Scholarly article on topic 'Effects of Melothria maderaspatana leaf extract on antioxidant status in sham-operated and uninephrectomized DOCA-salt hypertensive rats'

Effects of Melothria maderaspatana leaf extract on antioxidant status in sham-operated and uninephrectomized DOCA-salt hypertensive rats Academic research paper on "Chemical sciences"

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Abstract of research paper on Chemical sciences, author of scientific article — Chinnadurai Veeramani, Balakrishnan Aristatile, Ganesan Pushpavalli, Kodukkur Viswanathan Pugalendi

Abstract The present study was designed to investigate the antihypertensive and antioxidant effect of Melothria maderaspatana leaf extract (MME) on sham-operated and DOCA-salt (deoxycorticosterone acetate) induced hypertensive rats. Administration of DOCA-salt significantly increased the systolic (from 127 to 212mm Hg) and diastolic (from 91 to 174mm Hg) blood pressure compared to sham-operated control rats, while treatment with MME significantly reduced the systolic (from 212 to 135mm Hg) and diastolic (from 174 to 96mm Hg) blood pressure compared to hypertensive control. In DOCA-salt rats, the plasma and tissue concentration of thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxide (LOOH) significantly increased and administration of MME significantly reduced these parameters towards the levels in sham-operated control. In hypertensive rats, activities of the enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and levels of non-enzymatic antioxidants such as vitamin C, vitamin E and reduced glutathione (GSH) decreased significantly in the plasma and tissues. Administration of MME returned the enzymatic and non-enzymatic antioxidants towards sham-operated control. MME shows both antihypertensive and antioxidant properties in DOCA-salt hypertensive rats and, among the three different doses tested, 200mg/kg caused the maximum effect.

Academic research paper on topic "Effects of Melothria maderaspatana leaf extract on antioxidant status in sham-operated and uninephrectomized DOCA-salt hypertensive rats"

King Saud University Saudi Journal of Biological Sciences

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ORIGINAL ARTICLE

Effects of Melothria maderaspatana leaf extract on antioxidant status in sham-operated and uninephrectomized DOCA-salt hypertensive rats

Chinnadurai Veeramani a, Balakrishnan Aristatile b, Ganesan Pushpavalli Kodukkur Viswanathan Pugalendi a'*

a Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar 608 002, Tamilnadu, India

b Department of Food Science and Nutrition, College of Food and Agricultural Science, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia

Received 11 February 2010; revised 2 May 2010; accepted 8 May 2010 Available online 31 May 2010

KEYWORDS

DOCA-salt; Hypertension; Oxidative stress; Antioxidants; Melothria maderaspatana

Abstract The present study was designed to investigate the antihypertensive and antioxidant effect of Melothria maderaspatana leaf extract (MME) on sham-operated and DOCA-salt (deoxycorticosterone acetate) induced hypertensive rats. Administration of DOCA-salt significantly increased the systolic (from 127 to 212 mm Hg) and diastolic (from 91 to 174 mm Hg) blood pressure compared to sham-operated control rats, while treatment with MME significantly reduced the systolic (from 212 to 135 mm Hg) and diastolic (from 174 to 96 mm Hg) blood pressure compared to hypertensive control. In DOCA-salt rats, the plasma and tissue concentration of thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxide (LOOH) significantly increased and administration of MME significantly reduced these parameters towards the levels in sham-operated control. In hypertensive rats, activities of the enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and levels of non-enzymatic antioxidants such as vitamin C, vitamin E and reduced glutathione (GSH) decreased significantly in the plasma and tissues. Administration of MME returned the enzymatic and non-enzymatic antioxidants towards sham-operated control. MME shows both antihypertensive and antioxidant properties in DOCA-salt

* Corresponding author. Tel.: +91 4144 238343; fax: +91 4144 239141.

E-mail address: pugale@siffymail.com (K.V. Pugalendi).

1319-562X © 2010 King Saud University. All rights reserved. Peerreview under responsibility of King Saud University. doi:10.1016/j.sjbs.2010.05.002

hypertensive rats and, among the three different doses tested, 200 mg/kg caused the maximum effect.

© 2010 King Saud University. All rights reserved.

1. Introduction

Hypertension is a well-defined risk factor for cardiovascular diseases and recent analyses show that it is the single most important cause of strokes and coronary heart disease (Touyz and Schiffrin, 2004; Beswick et al., 2001) In hypertensive animals, ROS are increased and superoxide dismutase, which converts O2 to H2O2, is reduced, suggesting that an imbalance between oxidant and antioxidant mechanisms is a contributing factor (Beswick et al., 2001; Thakali et al., 2006). ROS play an important role in several responses involved in vascular remodeling, including proliferation, migration, and hypertrophy (Beswick et al., 2001; Taniyama and Griendling, 2003). Therefore, ROS have been implicated in the pathogenesis of vascular disease, including atherosclerosis, apoptosis and hypertension. The long-term administration of DOCA-salts to rats induces sodium retention and high salt intake, producing volume-dependent hypertension (Hollenberg et al., 1992). This experimental model of hypertension is also associated with an increase in vascular superoxide anion generation and altered endothelium-dependent vascular relaxation (Wu et al., 2001). The heart, kidney and blood vessels are all implicated in hypertension; consequences of exposure to oxidative stress may include myocardial and renal hypertrophy (Sawyer et al., 2002) and endothelial dysfunction (Mclntyre et al., 1999).

Traditional medicine has demonstrated its contribution to the reduction of excessive mortality, morbidity and disability due to diseases such as hypertension, hepatotoxicity and diabetes. In India, the different traditional medicine systems make use of a number of plants in the treatment of hypertension. Melothria maderaspatana (L.) is one such medicinal plant popularly used for the treatment of hypertension in traditional practice (Cooke, 1967). Decoctions of leaves of M. Maderaspatana have been used for the treatment of live stock diseases such as adenitis, proplasmosis, theileriasis, plague, anthrax, rabies, madness and anaplas-mosis (Jayaweera, 1982). This plant leaf extract have also shown to have hepatoprotective (Jayathilaka et al., 1990), immunomodulatory effect and antiarthritic activity properties. Therefore, in the present study the main aim was to investigate the effects of MME on blood pressure antioxi-dants status on DOCA-salt hypertensive rats and to compare such effects with those of the calcium channel blocker nifedipine.

filtered using a muslin cloth and concentrated at 40 ± 5 0C. The extract was kept in deep freezer until use.

2.2. Animals

Male albino Wistar rats (weighing 200-230 g) were purchased from the Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College and Hospital, Annamalai University, and maintained in an air-conditioned room (25 ± 1 0C) with a 12 h light/12 h dark cycle. Feed and water were provided ad libitum. The experimental study was approved by the Ethical Committee of Rajah Muthiah Medical College and Hospital (Reg No.160/1999/CPCSEA, Proposal No.444), Annamalai University, Annamalainagar.

2.3. Chemicals

DOCA-salt was obtained from Sigma-Aldrich Company (St. Louis, Missouri, USA). All other chemicals used were of analytical grade obtained from E. Merck or HIMEDIA, Mumbai, India.

2.4. Method of uninephrectomy

Animals were anesthetized by an intraperitonial injection of ketamine (75 mg/kg BW). A small patch of skin above the left kidney was shaved, cleaned and applied with iodine based antiseptic. 1 cm incisions were made at midscapular region. The kidney was freed from the surrounding tissues and pulled out gently. The adrenal glands, which is attached loosely to the anterior pole of the kidney by connective tissue and fat, was gently freed by tearing the attachments, and was put back into the abdominal cavity. The renal artery and ureter were tied by silk thread, and then the kidney was removed. The muscle and skin layers were closed separately by using a chromic sterile absorbable suture.

2.5. Experimental induction of hypertension

Animals were given weekly twice subcutaneous injections of DOCA-salt (25 mg/kg BW) in dimethyl formamide (vehicle) solution and salt was administered by substitution of 1% NaCl solution for drinking water ad libitum throughout the experimental period.

2. Materials and methods

2.1. Preparation of leaf extract

Leaf powder of M. maderaspatana was purchased from the local herbal market (Vinayaga herbals), Chidambaram, Cudda-lore district, Tamil Nadu, India. The leaf powder was sieved and kept in deep freezer until use. 100 g of dry fine powder was suspended in 300 ml of ethanol for 72 h. The extract was

2.6. Experimental design

The rats were randomly divided into six groups of six rats each. Group one served as sham-operated control and group two served as hypertensive control. Groups III to V were hypertensive rats which received different doses of MME 50, 100 and 200 mg/kg BW and group VI received nifedipine 20 mg/kg BW. MME or nifedipine were administered orally once a day for 6 weeks between 9:00 a.m. and 10:00 a.m.

Group I: Sham-operated control (0.5% DMSO). Group II: Sham-operated + DOCA-salt + 1% NaCl control. Group III: Sham-operated + DOCA (25 mg/kg BW) + 1% NaCl.

Group IV: Sham-operated + DOCA-salt + 1%

NaCl + 50 mg/kg BW MME in 0.5% DMSO. Group V: Sham-operated + DOCA-salt + 1%

NaCl + 100 mg/kg BW MME in 0.5% DMSO. Group VI: Sham-operated + DOCA-salt + 1%

NaCl + 200 mg/kg BW MME in 0.5% DMSO. Group VII: Sham-operated + DOCA-salt + 1%

NaCl + 20 mg/kg BW nifedipine in 0.5% DMSO.

After 6 weeks, the animals were anaesthetized between 8:00 a.m. and 9:00 a.m. using ketamine (24 mg/kg BW, intramuscular injection) and sacrificed by cervical dislocation. Blood was collected in tubes with a mixture of EDTA for the estimation biochemical markers.

2.7. Blood pressure measurements

Systolic and diastolic blood pressures were determined by the tail-cuff method (IITC, model 31, Woodland Hills, CA, USA). The animals were placed in a heated chamber at an ambient temperature of 30-34 0C for 15 min and from each animal, 1-9 blood pressure values were recorded. The lowest three readings averaged to obtain a mean blood pressure. All recordings and data analyses were done using a computerized data acquisition system and software.

2.8. Biochemical analysis

The concentration of TBARS was estimated by the method of Niehaus and Niehaus and Samuelson, 1968, Lipid hydroperoxide was estimated by the method of Jiang et al., 1992. The activities of enzymatic antioxidants SOD, CAT and GPx were measured by the method of Kakkar et al., 1984; Sinha, 1972 and Rotruck et al., 1973, respectively. The non-enzymatic anti-oxidants GSH, vitamin C and vitamin E were estimated by the method of Ellman (1959), Roe and Kuether (1943) and Baker et al. (1980), respectively.

2.9. Statistical analysis

Statistical evaluation was performed using a one-way analysis of variance (ANOVA), followed by Duncan's multiple range test (DMRT) using the statistical package of social science (SPSS) version 10.0. The significance level was set at p < 0.05.

3. Results

Table 1 shows the effect of MME at three different doses (50, 100 and 200 mg/kg BW) on systolic and diastolic blood pressure in sham-operated and DOCA-salt hypertensive rats. The DOCA-salt rats significantly increased the systolic and dia-stolic blood pressure while treatment with MME or nifedipine significantly reduced the systolic and diastolic blood pressure and the effect was more pronounced at 200 mg/kg BW dose. There was no significant difference between MME and nifedi-pine on blood pressure.

Tables 2 and 3 show the effect of MME on the levels of TBAR and LOOH in the plasma and tissues of sham-operated and DOCA-salt hypertensive rats. The DOCA-salt rats exhibited a significant increase in the level of TBARS and LOOH. The administration of MME or nifedipine reduced the levels of TBARS and LOOH significantly and the effect was more pronounced at 200 mg/kg BW dose. Compared with nifedi-pine, MME showed better effect.

Tables 4-6 show the effect of MME on the activities of SOD, CAT and GPx in the erythrocyte and tissues of sham-operated and DOCA- salt hypertensive rats. The activities of SOD, CAT and GPx decreased significantly in DOCA-salt rats and the administration of MME or nifedipine significantly increased these parameters with a maximum at 200 mg/kg BW. The MME showed better effect compared with nifedipine.

Tables 7-9 show the effect of MME on GSH, vitamin C and E levels in the plasma and tissues of sham-operated and DOCA-salt hypertensive rats. The levels of GSH, vitamin C and E decreased significantly in DOCA-salt rats and administration of MME or nifedipine increased significantly all these parameters with a maximum at 200 mg/kg BW. The MME showed better effect compared with nifedipine.

4. Discussion

The long-term administration of DOCA-salt induces sodium retention and in the presence of a high salt, it produces volume-dependent type of hypertension in rats (Zhou et al., 2008). In the present study, the DOCA-salt rats significantly increased the systolic and diastolic blood pressure. Oral administration of MME or nifedipine resulted in a significant reduction in systolic and diastolic blood pressure. Triterpenes are one of the most numerous and widespread group of phenolic compounds in higher plants (Ohara and Ohira, 2003). Recent studies have shown that phenolic phytochemicals have high antioxidant (Vattem et al., 2005), antihypertensive and anti-diabetic activity (Kwon et al., 2006). The plant leaf

Table 1 Effect of Melothria maderaspatana leaf extract (MME) on the systolic and diastolic blood pressure of sham-operated and uninephrectomized DOCA-salt hypertensive rats.

Name of the group Systolic blood pressure (mm Hg) 0th day 6th week Diastolic blood pressure (mm Hg) 0th day 6th week

Sham-operated control DOCA-salt + 1% NaCl control DOCA-salt + 1% NaCl + MME (50 mg/kg BW) DOCA-salt + 1% NaCl + MME (100 mg/kg BW) DOCA-salt + 1% NaCl + MME (200 mg/kg Bw) DOCA-salt + 1% NaCl + nifedipine (20 mg/kg BW) 120.67 ± 6.42 117.83 ± 4.50 120.17 ± 3.82 115.83 ± 4.85 120.67 ± 4.64 118.00 ± 5.04 127.17 ± 7.18a 212.00 ± 9.51b 192.67 ± 9.20c 172.67 ± 7.00d 135.50 ± 7.48a 131.67 ± 7.80a 81.83 ± 4.50 87.50 ± 3.75 88.17 ± 5.34 86.67 ± 4.44 84.20 ± 4.68 87.67 ± 4.45 91.00 ± 4.84a 174.67 ± 8.89b 160.83 ± 9.28c 140.67 ± 7.31d 96.33 ± 6.08a 93.50 ± 4.51a

Values are means ± SD for six rats. Values not sharing a common superscript differ significantly at p < 0.05 (DMRT).

Table 2 Effect of MME on TBARS in the plasma and tissues of sham-operated and uninephrectomized DOCA-salt hypertensive rats.

Name of the group Plasma (mmol/dL) TBARS (mmol/100 g wet tissue)

Liver Kidney Heart

Sham-operated control DOCA-salt + 1% NaCl control DOCA-salt + 1% NaCl + MME (50 mg/kg BW) DOCA-salt + 1% NaCl + MME (100 mg/kg BW) DOCA-salt + 1% NaCl + MME (200 mg/kg Bw) DOCA-salt + 1% NaCl + nifedipine (20 mg/kg BW) 0.153 ± 0.014a 0.445 ± 0.035b 0.395 ± 0.025c 0.320 ± 0.020c 0.165 ± 0.014a 0.180 ± 0.023a 0.854 ± 0.062a 2.600 ± 0.151b 2.050 ± 0.145c 1.950 ± 0.134d 0.905 ± 0.073a'd 1.030 ± 0.122d 1.450 ± 0.122a 4.000 ± 0.338b 3.350 ± 0.235c 2.750 ± 0.205d 1.525 ± 0.125a'e 1.775 ± 0.147e 0.525 ± 0.042a 3.175 ± 0.157b 2.850 ± 0.122c 1.450 ± 0.122d 0.600 ± 0.054a 0.825 ± 0.063a'e

Values are means ± SD for six rats. Values not sharing a common superscript differ significantly at p < 0.05 (DMRT).

Table 3 Effect of MME on lipid hydroperoxide in hypertensive rats. l the plasma and tissues of sham-operated and uninephrectomized DOCA-salt

Name of the group Plasma (mmol/dL) LOOH (mmol/100 g wet tissue)

Liver Kidney Heart

Sham-operated control DOCA-salt + 1% NaCl control DOCA-salt + 1% NaCl + MME (50 mg/kg BW) DOCA-salt + 1% NaCl + MME (100 mg/kg BW) DOCA-salt + 1% NaCl + MME (200 mg/kg Bw) DOCA-salt + 1% NaCl + nifedipine (20 mg/kg BW) 9.16 ± 0.89a 20.47 ± 1.31b 18.51 ± 1.18c 15.83 ± 1.50d 10.65 ± 0.82a 13.50 ± 1.12e 78.57 ± 4.51a 99.07 ± 8.78b 96.33 ± 8.35b'c 91.78 ± 7.74b'c'd 82.52 ± 5.83a'd 86.97 ± 4.74a'c'd 65.48 ± 6.20a 170.24 ± 8.04b 148.81 ± 10.02c 119.05 ± 9.22d 70.83 ± 6.15a 75.95 ± 5.00a 69.64 ± 6.68a 139.88 ± 11.38b 120.83 ± 11.60c 107.14 ± 5.05d 72.02 ± 6.15a 77.05 ± 6.25a

Values are means ± SD for six rats. Values not sharing a common superscript differ significantly at p < 0.05 (DMRT).

Table 4 Effect of MME on activities of SOD in the erythrocyte and tissues of sham-operated and uninephrectomized DOCA-salt hypertensive rats.

Name of the group Erythrocyte (U/mg Hb) SOD (U/mg protein)

Liver Kidney Heart

Sham-operated control DOCA-salt + 1% NaCl control DOCA-salt + 1% NaCl + MME (50 mg/kg BW) DOCA-salt + 1% NaCl + MME (l00 mg/kg BW) DOCA-salt + 1% NaCl + MME (200 mg/kg Bw) DOCA-salt + 1% NaCl + nifedipine (20 mg/kg BW) 7.47 ± 0.43a 3.09 ± 0.30b 3.80 ± 0.23c 4.20 ± 0.21c 6.93 ± 0.52a 6.49 ± 0.57a 7.94 ± 0.47a 4.21 ± 0.34b 4.47 ± 0.41b 5.51 ± 0.46c 6.96 ± 0.59a 5.66 ± 0.43c'a 14.48 ± 1.54a 8.55 ± 0.84b 8.35 ± 0.83b 10.39 ± 1.37c 12.91 ± 1.68a 12.35 ± 0.96a 5.10 ± 0.34a-d 2.81 ± 0.12b 3.15 ± 0.23b-c 3.62 ± 0.29c 4.83 ± 0.47a-d 4.24 ± 0.22d

Values are means ± SD for six rats. Values not sharing a common superscript differ significantly at p < 0.05 (DMRT). U = enzyme concentration required to inhibit the chromogen produced by 50% in one min under standard condition.

Table 5 Effect of MME on activities of CAT in the erythrocyte and tissues of sham-operated and uninephrectomized DOCA-salt hypertensive rats.

Name of the group Erythrocyte (U/mg Hb) CAT (U/mg protein)

Liver Kidney Heart

Sham-operated control DOCA-salt + 1% NaCl control DOCA-salt + 1% NaCl + MME (50 mg/kg BW) DOCA-salt + 1% NaCl + MME (100 mg/kg BW) DOCA-salt + 1% NaCl + MME (200 mg/kg Bw) DOCA-salt + 1% NaCl + nifedipine (20 mg/kg BW) 168.90 ± 8.03a 97.26 ± 6.40b 108.80 ± 8.40b 124.88 ± 10.09c 160.26 ± 10.53a'd 153.59 ± 9.49d 76.99 ± 6.87a 52.81 ± 3.16b 56.06 ± 3.11b'c 60.32 ± 3.48c 72.47 ± 6.67a'd 68.08 ± 4.28d 31.99 ± 2.43a 18.07 ± 1.02b 19.72 ± 0.88b 22.89 ± 1.87c 28.04 ± 1.72d 24.29 ± 2.45c 48.57 ± 3.71a'd 28.19 ± 2.40b 33.86 ± 2.62c 36.03 ± 1.81c'd 44.86 ± 2.52e 39.30 ± 3.25d

Values are means ± SD for six rats. Values not sharing consumed/min. a common superscript differ significantly at p < 0.05 (DMRT). U = imol of H2O2

possesses coumarine (a triterpene) have many pharmacological et al., 1992). The significant reduction of blood pressures in activities such as anti-clotting ( Suttie, 1987), hypotensive DOCA-salt rats may be due to one or few of the active princi-(Huang et al., 1992) and anti-inflammatory activities (Paya ples of the leaf extract. Furthermore, it has been suggested that

Table 6 Effect of MME on activities of GPx in the erythrocyte and tissues of sham-operated and uninephrectomized DOCA-salt hypertensive rats.

Name of the group Erythrocyte (U/mg Hb) GPx (U/mg protein)

Liver Kidney Heart

Sham-operated control DOCA-salt + 1% NaCl control DOCA-salt + 1% NaCl + MME (50 mg/kg BW) DOCA-salt + 1% NaCl + MME (l00 mg/kg BW) DOCA-salt + 1% NaCl + MME (200 mg/kg Bw) DOCA-salt + 1% NaCl + nifedipine (20 mg/kg BW) 15.47 ± 1.33a 6.64 ± 0.45b 8.43± 0.57b 10.06 ± 0.55c 13.95 ± 1.26d 10.65 ± 0.78c 7.25 ± 0.52a 4.48 ± 0.40b 4.80 ± 0.35b 5.44 ± 0.35c 7.02 ± 0.30a 5.56 ± 0.34c 8.11± 0.47a 3.52 ± 0.35b 4.16 ± 0.35c 5.33 ± 0.53d 7.15 ± 0.64a'd 5.93 ± 0.47d 6.93 ± 0.48a 3.73 ± 0.74b 3.95 ± 0.26b-c 4.16 ± 0.35b'c 5.98 ± 0.46d 4.94 ± 0.44c'd

Values are means ± SD for six rats. Values not sharing a utilized/min. common superscript differ significantly at p < 0.05 (DMRT). U = imole of GSH

Table 7 Effect of MME on levels of vitamin-E in hypertensive rats. the plasma and tissues of sham-operated and uninephrectomized DOCA-salt

Name of the group Plasma (mg/dL) Vitamin-E (ig/mg protein)

Liver Kidney Heart

Sham-operated control DOCA-salt + 1% NaCl control DOCA-salt + 1% NaCl + MME (50 mg/kg BW) DOCA-salt + 1% NaCl + MME (100 mg/kg BW) DOCA-salt + 1% NaCl + MME (200 mg/kg Bw) DOCA-salt + 1% NaCl + nifedipine (20 mg/kg BW) 1.88 ± 0.12a 0.96 ± 0.11b 1.14 ± 0.16c 1.33 ± 0.14d 1.79 ± 0.11a 1.51 ± 0.11e 6.01 ± 0.43a 3.45 ± 0.24b 4.06 ± 0.26c 4.45 ± 0.29c 5.84 ± 0.32a 4.92 ± 0.37d 4.06 ± 0.34a 1.52 ± 0.23b 2.06 ± 0.19c 2.60 ± 0.19d 3.86 ± 0.24a 2.96 ± 0.22e 4.13 ± 0.36a 1.67 ± 0.16b 2.06 ± 0.26c 2.40 ± 0.12d 3.98 ± 0.25a 2.87 ± 0.18e

Values are means ± SD for six rats. Values not sharing a common superscript differ significantly at p < 0.05 (DMRT).

Table 8 Effect of MME on levels of vitamin-C in hypertensive rats. the plasma and tissues of sham-operated and uninephrectomized DOCA-salt

Name of the group Plasma (mg/dL) Vitamin-C (ig/mg protein)

Liver Kidney Heart

Sham-operated control DOCA-salt + 1% NaCl control DOCA-salt + 1% NaCl + MME (50 mg/kg BW) DOCA-salt + 1% NaCl + MME (100 mg/kg BW) DOCA-salt + 1% NaCl + MME (200 mg/kg Bw) DOCA-salt + 1% NaCl + nifedipine (20 mg/kg BW) 2.07 ± 0.12a 0.91 ± 0.07b 1.18 ± 0.11c 1.35 ± 0.10d 2.03 ± 0.15a 1.82 ± 0.07e 0.77 ± 0.06a 0.56 ± 0.03b 0.60 ± 0.04b-c 0.64 ± 0.06c 0.74 ± 0.06a-d 0.69 ± 0.05c'd 0.65 ± 0.03a 0.39 ± 0.02b 0.46 ± 0.03c 0.56 ± 0.04d 0.62 ± 0.05a 0.57 ± 0.03d 0.54 ± 0.03a 0.27 ± 0.02b 0.30 ± 0.03b 0.36 ± 0.03c 0.50 ± 0.04a 0.45 ± 0.04d

Values are means ± SD for six rats. Values not sharing a common superscript differ significantly at p < 0.05 (DMRT).

cardiac and renal injury can be avoided or minimized by reducing oxidative stress through increased intake of antioxidants (Schnackenberg et al., 1999). A few studies reported that antioxidants such as vitamins and SOD normalize the endothelial dysfunction and improves vascular remodeling in experimental hypertension (Akpaffiong and Taylor, 1998). Raja et al. reported that M. maderaspatana possesses antioxidant property in vitro (Raja and Pugalendi, 2009). Thus, the antihypertensive effect of this extract may also be due to the decreased oxidative stress and enhanced antioxidant potential of MME.

Oxidative stress, characterized by increased bioavailability of reactive oxygen species (ROS), plays an important role in the development and progression of cardiovascular dysfunction associated with hypertensive disease. Several studies have been reported the increased level of ROS such as superoxide anion, hydrogen peroxide and lipid peroxides in hypertensive patients (Touyz, 2000). Salt-sensitive hypertension is characterized by endothelial dysfunction associated with increases in

ROS and local renin-angiotensin-aldosterone system activation (Zhou et al., 2003). In both spontaneously hypertensive rat and DOCA-salt hypertension models, reactive oxygen species production increased because of an increase in reduced NADPH oxidase activity (Zalba et al., 2000). In this study, the concentration of TBARS and LOOH significantly increased in the plasma and tissues of DOCA-salt rats as reported earlier in clinical and experimental hypertensive rats (Sundaram et al., 1996). The increased concentration of lipid peroxidative markers suggests that an increase in oxygen free radicals, either by increased production or decreased destruction (Kakkar et al., 1995). The levels of lipid peroxidative markers in MME treated rats decreased significantly, which might be due to the presence of phenolic compounds such as coumarins, flavonoids, steroids and triterpenes (Sinha et al., 1997; Bhattacharjee and Das, 1969). Earlier report also supports the in vitro antioxidant effect of M. maderaspatana (Raja and Pugalendi, 2009).

Table 9 Effect of MME on levels of GSH in hypertensive rats. the plasma and tissues of sham-operated and uninephrectomized DOCA-salt

Name of the group Plasma (mg/dL) GSH (ig/mg protein)

Liver Kidney Heart

Sham-operated control DOCA-salt + 1% NaCl control DOCA-salt + 1% NaCl + MME (50 mg/kg BW) DOCA-salt + 1% NaCl + MME (l00 mg/kg BW) DOCA-salt + 1% NaCl + MME (200 mg/kg Bw) DOCA-salt + 1% NaCl + nifedipine (20 mg/kg BW) 34.73 i 2.99a 21.86 i 1.99b 23.28 i 1.57b 26.31 i 1.98c 32.64 i 1.74a 31.36 i 1.94a 11.77 i 1.03a 7.73 i 0.52b 8.10 i 0.58b 8.65 i 0.59b,c 10.07 i 0.93d 9.12 i 0.62c,d 9.94 i 0.53a 4.65 i 0.43b 5.39 i 0.50b 6.45 i 0.63c 9.01 i 0.56a 8.53 i 0.68a,c 7.97 i 0.41a 3.78 i 0.58b 4.26 i 0.38b 5.12 i 0.55c 7.20 i 0.57a 7.17 i 0.63a

Values are means ± SD for six rats. Values not sharing a common superscript differ significantly at p < 0.05 (DMRT).

Antioxidants inhibit lipid peroxidation (LPO) by preventing peroxidation chain reaction or accumulation of the ROS (Bernard and Kathy, 2004). SOD, CAT and GPx are major free radical scavenging enzymes that have shown to be reduced in a number of pathophysiological processes and diseases such as hypertension (Halliwell and Gutteridge, 1999). In the present study, DOCA-salt rats caused a significant depletion of enzymatic antioxidants in erythrocyte and tissues. SOD is an enzymatic antioxidant which reduces superoxide radical to hydrogen peroxide. CAT is a heme protein located predominantly in peroxisomes and the inner mitochondrial membrane that catalyzes the conversion of H2O2 to water and molecular oxygen (Farombi et al., 2000). A decrease in the activities of these antioxidant enzymes in tissue leads to the formation of superoxide anion and hydrogen peroxide which can later form hydroxyl radical. GSH-metabolizing enzyme, GPx, work in concert with glutathione in the decomposition of hydrogen peroxide and other organic hydroperoxides to non-toxic products. Reduced activity of GPx was observed due to inactiva-tion of this enzyme by ROS. Treatment with MME show increased activities of these enzymatic antioxidants, which might be due to the presence of phenolic compounds. Thus, administration of this extract clearly shows the free radical scavenging activity, which could exert a beneficial action against pathophysiological alterations caused by superoxide anion and hydroxyl radicals.

Non-enzymic antioxidants such as reduced glutathione, vitamin C and vitamin E play an excellent role in protecting the cells from oxidative damage.43 Vitamin E is a well-known physiological antioxidant and membrane stabilizer. It interrupts the chain reaction of lipid peroxidation by reacting with lipid peroxy radicals, thus protecting the cell structures against damage (Scibior et al., 2008). In our study, the level of vitamin E decreased significantly in DOCA-salt hypertensive rats which may be due to the increased utilization for free radical scavenging reaction or could be due to the decreased vitamin C because there is a well established synergism between vitamin C and E. Vitamin C is a hydrophilic antioxidant, because it disappears faster than other antioxidants. In our study, vitamin C level significantly decreased in DOCA-salt rats which might be caused by increased utilization of vitamin C as an antioxidant defense against reactive oxygen species or by a decrease in GSH, which is required for the recycling of vitamin C. GSH is one of the most important endogenous antioxi-dants. It plays the role of a sulfhydryl (SH) group provider for direct scavenging reactions. Glutathione peroxidase (GPx) catalyzes peroxide reduction utilizing GSH as the substrate and converting it to GSSG. Decreased GSH concen-

tration may also contribute to decreased GPx activity because GSH is one of the substrates for GPx (Asahi et al., 1995). In our study, the plasma and tissue GSH concentration significantly decreased in DOCA-salt rats which may be due to an increased utilization of GSH (Mahdi, 2002). The treatment with MME has elevated the levels of these parameters in DOCA-salt rats may be responsible for the decreased level of lipid peroxidation.

In conclusions, the antihypertensive and antioxidant properties of MME support its ethno medical use in India. It can be concluded that the M. maderaspatana leaf extract possesses strong antihypertensive and antioxidant properties in DOCA-salt hypertensive rats as evidenced by a significant decrease the blood pressure and increase in the levels of enzymatic and non-enzymatic antioxidants.

Acknowledgement

The financial assistance to the corresponding author as a major research project by the Indian Council of Medical Research, New Delhi, is gratefully acknowledged.

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