Extract from Clerodendron colebrookianum Walp protects rat heart against oxidative stress induced by ischemic–reperfusion injury (IRI)

Life Sciences 77 (2005) 2999 – 3009 www.elsevier.com/locate/lifescie

Extract from Clerodendron colebrookianum Walp protects rat heart against oxidative stress induced by ischemic–reperfusion injury (IRI) R. Devi a, S.K. Banerjee b,1, S. Sood b,1, A.K. Dinda c, S.K. Maulik b,T a

b

Institute of Advanced Study in Science and Technology, Guwahati-22, Assam, India Department of Pharmacology, All India Institute of Medical Sciences, New Delhi-110029, India c Department of Pathology, All India Institute of Medical Sciences, New Delhi-110029, India Received 23 January 2004; accepted 17 November 2004

Abstract Reactive oxygen species (ROS) have pathogenic effects on ischemic–reperfusion injury of heart. Hence, it is important to identify natural antioxidative agents to mitigate such effects. Recently, it has been reported that Clerodendron colebrookianum (CC) leaf extract has antioxidant and hypolipidemic effects in experimental animals. The aim of this study was to examine whether acute treatment with CC extract offers protection against ischemic–reperfusion injury (IRI) and IRI-induced changes in endogenous antioxidant enzyme activities of rat heart. Isolated rat hearts were perfused using the Langendorff’s technique, and 20 min of global ischemia was followed by 40 min of reperfusion. Lipid peroxidation after the ischemic–reperfusion episode was significantly reduced in the CC extract-treated heart compared to the control group and suppressed the leakage of lactate dehydrogenase (LDH) during reperfusion. Moreover, CC extract diminished the depletion of myocardial antioxidant enzymes (SOD, Catalase, GSH and GPx) after ischemia–reperfusion. Furthermore, IRI-induced cellular damage was significantly less in CC extract treated myocytes. These results indicate that CC leaf extract protects against oxidative stress and cellular injury associated with ischemic–reperfusion injury of rat heart and suggests that the protective effects of CC extract depend on its antioxidant properties. D 2005 Published by Elsevier Inc. Keywords: Cardioprotection; Clerodendron colebrookianum; Ischemia; Reperfusion; Heart; Free radical; Antioxidant

T Corresponding author. Tel.: +91 11 26593540; fax: +91 11 26862663. E-mail address: [email protected] (S.K. Maulik). 1 Tel.: +91 11 26593540; fax: +91 11 26862663. 0024-3205/$ - see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.lfs.2004.11.042

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Introduction Clerodendron colebrookianum (CC) Walp (Family-Verbenaceae) is a perennial shrub indigenous to the northeastern region of India as well as in many Southeast Asian countries and grows up to an altitude of 1700 m (Nath and Bordoloi, 1991; Goswami et al., 1996; Wang et al., 2000). The Mizo people of northeast India claim that the low incidence of hypertension among their community members is due to the regular use of the tender leaves and shoot of CC in their diet (Nath and Bordoloi, 1991). The roots of the same plant have been reported to have anthelminthic (Banerjee, 1936) and antibacterial (Hosozowa et al., 1974) properties and are used to treat bronchitis, asthma, fever, stomach troubles, syphilis and gonorrhoea (Singh et al., 1995) in tribal populations. In China, it is used to induce diuresis (Yunnn Medical Material Corporation, 1993). The hypotensive effect of CC leaf extract has also been documented in rats (Gupta et al., 1994). The leaves and roots of CC contain many chemical compounds, like flavone and their glycosides (Lin et al., 1989; Jacke and Rimpler, 1983), which have significant antioxidant activity. Recently, we have reported that CC leaves possess both antioxidant (Devi et al., 2003) and hypolipidemic activity (Devi and Sharma, 2004). We studied both in vitro and in vivo antioxidant activity of CC leaves on animal model using water and organic extracts. In the in vitro study, all of the extracts inhibited the FeC12-ascorbic acid stimulated lipid peroxidation in rat liver homogenate, with the water extract exhibiting best response. In in vivo study, water extract of CC, following oral administration caused a significant reduction of lipid peroxidation both in liver and kidney. Moreover, lipid lowering activity of the different extracts of CC leaf have been studied and found that the leaf extract has significant lipid lowering activity which indicates that this leafy vegetable of Mizo people certainly possesses some medicinal potentiality and it needs some special attention to conduct further research to explain its ethnomedical use. In ischemia and reperfusion of the heart, oxygen derived free radicals are thought to play an important role in the genesis of tissue injury (Visioli et al., 2000; Banerjee et al., 2002; Thompson and Zweier, 1990; Zweier, 1988). Many reports have demonstrated that free radical scavengers reduced free radical injury in the ischemic–reperfused heart (Chambers et al., 1989; Gelvan et al., 1991; Janero and Burghardt, 1989; Packer et al., 1991; Pyke and Chan, 1990; Rezanick et al., 1992), which supports the potential therapeutic uses of the free radical scavengers in this condition. Presence of hypolipidemic as well as antioxidant agents in the plants species or in natural resources have been previously highlighted by many workers (Helen et al., 1999; Augusti et al., 2001; Sodimu et al., 1984; Helen et al., 2000). Therefore, the present study was designed to investigate the role of CC leaf extract in suppressing the oxidative stress and cell injury induced by ischemia–reperfusion of isolated rat heart.

Materials and methods Plant material Arial part of the CC was collected from Guwahati (Assam, India) during April to August, identified and authenticated in the Department of Botany, Gauhati University, Assam, using some references (Nath and Bordoloi, 1991).

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Preparation of extract The leaves were dried in shade and two different concentrations of water extract (0.01% and 0.05%) were prepared. Briefly 0.5 and 2.5 g of the dried powdered leaves were mixed with 10 and 50 mL of double distilled water respectively, heated (60 8C) and filtered off using Whatman filter paper (1.6 Am). The filtrate was lyophilized and stored at 4 8C and used as the plant extract. Animals The study was approved by the Institute Animals Ethics Committee (No185/IAEC/02). Laboratory bred Wistar rats of either sex (250–300 g) were used for this study. All animals were allowed at least 3 days for in house acclimatization with ad lib access to standard laboratory food and water. Experimental protocol Rats were randomly divided into four groups, each having 6 rats. The animals were heparinised [375 units / 200 g i.p.] 1 h prior to sacrifice, anaesthetized with sodium pentobarbitone [60 mg/kg i.p.], and subjected to the following protocol. Production of ischemic–reperfusion injury in isolated rat heart Hearts were rapidly excised and washed in ice-cold saline and then perfused by the non-recirculating Langendorff’s technique (Hufesco, Hungary), in constant pressure mode with a modified Kreb’s Henseleits solution (KH) (Borchgrevink et al., 1989) containing in mM: glucose 11.1; NaCl 118.5; NaHCO3 25; KCl 2.8; KH2PO4 1.2; CaCl2 1.2; MgSO4 0.6 with a pH 7.4. The buffer solution, equilibrated with 95% O2 + 5% CO2 was delivered to the aortic cannula at 37 8C under 60 mm Hg pressure. An initial 10 min equilibration period was followed by 20 min of zero-flow (ischemia) with 40 min of reflow (reperfusion). Two concentrations of water extract (0.01% and 0.05%) of CC leaf were used in the perfusion buffer. Groups studied Following groups were considered for the study: Group A: Rat hearts were subjected to 70 min perfusion only. Group B: Rat hearts were subjected to 10 min of perfusion, followed by 20 min of ischemia and 40 min of reperfusion. Group C: Rat hearts were subjected to 10 min of perfusion, followed by 20 min of ischemia and 40 min of reperfusion with 0.01% water extract. Group D: Rat hearts were subjected to 10 min of perfusion, followed by 20 min of ischemia and 40 min of reperfusion with 0.05% water extract. At the end of each experiment, myocardial tissue was stored in liquid nitrogen for biochemical estimations and 10% buffered formalin for histopathological studies.

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Biochemical parameters Myocardial thiobarbituric acid reactive substances (TBARS) (Okhawa et al., 1979) (a marker of lipid peroxidation), lactic dehydrogenase (LDH) (a marker of tissue injury) (Molders et al., 1978) and endogenous antioxidants e.g. superoxide dismutase (SOD) (Kakkar et al., 1984), catalase (CAT) (Aebi, 1974), glutathione (GSH) (Ellman, 1959) and glutathione peroxidase (GPx) (Wendel, 1981) were measured in all the groups. Preparation of rat heart homogenate Tissue homogenate was prepared in a ratio of 1 g of wet tissue to 10 times (w/v) 0.05 M-ice cold phosphate buffer (pH 7.4) and homogenized by using a teflon homogenizer. A 0.2 mL of homogenate was used for estimation of TBARS. The remaining part of the homogenate was divided into two parts, one part of which was mixed with 10% trichloroacetic acid (1 : 1), centrifuged at 5000 g (4 8C, for 10 min) and supernatant was used for GSH estimation. The other part of the homogenate was centrifuged at 15000 g at 4 8C for 60 min, the supernatants was used for SOD, catalase, GPx and protein estimation (Jiankang et al., 1990; Annadora and Michel, 1995). Thiobarbituric acid reactive substances (TBARS) assay (Okhawa et al., 1979) The 0.2 mL of tissue homogenate was mixed with 1.5 mL of 0.8% (w/v) 2-thiobarbituric acid, 1.5 mL of 20% acetic acid and 0.2 mL of 8.1% (w/v) sodium dodecyl sulfate (SDS). Then the volume of the mixture was increased up to 4.0 mL with distilled water and heated at 95 8C for 60 min. After cooling with tap water, 1.0 mL of distilled water and 5.0 mL of mixture of n-butanol and pyridine (15 : 1, v/v) were added. The mixture was shaken vigorously and centrifuged at 5000 g for 10 min. After centrifugation, the optical density of the butanol layer was measured at 532 nm in a Spectrophotometer (Backman, UK). Histopathology Myocardial tissue was fixed in 10% buffered formalin, routinely processed and embedded in paraffin. Paraffin sections (3 Am) were cut on slides and stained with hematoxylin and eosin (H and E), periodic acid Schiff (PAS) reagent and examined under a light microscope by a histopathologist blinded to the groups of study. Chemical used All chemicals were of analytical grade and were obtained from Sigma Chemicals, St Louis, USA. Double distilled water was used for all biochemical assays. Statistical analysis All values are expressed as mean F SE. Unpaired Student’s T-test was applied to test the significance of biochemical data of different groups. Significance was set at P b 0.05.

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Results Biochemical parameters The level of lipid peroxidation and endogenous antioxidants of heart in different groups are shown in Figs. 1–6. Myocardial TBARS (Fig. 1) There was a significant ( P b 0.05) increase in myocardial TBARS in group B as compared to that of group A (2.4 F 0.12 vs. 2.1 F 0.16 nmol/mg protein), while significant ( P b 0.01 and P b 0.001) decrease in myocardial TBARS was observed in groups C and D in comparison to that of the group B (IRI) (1.7 F 0.12 and 1.2 F 0.12 vs. 2.4 F 0.12 nmol/mg protein respectively). Myocardial LDH (Fig. 2) There was a significant ( P b 0.001) reduction in myocardial LDH activity occurred in group B when compared to that of group A (5.4 F 0.5 vs. 8.8 F 0.29 U of pyruvate release/min/mg protein), while the LDH activity increased significantly ( P b 0.001 and P b 0.01) after the treatment with CC extract i.e. in groups C and D in comparison to group B (IRI) (9.4 F 0.4 and 7.2 F 0.3 vs. 5.4 F 0.5 U of pyruvate release/min/mg protein). Myocardial SOD (Fig. 3) There was a significant ( P b 0.05) reduction in myocardial SOD activity in group B when compared to that of group A (4.7 F 0.5 vs. 6.6 F 0.5 U/mg protein), while it increased significantly ( P b 0.05) in group C only in comparison to group B (IRI) (5.9 F 0.2 vs. 4.7 F 0.5 U/mg protein). 3

+

nmol/mg protein

2.5

** 2

***

1.5

TBARS

1 0.5 0

A

B

C

D

Fig. 1. Changes in myocardial TBARS (nmol/mg protein) in different groups. A=only perfusion, B=ischemic–reperfusion injury (IRI), C=IRI + 0.01% water extract of CC leaf and D=IRI + 0.05% water extract of CC leaf. Values are mean F SE. No. of observation 6.+P b 0.05 vs. A, **P b 0.01 vs. B, ***P b 0.001 vs. B.

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U of pyruvate release/min /mg protein

3004 12

++ 10

+

8

*** LDH

6

4

2

0

A

B

C

D

Fig. 2. Changes in myocardial LDH (U of pyruvate release/min/mg protein) in different groups. A=only perfusion, B=ischemic– reperfusion injury (IRI), C=IRI + 0.01% water. Extract of CC leaf and D=IRI + 0.05% water extract of CC leaf. Values are mean F SE. No. of observation 6. ***P b 0.001 vs. A,++P b 0.001 vs. B, +P b 0.01 vs. B.

Myocardial CAT (Fig. 4) There was a significant ( P b 0.001) reduction in myocardial CAT activity in group B when compared to that of group A (4.6 F 0.22 vs. 8.8 F 0.29 U/mg protein), while CAT activity increased significantly ( P b 0.01) in both groups C and D in comparison to group B (IRI) (8.5 F 0.8 and 9 F 0.5 vs. 4.6 F 0.2 U/mg protein). Myocardial GSH (Fig. 5) There was no significant change in myocardial GSH level in group B when compared to that of group A (10 F 1.9 vs. 10.7 F 0.02 Ag/mg protein), while it was increased significantly ( P b 0.001) in group C and D in comparison to group B (IRI) (13.7 F 0.5 and 17 F 1 vs. 10 F 1.9 Ag/mg protein). 8

+

7

*

U/mg protein

6 5

SOD

4 3 2 1 0

A

B

C

D

Fig. 3. Changes in myocardial SOD (U/mg protein) in different groups. A=only perfusion, B=ischemic–reperfusion injury (IRI), C=IRI + 0.01% water extract of CC leaf and D=IRI + 0.05% water extract of CC leaf. Values are mean F SE. No. of observation 6. *P b 0.05 vs. A,+P b 0.05 vs. B.

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** ** 10 9 8

U/mg protein

7

***

6 5

CAT

4 3 2 1 0

A

B

C

D

Fig. 4. Changes in myocardial CAT (U/mg protein) in different groups. A=only perfusion, B=ischemic–reperfusion injury (IRI), C=IRI + 0.01% water extract of CC leaf and D=IRI + 0.05% water extract of CC leaf. Values are mean F SE. No. of observation 6. ***P b 0.001 vs. A, **P b 0.01 vs. B.

Myocardial GPx (Fig. 6) There was no significant change in myocardial GPx activity in group B when compared to group A (0.28 F 0.02 vs. 0.26 F 0.004 U/mg protein). No change has been observed in group C and D also. Histopathological results H and E stained heart sections from IRI group (group B) revealed the presence of large areas of necrosis (Fig. 7B) with neutrophil infiltration and interstitial edema. In group C, there was marked edema with focal loss of myofibres and inflammation but no myonecrosis was seen (Fig. 7C). In group **

20 18

**

micro g/mg protein

16 14 12

GSH

10 8 6 4 2 0

A

B

C

D

Fig. 5. Changes in myocardial GSH (Ag/mg protein) in different groups. A=only perfusion, B=ischemic–reperfusion injury (IRI), C=IRI + 0.01% water extract of CC leaf and D=IRI + 0.05% water extract of CC leaf. Values are mean F SE. No. of observation 6. **P b 0.001 vs. B.

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R. Devi et al. / Life Sciences 77 (2005) 2999–3009 0.35 0.3

U/mg protein

0.25 0.2

GPx 0.15 0.1 0.05 0

A

B

C

D

Fig. 6. Changes in myocardial GPx (U/mg protein) in different groups. A=only perfusion, B=ischemic–reperfusion injury (IRI), C=IRI + 0.01% water extract of CC leaf and D=IRI + 0.05% water extract of CC leaf. Values are mean F SE. No. of observation 6.

A

B

C

D

Fig. 7. Light micrograph of heart tissue. A] Rat heart subjected to 70 min perfusion only. No focal separation of myocardial fibre (H and E  400). B] Rat heart subjected to 20 min ischemia and 40 min reperfusion injury showing focal destruction of myocardial fibres with neutrophil infiltration and marked edema (H and E  400). C] Rat heart subjected to 20 min ischemia and 40 min of reperfusion with 0.01% water extract of CC (H and E  400) marked edema with focal loss of myofibre and neutrophil infiltration. D] Rat heart subjected to 20 min ischemia and 40 min of reperfusion with 0.05% water extract of CC (H and E  400) with mild edema and mild focal occasional neutrophil infiltration and occasional loss of muscle fibre.

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D, the interstitial edema as well as inflammation was very less as compared to group B without any myonecrosis.

Discussion There is a dynamic relationship between reactive oxygen species (ROS) and antioxidants in the human body (Harrison et al., 2003). Healthy cells can scavenge free radicals effectively by means of antioxidants. However, in pathological conditions, like ischemic–reperfusion injury (IRI), the sudden generation of ROS can dramatically upset this balance with an increased demand on the antioxidant defense system. Once generated, free radicals alter the structural and functional integrity of cells by a variety of mechanisms, including lipid peroxidation, proteolysis and shearing of the nuclear material (Sun et al., 2002). Endogenous antioxidants including, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) are depleted accompanied by accumulation of ROS. It may be possible to limit oxidative stress induced tissue damage and, hence, prevent or ameliorate disease progression by favoring the balance towards lower oxidative stress. In the present study, ischemic–reperfusion injury was associated with increased oxidative stress, as evidenced by increase in myocardial TBARS and depletion of myocardial endogenous antioxidants, like SOD and CAT with reduction in LDH activity (marker for cellular injury). TBARS signifies the level of lipid peroxidation, as a consequence of increased ROS generation. SOD and CAT are two important enzymes, which act in unison to scavenge the ROS. First SOD dismutases the superoxide radical to form hydrogen peroxide, which is then acted upon by CAT to form water molecule (Sun et al., 2002). No significant change in GSH and GPx levels might signify that the level of oxidative stress was not severe enough for these compounds to be involved. However, administration of CC extract caused a significant increase in the GSH level which might be due to the sulphahydryl group containing compounds present in the CC leaf (Cao and Prior, 1998). Structural changes, like loss of myocardial fibre along with edema occurred in the myocytes following ischemia reperfusion. Treatment with CC extract prevented increase in lipid peroxidation and depletion of endogeneous antioxidants along with significantly less cellular injury. The mechanism of such protection of CC leaf extract may be due to the direct antioxidant effects of the plant leaf, which was previously reported by us (Devi et al., 2003). In this study, chronic oral administration of CC leaf extract in rat was reported to cause increase in ferric reducing ability of plasma (FRAP). The significant increase in FRAP after oral administration of water extract (WE) indicated the presence of bio-available antioxidants in CC leaf. Besides this we also observed in our previous studies that oral administration of water extract of CC leaf caused a significant reduction of lipid peroxidation (TBARS) in some vital organs of the body, like liver and kidney (Devi et al., 2003). In the present study, lowering of myocardial LDH in the reperfused heart reconfirmed the damage of myocyte during ischemia reperfusion, which might be due to oxidative stress. In the CC treated rat hearts there was no decrease in myocardial LDH activity indicating protection against ischemia–reperfusion injury. However dose-dependent effect was not evident in respect to SOD and LDH activity. This might be due to plateauing of the antioxidant activity of the leaf extract. Otherwise dose–response effect was observed with TBARS and to some extent with CAT activity.

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The observation made in the present study showed for the first time that acute treatment with CC extract prevent oxidative stress induced by myocardial ischemia reperfusion injury in rat.

Conclusion The present study suggests that the CC leaf extract possesses significant antioxidant activity, which might be helpful in preventing or slowing the progress of various oxidative stress related cardiovascular diseases.

Acknowledgements The authors acknowledge the financial support from Department of Biotechnology (DBT), Government of India, New Delhi, India, (DBT-PDF/KM BC /022) for this work.

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