|Year : 2014 | Volume
| Issue : 2 | Page : 112-117
Effects of chrysin on free radicals and enzymatic antioxidants in Nω-nitro-l-arginine methyl ester: Induced hypertensive rats
Thekkumalai Malarvili, Ramanathan Veerappan
Department of Biochemistry, Rajah Serfoji Government College, Thanjavur, Tamil Nadu, India
|Date of Submission||07-Oct-2013|
|Date of Acceptance||04-Nov-2013|
|Date of Web Publication||29-Mar-2014|
Department of Biochemistry, Rajah Serfoji Government College, Thanjavur - 613 001,Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: To evaluate the effect of chrysin, a natural biologically active compound extracted from many plants, mushrooms, honey, and propolis on the tissue and circulatory antioxidant status and lipid peroxidation in Nω-nitro-L-ariginine methyl ester (L-NAME)-induced hypertensive rats. Materials and Methods: Hypertension was induced in adult male albino rats of the Wistar strain, weighing 180-220 g, by oral administration of l-NAME (40 mg/kg BWT/day) in drinking water for 4 weeks. Rats were treated with chrysin (25 mg/kg BWT/day) for 4 weeks. Results: The results showed significantly elevated levels of tissue malondialdehyde, protein carbonyl, and xanthine oxidase, and significantly lowered nonenzymatic antioxidant activity of glutathione reductase and glutathione-S-transferase in l-NAME-induced hypertensive rats compared with those in the control group. Supplementation of chrysin at the dosage of 25 mg/kg considerably decreased the levels of malondialdehyde, protein carbonyl, and xanthine oxidase and significantly increased the activities of glutathione reductase and glutathione-S-transferase in the tissues and blood compared with those in the unsupplemented l-NAME-induced hypertensive groups. Conclusions: Chrysin offers protection against free radical-mediated oxidative stress in rats with l-NAME-induced hypertension.
Keywords: Glutathione-S-transferase, malondialdehyde, nitric oxide, protein carbonyl, xanthine oxidase
|How to cite this article:|
Malarvili T, Veerappan R. Effects of chrysin on free radicals and enzymatic antioxidants in Nω-nitro-l-arginine methyl ester: Induced hypertensive rats. Int J Nutr Pharmacol Neurol Dis 2014;4:112-7
|How to cite this URL:|
Malarvili T, Veerappan R. Effects of chrysin on free radicals and enzymatic antioxidants in Nω-nitro-l-arginine methyl ester: Induced hypertensive rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2014 [cited 2022 Jul 1];4:112-7. Available from: https://www.ijnpnd.com/text.asp?2014/4/2/112/129600
| Introduction|| |
Hypertension is a major public health problem and a leading cause of death and disability in developing countries. It remains a major risk factor in cardiovascular mortality and morbidity, through its effects on important target organs such as the heart, liver, and kidney.  By the year 2000, about 972 million of the world's adult population was affected by hypertension. This number may rise to 1.56 billion by the year 2025.  Nω-nitro-L-ariginine methyl ester (L-NAME) is a nonspecific inhibitor with three nitric oxide synthase (NOS) isoforms (including neuronal nitric oxide synthase, inducible nitric oxide synthase, and endothelial nitric oxide synthase) and it causes increased level of blood pressure when administered to experimental animals in a dose-dependent manner.  Oxidation and production of free radicals are common processes in regular cellular metabolism. A free radical is defined as any atom or molecule owning unpaired electrons. Free radicals such as O 2− (superoxide anion), OH− (hydroxyl radical), and 1 O 2 (singlet oxygen) are usually formed as a part of the normal metabolic processes. The destruction of these radicals present in cells may be quantitatively determined by measuring statistically the level of malondialdehyde (MDA), a product of membrane lipid peroxidation. Certain enzymes play a significant part in antioxidant defense, converting free radicals or reactive oxygen intermediates to non-radical products.
Flavonoids are usually plant polyphenolic compounds that consist of several classes including flavanols, flavones, and flavans. Chrysin (5,7-dihydroxy flavones; structure shown in [Figure 1]) is a naturally occurring flavone present in flowers such as the blue passion flower (Passiflora caerulea) and the Indian trumpet flower, as well as in edible items such as mushroom,  honey, and propolis. 
Chrysin has been found to possess antioxidant,  anti-allergic,  anti-inflammatory,  anti-cancer,  antiestrogenic,  anxiolytic,  and antihypertensive  properties. The prospective adverse reactions of chrysin have not been studied well yet. Only limited studies have been carried out to investigate its antioxidant properties and its role as antihypertensive agent. Therefore, the present study aimed to evaluate the effect of chrysin on tissue MDA, protein carbonyl (PCO), xanthine oxidase (XO), glutathione-S-transferase (GST), glutathione reductase (GR), protein, albumin, and globulin in l-NAME-induced hypertensive rats.
| Materials and Methods|| |
Chrysin and l-NAME were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used in this study were of analytical grade and obtained from E-Merck or HIMEDIA, Mumbai, India.
All the animal handling and experimental procedures were approved by the Institutional Animal Ethics Committee of Bharathidasan University (registration no: 418/01/a/date 04.06.2001), and the animals were cared for in accordance with the Indian National Law on Animal Care and Use. Male Wistar rats (180-220 g) were purchased from the Indian Institute of Science, Bangalore, India. Rats were housed in plastic cages with filter tops under controlled conditions of a 12-h light-dark cycle, 50% humidity, and a temperature of 28ºC. All rats received a standard pellet diet (Lipton Lever, Mumbai, India) and water ad libitum (BDU/IAEC63/2013).
Induction of l-NAME-induced hypertension
l-NAME (40 mg/kg BWT) was dissolved in drinking water and given to rats at an interval of 24 h for 8 weeks. Mean arterial blood pressure (MAP) was measured using tail cuff method. MAP measurements were performed at 1-8 weeks.
Animals were divided into four groups of six rats each and all were fed the standard pellet diet. The rats were grouped as given below:
- Group I: Control
- Group II: Normal + chrysin (25 mg/kg of BWT) after 4 th week
- Group III: l-NAME-induced hypertension (40 mg/kg of BWT)
- Group IV: l-NAME-induced hypertension + chrysin (25 mg/kg of BWT).
Chrysin was administered orally once in a day in the morning for 4 weeks. The compound was suspended in 2% dimethyl sulfoxide solution and fed by intubation. After the 8 th week, the animals were sacrificed by cervical dislocation. The blood was collected in clean dry test tubes and allowed to coagulate at ambient temperature for 30 min. Serum was separated by centrifugation at 175 × g for 10 min. The blood collected in a heparinized centrifuge tube was centrifuged at 175 × g for 10 min and the plasma was separated by aspiration. After the separation of plasma, the buffy coat enriched in white cells was removed and the remaining erythrocytes were washed three times with physiological saline. A known volume of erythrocytes was lysed with hypotonic phosphate buffer at pH 7.4. The hemolysate was separated by centrifugation at 290 × g for 10 min and the supernatant was used for various estimations. The liver, heart, and kidney were immediately removed and washed in ice-cold saline to remove the blood. The tissues were sliced and homogenized in 0.1 M Tris-HCl buffer (pH 7.0). The homogenates were centrifuged at 48 × g for 10 min at 0ºC in a cold centrifuge. The supernatants were separated and used for the determination of various parameters.
Biochemical parameters and free radical markers
Measurement of MDA as an assay for membrane lipid peroxidation was carried out using the method of Wright et al.  PCO was assayed by the method of Levin et al.  The activity of XO was assayed by the method of Athar et al.  GST activity was estimated by the method of Habig et al.  GR activity was measured by the method of Carlberg and Mannervik.  Serum total protein, albumin, and globulin levels were estimated by Biuret method. 
Data were analyzed by one-way analysis of variance followed by a Duncan's multiple range tests using a commercially available statistics software package (SPSS for Windows, version 11.0; SPSS Inc., Chicago, IL, USA). Results were presented as mean ± standard deviation (SD) values. P values < 0.05 were regarded as statistically significant.
| Results|| |
MDA, PCO, and XO levels in the tissues and blood of control and experimental animals are given in [Table 1]. MDA, PCO, and XO levels in the liver, kidney, heart, and blood of l-NAME-induced hypertensive rats (group III) were significantly higher compared with those of the control rats (group I) (P < 0.05). Chrysin supplementation to rats treated with l-NAME (group IV) lowered the MDA, PCO, and XO levels significantly compared with those in unsupplemented l-NAME-induced hypertensive rats (group III). Treatment with chrysin to control rats (group II) did not alter the MDA, PCO, and XO levels significantly.
|Table 1: Effect of chrysin on MDA, PCO, and XO levels in tissues and plasma |
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The activities of glutathione-related enzymes such as GR and GST in the tissues and circulation of both the control and experimental animals are given in [Table 2]. The levels/activities of GR and GST were found to be significantly lower in the liver, kidney, heart, and blood of l-NAME-induced hypertensive rats (group III) when compared with those in control rats (group I) (P < 0.05). Supplementation of chrysin to l-NAME-induced hypertensive rats significantly elevated the activities of GR and GST in the tissues and blood compared with those in the unsupplemented l-NAME-induced hypertensive rats (group III). GR and GST values did not alter significantly on treatment with chrysin in the control rats (group II) compared with the untreated normal control rats (group I).
|Table 2: Effect of chrysin on GST and GR activities in tissues and lysate |
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[Table 3] shows the level of serum total protein, albumin, globulin, and A/G ratio in both the control and experimental animals. The levels of total protein, albumin, and A/G ratio were significantly lower in l-NAME-induced hypertensive rats (group III) compared with those in control rats (group I) (P < 0.05). Chrysin at a dose of 25 mg/kg administered to l-NAME-induced hypertensive rats significantly increased the activities of total protein, albumin, and A/G ratio compared with those in unsupplemented l-NAME-induced hypertensive rats (group III). Total protein, albumin, and A/G ratio did not alter significantly on treatment with chrysin in the control rats (group II) compared with the untreated normal control rats (group I). None of the groups showed significant changes in globulin levels.
|Table 3: Effect of chrysin on protein, albumin, globulin levels, and A/G ratio in serum |
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| Discussion|| |
This experiment provides evidence that chronic inhibition of nitric oxide (NO) synthesis in rats leads to marked elevation of MDA, PCO, XO, and peripheral vascular resistance, with alteration of vascular responsiveness. These vascular alterations were associated with noticeable oxidative stress. In a previous work, it was demonstrated that a daily oral dose (25 mg/kg) of chrysin for 6 weeks reduced the elevated blood pressure in l-NAME-induced hypertensive rats.  A contingency treatment with chrysin partially prevented the development of l-NAME-induced hypertension and partly restored normal vascular responses. These findings confirm the previous ones on chronic inhibition of NO synthesis in l-NAME-induced hypertension, which is primarily due to the loss of both basal and stimulated NO production that normally has a relaxant effect on the vasculature.  The study shows significant increases in the levels of MDA, PCO, and XO present in the liver, kidney, heart, and plasma of rats with l-NAME-induced hypertension (group III) compared with those in normal control rats (group I) (P < 0.05) Free radical-mediated lipid peroxidation can result in membrane disorganization and accompanying decrease in membrane fluidity. 
Detection of lipid hydroperoxides, conjugated dienes, and thiobarbituric acid reactive substances such as MDA is part of the research involving lipid peroxidation reactions.  MDA, a secondary product of lipid peroxidation, is takes as a sign of tissue damage.  Hypertension may result in generating reactive oxygen species (ROS). However, in this particular study, MDA in the blood of l-NAME-treated rats was not different compared to that in the control rats. Cracowski et al. observed that lipid peroxidation and oxidative stress were not increased in untreated mild-to-moderate hypertension. In addition, it is suggested that ROS may not be critical in the early stages of hypertension; however, it could be far more critical in severe hypertension. Healthy protein oxidation is an exothermic event in which peptides react with free radicals, resulting in modification of several amino acids and leading to their accumulation in tissues, in addition to fragmentation involving protein.  The present study showed a marked decrease of protein MDA and carbonyl levels in the tissue samples from l-NAME-induced hypertensive rats. Low levels of MDA and PCO have been reported in the liver, heart, and kidney tissue samples from l-NAME-induced hypertensive rats that had been treated with chrysin.
XO is an intracellular enzyme which is an important marker of free radical-induced toxicity. In purine catabolism, XO brings about the generation of free radicals (O 2− , OH− , and H2 O2 ) during the oxidative hydroxylation of hypoxanthine to xanthine and subsequently via conversion of xanthine to uric acid.  Our results show that there was a significant increase in XO activity in l-NAME-induced hypertensive rats as compared with the control group. Chrysin was successful in combating this elevation of XO activity. This point leads to another mechanism by which chrysin may protect the renal tissue from oxidative damage. The capability of chrysin to enhance the levels of antioxidants along with its anti-lipid peroxidative activity indicates that this compound may be useful in counteracting free radical-mediated injury which is involved in the development of tissue damage caused by l-NAME-induced hypertension. Oxidative stress is an imbalance between ROS and the antioxidant defense mechanisms of a cell or tissue, which leads to lipid peroxidation, DNA damage, and the inactivation of many enzymes.  The enzymatic antioxidant defense system is a natural protector against lipid peroxidation that includes superoxide dismutase, catalase, and glutathione peroxidase.
Endogenously generated ROS, such as hydrogen peroxide and hydroxyl radicals, are immediately detoxified by the endogenous antioxidant armory comprising glutathione-dependent enzymes (GST and GR), and this detoxification reaction is catalyzed by GST.  In this study, a significant decrease in the activity of antioxidant enzymes such as GST and GR was observed in l-NAME-induced hypertensive rats as compared with the control group. A dose of 25 mg/kg of chrysin administered to l-NAME-induced hypertensive rats significantly elevated the activities of GR and GST in these tissues and blood compared with those in unsupplemented l-NAME-induced hypertensive rats (group III). GR and GST values did not alter significantly on treatment with chrysin in the control rats (group II) compared with the normal untreated rats (group I). Chrysin is known to be a potent free radical scavenger. The hydroxyl groups present in the fifth and seventh positions may contribute to its potent antioxidant effects.  Thus, the inherent antioxidant potential of chrysin may empower the other antioxidants such as GST and GR. This may be the reason for the increased levels of these enzymatic antioxidants in the tissues and lysate of chrysin in l-NAME-induced hypertensive rats. Chrysin seems to preserve the structural integrity of the hepatocyte membrane, as evidenced from the significant reduction in the activities of these enzymes. This indicates that chrysin has antihypertensive effect in l-NAME-induced hypertensive rats.
The levels of total protein, albumin, and A/G ratio were significantly lowered in l-NAME-induced hypertensive rats (group III) compared with those in control rats (group I) (P < 0.05). Albumin and globulin are the two key components of serum protein. As albumin is synthesized in the liver, it is the major element that is used to monitor liver function.  Decreased levels of serum total protein and albumin were seen in l-NAME-induced hypertensive rats. This demonstrates the decreased functional ability of the liver and this is a sign of poor health. Significant increases in serum total protein, albumin, and A/G ratio were observed in chrysin administered rats. Stabilization of serum protein levels on the administration of chrysin is a clear indication of the improvement of the functional status of the liver cells.
| Conclusion|| |
The data obtained in this study suggest for the first time under in vivo conditions that the active compound of chrysin attenuates the development of hypertension, improves hemodynamic status, and restores vascular function in NO-deficient hypertensive rats. The antioxidant activities of chrysin may be attributable to many factors including the ability to scavenge ROS, the increase in protein bioavailability, and the strengthening of the antioxidant GST, GR defense system. Chrysin at 25 mg/kg BWT produced marked antihypertensive effects in l-NAME-induced hypertensive rats.
| References|| |
|1.||Saravanakumar M, Raja B. Protective effect of borneol in liver and kidney tissues in l-NAME induced hypertensive rats; a FTIR report; oral presentation. Int J Nutr Pharmacol Neurol Dis 2011;1:19-26. |
|2.||Kearney PM, Whelton M, Reynolds K, Muntner P, Whelton PK, He J. Global burden of hypertension: Analysis of worldwide data. Lancet 2005;365:217-23. |
|3.||Bernátová I, Pechánová O, Kristek F. Mechanism of structural remodelling of the rat aorta during long-term NG-nitro-L-arginine methyl ester treatment. Jpn J Pharmacol 1999;81:99-106. |
|4.||Jayakumar T, Thomas PA, Geraldine P. In-vitro antioxidant activities of an ethanolic extract of the oyster mushroom, Pleurotus ostreatus. Innov Food Sci Emerg Technol 2009;10:228-34. |
|5.||Williams CA, Harborne JB, Newman M, Greenham J, Eagles J. Chrysin and other leaf exudate flavonoids in the genus Pelargonium. Phytochemistry 1997;46:1349-53. |
|6.||Hecker M, Preiss C, Klemm P, Busse R. Inhibition by antioxidants of nitric oxide synthase expression in murine macrophages: Role of nuclear factor kappa B and interferon regulatory factor 1. Br J Pharmacol 1996;118:2178-84. |
|7.||Pearce FL, Befus AD, Bienenstock J. Mucosal mast cells. III. Effect of quercetin and other flavonoids on antigen-induced histamine secretion from rat intestinal mast cells. J Allergy Clin Immunol 1984;73:819-23. |
|8.||Ingrid P. Red wine, white wine, rosé wine, and grape juice inhibit angiotensin-converting enzyme in human endothelial cells. Int J Nutr Pharmacol Neurol Dis 2013;3:17-23. |
|9.||Karthikeyan S, Srinivasan R, Wani SA, Manoharan S. Chemopreventive potential of chrysin in 7,12-dimethylbenz (a) anthracene-induced hamster buccal pouch carcinogenesis. Int J Nutr Pharmacol Neurol Dis 2013;3:46-53. |
|10.||Kao YC, Zhou C, Sherman M, Laughton CA, Chen S. Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: A site-directed mutagenesis study. Environ Health Perspect 1998;106:85-92. |
|11.||Wolfman C, Viola H, Paladini A, Dajas F, Medina JH. Possible anxiolytic effects of chrysin, a central benzodiazepine receptor ligand isolated from Passiflora coerulea. Pharmacol Biochem Behav 1994;47:1-4. |
|12.||Veerappan RM, Malarvili T. Role of chrysin on hepatic and renal activities of L-NAME induced hypertensive rats. Int J Nutr Pharmacol Neurol Dis 2013;(In press). |
|13.||Wright JR, Colby HD, Miles PR. Cytosolic factors which affect microsomal lipid peroxidation in lung and liver. Arch Biochem Biophys 1981;206:296-304. |
|14.||Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, et al. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 1990;186:464-78. |
|15.||Athar M, Sharma SD, Iqbal M, Sultana S, Pandeya KB, Tripathi IP. Coordination of copperpolyamine complex with imidazoles potentiates it superoxide dismutase mimicking activity and abolishes its interaction with albumin. Biochem Mol Biol Int 1996;39:813-21. |
|16.||Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130-9. |
|17.||Carlberg I, Mannervik B. Glutathione level in rat brain. J Biol Chem 1975;250:5475-80. |
|18.||Reinhold JG. Manual determination of serum total protein, albumin and globulin fractions by Biuret method. In: Reiner M. editor. Standard Methods in Clinical Chemistry. New York: Academic Press; 1953. p. 88. |
|19.||Manning RD Jr, Hu L, Mizelle HL, Montani JP, Norton MW. Cardiovascular responses to long-term blockade of nitric oxide synthesis. Hypertension 1993;22:40-8. |
|20.||Recknagel RO, Ghoshal AK. Quantitative estimation of peroxidative degeneration of rat liver microsomal and mitochondrial lipids after carbon tetrachloride poisoning. Exp Mol Pathol 1966;5:413-26. |
|21.||Eryavuz A, Dundar Y, Aslan R, Cengiz N. Investigation of the blood glutathione and malondialdehyde levels in tannery workers. Kocatepe Týp Derg 2001;2:147-51. |
|22.||Ohkawa H, Ohishi N, Yagi K. Assay of lipid peroxides in animal tissue by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8. |
|23.||Cracowski JL, Baguet JP, Ormezzano O, Bessard J, Stanke-Labesque F, Bessard G, et al. Lipid peroxidation is not increased in patients with untreated mild-to-moderate hypertension. Hypertension 2003;41:286-8. |
|24.||Ramana BV, Kumar VV, Krishna PN, Kumar CS, Reddy PU, Raju TN. Effect of quercetin on galactose-induced hyperglycaemic oxidative stress in hepatic and neuronal tissues of Wistar rats. Acta Diabetol 2006;43:135-41. |
|25.||Vorbach C, Harrison R, Capecchi MR. Xanthine oxidoreductase is central to the evolution and function of the innate immune system. Trends Immunol 2003;24:512-7. |
|26.||Speaker SD, Fabris M, Ferrari V, Dalle Carbonare M, Leon A. Quercetin protects cutaneous tissue-associated cell types including sensory neurons from oxidative stress induced by glutathione depletion: Cooperative effects of ascorbic acid. Free Radic Biol Med 1997;22:669-78. |
|27.||Kiziltepe T, Anderson KC, Kutok JL, Jia L, Boucher KM, Saavedra JE, et al. JS-K has potent anti-angiogenic activity in vitro and inhibits tumor angiogenesis in a multiple myeloma model in vivo. J Pharm Pharmacol 2010;62:145-51. |
|28.||Sathiavelu J, Senapathy GJ, Devaraj R, Namasivayam N. Hepatoprotective effect of chrysin on prooxidant-antioxidant status during ethanol-induced toxicity in female albino rats. J Pharm Pharmacol 2009;61:809-17. |
|29.||Friedman RB, Anderson RE, Entine SM, Hirshberg SB. Effects of diseases on clinical laboratory tests. Clin Chem 1980;26(Suppl 4):1D-476D. |
[Table 1], [Table 2], [Table 3]
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