

Male Wistar albino rats, weighing 150-200g, were obtained from the Central Animal House Facility of All India Institute of Medical Sciences, New Delhi, India. The study protocol was reviewed and approved by the Institutional Animal Ethics Committee (IAEC, No 356/2007) and all study related activities conformed to the Indian National Science Academy Guidelines for the use and care of experimental animals in research. Animals were kept in the departmental animal house under controlled conditions of temperature at 25 [+ or -] 2 [degrees]C, relative humidity of 60 [+ or -] 5% and light-dark cycle of 12:12 h. They were fed food pellets (Ashirwad Industries Ltd., Chandigarh, India) and water ad libitum. Animals were maintained in polypropylene cages, each containing a maximum of four animals.

Induction of experimental cardiotoxicity

The cardiotoxicity was induced by subcutaneous (s.c.) administration of isoproterenol (85 mg/kg) to rats daily for two consecutive days at 24 h interval (Goyal et al. 2009).

Experimental groups

A total of 128 rats were used in the study. They were randomly divided into eight groups, with 16 animals in each group:

Group 1 (vehicle-control): Rats were administered saline orally (3 ml/kg/day) using intragastric tube for 21 days and on 20th and 21st day received 0.3 ml saline, s.c. at an interval of 24 h.

Groups 2-4 (crocin per se): Animals were treated orally with crocin (5, 10 and 20 mg/kg/day) for a period of 21 days and on 20th and 21 st day administered 0.3 ml saline, s.c. at an interval of 24 h.

Group 5 (ISO-control): Rats were administered saline orally (3 ml/kg/day) for 21 days along with ISO (85 mg/kg, s.c., at 24 h interval) on 20th and 21st day.

Groups 6-8 (crocin+ISO): Animals were treated with crocin (5, 10 and 20 mg/kg/day) orally for a period of 21 days along with ISO (85 mg/kg, s.c., at 24 h interval) on 20th and 21st day.

Surgery for recording hemodynamic parameters

The surgical procedure as described by Goyal et al. (2009) for recording of hemodynamic parameters has been followed. After recording of hemodynamic parameters, animals were sacrificed and their hearts were excised and processed for biochemical, histopathological and ultrastructural studies.

Biochemical Estimation

A 10% homogenate of myocardial tissue was prepared in ice-chilled phosphate buffer (pH 7.4) and an aliquot was used for the estimation of malondialdehyde (MDA) according to the method described by Okhawa et al. (1979) and reduced glutathione (GSH) content by the method of Moron et al. (1979). The homogenate was centrifuged at 5000 rpm for 20 min at 4 [degrees]C and the supernatant was used for enzyme assays. The activities of lactate dehydrogenase (LDH) (Cabaud and Wroblewski 1958), catalase (Aebi 1974), superoxide dismutase (SOD) (Marklund and Marklund 1974) and protein (Bradford 1976) were assessed. Creatine kinase-MB (CK-MB) isoenzyme was estimated spectro-photometrically using a kit from Logotech (Delhi, India).

Histopathology (Light microscopic study)

The tissues were fixed in 10% buffered formalin and embedded in paraffin. Serial sections (3 [micro]m thick) were cut using microtome (Leica RM 2125, Germany). Each section was stained with hematoxylin and eosin (H&E). The sections were examined under light microscope (Nikon, Tokyo, Japan). The pathologist performing histopathological evaluation was blinded to the treatment assignment of different study groups.

Ultrastructural studies by Transmission Electron Microscope (TEM)

The Karnovsky's fixed tissues were washed with chilled phosphate buffer (0.1 M, pH 7.4) and post fixed for 2h in 1% osmium tetraoxide in the same buffer at 4 [degrees]C. The specimens were further washed in phosphate buffer, dehydrated with graded acetone and then embedded in araldite CY212 to make tissue blocks. Semithin (1 [micro]m) as well as ultrathin sections (70-80 nm) were cut by ultramicrotome (Ultracut E, Reichert, Austria). The sections were stained with uranyl acetate and lead acetate and examined under TEM (Morgagni 268D, Fei Co., The Netherlands) operated at 60 kV by a morphologist blinded to the groups studied.

Statistical analysis

The data are presented as mean[+ or -]S.D. One way ANOVA followed by Scheffe Post-hoc test were used for analysis of hemodynamic and biochemical data of different groups. A value of p < 0.05 was considered significant.

Results

Mortality

The mortality rate in surgery for recording hemodynamic parameter was 5.4% due to bleeding or improper cannulation in right carotid artery.

Per se effect of the drug

Crocin per se treatment (5, 10 and 20 mg/kg) daily for a period of 21 days to normal control rats did not show any significant change in hemodynamic and biochemical parameters as compared to vehicle treated group.

Effect of crocin on cardiac function

Fig. 1 depicts the effect of crocin on arterial pressure of vehicle and drug treated rats. A significant decrease in systolic, diastolic and mean arterial blood pressure was seen in ISO-control animals as compared to vehicle-control group (p < 0.001). Twenty one days treatment with crocin (5-20 mg/kg/day) dose-dependently restored systolic, diastolic and mean arterial blood pressure in ISO treated rats. However, the effect was significant only at 20 mg/kg dose (p < 0.001). There was no significant change in heart rate in any of the groups observed (Data not shown).

[FIGURE 1 OMITTED]

Figs. 2 and 3 shows the deleterious effect of ISO on left ventricular function in vehicle and drug treated rats. Compared with vehicle-control group, ISO-control rats showed left ventricular dysfunction as indicated by a significant rise in LVEDP and fall in values of [+ or -]LVdp/[dt.sub.max]. Oral administration of crocin (10 and 20 mg/kg/day) significantly improved cardiac function of ISO-treated rats (p < 0.001).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Effect of crocin on myocardial injury markers and lipid peroxidation

Table 1 shows the activities of CK-MB isoenzyme, LDH and MDA level in the hearts of vehicle and drug treated rats. In ISO-control rats, the activities of these enzymes declined whereas MDA level was increased significantly (p < 0.001) when compared to vehicle-control rats. Crocin at both doses, 10 and 20 mg/kg/day significantly (p < 0.01) increased the activities of these enzymes while at dose of 20 mg/kg/day significantly (p <0.001) decreased the level of MDA when compared to ISO-control animals.
Table 1
Effect of crocin on activities of CK-MB isoenzyme, LDH and MDA level in
isoproterenol-induced myocardial infarction MI) in rats

Treatment groups       CK-MB (IU/mg protein)     LDH (IU/mg protein)

Vehicle-control        140.33 [+ or -] 4.08      88.07 [+ or -] 4.0

ISO-control            101.38 [+ or -] 9.5 **    51.01 [+ or -] 9.7 **

Crocin (5 mg/kg/day)   141.32 [+ or -] 5.9       87.16 [+ or -] 7.0

Crocin (10 mg/kg/day)  139.81 [+ or -] 2.0       88.17 [+ or -] 4.7

Crocin (20 mg/kg/day)  139.83 [+ or -] 4.49      89.50 [+ or -] 3.5

Crocin                 118.20 [+ or -] 2.27      55.23 [+ or -] 4.9
(5 mg/kg/day)+ ISO

Crocin                 127.16 [+ or -] 7.12 ###  64.74 [+ or -] 5.2 #
(10 mg/kg/day)+ISO

Crocin                 135.00 [+ or -] 6.4 ###   82.91 [+ or -] 1.8 ###
(20 mg/kg/day)+ISO

Treatment groups           MDA (nmol/g tissue)

Vehicle-control            31.94 [+ or -] 5.6

ISO-control                45.62 [+ or -] 4.8 **

Crocin (5 mg/kg/day)       30.39 [+ or -] 5.9

Crocin (10 mg/kg/day)      32.11 [+ or -] 3.9

Crocin (20 mg/kg/day)      29.83 [+ or -] 6.5

Crocin (5 mg/kg/day)+ISO   40.66 [+ or -] 1.0

Crocin (10 mg/kg/day)+ISO  37.96 [+ or -] 2.5

Crocin (20 mg/kg/day)+ISO  32.08 [+ or -] 5.0 ##

CK-MB isoenzyme- creatine kinase-MB isoenzyme, LDH- lactate
dehydrogenase, MDA- malondialdehyde, ISO- isoproterenol. All values are
expressed as mean [+ or -]S.D for each group (n = 8/group). Significance
was determined by One-Way ANOVA followed by Scheffe Post-hoc test:
**p<0.001 compared with vehicle-control; *p<0.01, **p<0.001.
###p<0.0001 compared with ISO-control.


Effect of crocin on the activities of CAT, SOD and GSH content

Table 2 shows content of CAT, SOD and GSH in the heart of vehicle and drug treated rats. In ISO-control group, the activities of these enzymes declined significantly (p < 0.001) when compared to vehicle-control group. Pretreatment with crocin (5, 10 and 20 mg/kg/day) dose-dependently increased the content of these antioxidants. Crocin at dose of 20 mg/kg/day significantly (p < 0.01) increased the activities of CAT (26.63%) and SOD (33.14%), whereas at dose of 10 and 20 mg/kg/day significantly augmented GSH level by 53.71% and 62.79% respectively (p < 0.0001).
Table 2
Effect of crocin on antioxidant parameters in heart of isoproterenol
(ISO)-induced myocardial infarction (Ml) in rats

Treatment groups           CAT (U/mg protein)       SOD (U/mg protein)

Vehicle-control            41.93 [+ or -] 5.58      12.00 [+ or -]2.0

ISO-control                28.62 [+ or -] 4.71 **    7.26 [+ or -]0.61 *
                           (31.74%) (a)            (39.5%) (a)

Crocin (5 mg/kg/day)       39.91 [+ or -] 5.51      11.50[+ or -]1.87
                           (4.81%) (a)              (4.16%) (a)

Crocin (10 mg/kg/day)      40.95 [+ or -] 4.35      11.00 [+ or -]2.60
                           (233%) (a)               (8.33) (a)

Crocin (20 mg/kg/day)      41.55 [+ or -] 2.36      12.83 [+ or -]2.78 *
                           (0.90%) (a)              (6.46%) (a)

Crocin (5 mg/kg/day)+ISO   34.87 [+ or -] 7.63       8.11 [+ or -]1.08
                           (16.83%) (a)            (32.41 %) (a)
                           (18%) (a)               (10.48%) (a)

Crocin (10 mg/kg/day)+ISO  36.31 [+ or -] 3.22       9.42 [+ or -]1.06
                           (13.40%) (a)            (215%)(a)
                           (21.17%) (a)            (22.93%) (b)

Crocin (20 mg/kg/day)+ISO  39.01 [+ or -] 2.08 #    10.86 [+ or -]0.97 #
                           (6.96%) (a)              (9.5%) (a)
                           (26.63%) (b)            (33.14%) (b)

Treatment groups           GSH ([mu]g/g tissue)

Vehicle-control            3.20 [+ or -] 0.29
ISO-control                1.12 [+ or -] 0.22 **
                           (65%) (a)

Crocin (5 mg/kg/day)       3.18 [+ or -] 0.38
                           (0.6%) (a)

Crocin (10 mg/kg/day)      3.22 [+ or -] 0.34
                           (0.62%) (a)

Crocin (20 mg/kg/day)      3.05 [+ or -] 0.32
                           (4.68%) (a)

Crocin (5 mg/kg/day)+ISO   1.70 [+ or -] 0.28
                           (46.8%) (a)
                           (34.11%) (b)

Crocin (10 mg/kg/day)+ISO  2.42 [+ or -] 0.35 ###
                           (24.37%) (a)
                           (53.71%) (b)

Crocin (20 mg/kg/day)+ISO  3.01 [+ or -] 0.34 ###
                           (5.93%) (a)
                           (62.79%) (b)

CAT- catalase. SOD- superdoxide dismutase, GSH- reduced glutathione
Data are expressed as mean [+ or -]S.D values (n = 8/group).
Significancewas determined by One-Way ANOVA followed by Scheffe
 Post-hoc test:
**p<0.001, *p<0.01 compared with vehicle-control; #p<0.05,
###p<0.0001 compared with ISO-control. Values in parentheses
indicate the percentage change versus  (a) vehicle-control or (b) ISO-
control.


Effect of crocin on histopathological changes of rat myocardium

Fig. 4A shows the light micrograph of vehicle treated heart showing normal architecture. Crocin (5-20 mg/kg) administration alone did not lead to any histopathological alterations in the myocardium (Figs. 4B-D). ISO-control group shows focal confluent necrosis of muscle fibers with inflammatory cell infiltration, edema with fibroblastic proliferation and myophagocytosis along with extravasations of red blood cells (Fig. 4E). The degree of myocardial damage at crocin 5 mg/kg dose in ISO-treated rats was similar to that of the ISO-control group (Fig. 4F). However, crocin (10 mg/kg) treated group showed myonecrosis with less edema and inflammatory cells (Fig. 4G). In ISO treated animals, crocin 20 mg/kg/day shows mild edema but no necrosis and myocardial fibers are almost in normal architecture (Fig. 4(H).

[FIGURE 4 OMITTED]

Effects of crocin on ultrastructural morphology of rat myocardium

TEM images of the heart showed normal architecture in vehicle treated (Fig. 5, Lane 1) and crocin (5, 10 and 20 mg/kg) alone treated rats (Fig. 5, Lanes 2-4). ISO-control hearts exhibited myonecrosis, swelling of mitochondria and disruption of cristae with vacuolation (Fig. 5, Lane 5). Similar ultrastructural changes were seen in crocin 5 and 10 mg/kg plus ISO groups, although the changes were milder in the 10 mg/kg dose (Fig. 5, Lane 6 and 7). Rats treated with crocin (20 mg/kg) and ISO showed mild separation of cristae without swelling and vacuolation. Almost normal ultrastructure was seen in this group (Fig. 5, Lane 8).

[FIGURE 5 OMITTED]

Discussion

Isoproterenol-induced myocardial necrosis is a well established model of MI in rats (Goyal et al. 2009; Zhou et al. 2008). The activities and capacities of antioxidant systems of heart declined following ISO challenge leading to the gradual loss of prooxidant/antioxidant balance which accumulates into oxidative damage of cardiac myocyte. Therefore, the present study evaluated the role of crocin a carotenoid pigment in combating ISO associated macromolecular damage in the myocardium of MI rats. Amongst various proposed mechanisms of ISO causing MI, generation of redundant free radicals is one of the important causative factor (Singal et al. 1982).

Lipid peroxidation is an important pathogenic event in myocardial necrosis (Halliwell and Chirico 1993). In present study, crocin significantly decreased MDA contents near to normal levels. Several earlier reports have demonstrated the reduction in MDA levels by aqueous extracts of Saffron or its active constituents (Crocin and/or Safranal) in renal ischemia-reperfusion induced-injury in rats (Hosseinzadeh et al. 2005) and in ischemia-reperfusion model of stroke (Zheng et al. 2007). Our results reconfirmed that crocin have potential to reduced lipid peroxidation in pathological condition.

A growing body of evidences has shown that ISO treatment produced marked systolic and diastolic dysfunction in heart (Loh et al. 2007) by increasing end-diastolic volume leading to increased end-diastolic pressure. These changes further lead to increased left ventricular wall thickness and ST-segment elevation (Zhou et al. 2008). Crocin dose-dependently prevented ISO-induced left ventricular systolic and diastolic dysfunction as evidenced by improvement in systolic, diastolic and mean arterial pressures. Crocin also improved left ventricular function by increasing inotropic .(+LVdp/dt, marker of myocardial contraction) and lusitropic (-LVdp/dt, marker of myocardial relaxation) states of the heart. It also ameliorated ISO-induced increased in LVEDP (a marker of pre-load) which again reflects an improvement of left ventricular function.

Apart from hemodynamic and ventricular function parameters, several diagnostic marker enzymes like CK-MB isoenzyme and LDH are present in myocardium that are used as a predictor for pathological changes. These enzymes are released into the extracellular fluid during myocardial injury (Suchalatha and Shyamala Devi 2004). Our experimental data showed a decrease in activities of these enzymes in hearts of ISO-control rats which is in consonance with previously reported studies (Sabeena Farvin et al. 2004) and indicates ISO-induced necrotic damage of the myocardial membrane. We observed that oral administration of crocin significantly restored the loss in activities of CK-MB isoenzyme and LDH in heart tissue following ISO insult and thus exhibited the protective effect on the myocardium cell membrane integrity.

Histopathological findings of the heart pretreated with crocin presents a well preserved normal morphology of cardiac muscle with no evidence of necrosis when compared to ISO-control heart. Crocin per se also exhibit normal cardiac fibers without any pathological changes, therefore indicating that crocin itself does not possess any adverse effects on myocardium. An ultrastructural study also reveals similar and supportive observation regarding histology of myocardium.

To conclude, the present results endorse our hypothesis that crocin has cardioprotective potential. Crocin pretreatment improved cardiac functions, the effect which can be attributed to its ability of maintaining redox status which is disturbed by ISO challenge, via restoration of endogenous antioxidants, controlling lipid peroxide formation and preserving activities of CK-MB, LDH enzymes. Preservation of histoarchitecture of myocyte by crocin pretreatment reconfirms these effects. Further studies are needed to explore other possible mechanisms and pathways that might be directly or indirectly involved in its cardioprotective effects.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgment

Authors are thankful to Mr. Brij Mohan Sharma and Deepak Sharma for his expert technical assistance during the course of study.

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S.N. Goyal (a), S. Arora (a), A.K. Sharma (a), S. Joshi (a), R. Ray (b), J. Bhatia (a), S. Kumari (c), D.S. Arya (a), *

(a) Department of Pharmacology. All India Institute of Medical Sciences, New Delhi 110029, India

(b) Department of Pathology, All India Institute of Medical Sciences, New Delhi 110029, India

(c) Division of Plant Physiology, Indian Agricultural Research Institute, Pusa, Delhi 110012, India

* Corresponding author. Tel.: +91 11 26594266; fax: +91 11 26584121. E-mail address: dsarya16@hotmail.com (D.S. Arya).

0944-7113/$-see front matter [C] 2009 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2009.08.009
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Author:Goyal, S.N.; Arora, S.; Sharma, A.K.; Joshi, S.; Ray, R.; Bhatia, J.; Kumari, S.; Arya, D.S.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Clinical report
Geographic Code:1USA
Date:Mar 1, 2010
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