International Journal of Nutrition, Pharmacology, Neurological Diseases

: 2014  |  Volume : 4  |  Issue : 1  |  Page : 58--63

Role of chrysin on hepatic and renal activities of Nω-nitro-l-arginine-methylester induced hypertensive rats

Veerappan Ramanathan, Malarvili Thekkumalai 
 Department of Biochemistry, Rajah Serfoji Government College, Thanjavur, Tamil Nadu, India

Correspondence Address:
Malarvili Thekkumalai
Department of Biochemistry, Rajah Serfoji Government College, Thanjavur - 613 001, Tamil Nadu


Objectives: The present study was undertaken to assess the antihypertensive, anti-hepatic and anti-renal activity of chrysin on Nω-nitro-l-arginine 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 the l-NAME (40 mg/kg B.W/day) in drinking water for 4 weeks. Rats were treated with chrysin (25 mg/kg B.W/day) for 4 weeks. Results and Discussion: Hypertension was manifested by considerably increased systolic and diastolic blood pressure and the toxic effect of 1-NAME was determined using the hepatic markers of lactate dehydrogenase, gamma glutamyl transpeptidase, renal markers of serum creatinine, creatinine clearance, urea, uric acid levels, urinary arachidonic acid metabolites of 6-keto-prostaglandin F 1α, thromboxane B2, 8-isoprostane-prostaglandin F and inflammatory parameters interleukin-6, tumor necrosis factor-alpha. Supplementation of chrysin at the dosage of 25 mg/kg considerably decreased systolic and diastolic blood pressure, hepatic markers, renal markers, urinary arachidonic acid metabolites and inflammatory parameters. Conclusion: These results suggest that chrysin decreases the blood pressure, significantly restores hepatic marker, renal markers, urinary arachidonic acid metabolites and inflammatory parameters and thus exhibits antihypertensive and anti-renal effects in l-NAME induced hypertensive rats.

How to cite this article:
Ramanathan V, Thekkumalai M. Role of chrysin on hepatic and renal activities of Nω-nitro-l-arginine-methylester induced hypertensive rats.Int J Nutr Pharmacol Neurol Dis 2014;4:58-63

How to cite this URL:
Ramanathan V, Thekkumalai M. Role of chrysin on hepatic and renal activities of Nω-nitro-l-arginine-methylester induced hypertensive rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2014 [cited 2021 Jan 28 ];4:58-63
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Hypertension is a major risk factor for cardiovascular mortality and morbidity, through its effects on target organs such as the heart, liver and kidney. [1] At the moment, hypertension afflicts more than 1 billion adults world-wide and 90-95% of these patients have essential hypertension. [2] Hypertension and its related developments such as coronary artery disease, stroke, heart failure and chronic kidney disease is a growing public health problem for which successful treatment often remains inadequate. [3] Hypertension is mainly attributable to endothelial dysfunction, which results from nitric oxide (NO) deficiency. In fact, it has been found that vascular endothelium of hypertensive patients produces less NO, a key regulator of the cardiovascular system and metabolic homeostasis. [4] NO is recognized as the endothelium-derived relaxing factor responsible for vascular dilation. Moreover, NO is known to be an anti-hypertrophic agent. [5] Nω-nitro-l-arginine methylester (l-NAME) is a non-specific inhibitor of all nitric oxide synthase (NOS) including neuronal nitric oxide synthase, inducible nitric oxide synthase and endothelial nitric oxide synthase. Supplementation of l-NAME can induce high blood pressure in animal models. [6] l-NAME-induced hypertension is thus a suitable model to study the cardiovascular effects of new active substances. The kidney is one of the organs targeted by hypertension. Renal disease secondary to hypertension progressively develops, culminating in chronic renal failure with a loss of glomeruli and several lots of morphological features and accompanying quantitative alterations. [7]

Flavonoids are usually plant polyphenolic compounds that consist of a number of classes, including flavanols, flavones and flavans. Chrysin (5,7-dihydroxy flavones [Figure 1] is a naturally occurring flavone contained in flowers such as the blue passion flower (Passiflora caerulea) and the Indian trumpet flower, as well as in edible items such as mushroom, [8] honey and propolis. [9] Chrysin has been found to possess antioxidant, [10] anti-allergic, [11] anti-inflammatory, [12] anti-cancer, [13] antiestrogenic, [14] anxiolytic [15] and antihypertension [16] properties.{Figure 1}

The prospective adverse reactions of chrysin have not been well studied. Only limited studies have been carried out to investigate its antioxidant properties and its role as an antihypertensive agent. The present study was indicate to know the association between hepatic markers of lactate dehydrogenase (LDH), gamma glutamyl transpeptidase (GGT), renal markers of serum creatinine, creatinine clearance, urea, uric acid (UA) levels, 8-isoprostane-prostaglandin F2α(8-iso-PGF2α), 6-keto-prostaglandin F1α(6-keto-PGF1α) , thromboxane B2 (TXB2) and Inflammatory parameters interleukin-6 (Il-6), tumor necrosis factor-alpha (TNF-α) in l-NAME induced hypertensive rats.

 Materials and Methods


Chrysin and l-NAME was 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 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 temperature of 28°C. 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 B.W) 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 the time of 1-8 weeks.

Study design

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 : ControlGroup II : Normal + chrysin (25 mg/kg of B.W) after 4 th weekGroup III : l-NAME induced hypertension (40 mg/kg of B.W)Group IV : l-NAME induced hypertension + Chrysin (25 mg/kg of B.W).

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 morning, 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 minutes. Serum was separated by centrifugation at 2000 rpm for 10 minutes. The blood, collected in a heparinized centrifuge tube, was centrifuged at 2000 rpm for 10 minutes and the plasma separated by aspiration was used for estimations.

Experimental methods

The serum GGT; EC was assayed according to the method of Rosalki and Rau. [17] The activity of LDH; EC was estimated by the method of King. [18] The serum urea, UA and creatinine, creatinine clearance were estimated by using diagnostic kits based on the methods of Fawcett and Scott, [19] Caraway [20] and Jaffe [21] respectively. Urinary 6-keto-PGF2α, TXB2, 8-iso-PGF2α, Il-6 and TNF-α were assayed according to the general principles of the enzyme-linked immunosorbant assay technique [22] using specific and sensitive kits (Cayman Chemical, Ann Arbor, MI, USA).

Statistical analysis

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 of P < 0.05 were regarded as statistically significant.


[Table 1] shows the activities of hepatic and renal functional markers such as GGT, LDH, creatinine, cratinine clearance, urea and UA in control and l-NAME induced hypertensive rats. The activities of these markers were increased significantly in l-NAME induced hypertensive rats and administration of chrysin significantly decreased the activities of these markers.{Table 1}

Urinary arachidonic acid metabolites such as 6-keto-PGF 1α were slightly higher (P < 0.05), and the excretion of TXB2 and 8-iso-PGF 2α was more pronounced (P < 0.05 respectively) as despicted in [Table 2]. Supplementation of chrysin significantly reduced the 6-keto-PGF 1α, TXB2 and 8-iso-PGF 2α compared to untreated l-NAME induced hypertensive rats.

[Table 3] shows elevated serum levels of Il-6 and TNF-α in l-NAME induced hypertensive rats (Group III) when compared to control. Supplementation of chrysin in (Group IV) reduced serum levels of Il-6 and TNF-α compared to l-NAME induced hypertensive rats. There is no significant variance between the control (Group I) and chrysin supplementation of the control (Group II).{Table 2}{Table 3}


Serum GGT and LDH are membrane bound enzymes, which may be secreted unequally depending on the pathological phenomenon. LDH is a cytosolic enzyme, which exists in all the tissues involved in glycolysis and prevails in five different isoforms specific as LDH1-LDH5. [23] GGT and LDH is excreted by the liver through bile and hence when liver is damaged, the serum enzyme level increases due to defective excretion. In our study, the action of GGT and LDH in the serum significantly increased in l-NAME induced hypertensive rats. The increase in the activities of GGT and LDH enzymes in the serum and pursuing fall in the tissue might be due to the leak of these cytosolic enzymes into the circulatory system resulting from liver as well as renal damage during l-NAME induced hypertensive rats. It is a measure of the renal as well as hepato-cellular damage due to kidney and liver dysfunction and disturbance in the biosynthesis of these enzymes, with alteration in the membrane permeability. Administration of chrysin prevented l-NAME induced renal toxicity and hepatotoxicity as indicated by as precipitous drop in serum GGT, LDH, creatinine and creatinine clearence levels, possibly by maintaining the integrity or renal cellular and hepato-cellular membrane. This is an indicator of possible nephro- and hepato-protective efficacy offered by chrysin.

Urea could be major nitrogen containing metabolic compound of necessary for protein metabolism; UA is the major product of purine nucleotides; creatinine is endogenously produced and released into body fluids and its clearance is a measure of the glomerular filtration rate. [24] The outcomes from the existing research reveal that l-NAME induced hypertensive rats show significantly increased levels of urea, UA and creatinine in serum as shown in earlier findings. [25] In addition to dyslipidemia, hypertensive rats suffered renal damages as evidenced by the elevation in serum urea and creatinine levels. [26] This is explained which there seemed to be clearance of blood urea and creatinine by the kidney, serum or even which right now there in which lowered necessary protein degradation.

UA could be the end product of endogenous as well as dietary purine nucleotide metabolism in humans. A growing body of data suggests a putative pathogenetic role for hyperuricemia in atherosclerosis and cardiovascular disease, especially in patients with diabetes mellitus, heart failure and hypertension. [27] Prospective studies have confirmed separate and important organizational associations of increased UA levels and cardiovascular events. [28] As regards blood pressure, elevated levels of UA have been identified as a forecaster of hypertension incidence and progression. [29] In the present study, serious self-consciousness of NOS by l-NAME caused an increase in serum urea UA level. Serum urea and UA is a sensitive and reliable biochemical index for evaluation of renal function in an animal model. The increased serum urea and UA indicates impairment of kidney function. Hypertensive rats also presented renal damages that were evidenced by the elevation in serum urea, UA, creatinine and creatinine clearance levels, which are considered as significant markers of renal dysfunction. Supplementation of chrysin prevented the increase in the levels of serum urea, UA, creatinine and creatinine clearance levels in l-NAME -induced hypertensive treated rats. This may due to chrysin having antireanl damage activity.

Arachidonic acid metabolites, a potent vasoconstrictor acting through TXA2α receptor activation, has been recommended as a paying attention of oxidative stress. [30] Urinary levels of 6-keto-PGF1α only increased a little bit, in line with the results of Tomida et al., [31] indicating that in l-NAME-hypertensive rats the levels of cyclooxygenase-2 (COX-2) messenger ribonucleic acid and necessary protein were increased in the kidneys and the thoracic aorta, suggesting that an increase in COX-2 expression might have a hypertensive effect partly associated with 8-iso-PGF2α formation in l-NAME rats. The present study shows supplementation of chrysin reduces the, TXB2, 6-keto-PGF1α and 8-iso-PGF2α in l-NAME induced hypertensive rats. The l-NAME induced group developed hypertension after the end of the first induction week. At the end of the experiment, an increase in inflammatory parameters (the serum Il-6 and TNF-α levels).

Il-6 is a pleiotropic cytokine having an array of biologic routines in immune regulation, hematopoiesis, inflammation and oncogenesis. Recent evidence indicates a pathological role for Il-6 in promoting proliferation of both smooth muscle and endothelial cells in the pulmonary arterioles, resulting in development of pulmonary arterial hypertension. [32] Biochemical markers, especially markers of vascular inflammation, such as high-sensitivity C-reactive protein (hs-CRP), have been recommended to be predictive of cardiovascular events. [33] The primary proinflammatory cytokines TNF-α and Il-6 include the major inducers of hs-CRP. On top of that, according to Navarro and Mora-Fernαndez, [34] TNF-α and Il-6 are regarded as causative of direct renal effects. In this particular study, treatment with chrysin reduced the serum Il-6 and TNF-α level. Experimental and clinical studies have demonstrated the pathogenic role of TNF-α in the development of renal injury and the potential benefit of modulating TNF-α activity as a therapeutic target in diverse renal diseases. Il-6 has been correlated with an increased width of the glomerular basement membrane. [35] It also enhances fibronectin expression, affects extracellular matrix dynamics at both the mesangial and podocyte levels, stimulates mesangial cell proliferation, and increases endothelial permeability. [36] Il-6 plays an important role in vascular remodeling and has been reported to be a useful biomarker in predicting cardiovascular events.

TNF-α is cytotoxic to glomerular, mesangial and epithelial cells, and it is able to induce direct renal damage. In addition, TNF-α stimulates endothelial generation of reactive oxygen species by activating the nicotinamide adenine dinucleotide phosphate-oxidase subunits gp91phox, NOX-1, p47phox, and p22phox. TNF-α also activates the transcription of nuclear factor-kappaB, which regulates the expression of genes involved in inflammation, oxidative stress and endothelial dysfunction. [37] Chrysin appears to alleviate adverse effects in l-NAME induced hypertensive rats by enhancing hepatic and inflammatory markers.


Chrysin prevented progression of hypertension in rats produced by administration of l-NAME, which may be due to its diuretic, nephroprotective, antihyperlipidaemic and antioxidant effects. It can be concluded that the chrysin possesses strong antihypertensive properties in l-NAME induced hypertensive rats as evidenced by a significant decrease in the hepatic, renal markers, urinary arachidonic acid metabolites and inflammatory markers.


1Saravanakumar 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.
2Whitworth JA, World Health Organization, International Society of Hypertension Writing Group. 2003 World Health Organization (WHO)/International Society of Hypertension (ISH) statement on management of hypertension. J Hypertens 2003;21:1983-92.
3Bilge M, Tolunay H, Kurmuþ O, Köseoðlu C, Alemdar R, Ali S. Percutaneous renal denervation in patients with resistant hypertension-first experiences in Turkey. Anadolu Kardiyol Derg 2012;12:79-80.
4Razny U, Kiec-Wilk B, Wator L, Polus A, Dyduch G, Solnica B, et al. Increased nitric oxide availability attenuates high fat diet metabolic alterations and gene expression associated with insulin resistance. Cardiovasc Diabetol 2011;10:68.
5Garcia JA, Incerpi EK. Factors and mechanisms involved in left ventricular hypertrophy and the anti-hypertrophic role of nitric oxide. Arq Bras Cardiol 2008;90:409-16.
6Bernátová 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.
7Caetano ER, Zatz R, Saldanha LB, Praxedes JN. Hypertensive nephrosclerosis as a relevant cause of chronic renal failure. Hypertension 2001;38:171-6.
8Jayakumar 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.
9Williams CA, Harborne JB, Newman M, Greenham J, Eagles J. Chrysin and other leaf exudate flavonoids in the genus Pelargonium. Phytochemistry 1997;46:1349-53.
10Hecker 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.
11Pearce 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.
12Fishkin RJ, Winslow JT. 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.
13Karthikeyan S, Srinivasan R, Wani SA, Manoharan S. Chemopreventive potential of chrysin in 7, 12 dimethylbenzyl (a) anthracene-induced hamster buccal pouch carcinogenesis. Int J Nutr Pharmacol Neurol Dis1997;60:775-8.
14Kao 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.
15Wolfman 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.
16Villar IC, Jiménez R, Galisteo M, Garcia-Saura MF, Zarzuelo A, Duarte J. Effects of chronic chrysin treatment in spontaneously hypertensive rats. Planta Med 2002;68:847-50.
17Rosalki SB, Rau D. Serum γ-glutamyl transpeptidase activity in alcoholism. Clin Chim Acta 1972;39:41-7.
18King J. The dehydrogenases or oxidoreductases-lactate dehydrogenase. In: Van D, editor. Practical Clinical Enzymology. London: Nostrand Company Limited; 1965. p. 83-93.
19Fawcett JK, Scott JE. A rapid and precise method for the determination of urea. J Clin Pathol 1960;13:156-9.
20Caraway WT. Determination of uric acid in serum by a carbonate method. Am J Clin Pathol 1955;25:840-5.
21Jaffe M. Concerning the precipitate produced in normal urine by picric acid and a new reaction of creatinine. Z Physiol Chem 1886;10: 91-400.
22Porstmann T, Kiessig ST. Enzyme immunoassay techniques. An overview. J Immunol Methods 1992;150:5-21.
23Plaa GL, Zimmerson HJ. Evaluation of hepatotoxicity: Physiological and biochemical measures of hepatic function. In: McCuskey RS, Earnest DL, editors. Comprehensive Toxicology. Cambridge: Cambridge University Press; 1997. p. 97-109.
24Burtis CA, Ashwood ER. Enzymes, Tietz Fundamental of Clinical Chemistry. Philadelphia, USA: N.B. Saunders Company; 1996. p. 4312-35.
25Saravanakumar M, Raja B. Veratric acid, a phenolic acid attenuates blood pressure and oxidative stress in L-NAME induced hypertensive rats. Eur J Pharmacol 2011;671:87-94.
26Stevens LA, Levey AS. Measurement of kidney function. Med Clin North Am 2005;89:457-73.
27Gagliardi AC, Miname MH, Santos RD. Uric acid: A marker of increased cardiovascular risk. Atherosclerosis 2009;202:11-7.
28Meisinger C, Koenig W, Baumert J, Döring A. Uric acid levels are associated with all-cause and cardiovascular disease mortality independent of systemic inflammation in men from the general population: The MONICA/KORA cohort study. Arterioscler Thromb Vasc Biol 2008;28:1186-92.
29Sundström J, Sullivan L, D'Agostino RB, Levy D, Kannel WB, Vasan RS. Relations of serum uric acid to longitudinal blood pressure tracking and hypertension incidence. Hypertension 2005;45:28-33.
30Takahashi K, Nammour TM, Fukunaga M, Ebert J, Morrow JD, Roberts LJ 2 nd , et al. Glomerular actions of a free radical-generated novel prostaglandin, 8-epi-prostaglandin F2 alpha, in the rat. Evidence for interaction with thromboxane A2 receptors. J Clin Invest 1992;90:136-41.
31Tomida T, Numaguchi Y, Nishimoto Y, Tsuzuki M, Hayashi Y, Imai H, et al. Inhibition of COX-2 prevents hypertension and proteinuria associated with a decrease of 8-iso-PGF2alpha formation in L-NAME-treated rats. J Hypertens 2003;21:601-9.
32Furuya Y, Satoh T, Kuwana M. Interleukin-6 as a potential therapeutic target for pulmonary arterial hypertension. Int J Rheumatol 2010;2010:720305.
33Ridker PM, Danielson E, Fonseca FA, Genest J, Gotto AM Jr, Kastelein JJ, et al. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. N Engl J Med 2008;359:2195-207.
34Navarro JF, Mora-Fernández C. The role of TNF-alpha in diabetic nephropathy: Pathogenic and therapeutic implications. Cytokine Growth Factor Rev 2006;17:441-50.
35Bourraindeloup M, Adamy C, Candiani G, Cailleret M, Bourin MC, Badoual T, et al. N-acetylcysteine treatment normalizes serum tumor necrosis factor-alpha level and hinders the progression of cardiac injury in hypertensive rats. Circulation 2004;110:2003-9.
36Dalla Vestra M, Mussap M, Gallina P, Bruseghin M, Cernigoi AM, Saller A, et al. Acute-phase markers of inflammation and glomerular structure in patients with type 2 diabetes. J Am Soc Nephrol 2005;16 Suppl 1:S78-82.
37dela Paz NG, Simeonidis S, Leo C, Rose DW, Collins T. Regulation of NF-kappaB-dependent gene expression by the POU domain transcription factor Oct-1. J Biol Chem 2007;282:8424-34.