|Year : 2014 | Volume
| Issue : 5 | Page : 17-22
Effects on chrysin on lipid and xenobiotic metabolizing enzymes in l-NAME-induced hypertension
Ramanathan Veerappan1, Thekkumalai Malarvili1, Govindaraju Archunan2
1 Department of Biochemistry, Rajah Serfoji Government College, Thanjavur, India
2 Department of Animal Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India
|Date of Web Publication||19-Dec-2014|
Department of Biochemistry, Rajah Serfoji Government College, Thanjavur - 613 001, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Clinical trial registration ijnpnd_56_14R1_Ref
| Abstract|| |
Objective: Nω-nitro-l-arginine methyl ester (l-NAME) is a non-specific nitric oxide (NO) synthase inhibitor, commonly used for the induction of NO-deficient hypertension. Hypertension is a significant risk factor in cardiovascular complications. Materials and Methods: This study was undertaken to investigate the effects of chrysin on lipid metabolizing enzymes, xenobiotic metabolizing enzymes, and microalbuminuria and N-acetyl-β-d-glucosaminidase (NAG) in urine in l-NAME-induced hypertensive rats. Hypertension was induced in adult male Wistar rats weighing 180-220 g by oral administration of l-NAME (40 mg/kg BW) in drinking water for 4 weeks. Rats were treated with chrysin (25 mg/kg BW) for 4 weeks. Results: The activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase increased significantly in the liver and kidney, whereas the activities of lipoprotein lipase and lecithin cholesterol acyl transferase decreased significantly in the plasma of hypertensive rats. Conclusions: The xenobiotic phase I enzymes, microalbuminuria, and NAG significantly increased, whereas xenobiotic phase II enzymes decreased in l-NAME-treated rats. Oral administration of chrysin reduced hyperlipidemia-related risk of hypertension.
Keywords: 3-hydroxy-3-methylglutaryl coenzyme A, cytochrome P450, lecithin cholesterol acyl transferase, lipoprotein lipase, nitric oxide
|How to cite this article:|
Veerappan R, Malarvili T, Archunan G. Effects on chrysin on lipid and xenobiotic metabolizing enzymes in l-NAME-induced hypertension. Int J Nutr Pharmacol Neurol Dis 2014;4, Suppl S1:17-22
|How to cite this URL:|
Veerappan R, Malarvili T, Archunan G. Effects on chrysin on lipid and xenobiotic metabolizing enzymes in l-NAME-induced hypertension. Int J Nutr Pharmacol Neurol Dis [serial online] 2014 [cited 2020 Jan 28];4, Suppl S1:17-22. Available from: http://www.ijnpnd.com/text.asp?2014/4/5/17/147459
| 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.  It is mainly caused by endothelial dysfunction which is caused by nitric oxide (NO) deficiency. An inhibitor of nitric oxide synthase in vitro, Nω -nitro-l-arginine methyl ester (l-NAME), also inhibits the release of NO from endothelial cells and aortic rings. In vivo l-NAME supplementation to rodents is associated with the development of hypertension and a generalized decrease in peripheral blood flow, NO synthesis and release by endothelial cells play an important vascular relaxation effect, contributing to the modulation of vascular tone.  The obstruction of NO supplementation synthase by l-NAME seems to be involved in lipid metabolism alterations of increases in serum cholesterol levels in rats  and also affects the endothelium function in hypercholesterolemic rabbits,  through which it causes atherosclerosis.  So, the l-NAME-induced animal model is a well-consistent and commonly used model of investigations on hypertension. 
Besides their particular lipid-lowering effects, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) crank out a number of pleiotropic actions that have been recently studied in the area of cardiovascular and renal protection. Microalbuminuria is a significant indicator of the early stage of hypertensive nephropathy. Moreover, it is an important intermediary end-point which correlates strongly with advanced renal disease.  N-acetyl-β-d-glucosaminidase (NAG) is a lysosomal enzyme that is present in high concentrations in the renal proximal tubular cells. Urinary NAG excretion has been recommended as one of the useful marker for detection of changes in proximal tubular function long before elevation in other markers as proteinuria and rise in serum creatinine.  Elevated urinary NAG excretion has been reported as a cause of renal damage in hypertension.  Many clinical and experimental studies have demonstrated a relation between urinary NAG excretion and renal oxidative damage. 
Flavonoids are plant polyphenolic compounds that consist of a number of classes, such as flavonols, flavones, and flavans. Chrysin (5,7-dihydroxy flavone, structure shown in [Figure 1]) is a naturally occurring flavone present in flowers such as 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. However, there are no scientific reports available on the effects of chrysin on l-NAME-induced hypertensive rats.
The present study was aimed to study the effects of chrysin on urinary NAG and microalbuminuria levels, lipid metabolizing enzymes such as lecithin cholesterol acyl transferase (LCAT), lipoprotein lipase (LPL), and HMG-CoA reducatse, and hepatic xenobiotic metabolizing enzymes as markers in l-NAME-treated 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. They were housed in plastic cages with filter tops under controlled conditions of a 12-h light-dark cycle, 50% humidity, and a temperature of 28°C. All the rats received a standard pellet diet (Lipton Lever, Mumbai, India) and water ad libitum (BDU/IAEC63/2013).
Induction of l-NAME-induced arterial hypertension
l-NAME (40 mg/kg BW) was dissolved in drinking water and given to rats at an interval of 24 h for 8 weeks. The mean arterial blood pressure (MAP) was measured using tail cuff method. MAP measurements were performed at 1-8 weeks.
MAP was determined by the tail cuff method (Model 31; IITC, Woodland Hills, CA, USA). Each animal was introduced into a restrainer and kept quiet for 5 to 10 minutes. This procedure was repeated for 2-3 days in order to familiarize the rats with the restrainer. A rubber cuff (proximally) and a photoelectric sensor of pulsations (more distally) were placed around the tail. The sensor was connected to an amplifier and pulsations were recorded on a power lab-recording unit. Peaks and troughs in the blood pressure curve were detected. A large number of recordings were taken; each value recorded was derived from eight to ten consecutive measurements (within approximately 15 minutes), which were then averaged to give one value representative of each experimental condition.
The animals were placed in a heated chamber at an ambient temperature of 30-34°C for 15 min, and for each animal, BP values (1-9) were recorded. The lowest three readings were averaged to obtain a mean BP. All recordings and data analyses were done using a computerized data acquisition system and software.
Design of this study
Animals were divided into four groups of six rats each and all were fed the standard pellet diet. Grouping of rats is as given below.
- Group I: Control
- Group II: Control + chrysin (25 mg/kg of BW) after 4 th week
- Group III: l-NAME-induced hypertension (40 mg/kg of BW)
- Group IV: l-NAME-induced hypertension + chrysin (25 mg/kg of BW).
Chrysin were administered orally once in a day in the morning for 4 weeks. The compound was suspended in 2% dimethyl sulfoxide vehicle solution and fed to animals by intubation. After 8 th week, the animals were sacrified by cervical dislocation. Blood was collected in clean dry test tubes and allowed to coagulate at an ambient temperature for 30 min. The serum was separated by centrifugation at 2000 rpm for 10 min. The blood, collected in a heparinized centrifuge tube, was centrifuged at 2000 rpm for 10 min and the plasma separated by aspiration was used for the estimations. The liver and kidney were immediately removed and washed in ice-cold saline to remove blood. The tissues were sliced and homogenized in 0.1 M Tris-HCl buffer (pH 7.0). The homogenates were centrifuged at 1000 rpm for 10 min at 0°C in a cold centrifuge.
Estimation of lipid marker enzymes
The activities of HMG-CoA reductase in liver and kidney, plasma LPL, and plasma LCAT were assayed by the method of Rao and Ramakrishnan,  Korn,  and Hitz et al.,  respectively. Control tubes containing only the substrate were treated similarly to check for complete inactivation of plasma during substrate preparations. LCAT activity was expressed as a function of the disappearance of free cholesterol during the incubation period.
Cytochrome P450 (CYP) and cytochrome b5 were assayed by the method of Omura and Sato.  NADPH-cytochrome P450 reductase (EC 184.108.40.206) was assayed by the method of Omura and Takesue.  Cytochrome P4502E1 (CYP4502E1) activity was assayed by the method of Watt et al.  NADH-cytochrome b5 reductase (EC 220.127.116.11) activity was assayed by the method of Mihara and Sato.  DT-diaphorase (EC 18.104.22.168) activity was assayed by the method of Ernster et al. 
Urinary NAG level was measured according to the method of Yakata et al.,  at 580 nm as 3-cersol sulfonphthalein released from 3-cresol sulfonphthaleinyl β-d-glucosaminide. Creatinine level in urine was measured using standard spectrophotometric methods (autoanalyzer, Abbott Aeroset; Abbott Diagnostics, IL, USA). Quantitative determination of albumin in the urine was performed using competitive chemiluminescent enzyme immunoassay using kits (Immulite 2000 Albumin) and Immulite Analyzer (DPC, Los Angeles, CA, USA). Urinary NAG excretion was expressed as U/g urinary creatinine to rule out the influence of urinary dilution or concentration. In addition, the final value of microalbuminuria was represented by the ratio of albumin and creatinine in urine (l g/mg creatinine). NO in the plasma samples was measured based on Griess reaction. 
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 and P < 0.05 were regarded as statistically significant.
| Results|| |
The activity of HMG-CoA reductase in control rats and l-NAME-induced hypertensive rats is shown in [Table 1]. Increased activity of HMG-CoA reductase was observed in the liver and kidney of l-NAME-treated hypertensive rats (group III). Treatment with chrysin (group IV) significantly (P < 0.05) decreased the activity of HMG-CoA reductase in these tissues when compared to l-NAME-treated hypertensive control rats (group III). The activities of LCAT and LPL of control rats and l-NAME-induced hypertensive rats are shown in [Table 2]. Decreased activities of LCAT and LPL were observed in the plasma of l-NAME-treated hypertensive rats (group III) as compared to control rats. Administration of chrysin (group IV) to hypertensive rats significantly (P < 0.05) increased the activities of LCAT and LPL when compared to hypertensive control rats (group III).
|Table 1: Effect of chrysin on the activities of HMG-CoA reducatse in liver and kidney |
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l-NAME (group III) treated rats showed increased activities of hepatic microsomal phase I enzymes, such as CYP, cytochrome b5, cytochrome P4502E1, NADPH-cytochrome P450 reductase, and NADH-cytochrome b5 reductase, as compared to the control rats (group I). Chrysin supplementation to l-NAME (group IV) treated rats reduced the activities of all the above phase I enzymes in the liver as compared to l-NAME alone (group III) treated rats [Table 3]. The activities of the liver cytosolic phase II enzymes such as DT-diaphorase showed decreased activities in the l-NAME (group III) treated rats as compared to the controls. Chrysin supplementation to l-NAME rats (group IV) increased the activities of DT-diaphorase as compared to the l-NAME alone (group III) treated rats [Table 3].
|Table 3: Effect of chrysin on hepatic xenobiotic metabolizing enzymes of control and experimental rats |
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[Table 4] shows that the level of urinary NAG or microalbumin excretion increased and plasma NOx decreased in l-NAME-administered rats (group III) compared to the control group (group I) (P < 0.05). Interestingly, in rats administered l-NAME plus chrysin (group IV), the urinary NAG or microalbumin excretion was significantly (P < 0.05) reduced and increased plasma NOx was found, compared with the l-NAME alone treated rats (group III).
|Table 4: Effect of chrysin on urinary microalbuminuria, urinary NAG/Cr, and NOx plasma |
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| Discussion|| |
HMG-CoA reductase has an important function in the regulation of cholesterol metabolism and it is a rate-limiting enzyme in the pathway of cholesterol biosynthesis. The present study shows a significant increase in the activity of HMG-CoA reductase in the liver and kidney of l-NAME-induced hypertensive rats. The enhanced activity of HMG-CoA reductase might be due to increased lipid peroxidation in l-NAME-treated rats in our study. An increase in HMG-CoA reductase activity leads to excessive production and accumulation of cholesterol, resulting in the formation of foam cells, a prerequisite step in the development of atherosclerosis caused by NO deficiency.  Supplementation of chrysin controlled the activity of HMG-CoA reductase in l-NAME-treated rats. This might be due to the inhibition of lipid peroxidation.
Liver is the major organ of LCAT synthesis. LCAT is an enzyme bound to high-density lipoproteins (HDLs) and low-density lipoproteins (LDLs) in the plasma. LCAT catalyzes the formation of cholesterol esters in lipoproteins. Decreased HDL-cholesterol (HDL-C) may be due to diminished LCAT activity and it contributes to increased cholesterol levels. An interesting observation was that chrysin significantly decreased the activity of LPL and LCAT in l-NAME-induced hypertensive rats. This shows the antilipidemic effect of chrysin in l-NAME-treated rats.
Phase I detoxification enzymes, CYP, comprise a multigene superfamily of microsomal heme-thiolate proteins that play critical roles in endogenous as well as xenobiotic metabolism and their detoxification.  The elevated activities of cytochrome P450 enzymes both in the liver and extra hepatic tissues can result in extremely low bioavailability of a number of orally administered phytochemicals.  In the present study, an increased activity of the cytochrome P450 family enzymes was observed in the liver of l-NAME-treated rats which was reversed on supplementation with chrysin. This strategic inhibition of P450 enzymes could be used to improve bioavailability of highly metabolized drugs. Chrysin is known to inhibit CYP1A activity, resulting in the modulation of drug metabolizing enzymes and regulation of cellular oxidation process. Phase II enzyme such as DT-diaphorase help in conjugating the xenobiotics to endogenous ligands like glutathione (GSH), glucuronic acid, acetic acid, or sulfuric acids, thus enhancing their solubility and excretion. Generally, inhibition of phase 1 enzymes concomitantly with induction of phase II enzymes is considered a logical strategy in chemoprevention.  l-NAME-treated rats showed decreased activities of the phase II enzymes. Supplementation with chrysin enhanced the phase II enzymes' activities, thereby helping in the regulation of xenobiotic metabolism.
In hypertensive patients, high urinary NAG and microalbumin excretion might reflect lysosomal dysfunction of both glomerular and proximal tubular epithelial cells.  In the present study, increased urinary NAG and microalbumin excretion was observed in hypertensive rats. This significant increase of the excretion of urinary NAG and microalbuminuria accounts for the occurrence of tubular cell damage due to oxidative stress in hypertensive rats. Increased peroxynitrite levels have been reported in the proximal tubules of patients with hypertensive nephropathy. Peroxynitrite generated in the tubular epithelium during renal hypertension has a potential to impair the structure of tubular cells.  By interfering with peroxynitrite-related pathways, chrysin proved to be effective in the prevention of hypertension and oxidative tubular renal injury. Furthermore, long-term chrysin administration resulted in decline in urinary NAG and microalbumin excretion. Also, we found a positive correlation among those parameters. These findings showed that chrysin may have beneficial effects in preventing tubular damage in hypertensive rat kidney.
NO synthesis and release by endothelial cells plays an important vascular relaxation effect,  contributing to the modulation of vascular tone. In addition, NO has been identified as important in other cellular events, such as vascular smooth muscle cell proliferation.  NO, synthesized by endothelial NO synthase (eNOS), is a major regulator of the vascular tone. Arterial hypertension, an important risk factor of cardiovascular diseases, is characterized by the production of excessive reactive oxygen species interacting with NO and reducing its bioavailability; oxidation of NO to peroxynitrite by reactive oxygen species induces oxidative stress.  Bioavailability of NO can be maintained by inhibition of oxidative stress, and therefore the agents with antioxidant properties inactivating free radicals increase NO bioavailability and can improve regulation of the vascular tone. Our findings also confirmed that chrysin-treated l-NAME groups significantly increased the NO level in plasma. These findings most likely reflect the diminished vasodilator action of endogenous NO, where l-NAME may have interfered with the activity of eNOS.  Administration of chrysin protects against renal and hepatic damage.
| Conclusion|| |
In conclusion, inhibition of NO synthase by chrysin has been shown to protect kidney and liver against oxidative damage induced by hypertension. Chrysin supplementation ameliorates the biochemical changes in the liver and kidney. Chrysin at 25 mg/kg BW offered marked renal and hepatoprotection by reversing the changes and exbited its protective role in reducing hyperlipidemia in l-NAME-induced hypertensive rats.
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[Table 1], [Table 2], [Table 3], [Table 4]