Subscribe to RSS
DOI: 10.1055/s-2002-34400
Effects of Chronic Chrysin Treatment in Spontaneously Hypertensive Rats
Prof. Dr. Juan Duarte
Department of Pharmacology
School of Pharmacy
University of Granada
18071 Granada
Spain
Phone: +34-958243889
Fax: +34-958248964
Email: jmduarte@ugr.es
Publication History
Received: December 10, 2001
Accepted: March 29, 2002
Publication Date:
30 September 2002 (online)
Abstract
The effects of an oral daily dose (20 mg kg-1) of the flavonoid chrysin for 6 weeks in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY) were analysed. Chrysin reduces SHR elevated blood pressure, cardiac hypertrophy and functional vascular changes, but is without effect in WKY. These protective effects were associated with a reduced oxidative status due to the antioxidant properties of the drug.
#Abbreviations
MDA:total plasma malondialdehyde
SBP:systolic blood pressure
SHR:spontaneously hypertensive rat
WKY:Wistar Kyoto rat
Reactive oxygen species have been suggested to contribute to the genesis of hypertension [1]. In vessels from spontaneously hypertensive rats (SHR) and essential hypertensives, enhanced endothelial superoxide anions (O2 -) production has been described [2], [3], and this effect has been related to the impairment of endothelium-dependent relaxation [5]. Through its interaction with nitric oxide (NO), O2 - is now emerging as a molecule of equal if not greater importance in cardiovascular pathology. Pharmacological intervention to tip the balance between NO and O2 - in favour of NO may be useful in the prevention and treatment of hypertension. Flavonoids comprise a large group of secondary metabolites with antioxidant properties [5]. The flavone chrysin (5,7-dihydroxyflavone) is present in honey and propolis and, in low concentrations, in fruits, vegetables and beverages [6]. In vitro experiments have revealed that chrysin was able to induce endothelium- and NO-dependent vasorelaxation [7]. However, there is little information about the effects of chrysin on in vivo animal models of systemic hypertension. Therefore, in the present study we have analysed the effects of chronic administration of an oral daily dose of chrysin (20 mg/kg) on systolic blood pressure (SBP), endothelial function and oxidative status in SHR and normotensive Wistar Kyoto rats (WKY).
Long-term chrysin administration induced a progressive reduction in SHR SBP while no changes were observed in WKY (Fig. [1]). At the end of the 6 weeks of treatment only a small reduction (-9 %) in SBP was observed in SHR-chrysin group as compared with SHR-vehicle. To our knowledge this is the first report showing the chronic antihypertensive effect of this flavonoid in this rat model of hypertension.
Chrysin, at low concentrations, shows endothelium- and NO-dependent vasodilator effects in isolated aortae [7]. Thus, this direct vasodilator effect might contribute to its antihypertensive effect observed in the present study. However, blood pressure measurements were carried out 42 - 48 hours after the last administration of chrysin, when chrysin and its metabolites (chrysin sulphate and glucuronide) had disappeared from plasma [8], suggesting that the direct vasodilator effect would play a role, but is not essential for the maintenance of low blood pressure.
The SHR model is characterised by an increased oxidative stress. Urinary levels of isoprostane F2 α, a prostaglandin-like compound produced in a non enzymatic reaction between arachidonic acid and O2 -, as well as plasma levels of malondialdehyde (MDA) have been proposed to be reliable markers of lipid peroxidation and oxidative stress [9]. In the present study, these parameters were enhanced in SHR as compared to normotensive WKY (Figs. [2] A and 2B, respectively). Chrysin treatment reduced both markers in SHR to levels similar to those of WKY (Fig. [2]), indicating that this drug reduced SHR oxidative stress. Furthermore, similarly as in in vitro studies [7], chronic chrysin treatment restored the endothelium-dependent vasodilatation to acetylcholine observed in ex vivo aortic rings from SHR but not in WKY (Fig 3A), while it had no effect on the relaxant effects of nitroprusside (Table [1]). This endothelium-dependent effect might result from the O2 - scavenging properties of chrysin, preventing the O2 --induced NO degradation and thus prolonging its half-life. Therefore, the restoration of NO-induced vasodilatation together with the reduced production of the vasoconstrictor 8-iso-prostaglandin F2 α may explain, at least partially, the antihypertensive effects of chrysin.
Sustained high blood pressure is one of the most powerful determinants of the development of cardiac hypertrophy [10]. In our study, the left ventricular weight index was significantly reduced in chrysin-treated SHR as compared to vehicle-treated SHR, following the trend of blood pressure reduction (Table [2]). Thus, the beneficial effect of chrysin on cardiac structure seems to be related to its blood pressure lowering effect. However, other effects beyond the antihypertensive properties, such as the antioxidant effects and the protection from NO breakdown, might also play a role in the prevention of morphological changes. When comparing its antihypertensive effect with that of the flavonoid quercetin [11], chrysin was less potent and effective. The reasons for this profile might be the different degree of systemic bioavailability. Chrysin has low oral bioavailability, mainly due to extensive intestinal metabolism [8]. Furthermore, our results are in agreement with previous data revealing that in vivo metabolism of flavonoids also gives rise to metabolites which, in general, are better antioxidants than the parent compound [5].
In conclusion, these data demonstrate that chrysin reduces the elevated blood pressure, the cardiac hypertrophy and the functional vascular changes in SHR rats. These effects were associated with a reduced oxidative status due to the antioxidant properties of the drug.
Fig. 1 Effects of chrysin on systolic arterial pressure as measured by tail-cuff plethysmography in the SHR vehicle (○), SHR chrysin (•), WKY vehicle (□) and WKY chrysin () groups. Values are expressed as means ± s. e. mean. * P < 0.05 , ** P < 0.01 vs. the SHR vehicle group (Bonferroni's test).
Fig. 2 Oxidative status. Urinary iso-PGF2 α excretion (A) and total plasma malondialdehyde (MDA) content (B) in the vehicle- (open bars) and chrysin-treated rats (solid bars). Data are means ± s. e. mean (n = 10). * P < 0.05, ** P < 0.01, ns not significant (Bonferroni's test).
Fig. 3 Endothelium-dependent relaxation (A) and endothelium-dependent contractions (B) induced by acetylcholine (ACh) in aortae from the SHR vehicle (○), SHR chrysin (•), WKY vehicle (□) and WKY chrysin () groups. ACh-induced relaxations were induced in arteries contracted by 10-7 M noradrenaline. ACh-induced contractions were evoked in arteries treated with L-NAME (10-4 M) and expressed as a percent of the response to 80 mM KCl. Values are expressed as means ± s. e. mean. * P < 0.05 and ** P < 0.01 SHR chrysin vs. the SHR vehicle group (Bonferroni's test).
| WKY vehicle (n = 10) | WKY chrysin (n = 10) | SHR vehicle (n = 10) | SHR chrysin (n = 10) | ||||||
| pD2 | Emax | pD2 | Emax | pD2 | Emax | pD2 | Emax | ||
| Noradrenaline (10-9 M - 10-6 M) |
8.03 ± 0.07 | 2 491 ± 333 | 8.09 ± 0.05 | 2 383 ± 244 | 8.00 ± 0.05 | 2 047 ± 215 | 7.87 ± 0.05 | 2 002 ± 182 | |
| KCl (8 - 80 mM) |
1.89 ± 0.04 | 2 372 ± 271 | 1.96 ± 0.05 | 2 343 ± 272 | 1.88 ± 0.03 | 2 168 ± 154 | 1.82 ± 0.02 | 2 078 ± 207 | |
| Sodium nitroprusside (10-10 M - 10-5 M) |
7.95 ± 0.08 | 103 ± 2 | 7.91 ± 0.11 | 109 ± 4 | 8.44 ± 0.05* | 108 ± 3 | 8.37 ± 0.06 | 104 ± 3 | |
| Values are means ± s. e. mean. Emax (maximal effect) for noradrenaline and KCl are expressed as mg of contraction and for sodium nitroprusside as a percentage of relaxation of the pre-contraction with noradrenaline. pD2 is the drug concentration exhibiting 50 % of the Emax expressed as negative log molar. | |||||||||
| *The pD2 values for sodium nitroprusside were significantly (P < 0.01) different in WKY as compared to SHR. No significant differences were found in chrysin- vs vehicle-treated groups (two way ANOVA). | |||||||||
| Group | BW, (g) | HW, mga | LVW, mga,c | KW, mga | HW/BW Ratioa,b |
LVW/BW Ratioa,c |
KW/BW Ratioa,b |
| WKY vehicle (n = 10) | 309 ± 14 | 895 ± 29 | 629 ± 23 | 808 ± 29 | 2.91± 0.05 | 2.04 ± 0.03 | 2.63 ± 0.05 |
| WKY chrysin (n = 10) | 307 ± 6 | 898 ± 29 | 637 ± 21 | 829 ± 14 | 2.92 ± 0.05 | 2.07 ± 0.04 | 2.70 ± 0.02 |
| SHR vehicle (n = 10) | 317 ± 9 | 1 086 ± 30 | 810 ± 20 | 872 ± 26 | 3.44 ± 0.06 | 2.56 ± 0.03 | 2.76 ± 0.04 |
| SHR chrysin (n = 10) | 303 ± 6 | 1 002 ± 21 | 736 ± 15* | 867 ± 23 | 3.31 ± 0.04 | 2.43 ± 0.03* | 2.86 ± 0.05 |
| BW indicates body weight; HW, heart weight; LVW, left ventricular weight; KW, kidney weight. | |||||||
| a P < 0.05 WKY vs. SHR. | |||||||
| b P < 0.05 interaction strain x treatment. | |||||||
| c P < 0.05 vehicle vs chrysin (two way ANOVA). | |||||||
| * P < 0.05 (Bonferroni) vs. SHR vehicle. | |||||||
Materials and Methods
Twelve-week old, male SHR and WKY were obtained from Harlan Laboratories (Barcelona, Spain). Twenty WKY and twenty SHR were randomly assigned to a control group (vehicle, 1 ml of 1 % methylcellulose) or a chrysin group (20 mg kg-1, mixed in vehicle). Rats were treated orally by gavage for 6 weeks. Body weight was measured every week. Systolic blood pressure (SBP) was measured weekly in conscious, prewarmed, restrained rats by tail-cuff plethysmography. The last day of the experimental period the animals were placed in metabolic cages to collect urine. The chrysin treatment was stopped 2 days before the end of the experiment in order to study the long-term effects of chrysin without the involvement of the effects of acute administration (half live time aproximately 4.6 h). All the experiments were performed in accordance with Institutional Guidelines for the ethical care of animals.
Immediately after exsanguination, heart and kidneys were excised, cleaned and weighed. The atria and the right ventricle were then removed and the remaining tissue (left ventricle plus septum) weighed. The heart weight index, the left ventricular weight index and the kidney weight index were calculated by dividing the heart weight, the left ventricular weight and the kidney weight by the body weight. Descending thoracic aortic rings (3 mm) were dissected and mounted in organ chambers as previously described [7], [11]. In endothelium denuded rings concentration-response curves to noradrenaline or KCl were constructed. The concentration-relaxation response curves to acetylcholine were performed in rings pre-contracted with 10-7 M noradrenaline. The concentration-relaxation response curves to nitroprusside were performed in the dark in rings pre-contracted with 10-6 M noradrenaline. Endothelium-dependent contractions to acetylcholine were tested in rings which were initially stimulated with 80 mM KCl. After washing in Krebs solution and incubation for 30 min with L-NAME (10-4 M), acetylcholine was added in a cumulative fashion. In these experiments, the contractile responses to acetylcholine were expressed as a percentage of the response to KCl. The total urine 8-iso-prostaglandin F2 α concentration was measured by a competitive enzyme immunoassay (R&D Systems, Inc., Minneapolis, USA). Plasma MDA content was evaluated spectrophotometrically as described by Duarte et al. [11]. Chrysin (98 % purity) was from Aldrich and other reagents were from Sigma.
Results are expressed as means ± s. e.means of measurements. The evolution of tail SBP was compared using the nested design, with groups treatment and days as fixed factors and the rat as random factor. When the overall difference was significant, comparisons were made using Bonferroni’s method with an appropriate error. Analysis of the nested design was also carried out with groups and concentrations to compare the concentration-response curves to acetylcholine. The rest of variables were compared using a two way factor design, where group and treatment were fixed effect factors with unequal sample sizes in the different groups. When interaction was significant Bonferroni's method was used for pairwise comparisons. P < 0.05 was considered statistically significant.
#Acknowledgements
This work was supported by CICYT (SAF 98-0160, SAF-2001-2953) Grants. Milagros Galisteo was a recipient of a research contract from the University of Granada (Spain).
#References
- 1 Griendling K K, Alexander R W. Oxidative stress and cardiovascular disease. Circulation. 1997; 96 3264-5
- 2 Grunfeld S, Hamilton C A, Mesaros S, Mcclain S W, Dominiczak A F, Bohr D F. et al . Role of superoxide in the depressed nitric oxide production by the endothelium of genetically hypertensive rats. Hypertension. 1995; 26 854-7
- 3 Berry C, Hamilton C A, Brosnan M J, Magill F G, Berg G A, McMurray J JV. et al . Investigation into the sources of superoxide in human blood vessels. Angiotensin II increases superoxide production in human internal mammary arteries. Circulation. 2000; 101 2206-12
- 4 Jameson M, Dai F -X, Lüscher T, Skopec J, Diederich A, Diederich D. Endothelium-derived contracting factors in resistence arteries of young spontaneously hypertensive rats before development of overt hypertension. Hypertension. 1993; 21 280-8
- 5 Rice-Evans C A, Miller N J, Paganda G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Bio Med. 1996; 20 933-56
- 6 Duarte J, Jiménez R, Villar I C, Pérez-Vizcaino F, Jiménez J, Tamargo J. Vasorelaxant effects of the bioflavonoid chrysin in isolated rat aorta. Planta Med. 2001; 67 567-9
- 7 Siess M H, Le Bon A M, Canivenc-Lavier M C, Amiot M J, Sabatier S, Aubert S Y. et al . Flavonoids of honey and propolis: Characterization and effects on hepatic drug-metabolizing enzymes and benzo[α]pyrene-DNA binding in rats. J Agric Food Chem. 1996; 44 2297-301
- 8 Walle T, Otake Y, Brubaker J A, Walle U K, Halushka P V. Disposition and metabolism of the flavonoid chrysin in normal volunteers. Br J Clin Pharmacol. 2001; 51 143-6
- 9 Morrow J D, Roberts L J. The isoprostanes. Current knowledge and directions for future research. Biochem Pharmacol. 1996; 51 1-9
- 10 Frochlich E, Apstein C, Chobanian A, Devereux R, Dustan H, Dzau V. et al . The heart in hypertension. New Engl J Med. 1993; 327 998-1008
- 11 Duarte J, Pérez-Palencia R, Vargas F, Ocete M A, Pérez-Vizcaino F, Zarzuelo A. et al . Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats. Br J Pharmacol. 2001; 133 117-24
Prof. Dr. Juan Duarte
Department of Pharmacology
School of Pharmacy
University of Granada
18071 Granada
Spain
Phone: +34-958243889
Fax: +34-958248964
Email: jmduarte@ugr.es
References
- 1 Griendling K K, Alexander R W. Oxidative stress and cardiovascular disease. Circulation. 1997; 96 3264-5
- 2 Grunfeld S, Hamilton C A, Mesaros S, Mcclain S W, Dominiczak A F, Bohr D F. et al . Role of superoxide in the depressed nitric oxide production by the endothelium of genetically hypertensive rats. Hypertension. 1995; 26 854-7
- 3 Berry C, Hamilton C A, Brosnan M J, Magill F G, Berg G A, McMurray J JV. et al . Investigation into the sources of superoxide in human blood vessels. Angiotensin II increases superoxide production in human internal mammary arteries. Circulation. 2000; 101 2206-12
- 4 Jameson M, Dai F -X, Lüscher T, Skopec J, Diederich A, Diederich D. Endothelium-derived contracting factors in resistence arteries of young spontaneously hypertensive rats before development of overt hypertension. Hypertension. 1993; 21 280-8
- 5 Rice-Evans C A, Miller N J, Paganda G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Bio Med. 1996; 20 933-56
- 6 Duarte J, Jiménez R, Villar I C, Pérez-Vizcaino F, Jiménez J, Tamargo J. Vasorelaxant effects of the bioflavonoid chrysin in isolated rat aorta. Planta Med. 2001; 67 567-9
- 7 Siess M H, Le Bon A M, Canivenc-Lavier M C, Amiot M J, Sabatier S, Aubert S Y. et al . Flavonoids of honey and propolis: Characterization and effects on hepatic drug-metabolizing enzymes and benzo[α]pyrene-DNA binding in rats. J Agric Food Chem. 1996; 44 2297-301
- 8 Walle T, Otake Y, Brubaker J A, Walle U K, Halushka P V. Disposition and metabolism of the flavonoid chrysin in normal volunteers. Br J Clin Pharmacol. 2001; 51 143-6
- 9 Morrow J D, Roberts L J. The isoprostanes. Current knowledge and directions for future research. Biochem Pharmacol. 1996; 51 1-9
- 10 Frochlich E, Apstein C, Chobanian A, Devereux R, Dustan H, Dzau V. et al . The heart in hypertension. New Engl J Med. 1993; 327 998-1008
- 11 Duarte J, Pérez-Palencia R, Vargas F, Ocete M A, Pérez-Vizcaino F, Zarzuelo A. et al . Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats. Br J Pharmacol. 2001; 133 117-24
Prof. Dr. Juan Duarte
Department of Pharmacology
School of Pharmacy
University of Granada
18071 Granada
Spain
Phone: +34-958243889
Fax: +34-958248964
Email: jmduarte@ugr.es
Fig. 1 Effects of chrysin on systolic arterial pressure as measured by tail-cuff plethysmography in the SHR vehicle (○), SHR chrysin (•), WKY vehicle (□) and WKY chrysin () groups. Values are expressed as means ± s. e. mean. * P < 0.05 , ** P < 0.01 vs. the SHR vehicle group (Bonferroni's test).
Fig. 2 Oxidative status. Urinary iso-PGF2 α excretion (A) and total plasma malondialdehyde (MDA) content (B) in the vehicle- (open bars) and chrysin-treated rats (solid bars). Data are means ± s. e. mean (n = 10). * P < 0.05, ** P < 0.01, ns not significant (Bonferroni's test).
Fig. 3 Endothelium-dependent relaxation (A) and endothelium-dependent contractions (B) induced by acetylcholine (ACh) in aortae from the SHR vehicle (○), SHR chrysin (•), WKY vehicle (□) and WKY chrysin () groups. ACh-induced relaxations were induced in arteries contracted by 10-7 M noradrenaline. ACh-induced contractions were evoked in arteries treated with L-NAME (10-4 M) and expressed as a percent of the response to 80 mM KCl. Values are expressed as means ± s. e. mean. * P < 0.05 and ** P < 0.01 SHR chrysin vs. the SHR vehicle group (Bonferroni's test).