Effects of Dietary Chrysin Supplementation on Blood Pressure and Oxidative Status of Rats Fed a High-Fat High-Sucrose Diet

Abstract

Effects of dietary chrysin supplementation on blood pressure and oxidative status of rats were studied in comparison with quercetin. Rats were fed a control diet or a high-fat high-sucrose (HFS) diet with or without 0.25% flavonoids (chrysin or quercetin) for 4 weeks. In rats fed the HFS diet without flavonoids, there was a significant elevation of blood pressure, increase in aortic NADPH oxidase but not xanthine oxidase, an increase in plasma lipid peroxides, and a decrease in liver glutathione, compared to rats fed the control diet. Chrysin suppressed the elevation of blood pressure in rats fed the HFS diets at the same level as quercetin. Chrysin supplementation did not suppress the increase in NADPH oxidase activity and plasma lipid peroxides or the decrease in liver glutathione, whereas these effects were significant with quercetin. Also, the results of an in vitro experiment suggested that chrysin did not exert antioxidative effects on lipid peroxidation in a linoleic acid emulsion. These results suggest that chrysin has an antihypertensive effect similar to quercetin, but unlike quercetin is not antioxidative in normotensive rats fed a HFS diet. Thus, it is probable that a mechanism(s) other than antioxidative activity is responsible for the suppression of hypertension by chrysin.

Introduction

The pathogenesis of a wide range of lifestyle diseases such as hypertension and cardiovascular disease is not fully understood. However, the role of oxidative stress on the genesis and maintenance of these diseases has been revealed in experiments involving administration of several types of antioxidants, with a consequent lowering of the risk of these diseases (Rodríguez et al., 2002; Vasdev et al., 2001; Chen et al., 2001; Schnackenberg and Wilcox, 1999). Oxidative stress initiated by reactive oxygen species (ROS) might be a factor in these pathological conditions via oxidation of biological macromolecules such as lipids, protein, and DNA. Thus, there is growing interest in the many antioxidative components in foods for the prevention of ROS-induced oxidative damage.

Flavonoids are widely distributed in many kinds of plant food sources. The beneficial effects of flavonoids on human health have been widely studied, and antioxidative activity was proposed as one of the most important causes of these effects (Duarte et al., 1993; Duarte et al., 2001; Yamamoto and Oue, 2006; Gryglewski et al., 1987; Yu et al., 2006; Villar et al., 2002). Quercetin is the most abundant flavonol type of flavonoid, and a wide range of biological actions including vasodilation, antihypertensive, and antithrombotic effects have been reported (Duarte et al., 1993; Duarte et al., 2001; Yamamoto and Oue, 2006). Chrysin is a flavone-type flavonoid, and is present in foods like honey and propolis, and in many kinds of fruits and vegetables at low concentrations. Additionally, chrysin is commercially available as an androgen-boosting supplement (Kao et al., 1998). Previous experiments have suggested that chrysin possesses in vitro antioxidative and free radical scavenging activities (Rackova et al., 2005; Burda and Oleszek, 2001; Vinson et al., 1995). In assessing the in vivo physiological effects of chrysin, Villar et al. observed an antihypertensive effect in spontaneously hypertensive rats (SHR) (Villar et al., 2002), which has not been found in any study on normotensive rats.

The consumption of a high-fat diet or a high-fat high-sucrose (HFS) diet induces a significant rise in oxidative stress, accompanied by hyperlipidemia, hypercholesterolemia, and hypertensive effects (Wilde et al., 2000; Roberts et al., 2000; Roberts et al., 2005). These diet-induced adverse effects can be used as a model of lifestyle-related diseases. In the present study, the effects of dietary chrysin on blood pressure and oxidative status of normotensive rats fed a HFS diet was examined in comparison with quercetin. Also, the effect of chrysin on in vitro antioxidative activity was also investigated.

Materials and Methods

Animals and experimental diet    Twenty-eight 6-week-old male Sprague-Dawley rats were obtained from SLC Japan, Inc (Hamamatsu, Japan), and were housed individually in cages with wire mesh bottoms in a room kept at 22 ± 1°C and under a 12:12-h light-dark cycle (light period from 8:00 to 20:00 h). The animals were given free access to water and a basal diet for the first 5 days, and were subsequently randomly divided into four groups of seven animals each to average the initial body weight and systolic blood pressure (SBP). One group received a control diet (CON group), and the other four groups were fed a high-fat high-sucrose (HFS) diet with or without chrysin or quercetin (HFS, HFSC, and HFSQ groups). The concentration of added flavonoid was 0.25%, as 0.2% or 0.5% quercetin was shown to be antioxidative and antihypertensive in our previous experiment (Yamamoto and Oue, 2006). The composition of the control diet was equal to that of the basal diet and was as follows (wt%): soybean oil, 7.0; mineral mixture, 3.5; vitamin mixture, 1.0; choline bitartrate, 0.25; casein, 20.0; cellulose, 5.0, and α-cornstarch to make 100. The composition of the HFS diet was as follows (wt%): soybean oil, 7.0; lard, 15.0; cholesterol, 0.5, mineral mixture, 3.5; vitamin mixture, 1.0; choline bitartrate, 0.25; casein, 20.0; sucrose, 40.0, cellulose, 5.0, and α-cornstarch to make 100. The composition of the flavonoid-added diets was the same as the HFS diet except that 0.25% of the cellulose was replaced with chrysin or quercetin. Chrysin and quercetin were purchased from Wako Pure Chemicals (Osaka, Japan) and Tokyo Chemical Industry (Tokyo, Japan), respectively. The mineral mixture was AIN-93G-MX and the vitamin mixture was AIN-93-VX (Reeves, 1997). Freshly prepared experimental diets were served, and food and water were given ad libitum for the 4-week duration of the experiment. All experiments were conducted according to the Ethics Guidelines for Animal Experiments of Osaka City University.

Sampling procedures    After the 4-week experimental period, the rats were fasted overnight, anesthetized, and their blood was collected in a heparinized syringe from the abdominal aorta. The plasma was separated by centrifugation at 10,000 × g at 4°C for 15 min and was used to measure lipid peroxide levels. Immediately after blood sampling, the thoracic aorta was excised, rinsed gently with chilled saline, blotted, and then stored at −80°C while awaiting analysis of the NADPH oxidase and xanthine oxidase activities. The liver was harvested, cleaned, blotted, and stored at −80°C until analysis. The epididymal fat tissue was harvested and weighed.

Blood pressure    The SBP was measured at the start of the experiment and at 2-week intervals using the tail-cuff method with a blood pressure monitor (BP98; Softron, Tokyo, Japan).

Lipid peroxide levels in the plasma    Plasma lipid peroxide levels were measured as thiobarbituric acid reactive substances (TBARS) by the spectrometric method (Ohkawa et al., 1979) and were expressed as the amount of malondialdehyde in the plasma.

NADPH oxidase and xanthine oxidase in the aortic homogenate    Activities of NADPH oxidase and xanthine oxidase were measured by the chemiluminescent superoxide anion probe method (Rajagopalan et al., 1996). Aortic segments (3 to 4 cm) were homogenized with a glass-to-glass homogenizer on ice in 50 mmol/L phosphate-buffered saline (pH 7.0) with 0.01 mmol/L EDTA. The homogenate was centrifuged for 10 min at 1,000 × g, and the supernatant was used for measurement of oxidase activities. The reaction mixture contained 100 µmol/L NADPH or xanthine and 50 µmol/L lucigenin (Bis-N-methylacridinium), and the reaction was started by the addition of the aortic supernatant. The activity was measured by lucigenin-enhanced chemiluminescence using a Luminescensor AB-2200 (Atto Corp. Tokyo, Japan). Activities were expressed as the amount of generated superoxide anion per protein. Protein was measured by the method of Lowry et al. (Lowry et al., 1951).

Determination of total glutathione    The liver was homogenized with 5 volumes of 10% trichloroacetic acid and then centrifuged at 3,000 × g for 10 min. A portion of the supernatant was subjected to total glutathione measurement using 5,5′-dithio-bis-2-nitrobenzoic acid according to Anderson (Anderson, 1985).

In vitro antioxidative activity    For the in vitro measurement of antioxidative activity on lipid peroxidation, an aqueous emulsion was prepared with 25 mg of linoleic acid and 10 mL of 0.1 mol/L sodium phosphate buffer (pH 7.0) containing 100 mg of Tween 20 by sonicating in ice-cold water for 5 min. The emulsion with or without added flavonoids (final concentration: 50 µmol/L) was incubated at 40°C for 4 days. Oxidation was spectrophotometrically measured as the amount of produced TBARS (Ohkawa et al., 1979), and the results were expressed as increased optical density at 550 nm. Chrysin and quercetin were employed as the flavonoids, and were first dissolved in dimethyl sulfoxide and diluted with water.

Statistical analysis    Each data value for the animal experiments and the in vitro experiment were expressed as the mean ± SEM. Differences between the four groups in the animal experiment and the three groups in the in vitro experiment were assessed by a one-way analysis of variance (ANOVA) and multiple-range comparison by Tukey's honestly significant difference (HSD) test. A p value of < 0.05 was considered significantly different.

Results

Growth and dietary intake    Body weight gain and epididymal fat weight of the four experimental groups did not differ (Table 1). Food intake in the HFS group was lower, while food efficiency and liver weight were higher, than the CON group. Food efficiency and liver weight in the HFSC and HFSQ groups did not differ from those of the HFS group.

Table 1. Effects of dietary flavonoids on growth, dietary intake, and tissue weight in rats fed high-fat and high-sucrose diets for 4 weeks

	CON	HFS	HFSC	HFSQ
Body weight gain (g/day)	6.0 ± 0.3	6.4 ± 0.2	6.6 ± 0.3	6.6 ± 0.3
Food intake (g/day)	21.0 ± 0.7a	17.3 ± 0.4b	19.7 ± 0.4ab	17.6 ± 0.6b
Food efficiency (%)1	28.4 ± 0.9a	36.8 ± 1.01bc	33.6 ± 0.8b	37.7 ± 1.4c
Liver weight (g/100g BW)	2.96 ± 0.07a	4.02 ± 0.24bc	4.44 ± 0.15b	3.83 ± 0.09c
Epididymal fat weight (g/100g BW)	1.82 ± 0.08	1.94 ± 0.17	2.08 ± 0.08	1.99 ± 0.16

Rats were fed a control diet or a HFS diet with or without 0.25% chrysin or quercetin for 4 weeks. Each value represents the mean ± SEM of seven rats. Values within a row with different superscript letters are significantly different from each other by Tukey's HSD test (p < 0.05).

¹(Body weight gain/food intake) × 100.

Systolic blood pressure    The SBP of the HFS group was significantly higher than that of the CON group at 4 weeks, and the SBP of the HFSC and HFSQ groups was significantly lower than that of the HFS group (Fig. 1).

Fig. 1.

Effect of chrysin or quercetin dietary supplementation on the systolic blood pressure (SBP) of rats fed a HFS diet.

Rats were fed a control diet or a HFS diet with or without 0.25% chrysin or quercetin for 4 weeks, and SBP was measured at the start of the experiment and at 2-week intervals using the tail-cuff method. Values with different superscript letters in each assay are significantly different from each other by Tukey's HSD test (p < 0.05).

○: control, □: HFS, ●: HFSC, ■: HFSQ

In vivo antioxidative activity  In vivo antioxidative activity of chrysin was estimated by the change in NADPH oxidase and xanthine oxidase activities in the aortic homogenate, the plasma TBARS value, and liver total glutathione.

The NADPH oxidase activity in the HFS group was significantly higher than the CON group and lower in the HFSQ group than the HFS group, whereas the activity of the HFSC group did not differ from the HFS group (Fig. 2A). Xanthine oxidase activity in the HFS group with or without flavonoids did not differ from that of the CON group (Fig. 2B).

Fig. 2.

Effects of chrysin or quercetin dietary supplementation on NADPH oxidase (A) and xanthine oxidase (B) activities in the aorta of rats fed a HFS diet.

Rats were fed a control diet or a HFS diet with or without 0.25% chrysin or quercetin for 4 weeks, and activities of NADPH oxidase and xanthine oxidase in an aortic suspension were measured by the chemiluminescent superoxide anion probe method. Activities are expressed as the amount of generated superoxide anion per protein. Values with different superscript letters in each assay are significantly different from each other by Tukey's HSD test (p < 0.05).

The plasma TBARS value in the HFS group was higher than the CON group (Fig. 3). The value of the HFSQ group was lower than the HFS group, but the value of the HFSC group did not differ from that of the HFS group.

Fig. 3.

Effects of chrysin or quercetin dietary supplementation on plasma TBARS of rats fed a HFS diet.

Rats were fed a control diet or a HFS diet with or without 0.25% chrysin or quercetin for 4 weeks, and lipid peroxides in the blood plasma were measured as thiobarbituric acid reactive substances (TBARS) and are expressed as the amount of malondialdehyde in the plasma. Values with different superscript letters in each assay are significantly different from each other by Tukey's HSD test (p < 0.05).

Liver total glutathione concentration of the HFS group was significantly lower than the CON group. The concentration of the HFSQ group was higher than that of the HFS group, but the value of the HFSC group did not differ from that of the HFS group (Fig. 4).

Fig. 4.

Effects of chrysin or quercetin dietary supplementation on liver glutathione concentration in rats fed a HFS diet.

Rats were fed a control diet or a HFS diet with or without 0.25% chrysin or quercetin for 4 weeks, and total liver glutathione level was measured using 5,5′-dithio-bis-2-nitrobenzoic acid. Values with different superscript letters are significantly different from each other by Tukey's HSD test (p < 0.05).

In vitro antioxidative activity    Accumulation of lipid peroxidation products in the linoleic acid emulsion was not altered by the addition of chrysin but was significantly suppressed by the addition of quercetin (Fig. 5).

Fig. 5.

Effect of chrysin or quercetin on in vitro lipid peroxidation of a linoleic acid emulsion. Values with different letters are significantly different from each other (p < 0.05) by Tukey's HSD test.

○: not added, ●: chrysin, ■: quercetin

Discussion

Flavonoids have been reported to exert a wide range of biological actions, including vasodilation, antithrombotic, and antihypertensive effects (Duarte et al., 1993; Duarte et al., 2001; Yamamoto and Oue, 2006; Gryglewski et al., 1987; Yu et al., 2006; Villar et al., 2002). Most of these reports dealt with quercetin, and only one report focused on the in vivo biological effects of chrysin. Villar et al. showed the antihypertensive effect of orally administered chrysin on SHR (Villar et al., 2002); however, the effects of chrysin on normotensive rats remained unknown. In the present study, dietary chrysin suppressed the increase in blood pressure of normotensive rats fed a HFS diet. The effect of chrysin was similar to that observed for quercetin. From these results, dietary chrysin is expected to alleviate diet-induced hypertension.

One of the proposed mechanisms to suppress the elevation of blood pressure involves antioxidant activity (Vasdev et al., 2001; Chen et al., 2001; Schnackenberg and Wilcox, 1999). Superoxide is known to react quickly with NO (Thomson et al., 1995; Huie and Padmaja, 1993), and its consequent deactivation of NO is responsible for the retardation of acetylcholine-mediated vascular relaxation. Thus, the possibility that this antioxidative activity might be a mechanism of the antihypertensive effects of chrysin and quercetin was investigated in this experiment.

NADH/NADPH oxidase is well known as a major source of vascular superoxide in addition to two other enzyme systems, namely xanthine oxidase and NO synthase uncoupling (Cai and Harrison, 2000). Among these three systems, the importance of NADH/NADPH oxidase for the production of superoxide in the vessel wall has been suggested (Mohazzab et al., 1994; Guzik et al., 2000). In this study, accelerated oxidative stress in rats fed a HFS diet was observed using indicators such as elevated aortic NADPH oxidase activity, increased plasma lipid peroxides, and decreased liver total glutathione. The increase in NADPH oxidase activity was inhibited by the dietary addition of quercetin. However, chrysin supplementation did not significantly affect NADPH oxidase activity, suggesting that the suppression of NADH/NADPH oxidase-dependent superoxide production was not the mechanism responsible for the antihypertensive effect of chrysin.

An in vitro experiment revealed that chrysin and quercetin strongly inhibited xanthine oxidase activity, and suggested that a hydroxyl group at C-5 and/or C-7 of the A-ring of the flavonoid was essential for the inhibitory activity (Lin et al., 2002). In contrast, aortic xanthine oxidase activity was not inhibited by the addition of chrysin or quercetin in this experiment. These differences in the in vivo and in vitro effects of flavonoids on xanthine oxidase activity might be attributable to the low physiological bioavailability of chrysin (Walle et al., 2001) and quercetin (Hollman and Katan, 1999).

As chrysin did not significantly decrease NADPH oxidase and xanthine oxidase activities, the antihypertensive effect of chrysin was not explained by the inhibition of superoxide production. Another possible mechanism is a decrease in NO availability due to its radical scavenging activity. There are few reports dealing with the effects of chrysin on the in vivo oxidative status of rats. Free radical scavenging activity of flavones were relatively weak, and their antioxidative activities against lipid peroxidation were not detected in previous in vitro experiments (Burda and Oleszek, 2001; Vinson et al., 1995). Also, the results of this experiment suggested that chrysin was not antioxidative against the increase in lipid peroxidation products in the linoleic acid emulsion.

Many previous studies have investigated and postulated a number of hypotheses on the relationship between structure and antioxidative activity of flavonoids. The ortho-hydroxylation on the B-ring, the number of free hydroxyl groups, and a C2–C3 double bond in the C-ring are all proposed as conditions of radical scavenging activity (Rackova et al., 2005; Cao et al., 1997; Foti et al., 1996). Also, a free hydroxyl group at the C-3 position in flavonol has been proposed as a structural contributor to the high antioxidative activity against the progress of lipid peroxidation and the increase in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (Burda and Oleszek, 2001). The flavonol quercetin has five hydroxyl groups, the 3′, 4′-hydroxylation on the B-ring and free hydroxyl groups at C-3, C-5, and C-7, with all of these structural characteristics, and is the most potent antioxidant flavonoid. On the other hand, the flavone chrysin has only two hydroxyl groups, at C-5 and C-7, and does not possess a hydroxyl group at the C-3 position and in the B-ring. From these structural characteristics, chrysin was not expected to express efficient antioxidative activity.

A probable role of other mechanism(s) was suggested for chrysin's suppressive effect on the genesis and maintenance of hypertension in rats fed a high-fat high-sucrose diet regime. The vasoconstrictor angiotensin II is important in blood pressure control, and the effect of chrysin on angiotensin II production may be another possible mechanism for the suppression of blood pressure elevation. To our knowledge, there are no reports showing the suppression of angiotensin II production by chrysin. Further studies are needed to elucidate whether chrysin would be effective for suppressing angiotensin II production.

In summary, it was demonstrated that the dietary addition of the chrysin suppressed the elevation of blood pressure in normotensive rats fed a high-fat high-sucrose diets. Further studies are needed to clarify the mechanisms, other than antioxidative activity, involved in the antihypertensive effect of chrysin. Furthermore, studies employing other flavonoids should be conducted to elucidate the relationship between structure and antihypertensive activity of flavonoids.

References

•  Anderson,  M.E. (1985). Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol., 113, 548-555.
•  Burda,  S. and  Oleszek,  W. (2001). Antioxidant and antiradical activities of flavonoids. J. Agric. Food Chem., 49, 2774-2779.
•  Cai,  H. and  Harrison,  D.G. (2000). Endothelial dysfunction in cardiovascular diseases. The role of oxidant stress. Circ. Res., 87, 840-844.
•  Cao,  G.,  Sofic,  E., and  Prior,  R.L. (1997). Antioxidant and prooxidant behaviour of flavonoids: Structure-activity relationships. Free Radic. Biol. Med., 22, 749-760.
•  Chen,  X.,  Touyz,  R.M.,  Park,  J.B., and  Schiffrin,  E.L. (2001). Antioxidant effects of vitamins C and E are associated with altered activation of vascular NADPH oxidase and superoxide dismutase in stroke-prone SHR. Hypertension, 38(part 2), 606-611.
•  Duarte,  J.,  Galisteo,  M.,  Ocete,  M.A.,  Pérez-Vizcaino,  F.,  Zarzuelo,  F., and  Tamargo,  J. (2001). Effects of chronic quercetin treatment on hepatic oxidative status of spontaneously hypertensive rats. Mol. Cell. Biochem., 221, 155-160.
•  Duate,  J.,  Pérez-Vizcaino,  F.,  Zarzuelo,  A.,  Jiménez,  J., and  Tamargo,  J. (1993). Vasodilator effects of quercetin in isolated rat vascular smooth muscle. Eur. J. Pharmacol., 239, 1-7.
•  Foti,  M.,  Piattelli,  M.,  Baratta,  M.T., and  Ruberto,  G. (1996). Flavonoids, coumarins, and cinnamic acids as antioxidants in a micellar system. Structure-activity relationship. J. Agric. Food Chem., 44, 497-501.
•  Gryglewski,  R.J.,  Korbut,  R.,  Robak,  J., and  Swies,  J. (1987). On the mechanism of antithrombotic action of flavonoids. Biochem. Pharmacol., 36, 317-322.
•  Guzik,  T.J.,  West,  N.E.J.,  Black,  E.,  McDonald,  D.,  Ratnatunga,  C.,  Pillai,  R., and  Channon,  K.M. (2000). Vascular superoxide production by NAD(P)H oxidase. Association with endothelial dysfunction and clinical risk factors. Circ. Res., 86, e85-e90.
•  Hollman,  P.C.H. and  Katan,  M.B. (1999). Dietary flavonoids: intake, health effects and bioavailability. Food Chem. Toxicol., 37, 937-942.
•  Huie,  R.E. and  Padmaja,  S. (1993). Reaction of NO with O₂-. Free Radic. Res. Commun., 18, 195-199.
•  Kao,  Y.-C.,  Zhou,  C.,  Sherman,  M.,  Laughton,  C.A., and  Chen,  S. (1998). Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens. Environ. Health Perspect., 106, 85-92.
•  Lin,  C.M.,  Chen,  C.S.,  Chen,  C.T.,  Liang,  Y.C., and  Lin,  J.K. (2002). Molecular modeling of flavonoids that inhibits xanthine oxidase. Biochem. Biophys. Res. Commun., 294, 167-172.
•  Lowry,  O.H.,  Rosebrough,  N.J.,  Farr,  A.L., and  Randall,  R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275.
•  Mohazzab,  K.M.,  Kaminski,  P.M., and  Wolin,  M.S. (1994). NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am. J. Physiol., 266, H2568-H2572.
•  Ohkawa,  H.,  Ohishi,  N., and  Yagi,  K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem., 95, 351-358.
•  Rackova,  L.,  Firakova,  S.,  Kostalova,  D.,  Stefek,  M.,  Sturdik,  E., and  Majekova,  M. (2005). Oxidation of liposomal membrane suppressed by flavonoids: Quantitative structure-activity relationship. Bioorg. Medic. Chem., 13, 6477-6484.
•  Rajagopalan,  S.,  Kurz,  S.,  Münzel,  T.,  Tarpey,  M.,  Freeman,  B.A.,  Griendling,  K.K., and  Harrison,  D.G. (1996). Angiotensin II-mediated hypertension in the rat increased vascular superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations of vasomotor tone. J. Clin. Invest., 97, 1916-1923.
•  Reeves,  P.G. (1997). Components of the AIN-93 diets as improvements in the AIN-76A diet. J. Nutr., 127, 838S-841S.
•  Roberts,  C.K.,  Vaziri,  N.D.,  Wang,  X.Q., and  Barnard,  R.J. (2000). Enhanced NO inactivation and hypertension induced by a high-fat, refined-carbohydrate diet. Hypertension, 36, 423-429.
•  Roberts,  C.K.,  Barnard,  R.J.,  Sindhu,  R.K.,  Jurczak,  M.,  Ehdaie,  A., and  Vaziri,  N.D. (2005). A high-fat, refined-carbohydrate diet induces endothelial dysfunction and oxidant/antioxidant imbalance and depresses NOS protein expression. J. Appl. Physiol., 98, 203-210.
•  Rodríguez,  J.A.,  Grau,  A.,  Eguinoa,  E.,  Nespereira,  B.,  Pérez-Ilzarbe,  M.,  Arias,  R.,  Belzunce,  M.S.,  Páramo,  J.A., and  Martínez-Caro,  D. (2002). Dietary supplementation with vitamins C and E prevents downregulation of endothelial NOS expression in hypercholesterolemia in vivo and in vitro. Atherosclerosis, 165, 33-40.
•  Schnackenberg,  C.G. and  Wilcox,  C.S. (1999). Two-week administration of tempol attenuates both hypertensive and renal excretion of 8-iso prostaglandin F2α. Hypertension, 33(part 2), 424-428.
•  Thomson,  L.,  Trujillo,  M.,  Telleri,  R., and  Radi,  R. (1995). Kinetics of cytochrome c²⁺ oxidation by peroxinitrite: Implications for superoxide measurements in nitric oxide-producing biological systems. Arch. Biochem. Biophys., 319, 491-497.
•  Vasdev,  S.,  Ford,  C.A.,  Parai,  S.,  Longerich,  L., and  Gadag,  V. (2001). Dietary vitamin C supplementation lowers blood pressure in spontaneously hypertensive rats. Mol. Cell Biochem., 218, 97-103.
•  Villar,  I.C.,  Jimenez,  R.,  Galisteo,  M.,  Garcia-Saura,  M.F.,  Zarzuelo,  A., and  Duarte,  J. (2002). Effects of chronic chrysin treatment in spontaneously hypertensive rats. Letter Planta Med., 68, 845-847.
•  Vinson,  J.A.,  Dabbagh,  Y.A.,  Serry,  M.M., and  Jang,  J. (1995). Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease. J. Agric. Food Chem., 43, 2800-2802.
•  Walle,  T.,  Otake,  Y.,  Brubaker,  J.A.,  Walle,  U.K., and  Halushka,  P.V. (2001). Disposition and metabolism of the flavonoid chrysin in normal volunteers. Br. J. Clin. Pharmacol., 51, 143-146.
•  Wilde,  D.W.,  Massey,  K.D.,  Walker,  G.K.,  Vollmer,  A., and  Grekin,  R.J. (2000). High-fat diet elevates blood pressure and cerebrovascular muscle Ca²⁺ current. Hypertension, 35, 832-837.
•  Yamamoto,  Y. and  Oue,  E. (2006). Antihypertensive effect of quercetin in rats fed with a high-fat high-sucrose diet. Biosci. Biotechnol. Biochem., 70, 933-939.
•  Yu,  Z.,  Fong,  W.P., and  Cheng,  C.H.K. (2006). The dual actions of morin (3,5,7,2′,4′-pentahydroxyflavone) as a hypouricemic agent: Uricosuric effect and xanthine oxidase inhibitory activity. J. Pharmacol. Exp. Therap., 316, 169-175.