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Inhibitory effect of Ruta graveolens L. on oxidative damage, inflammation and aortic pathology in hypercholesteromic rats
Experimental and Toxicologic Pathology 63 (2011) 285–290
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Inhibitory effect of Ruta graveolens L. on oxidative damage, inflammation and aortic pathology in hypercholesteromic rats M. Ratheesh, G.L. Shyni, G. Sindhu, A. Helen n Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram 695 581, India
a r t i c l e in fo
abstract
Article history: Received 1 April 2009 Accepted 26 January 2010
The purpose of the study was to investigate the efficacy of methanolic extract of Ruta graveolens L. in reducing oxidative damage, inflammation and aortic pathology in hypercholesteremic rats. For the study rats were divided into three groups – control group, hypercholesteremic group and treatment group (20 mg MER/kg/d orally) – and were fed for 90 days. Serum total cholesterol, LDL-C, total WBC count, CRP level, TBARS, atherogenic index, activities of COX, 15 LOX in monocyte and serum myeloperoxidase were increased in cholesterol fed rats. Activities of antioxidant enzymes and the concentration reduced glutathione in liver and heart tissue and serum HDL-C were decreased in cholesterol fed rats. The results showed that level of total cholesterol, LDL-C, atherogenic index was decreased and HDL-C was increased in MER treated rats. Activities of antioxidant enzymes were found to be increased and the activity of MPO, COX and 15 LOX were decreased on supplementation with MER. Concentration of TBARS and total WBC count were decreased and GSH was increased on supplementation with MER. Histopathology of aorta of cholesterol fed rat showed marked alterations whereas the aorta of MER administrated rat showed no significant changes. These results suggested that MER reduces oxidative stress, inflammation and aortic pathology in hypercholesteremic rats. Thus the plant may therefore be useful for therapeutic treatment of clinical conditions associated atherosclerosis. & 2010 Elsevier GmbH. All rights reserved.
Keywords: Anti-inflammatory Antioxidant Atherosclerosis Cyclooxygenase Lipoxygenase Aortic Pathology
Introduction Atherosclerosis is currently considered as a chronic and progressive disease arising from the inflammatory processes and oxidative stress within vessel wall (Hansson, 2005; Doria et al., 2005). Numerous epidemiologic studies have evaluated several inflammatory markers, including C-reactive protein (CRP), various cytokines, adhesion molecules, ceruloplasmin and white blood cell (WBC) count for their clinical usefulness in predicting risk of cardiovascular disease (Ridker et al., 1997; Ernst et al., 1987). Recent investigations have suggested that myeloperoxidase (MPO), an abundant enzyme secreted from activated neutrophils, monocytes, and certain tissue macrophages, may be involved in the development of coronary artery disease (CAD). Prostaglandin (PG) biosynthesis has been implicated in the pathophysiology of cardiovascular processes and a variety of inflammatory diseases (Dubois et al., 1998). The rate-limiting enzyme in the biosynthesis of PGs is prostaglandin-H2-synthase or cyclooxygenase (COX). COX enzymes are essential in the inflammatory process and control the downstream regulation of immune cell activation and inflammatory cytokine induction (Hoozemans and O’Banion,
n
Corresponding author. Tel.: + 91471 2418078; fax: + 91 471 2447158. E-mail address: [email protected] (A. Helen).
0940-2993/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2010.01.007
2005). Lipoxygenases (LOXs) comprise a family of non-heme iron-containing dioxygenases, representing the key enzymes in the biosynthesis of leukotrienes that have been postulated to play an important role in the pathophysiology of several inflammatory diseases such as asthma, atherosclerosis, diabetes and hypertension (Anning et al., 2005; Cyrus et al., 1999). Raised serum lipid levels, particularly of cholesterol along with generation of reactive oxygen species (ROS) play a key role in the development of coronary artery disease and atherosclerosis (Ross, 1999). The body has evolved a complex defense strategy to minimize the damaging effects of various oxidants. Central to this defense are the antioxidant enzymes. They include superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) which act in concert to protect the organism from oxidative damage (Gutteridge and Halliwell, 1996). Ruta graveolens L. (Rutaceae) commonly known as rue is known as medicinal plant since ancient times and currently used for treatment of various disorders such as aching pain, eye problems, rheumatism and dermatitis (Conway and Slocumb, 1979; Miguel, 2003) etc. Rue is a native of the Mediterranean region but cultivated throughout Europe and many Asian countries, including China, India and Japan. The plant contains more than 120 compounds of different classes of natural products such as acridone alkaloids, coumarins, essential oils, flavonoids and furoquinolines (Kuzovkina et al., 2004). The components of
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M. Ratheesh et al. / Experimental and Toxicologic Pathology 63 (2011) 285–290
Ruta species are of great interest in medicinal chemistry, as these compounds show a broad range of biological activity and a number of them are used in medicine (Ulubelen et al., 1986). Traditionally R. graveolens L. is used against many inflammatory diseases, based on its traditional use; the present study was to assess the possible ability of methanolic extract of R. graveolens in limiting oxidative damage, inflammation and aortic pathology in hypercholesterolemic male albino rats.
Materials and method Chemicals and solvents All biochemicals used in this study were purchased from Sigma Chemical Company, St. Louis, MO, USA and other chemicals and solvents of analytical grade were from SRL chemicals, Mumbai, India.
Biochemical analysis Serum total cholesterol (TC) and HDL-C concentrations were estimated using diagnostic kit available from Monozyme India Limited Company. The concentration of serum LDL-C was calculated by using the Friedewald’s formula (Friedewald et al., 1972). The activity of SOD in tissue was assayed by the method of Kakkar et al. (1984). Catalase was assayed by the method of Takahara et al. (1960). Glutathione peroxidae (GPx) was assayed by the method of Rotruck et al. (1973). Reduced glutathione (GSH) was estimated by the method of Ellman (1959) and the concentration of TBARS by the method of Ohkawa et al. (1979) was done in heart and liver of the experimental animals. Protein was determined by the methods of Lowry et al. (1951). Serum myeloperoxidase activity measured by O-dianisidine dihydrochloride oxidase method (Schosinsky et al., 1974). CRP in serum was determined by using Immunoturbidimetric kit (Diasys Diagnostics, Germany). Total WBC count was determined by using haemocytometer.
1.1. Plant material Fresh plant material was collected from kannur district in Kerala, India. The plant was authenticated by a botanist (Dr. G.Valsaladevi, Department of Botany, Kariavattom campus, University of Kerala). Preparation of the extract The collected plant material was washed thoroughly and dried in shade. The powdered plant material (100 g) was soaked in 70% methanol and stand for 3 days. The extract was concentrated to dryness by rotary evaporator in low pressure. The extract was cleared of low polarity contaminants such as fats, chlorophyll and xanthophyll by repeated extraction with petroleum ether (60–80 1C). This is used as R. graveolens methanolic extract (MER) Animals Male albino rats (Sprague–Dawley strain) weighing 70–100 g were selected from the animal house, Department of Biochemistry, and used with the approval of Animal Ethics Committee. Animals were housed individually in a well-ventilated animal unit with normal daylight. The rats were fed with a commercial rat feed and were accommodated in well-ventilated spacious cages. Food and water were given ad libitum. The temperature of the house was maintained around 27 1C. The rats were randomly divided into three groups of six rats each. Group I: Control (normal laboratory diet). Group II: Cholesterol fed (CF) group (normal laboratory diet +1.5 % cholesterol+ 0.5% cholic acid). Group III: Treated group (cholesterol diet+ MER at a dose of 20 mg/kg body weight dissolved in saline given orally by gastric intubation everyday). The rats were maintained on their respective diet for 90 days. At the end of the experimental period, rats were killed after overnight fasting by euthanasia. Blood and tissues were removed to ice cold containers for various biochemical analyses. Toxicity studies Toxicity were evaluated by measuring the activities of glutamic oxaloacetic transaminase (GOT) (E.C.2.6.1.1) and glutamic pyruvic transaminase (GPT) (E.C.2.6.1.2) by using diagnostic kit from Agappe Diagnostic Pvt. Ltd. India
Activity of cyclooxygenase and lipoxygenase in peripheral blood mononuclear cells (PBMC) Mononuclear cells were prepared as described by Radhika et al. (2007). A 3 ml volume of Histopaque 1083-solution was placed in a 15 ml tube and 3 ml blood was layered on top of this density gradient. After the centrifugation (400g for 30 min at room temperature) the blood cells were separated into two fractions: an upper white layer consisting of mononuclear cells plus the majority of platelets at the interface region, and a lower layer containing erythrocytes and granulocytes. The plasma layer on top was clear and contained no cells. First the plasma layer was removed and discarded. From the buffy coat, the monocytes were carefully taken off by aspiration and washed with phosphate buffered saline (PBS). This was repeated twice. After that the pellet was resuspended in PBS-Tween and subjected to freeze thaw in three times. The resulting lysate was used as the enzyme source. COX activity was assayed by the method of Shimizu et al. (1984) and 15- LOX activity was done by the method of Axelrod et al. (1981).
Histopathological analysis of aorta The entire aortas were rapidly dissected out and tissue sections (5 mm) fixed by immersion at room temperature in 10% formalin solution. For the histological examinations, paraffinembedded tissue sections of aorta were stained with hematoxylin–eosin (H&E). The tissue samples were then examined and photographed under a light microscope for observation of structural abnormality. The severity of aortic atherosclerosis as judged by two-independent observers blinded to the experimental protocol.
Statistical analysis All statistical calculations were carried out with the statistical package for social sciences (SPSS) software program (version 11. 0 for windows). The values are expressed as the mean 7SEM. Statistical evaluation was done using the one-way ANOVA and significant difference was determined using Duncan’s test at the level of Po0.05.
M. Ratheesh et al. / Experimental and Toxicologic Pathology 63 (2011) 285–290
Results The activities of GOT and GPT in serum was significantly (Po0.05) increased in CF rats when compared with normal group. Treatment with MER of 20 mg/kg showed significant decrease in the activity of GOT and GPT when compared to CF rats (Table 1). Concentration of serum TC, LDL-C and atherogenic index were decreased significantly (Po0.05) and HDL-C level increased significantly in MER treated group as compared to CF group (Table 2). Activities of antioxidant enzymes SOD, catalase, GPx were increased significantly in liver and heart of MER treated rats as compared to CF rats (Table 3). Significantly increased concentration of GSH and decreased level of TBARS was observed in liver and heart of MER treated rats as compared to the CF rats (Table 4). COX and 15 LOX activity in PBMC was significantly increased in CF rats when compared with normal rats. Treatment with MER showed significant decrease in COX and 15 LOX activity when compared to CF rats (Figs. 1and 2). MPO activity in serum was significantly increased in CF rats when compared with normal group. Treatment with MER showed significant decrease in MPO activity as compared to CF rats (Table 5). Serum CRP level and total WBC counts were significantly increased in CF rats than normal rats. Supplementation with MER significantly decreased the serum CRP level and total WBC counts (Table 5). Histopathology of aorta of cholesterol fed rat showed increased number of myointimal cells, thickening of
Table 1 Effects of MER on GOT and GPT levels. Groups
GPT (units/liter)
GOT (units/liter)
Control Cholesterol fed MER treated
18.1 7 2.2 37.1 7 3.4a 22.46 7 2.1b
24.9 73.0 41.2 73.1a 25.1 72.3b
GPT: glutamate pyruvate transaminase; GOT: glutamate oxaloacetic transaminase. Values expressed as average of 6 samples 7SEM in each group. a - Statistical difference with control group at P o0.05. b–Statistical difference between cholesterol fed group and MER treated group at P o 0.05.
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underlying media and loss of normal arrangement of elastic lamellae of the media where as in aorta of MER supplemented rat showed no significant changes (Fig. 3).
Discussion The effect of MER on liver function was evaluated by measuring the activities of serum GOT and GPT. These enzymes leak into the circulation when hepatocytes are damaged (Kew, 2000). It is believed that high serum cholesterol level can cause liver damages (Bolkent et al., 2004). Our results show that highfat diet has caused significant increase in serum GOT and GPT levels. However, rats treated with MER had lower serum GOT and GPT levels. High concentration of serum cholesterol and LDL-C increases the risk of developing CHD (Libby et al., 2000; Bbrliner and Hetnecke, 1996). Oral administration of MER reduced the level of total cholesterol and LDL-C in hypercholesterolemic rats. Another risk factor for developing atherosclerosis is the reduced serum level of HDL-C. This effect of HDL is largely attributed to its central function in the reverse cholesterol. Supplementation of MER significantly increased the serum level of HDL-C. Atherosclerotic index (AI), defined as the ratio of LDL-C to HDL-C also decreased by MER treatment. Many of the biological action of flavonoids have been attributed to their powerful hypolipidemic properties (Koshy and Vijayalakshmi, 2001; Wang and Ng, 1999). Several clinical trials have documented beneficial modifications of the LDL/HDL ratio after intake of flavonoid containing food products (Weggemans and Trautwein, 2003). Independent studies have confirmed the presence of antioxidant phenolic compounds in the aerial part of R. graveolens L. (Saieed et al., 2006). These results strongly suggest that the hypolipidemic activity of this medicinal plant could be attributed to the presence of the valuable polyphenolic compounds. Inflammation plays an essential role in the development and rupture of atherosclerotic plaques (Willerson and Ridker, 2004). C-reactive protein (CRP), which is a classic downstream marker of inflammation and mediates the initiation and progression of
Table 2 Concentration of Total cholesterol, HDL-cholesterol, LDL-cholesterol, in serum of experimental groups. Groups
Total cholesterol (mg/dl)
HDL-C (mg/dl)
LDL-C (mg/dl)
Atherogenic index
Control Cholesterol fed MER treated
73.95 72.8 107.43 74.2a 87.6 73.6a,b
26.1 70.47 17.55 70.7a 22.6 70.9a,b
34.88 7 1.37 72.31 7 2.8 a 51.6 7 0.4a,b
1.33 7 0.006 4.27 0.02a 2.26 7 0.004a,b
Values expressed as average of 8 samples 7 SEM in each group. a – Statistical difference with control group at P o0.05. b – Statistical difference between cholesterol fed group and MER treated group at P o0.05
Table 3 Effects of MER on the activities of SOD, CAT and GPx in liver and heart. Groups
Control Cholesterol fed MER treated
SOD (U/mg1)
CAT( U/mg2)
GPx(U/mg3)
Liver
Heart
Liver
Heart
Liver
Heart
11.7 70.43 2.86 7 0.11a 7.1 7 0.8a,b
13.047 0.53 5.25 70.73a 12.03 7 0.33b
60.57 71.8 10.91 7 0.5a 25.3 7 0.9a,b
11.75 7 0.45 2.7 7 0.08a 6.037 17a,b
9.1 7 0.38 5.65 7 0.23a 6.6 7 0.23a,b
14.83 70.3 6.8 70.28a 14.37 0.3b
SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase. Values expressed as average of 6 samples 7 SEM in each group. a – Statistical difference with control group at Po 0.05. b – Statistical difference between cholesterol fed group and MER treated group at P o0.05. 1 2 3
Enzyme concentration required to inhibit chromogen production by 50% in 1 min. Micromole H2O2 decomposed/min/mg protein. 1 m mol NADPH oxidized/min/mg protein.
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Table 4 Effects of MER on the concentration of TBARS and GSH in liver and heart. Groups
TBARS(mM/g) Liver
GSH (mM/100 g) Heart
Liver
Heart
Control 0.5217 0.017 0.347 0.006 475.7 7 7.7 409 78.9 0.677 0.009a 312.6 7 8.2a 326.3 79.3a Cholesterol fed 0.987 0.009a a,b a,b b MER treated 0.637 0.025 0.437 0.008 481.6 7 10.4 412.6 78.2b GSH, reduced glutathione; TBARS, thiobarbituric acid reactive substance. Values expressed as average of 6 samples 7 SEM in each group. a – Statistical difference with control group at Po 0.05. b – Statistical difference between cholesterol fed group and MER treated group at P o 0.05.
Fig. 2. Effect of MER on 15-lipoxygenase activity in isolated monocytes. Values expressed as average of 6 samples7 SEM in each group. a – Statistical difference with control group at P o 0.05. b – Statistical difference between cholesterol fed group and MER treated group at Po 0.05.
Table 5 Effects of MER on the level of CRP, total WBC count and myeloperoxidase activity in experimental animals. Groups
CRP (mg/ml)
Total WBC count ( 103/ml)
Myeloperoxidase (mm/min/ mg)
Control Cholesterol fed MER treated
50.7 70.33 75.5 70.3a
3.6 7 0.09 6.4 7 0.4a
352.2 72.6 872.3 76.2a
56.3 70.3a,b
3.7 7 0.06b
Fig. 1. Effect of MER on cyclooxygenase activity in isolated monocytes. Cyclooxygenase activity was measured as nanomoles of arachidonic acid converted per milligram of cyclooxygenase protein during 1 min. Values expressed as average of 6 samples 7SEM in each group. a – Statistical difference with control group at Po 0.05. b – Statistical difference between cholesterol fed group and MER treated group at P o0.05.
CRP, C-reactive protein. Values expressed as average of 6 samples7 SEM in each group. a – Statistical difference with control group at Po 0.05. b – Statistical difference between cholesterol fed group and MER treated group at Po 0.05.
atherosclerotic plaques by numerous molecular mechanisms (Venugopal et al., 2005). An elevated total white blood cell (WBC) count is a risk factor for atherosclerotic vascular disease. WBC-derived macrophages and other phagocytes are believed to contribute to vascular injury and atherosclerotic progression (Ernst et al., 1987; Fuster and Lewis 1994). In our study the level of CRP and total WBC count was found to be decreased significantly in MER administrated rats. COX is the rate limiting enzyme in the biosynthesis of prostaglandin. Prostaglandin (PG) biosynthesis has been implicated in the pathophysiology of cardiovascular processes and a variety of inflammatory diseases (Dubois et al., 1998). Supplementation with MER decreases the activity of COX in monocyte. These results suggests that MER, protect against the inflammation induced by high cholesterol diet. Oxidative modification of LDL is one of the critical steps for the development of atherosclerosis. Accumulating studies have indicated that 15-lipoxygenase highly expressed in macrophages plays an essential role in the oxidation of circulating LDL (Yoshitaka Takahashi et al., 2005). Also myeloperoxidase (MPO), a leukocyte enzyme that promotes oxidation of lipoproteins in atheroma, has been proposed as a possible mediator of
atherosclerosis. MPO also promotes oxidative damage of host tissues at sites of inflammation, including atherosclerotic lesions (Heinecke, 1999; Podrez et al., 2000). On treatment with MER decreases the activity of 15 LOX and MPO and thereby reduces the development of atherosclerosis by inhibiting oxidation of LDL. Histopathological examination of MER fed rat revealed a lesser degree of aortic atherosclerotic changes when compared with the aorta of the hypercholesterolemic rats. This showed the protective effects of MER on aortic pathology. Hypercholesterolemia increases endothelial O2 production and vascular oxidative stress, which may in turn contribute to impaired endothelial damage and atherogenesis (Guzik et al., 2000). SOD catalyses the dismutation of O21 – radical anions to H2O2 and O2 (Okado-Matsumoto and Fridovich, 2001). Our study has shown a significant increase in SOD activity in liver and heart during MER administration whereas in cholesterol fed rats it was decreased. This decrease could be due to a feedback inhibition or oxidative inactivation of enzyme protein due to excess ROS generation. Catalase, which acts as preventative antioxidant plays an important role in protection against the deleterious effects of lipid peroxidation. The activity of catalase in tissues was found to
437 77.51a,b
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were found to be increased significantly in MER administrated rats. The increased activity of these enzymes may be due to the decreased levels of peroxides, increased level of reduced glutathione content, which may results in the decreased formation of toxic intermediates. There are many reports on the ability of plant products to enhance the activity of antioxidant enzymes in vivo (Aviram et al., 2002), which support our results. Lipid peroxidation is a fundamental process in atherogenesis. Lipid peroxidation products may contribute to tissue damage through direct cytotoxic actions on endothelial cells or via reactions in which modified LDL is generated and is selectively bound by scavenger receptors (Witzum, 1994). In our study TBARS, the lipid peroxidation product was found to be decreased significantly in the tissues of MER administrated rats. To conclude, feed supplementation with MER reduced oxidative stress, inflammation and aortic pathology in hypercholesterolemic rats. Thus the plant may therefore be useful for therapeutic treatment of clinical conditions associated atherosclerosis. Further studies are in progress to elucidate the exact mechanism of action of active ingredient in the MER that mediates the protective effect.
Acknowledgements Financial assistance from UGC in the form of RGNF is gratefully acknowledged. We express gratitude to Dr. Suja Mary Koshy, Veterinary Doctor, Department of Biochemistry, Kariavattom for helping us with the animal experiments. We also express our gratitude to Jayaram V. Cytotechnolologist, Dr. Girija’s diagnostic laboratory, Attingal, Trivandrum for helping us with the histopathological studies.
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Fig. 3. Histopathology of aorta of experimental animals
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Inhibitory effect of Ruta graveolens L. on oxidative damage, inflammation and aortic pathology in hypercholesteromic rats M. Ratheesh, G.L. Shyni, G. Sindhu, A. Helen n Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram 695 581, India
a r t i c l e in fo
abstract
Article history: Received 1 April 2009 Accepted 26 January 2010
The purpose of the study was to investigate the efficacy of methanolic extract of Ruta graveolens L. in reducing oxidative damage, inflammation and aortic pathology in hypercholesteremic rats. For the study rats were divided into three groups – control group, hypercholesteremic group and treatment group (20 mg MER/kg/d orally) – and were fed for 90 days. Serum total cholesterol, LDL-C, total WBC count, CRP level, TBARS, atherogenic index, activities of COX, 15 LOX in monocyte and serum myeloperoxidase were increased in cholesterol fed rats. Activities of antioxidant enzymes and the concentration reduced glutathione in liver and heart tissue and serum HDL-C were decreased in cholesterol fed rats. The results showed that level of total cholesterol, LDL-C, atherogenic index was decreased and HDL-C was increased in MER treated rats. Activities of antioxidant enzymes were found to be increased and the activity of MPO, COX and 15 LOX were decreased on supplementation with MER. Concentration of TBARS and total WBC count were decreased and GSH was increased on supplementation with MER. Histopathology of aorta of cholesterol fed rat showed marked alterations whereas the aorta of MER administrated rat showed no significant changes. These results suggested that MER reduces oxidative stress, inflammation and aortic pathology in hypercholesteremic rats. Thus the plant may therefore be useful for therapeutic treatment of clinical conditions associated atherosclerosis. & 2010 Elsevier GmbH. All rights reserved.
Keywords: Anti-inflammatory Antioxidant Atherosclerosis Cyclooxygenase Lipoxygenase Aortic Pathology
Introduction Atherosclerosis is currently considered as a chronic and progressive disease arising from the inflammatory processes and oxidative stress within vessel wall (Hansson, 2005; Doria et al., 2005). Numerous epidemiologic studies have evaluated several inflammatory markers, including C-reactive protein (CRP), various cytokines, adhesion molecules, ceruloplasmin and white blood cell (WBC) count for their clinical usefulness in predicting risk of cardiovascular disease (Ridker et al., 1997; Ernst et al., 1987). Recent investigations have suggested that myeloperoxidase (MPO), an abundant enzyme secreted from activated neutrophils, monocytes, and certain tissue macrophages, may be involved in the development of coronary artery disease (CAD). Prostaglandin (PG) biosynthesis has been implicated in the pathophysiology of cardiovascular processes and a variety of inflammatory diseases (Dubois et al., 1998). The rate-limiting enzyme in the biosynthesis of PGs is prostaglandin-H2-synthase or cyclooxygenase (COX). COX enzymes are essential in the inflammatory process and control the downstream regulation of immune cell activation and inflammatory cytokine induction (Hoozemans and O’Banion,
n
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2005). Lipoxygenases (LOXs) comprise a family of non-heme iron-containing dioxygenases, representing the key enzymes in the biosynthesis of leukotrienes that have been postulated to play an important role in the pathophysiology of several inflammatory diseases such as asthma, atherosclerosis, diabetes and hypertension (Anning et al., 2005; Cyrus et al., 1999). Raised serum lipid levels, particularly of cholesterol along with generation of reactive oxygen species (ROS) play a key role in the development of coronary artery disease and atherosclerosis (Ross, 1999). The body has evolved a complex defense strategy to minimize the damaging effects of various oxidants. Central to this defense are the antioxidant enzymes. They include superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) which act in concert to protect the organism from oxidative damage (Gutteridge and Halliwell, 1996). Ruta graveolens L. (Rutaceae) commonly known as rue is known as medicinal plant since ancient times and currently used for treatment of various disorders such as aching pain, eye problems, rheumatism and dermatitis (Conway and Slocumb, 1979; Miguel, 2003) etc. Rue is a native of the Mediterranean region but cultivated throughout Europe and many Asian countries, including China, India and Japan. The plant contains more than 120 compounds of different classes of natural products such as acridone alkaloids, coumarins, essential oils, flavonoids and furoquinolines (Kuzovkina et al., 2004). The components of
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Ruta species are of great interest in medicinal chemistry, as these compounds show a broad range of biological activity and a number of them are used in medicine (Ulubelen et al., 1986). Traditionally R. graveolens L. is used against many inflammatory diseases, based on its traditional use; the present study was to assess the possible ability of methanolic extract of R. graveolens in limiting oxidative damage, inflammation and aortic pathology in hypercholesterolemic male albino rats.
Materials and method Chemicals and solvents All biochemicals used in this study were purchased from Sigma Chemical Company, St. Louis, MO, USA and other chemicals and solvents of analytical grade were from SRL chemicals, Mumbai, India.
Biochemical analysis Serum total cholesterol (TC) and HDL-C concentrations were estimated using diagnostic kit available from Monozyme India Limited Company. The concentration of serum LDL-C was calculated by using the Friedewald’s formula (Friedewald et al., 1972). The activity of SOD in tissue was assayed by the method of Kakkar et al. (1984). Catalase was assayed by the method of Takahara et al. (1960). Glutathione peroxidae (GPx) was assayed by the method of Rotruck et al. (1973). Reduced glutathione (GSH) was estimated by the method of Ellman (1959) and the concentration of TBARS by the method of Ohkawa et al. (1979) was done in heart and liver of the experimental animals. Protein was determined by the methods of Lowry et al. (1951). Serum myeloperoxidase activity measured by O-dianisidine dihydrochloride oxidase method (Schosinsky et al., 1974). CRP in serum was determined by using Immunoturbidimetric kit (Diasys Diagnostics, Germany). Total WBC count was determined by using haemocytometer.
1.1. Plant material Fresh plant material was collected from kannur district in Kerala, India. The plant was authenticated by a botanist (Dr. G.Valsaladevi, Department of Botany, Kariavattom campus, University of Kerala). Preparation of the extract The collected plant material was washed thoroughly and dried in shade. The powdered plant material (100 g) was soaked in 70% methanol and stand for 3 days. The extract was concentrated to dryness by rotary evaporator in low pressure. The extract was cleared of low polarity contaminants such as fats, chlorophyll and xanthophyll by repeated extraction with petroleum ether (60–80 1C). This is used as R. graveolens methanolic extract (MER) Animals Male albino rats (Sprague–Dawley strain) weighing 70–100 g were selected from the animal house, Department of Biochemistry, and used with the approval of Animal Ethics Committee. Animals were housed individually in a well-ventilated animal unit with normal daylight. The rats were fed with a commercial rat feed and were accommodated in well-ventilated spacious cages. Food and water were given ad libitum. The temperature of the house was maintained around 27 1C. The rats were randomly divided into three groups of six rats each. Group I: Control (normal laboratory diet). Group II: Cholesterol fed (CF) group (normal laboratory diet +1.5 % cholesterol+ 0.5% cholic acid). Group III: Treated group (cholesterol diet+ MER at a dose of 20 mg/kg body weight dissolved in saline given orally by gastric intubation everyday). The rats were maintained on their respective diet for 90 days. At the end of the experimental period, rats were killed after overnight fasting by euthanasia. Blood and tissues were removed to ice cold containers for various biochemical analyses. Toxicity studies Toxicity were evaluated by measuring the activities of glutamic oxaloacetic transaminase (GOT) (E.C.2.6.1.1) and glutamic pyruvic transaminase (GPT) (E.C.2.6.1.2) by using diagnostic kit from Agappe Diagnostic Pvt. Ltd. India
Activity of cyclooxygenase and lipoxygenase in peripheral blood mononuclear cells (PBMC) Mononuclear cells were prepared as described by Radhika et al. (2007). A 3 ml volume of Histopaque 1083-solution was placed in a 15 ml tube and 3 ml blood was layered on top of this density gradient. After the centrifugation (400g for 30 min at room temperature) the blood cells were separated into two fractions: an upper white layer consisting of mononuclear cells plus the majority of platelets at the interface region, and a lower layer containing erythrocytes and granulocytes. The plasma layer on top was clear and contained no cells. First the plasma layer was removed and discarded. From the buffy coat, the monocytes were carefully taken off by aspiration and washed with phosphate buffered saline (PBS). This was repeated twice. After that the pellet was resuspended in PBS-Tween and subjected to freeze thaw in three times. The resulting lysate was used as the enzyme source. COX activity was assayed by the method of Shimizu et al. (1984) and 15- LOX activity was done by the method of Axelrod et al. (1981).
Histopathological analysis of aorta The entire aortas were rapidly dissected out and tissue sections (5 mm) fixed by immersion at room temperature in 10% formalin solution. For the histological examinations, paraffinembedded tissue sections of aorta were stained with hematoxylin–eosin (H&E). The tissue samples were then examined and photographed under a light microscope for observation of structural abnormality. The severity of aortic atherosclerosis as judged by two-independent observers blinded to the experimental protocol.
Statistical analysis All statistical calculations were carried out with the statistical package for social sciences (SPSS) software program (version 11. 0 for windows). The values are expressed as the mean 7SEM. Statistical evaluation was done using the one-way ANOVA and significant difference was determined using Duncan’s test at the level of Po0.05.
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Results The activities of GOT and GPT in serum was significantly (Po0.05) increased in CF rats when compared with normal group. Treatment with MER of 20 mg/kg showed significant decrease in the activity of GOT and GPT when compared to CF rats (Table 1). Concentration of serum TC, LDL-C and atherogenic index were decreased significantly (Po0.05) and HDL-C level increased significantly in MER treated group as compared to CF group (Table 2). Activities of antioxidant enzymes SOD, catalase, GPx were increased significantly in liver and heart of MER treated rats as compared to CF rats (Table 3). Significantly increased concentration of GSH and decreased level of TBARS was observed in liver and heart of MER treated rats as compared to the CF rats (Table 4). COX and 15 LOX activity in PBMC was significantly increased in CF rats when compared with normal rats. Treatment with MER showed significant decrease in COX and 15 LOX activity when compared to CF rats (Figs. 1and 2). MPO activity in serum was significantly increased in CF rats when compared with normal group. Treatment with MER showed significant decrease in MPO activity as compared to CF rats (Table 5). Serum CRP level and total WBC counts were significantly increased in CF rats than normal rats. Supplementation with MER significantly decreased the serum CRP level and total WBC counts (Table 5). Histopathology of aorta of cholesterol fed rat showed increased number of myointimal cells, thickening of
Table 1 Effects of MER on GOT and GPT levels. Groups
GPT (units/liter)
GOT (units/liter)
Control Cholesterol fed MER treated
18.1 7 2.2 37.1 7 3.4a 22.46 7 2.1b
24.9 73.0 41.2 73.1a 25.1 72.3b
GPT: glutamate pyruvate transaminase; GOT: glutamate oxaloacetic transaminase. Values expressed as average of 6 samples 7SEM in each group. a - Statistical difference with control group at P o0.05. b–Statistical difference between cholesterol fed group and MER treated group at P o 0.05.
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underlying media and loss of normal arrangement of elastic lamellae of the media where as in aorta of MER supplemented rat showed no significant changes (Fig. 3).
Discussion The effect of MER on liver function was evaluated by measuring the activities of serum GOT and GPT. These enzymes leak into the circulation when hepatocytes are damaged (Kew, 2000). It is believed that high serum cholesterol level can cause liver damages (Bolkent et al., 2004). Our results show that highfat diet has caused significant increase in serum GOT and GPT levels. However, rats treated with MER had lower serum GOT and GPT levels. High concentration of serum cholesterol and LDL-C increases the risk of developing CHD (Libby et al., 2000; Bbrliner and Hetnecke, 1996). Oral administration of MER reduced the level of total cholesterol and LDL-C in hypercholesterolemic rats. Another risk factor for developing atherosclerosis is the reduced serum level of HDL-C. This effect of HDL is largely attributed to its central function in the reverse cholesterol. Supplementation of MER significantly increased the serum level of HDL-C. Atherosclerotic index (AI), defined as the ratio of LDL-C to HDL-C also decreased by MER treatment. Many of the biological action of flavonoids have been attributed to their powerful hypolipidemic properties (Koshy and Vijayalakshmi, 2001; Wang and Ng, 1999). Several clinical trials have documented beneficial modifications of the LDL/HDL ratio after intake of flavonoid containing food products (Weggemans and Trautwein, 2003). Independent studies have confirmed the presence of antioxidant phenolic compounds in the aerial part of R. graveolens L. (Saieed et al., 2006). These results strongly suggest that the hypolipidemic activity of this medicinal plant could be attributed to the presence of the valuable polyphenolic compounds. Inflammation plays an essential role in the development and rupture of atherosclerotic plaques (Willerson and Ridker, 2004). C-reactive protein (CRP), which is a classic downstream marker of inflammation and mediates the initiation and progression of
Table 2 Concentration of Total cholesterol, HDL-cholesterol, LDL-cholesterol, in serum of experimental groups. Groups
Total cholesterol (mg/dl)
HDL-C (mg/dl)
LDL-C (mg/dl)
Atherogenic index
Control Cholesterol fed MER treated
73.95 72.8 107.43 74.2a 87.6 73.6a,b
26.1 70.47 17.55 70.7a 22.6 70.9a,b
34.88 7 1.37 72.31 7 2.8 a 51.6 7 0.4a,b
1.33 7 0.006 4.27 0.02a 2.26 7 0.004a,b
Values expressed as average of 8 samples 7 SEM in each group. a – Statistical difference with control group at P o0.05. b – Statistical difference between cholesterol fed group and MER treated group at P o0.05
Table 3 Effects of MER on the activities of SOD, CAT and GPx in liver and heart. Groups
Control Cholesterol fed MER treated
SOD (U/mg1)
CAT( U/mg2)
GPx(U/mg3)
Liver
Heart
Liver
Heart
Liver
Heart
11.7 70.43 2.86 7 0.11a 7.1 7 0.8a,b
13.047 0.53 5.25 70.73a 12.03 7 0.33b
60.57 71.8 10.91 7 0.5a 25.3 7 0.9a,b
11.75 7 0.45 2.7 7 0.08a 6.037 17a,b
9.1 7 0.38 5.65 7 0.23a 6.6 7 0.23a,b
14.83 70.3 6.8 70.28a 14.37 0.3b
SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase. Values expressed as average of 6 samples 7 SEM in each group. a – Statistical difference with control group at Po 0.05. b – Statistical difference between cholesterol fed group and MER treated group at P o0.05. 1 2 3
Enzyme concentration required to inhibit chromogen production by 50% in 1 min. Micromole H2O2 decomposed/min/mg protein. 1 m mol NADPH oxidized/min/mg protein.
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Table 4 Effects of MER on the concentration of TBARS and GSH in liver and heart. Groups
TBARS(mM/g) Liver
GSH (mM/100 g) Heart
Liver
Heart
Control 0.5217 0.017 0.347 0.006 475.7 7 7.7 409 78.9 0.677 0.009a 312.6 7 8.2a 326.3 79.3a Cholesterol fed 0.987 0.009a a,b a,b b MER treated 0.637 0.025 0.437 0.008 481.6 7 10.4 412.6 78.2b GSH, reduced glutathione; TBARS, thiobarbituric acid reactive substance. Values expressed as average of 6 samples 7 SEM in each group. a – Statistical difference with control group at Po 0.05. b – Statistical difference between cholesterol fed group and MER treated group at P o 0.05.
Fig. 2. Effect of MER on 15-lipoxygenase activity in isolated monocytes. Values expressed as average of 6 samples7 SEM in each group. a – Statistical difference with control group at P o 0.05. b – Statistical difference between cholesterol fed group and MER treated group at Po 0.05.
Table 5 Effects of MER on the level of CRP, total WBC count and myeloperoxidase activity in experimental animals. Groups
CRP (mg/ml)
Total WBC count ( 103/ml)
Myeloperoxidase (mm/min/ mg)
Control Cholesterol fed MER treated
50.7 70.33 75.5 70.3a
3.6 7 0.09 6.4 7 0.4a
352.2 72.6 872.3 76.2a
56.3 70.3a,b
3.7 7 0.06b
Fig. 1. Effect of MER on cyclooxygenase activity in isolated monocytes. Cyclooxygenase activity was measured as nanomoles of arachidonic acid converted per milligram of cyclooxygenase protein during 1 min. Values expressed as average of 6 samples 7SEM in each group. a – Statistical difference with control group at Po 0.05. b – Statistical difference between cholesterol fed group and MER treated group at P o0.05.
CRP, C-reactive protein. Values expressed as average of 6 samples7 SEM in each group. a – Statistical difference with control group at Po 0.05. b – Statistical difference between cholesterol fed group and MER treated group at Po 0.05.
atherosclerotic plaques by numerous molecular mechanisms (Venugopal et al., 2005). An elevated total white blood cell (WBC) count is a risk factor for atherosclerotic vascular disease. WBC-derived macrophages and other phagocytes are believed to contribute to vascular injury and atherosclerotic progression (Ernst et al., 1987; Fuster and Lewis 1994). In our study the level of CRP and total WBC count was found to be decreased significantly in MER administrated rats. COX is the rate limiting enzyme in the biosynthesis of prostaglandin. Prostaglandin (PG) biosynthesis has been implicated in the pathophysiology of cardiovascular processes and a variety of inflammatory diseases (Dubois et al., 1998). Supplementation with MER decreases the activity of COX in monocyte. These results suggests that MER, protect against the inflammation induced by high cholesterol diet. Oxidative modification of LDL is one of the critical steps for the development of atherosclerosis. Accumulating studies have indicated that 15-lipoxygenase highly expressed in macrophages plays an essential role in the oxidation of circulating LDL (Yoshitaka Takahashi et al., 2005). Also myeloperoxidase (MPO), a leukocyte enzyme that promotes oxidation of lipoproteins in atheroma, has been proposed as a possible mediator of
atherosclerosis. MPO also promotes oxidative damage of host tissues at sites of inflammation, including atherosclerotic lesions (Heinecke, 1999; Podrez et al., 2000). On treatment with MER decreases the activity of 15 LOX and MPO and thereby reduces the development of atherosclerosis by inhibiting oxidation of LDL. Histopathological examination of MER fed rat revealed a lesser degree of aortic atherosclerotic changes when compared with the aorta of the hypercholesterolemic rats. This showed the protective effects of MER on aortic pathology. Hypercholesterolemia increases endothelial O2 production and vascular oxidative stress, which may in turn contribute to impaired endothelial damage and atherogenesis (Guzik et al., 2000). SOD catalyses the dismutation of O21 – radical anions to H2O2 and O2 (Okado-Matsumoto and Fridovich, 2001). Our study has shown a significant increase in SOD activity in liver and heart during MER administration whereas in cholesterol fed rats it was decreased. This decrease could be due to a feedback inhibition or oxidative inactivation of enzyme protein due to excess ROS generation. Catalase, which acts as preventative antioxidant plays an important role in protection against the deleterious effects of lipid peroxidation. The activity of catalase in tissues was found to
437 77.51a,b
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were found to be increased significantly in MER administrated rats. The increased activity of these enzymes may be due to the decreased levels of peroxides, increased level of reduced glutathione content, which may results in the decreased formation of toxic intermediates. There are many reports on the ability of plant products to enhance the activity of antioxidant enzymes in vivo (Aviram et al., 2002), which support our results. Lipid peroxidation is a fundamental process in atherogenesis. Lipid peroxidation products may contribute to tissue damage through direct cytotoxic actions on endothelial cells or via reactions in which modified LDL is generated and is selectively bound by scavenger receptors (Witzum, 1994). In our study TBARS, the lipid peroxidation product was found to be decreased significantly in the tissues of MER administrated rats. To conclude, feed supplementation with MER reduced oxidative stress, inflammation and aortic pathology in hypercholesterolemic rats. Thus the plant may therefore be useful for therapeutic treatment of clinical conditions associated atherosclerosis. Further studies are in progress to elucidate the exact mechanism of action of active ingredient in the MER that mediates the protective effect.
Acknowledgements Financial assistance from UGC in the form of RGNF is gratefully acknowledged. We express gratitude to Dr. Suja Mary Koshy, Veterinary Doctor, Department of Biochemistry, Kariavattom for helping us with the animal experiments. We also express our gratitude to Jayaram V. Cytotechnolologist, Dr. Girija’s diagnostic laboratory, Attingal, Trivandrum for helping us with the histopathological studies.
References
Fig. 3. Histopathology of aorta of experimental animals
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