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Journal of the American College of Nutrition, Vol. 22, No. 3, 217-223 (2003)
Published by the American College of Nutrition

Original Research

Effects of Phenol-Depleted and Phenol-Rich Diets on Blood Markers of Oxidative Stress, and Urinary Excretion of Quercetin and Kaempferol in Healthy Volunteers

Hwa-Young Kim, MSc, Ok-Hee Kim, PhD and Mi-Kyung Sung, PhD

Department of Food and Nutrition, Sookmyung Women’s University (H.-Y.K., M.-K.S.), Seoul, KOREA
National Institute of Toxicological Research, Korea Food and Drug Administration (O.-H.K.), Seoul, KOREA

Address reprint requests to: Mi-Kyung Sung, Ph.D., Associate Professor, Department of Food and Nutrition, Sookmyung Women’s University, 53-12 Chungpa-dong 2-ka, Yongsan-ku Seoul, 140-742, KOREA. Email: mksung{at}sookmyung.ac.kr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: Epidemiological studies have suggested beneficial effects of dietary polyphenols in reducing the risk of chronic diseases. This study was performed to investigate the effects of polyphenol-depleted and polyphenol-rich diets on blood oxidative stress markers and urinary excretions of major phenols.

Methods: Nineteen healthy female non-smokers 19 to 21 years of age took part in the study, which consisted of two dietary intervention periods separated by three days. Experimental diets were composed of common foods selected to comply with low contents of polyphenols for phenol-depleted intervention and high contents of polyphenols for phenol-rich diets. Blood and urine samples were collected on day 0, 3 and 6 of each intervention. Duplicate portions of foods provided to the subjects were also collected. Blood oxidative stress markers included plasma antioxidant vitamins, erythrocyte superoxide dismutase (SOD) activity and lymphocyte DNA damage. Urinary excretions of major phenols were measured to affirm bioavailability of dietary phenols.

Results: Plasma {alpha}-tocopherol and ß-carotene concentrations were slightly decreased on day 3 and 6 of the phenol-depleted dietary intervention period, although no change was observed with phenol-rich diets. The erythrocyte SOD activity was also slightly decreased during phenol-depleted dietary intervention. However, at day 6 of the phenol-rich intervention, the activity of SOD was significantly increased by 41%. Tail moment and tail length of lymphocyte DNA as markers of DNA damage were higher on day 6 of phenol-depleted intervention, although only tail moment showed a statistical significance. The average intakes of quercetin and kaempferol during the phenol-rich intervention were 21 mg/day and 9 mg/day, respectively. The average urinary excretion rates during phenol-rich intervention were 2.06% for quercetin and 0.46% for kaempferol. There were positive correlations between erythrocyte SOD activity and urinary concentration of quercetin or kaempferol.

Conclusions: These results suggest that polyphenol-rich diets may decrease the risk of chronic diseases by reducing oxidative stress.

Key words: polyphenols, antioxidants, DNA damage, quercetin, kaempferol


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Polyphenols occur ubiquitously in most foods of plant origin and can be categorized into flavonoids, phenolic acids and tannins. Recently, polyphenolic compounds have attracted much attention as potent antioxidants due to their ability to scavenge free radical and form relatively inert phenoxy radical intermediates [1]. A number of in vitro studies have shown individual food polyphenol possesses antioxidant activity, and the proposed mechanisms by which polyphenols exert their effects were summarized as 1) scavenging free radicals, 2) inhibiting the oxidation of {alpha}-tocopherol in LDL, 3) recycling oxidized {alpha}-tocopherol and 4) scavenging metal ions [2]. Free radicals, generated in vivo, are responsible for the oxidative damage of biological molecules such as carbohydrate, lipid, protein and DNA. This damage is involved in the pathogenic processes of various chronic diseases including cancer, cardiovascular heart diseases and arthritis [3].

A number of epidemiological studies have suggested that the consumption of polyphenol-rich foods reduces the risk of developing chronic diseases. For example, red wine consumption might prevent cardiovascular heart disease [4,5], while frequent green tea consumption has been suggested to have an association with a lower incidence of cardiovascular diseases and cancer [6]. Similarly, fruit and vegetable consumption prevents different cancers [7].

However, the physiological significance of dietary polyphenols needs to be evaluated more carefully since bioavailability of polyphenols is relatively low [8]. A limited number of human intervention studies have been conducted to investigate the significance of polyphenol-rich foods in reducing oxidative stress [9,10]. Although increases in urinary excretion of polyphenols followed by intervention were observed, the effects of polyphenol intakes on oxidative stress markers were not conclusive.

The objective of this study was to evaluate the effects of polyphenol-depleted and polyphenol-rich diets on blood oxidative stress markers by a randomized dietary controlled intervention trial. Indicators of oxidative stress included erythrocyte SOD activity, plasma {alpha}-tocopherol and ß-carotene concentrations and lymphocyte DNA damage. To affirm the availability of major dietary polyphenols, dietary intake and urinary excretion of major dietary polyphenols were assessed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects
The subjects, 19 healthy, non-smoking females, between 19 and 21 years of age, were recruited through an in-campus advertisement. None of the subjects was taking any medication including oral contraceptives or supplements at the time of the study. The average BMI was 21.5 (range 18.1–28.8) kg/m2. Details of the study were fully explained to the subjects who gave their written consents. The study was approved by the University Research Ethics Committee.

Study Design and Diets
The study had two six-days intervention periods separated by three days (Fig. 1). Subjects were given a list of foods with high polyphenol contents, which they were to avoid for three days prior to the experimental diet. The experimental diet consisted of common foods selected to comply with low total phenol contents for phenol-depleted diets and high total phenol contents for phenol-rich diets. To compose the diet, we selected 30 phenol-rich foods based on previous reports [2,11,12] and analyzed them for their total phenol content. Twelve food items with higher total polyphenol contents (red cabbage, red chicory, onions, red lettuce, mushrooms, apples, grapes, small red beans, kidney beans, sorghum, black rice, wild grape juice) were used to compose parts of phenol-rich diets. Phenol-depleted diets were deprived of any known phenol-rich food and mainly consisted of potatoes, rice, fish, seaweed, low-phenol containing vegetables, low-fat breads, milk and cheese. The calculated average macronutrients contents and selected antioxidants of the phenol-depleted and phenol-rich diets are shown in Table 1. All meals were prepared and consumed at the metabolic kitchen in the department. Subjects were allowed to drink water at any time.



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  		Fig. 1. Experimental design.

 

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  		Table 1. Estimated Daily Intake of Energy, Macronutrients and Selected Antioxidant Nutrients in Phenol-Depleted Diets and Phenol-Rich Diets

 
Collection of Blood and Urine Samples
Fasting blood samples were taken on day 0, 3, 6 of each intervention period. Four mL of blood samples were used to separate lymphocytes. The rest of the blood sample was centrifuged at 2100 x g for 15 minutes at room temperature, and the plasma was stored at -20°C until analyzed. The washed erythrocytes were resuspended in 1 volume of saline and then stored at -80°C. Twenty-four hour urine samples were collected in 1.5 L bottles containing thymol on day 0, 3, 6 of each intervention period. The total amount of urine was measured, and aliquots were frozen at -20°C until analyzed. Duplicate portions of the foods provided to the participants were collected.

Analysis of Plasma {alpha}-Tocopherol
Plasma {alpha}-tocopherol was measured by HPLC [13]. In a vial containing 200 µL of plasma sample, tocopherol acetate was added as an internal standard and extracted with hexane twice. The samples were then evaporated under nitrogen and injected into the HPLC. Separation of {alpha}-tocopherol was performed on microBondapack C18 column (Waters, Milford, MA). The mobile phase was composed of methanol and water (95:5) and the flow rate was 1.5 mL/minute. Ultraviolet detection was done at 292 nm.

Analysis of Plasma ß-Carotene
Plasma ß-carotene was measured based on the method of Lee et al. [14]. Briefly, 200 µL of plasma sample was mixed with 200 µL of tocopherol acetate as an internal standard, followed by extraction with 400 µL of butanol/ethyl acetate (1:1). Twenty µL of the sample mixed with sodium sulfate was injected into the HPLC. Separation of ß-carotene was performed on HAISIL 100 C18 column (Higgins Analytical, Inc., Mountain View, CA). The mobile phase was composed of methanol/butanol/water (89.5/5/5.5) and the flow rate was 1.5 mL/minute. Ultraviolet detection was done at 450 nm.

Measurement of Erythrocyte SOD Activity
SOD activity was determined based on the method of Flohé et al. [15]. Hemoglobin was removed from the erythrocyte lysate by precipitating with chloroform:ethanol (1:1), and the supernatant was used to measure enzyme activity. Fifty µL of the sample was mixed with 2 mL of phosphate buffer containing 0.5 mM xanthine and 20 µM of cytochrome in an equilibrated 3 ml cuvette. Reaction was started with 50 µL xanthine oxidase (0.2 U/mL), and absorbance changes at 550 nm were recorded using a UV detector.

Lymphocyte DNA Damage Measurement
Four mL of blood was suspended in 4 mL of RPMI 1640 and 4 mL of Histopaque®-1077 (Sigma Chemical Co., St. Louis, MO). The suspension was centrifuged at 700 x g for 30 minutes. The buffy coat layer was collected, washed with RPMI 1640 twice and centrifuged at 500 x g for 5 minutes. Cell pellets were suspended in media (10% DMSO, 40% RPMI, 50% FBS) and stored in a liquid nitrogen tank until analysis. Comet assay was performed based on the method of Singh [16] to measure lymphocyte DNA damage. Damage was assessed as tail length and tail moment (tail length x % migrated DNA) using a fluorescent microscope (Nikon Biophot II) with an image analysis program (Komet 3.1, Kinetic Imaging Ltd., UK).

Analysis of Dietary and Urinary Quercetin and Kaempferol
Dietary quercetin and kaempferol were determined based on the methods of Arai et al. [17]. Briefly, a 0.25 g freeze-dried food sample was extracted with 25 mL of 50% methanol containing 1.2 mol/L HCl and 1.6 g/L tert-butylhydroquinone for 2 hours at 90°C. The extract was diluted to 100 mL with methanol. After centrifugation (1000 x g, 5 minutes, 4°C), a 2 mL aliquot was dried by evaporation under nitrogen gas flow. The residue was dissolved in 100 µL methanol, of which 10 µL was used for HPLC analysis. Mobile phase was acetonitrile/phosphate buffer (pH 2.4, 40/60) and flow rate was 0.6 ml/min. Quantification was made using a UV detector at 370 nm.

Urinary quercetin and kaempferol were determined based on the method of Young et al. [10]. An aliquot of urine samples was centrifuged at 2100 x g for 5 minutes. To 3 mL of supernatant, 1 mL of acetate buffer was added with 100 µg/mL of morin as internal standard, and mixed. Twenty µL of ß-glucuronidase and sulfatase (Helix pomatia, 5.5 x 103 U/L and 2.6 x 103 U/L) respectively were added and incubated for 1 hour at 37°C. One mL of ethyl acetate was added and centrifuged at 2100 x g for 15 minutes. The supernatant was subjected to HPLC analysis.

Statistical Analysis
Statistical analysis was performed using SAS package (SAS Institute Inc. 2001). Data was expressed as mean ± standard deviation. Duncan’s multiple range test was used for comparisons. Regression analysis was performed to evaluate the relationship between SOD and urinary concentrations of phenol compounds.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
ACKNOWLEDGMENTS
 REFERENCES
 
Plasma {alpha}-Tocopherol and ß-Carotene
As shown in Table 2, plasma concentrations of {alpha}-tocopherol and ß-carotene were slightly decreased on day 3 and day 6 of the phenol-depleted diet period compared to the baseline concentrations, although no statistical significance was found. Polyphenol-rich dietary intervention did not affect the levels of plasma antioxidants.


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  		Table 2. Plasma Antioxidant Vitamin Concentrations

 
Erythrocyte SOD Activity
The erythrocyte SOD activity was slightly decreased after the phenol-depleted diet was introduced (Table 3). However, at day 6 of phenol-rich dietary intervention, erythrocyte SOD activity was significantly increased by 41%.


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  		Table 3. Erythrocyte SOD Activity

 
Lymphocyte DNA Damage
Tail moment and tail length of lymphocytes were measured on day 0 and day 6 of both phenol-depleted and phenol-rich diet periods (Table 4). Both tail moment and tail length were higher on day 6 of phenol-depleted dietary intervention compared to those on day 0. However, only tail moment showed a statistical significance. Phenol-rich diet did not affect both damage markers although a slight decrease in tail moment was observed with phenol-rich diets.


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  		Table 4. Lymphocyte DNA Damage of Study Subjects on Phenol-Depleted and Phenol-Rich Diets

 
Dietary and Urinary Phenols
Dietary concentrations of quercetin and kaempferol were measured on day 3 and 6 of phenol-depleted dietary intervention and on day 0, 3 and 6 of phenol-rich dietary intervention (Fig. 2). Urinary concentrations of quercetin and kaempferol were measured on day 0, 3 and 6 of each intervention period. The average intakes of quercetin and kaempferol during phenol-rich dietary intervention were 21 mg/day and 9 mg/day, respectively, which were significantly higher than 0.2 mg quercetin/day and 0.7 mg kaempferol/day during phenol-depleted dietary intervention.



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  		Fig. 2. Dietary and urinary concentrations of quercetin (a) and kaempferol (b) in study subjects.

 
Urinary excretions of both quercetin and kaempferol during the phenol-depleted diet period were not significantly different from baseline excretion values. Also, kaempferol excretion was not detected in a few samples during phenol-depletion. However, quercetin and kaempferol excretions were increased during phenol-rich dietary intervention compared to the baseline values. Urinary excretion rates (excretion/intake x 100) of unmodified quercetin and kaempferol were 2.06% and 0.46%, respectively, during phenol-rich dietary intervention. As shown in Fig. 3, regression analysis indicates that positive relationships are present between erythrocyte SOD activity and urinary excretion of quercetin (p = 0.057) or kaempferol (p = 0.0005) indicating bioavailable phenols possibly scavenge radicals and spare SOD. However, urinary quercetin possessed a much weaker relationship with erythrocyte SOD activity compared to that of kaempferol. R2 value between erythrocyte SOD activity and the sum of urinary quercetin and urinary kaempferol was 0.075 (p = 0.006).



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  		Fig. 3. Regression of erythrocyte SOD activity and urinary excretion of quercetin (a) and kaempferol (b).

 

   DISCUSSION
 
Dietary phenols have been suggested as potent antioxidants to delay pathogenic processes originating from oxidative damage in vivo. Early studies hypothesized that flavonoids would not enter circulation, but would be cleaved by intestinal bacteria to produce metabolites with no antioxidant activity [18,19]. However, recent studies showed urinary excretion of dietary polyphenols in humans either as free aglycones or as conjugates [20]. Our study used urinary quercetin and kaempferol as biomarkers of dietary phenol absorption, although the urinary content of phenols does not reflect absolute absorptive efficiency because absorbed phenols may be metabolized, stored or excreted through different routes. The average concentrations of urinary phenols were two to three times higher during phenol-rich dietary intervention compared to the baseline values indicating dietary phenols are absorbed and utilized in the body. Although urinary quercetin does not reflect the total quercetin absorbed, Young et al. [10] stated urinary excretion reflects the absorption and, therefore, can be used as a better marker of bioavailability than dietary intake. De Vries et al. [21] also showed urinary flavonols as biomarkers of dietary consumption.

Previous studies indicated the elimination half-life of apple quercetin, onion quercetin and parsley apigenin was 23 hours, 28 hours and 12 hours, respectively [22,9]. Bourne and Rice-Evance [20] also showed urinary excretion of ferulic acid ceased 8 to 10 hours after 360–720 g of tomato consumption. Therefore, we decided to choose six days of phenol-rich intervention, which would be enough to raise and maintain the body pools of phenols. However, unlike with quercetin, urinary excretion of kaempferol was not completely equilibrated in this study, indicating longer intervention may be required to observe definite effects of dietary kaempferol on oxidative stress markers.

Results from the present study showed phenol-depleted diets slightly reduced plasma concentrations of ß-carotene and {alpha}-tocopherol without statistical significance. In spite of the high content of ß-carotene in phenol-rich diets during the intervention period, plasma ß-carotene and {alpha}-tocopherol concentrations were not affected. Van het Hof et al. [23] showed consumption of the spinach-supplemented meal did not affect plasma levels of ß-carotene, while broccoli and green pea consumption induced significant increases in plasma ß-carotene levels, indicating the bioavailability of ß-carotene is substantially different among different vegetables. The conversion of ß-carotene to retinol is another possible explanation for no observable change in ß-carotene levels as observed in children fed yellow green vegetables [24]. A carrot supplementation study also failed to increase plasma ß-carotene concentrations [25]. Also, the consumption of red wine-derived phenol compounds did not improve blood ß-carotene levels, while plasma LDL oxidation was decreased [26]. Similar results were observed when healthy subjects consumed 400 mL/day of red wine [27]. A recent human intervention study failed to increase erythrocyte antioxidant enzyme activities and antioxidant vitamins with ß-carotene supplementation [28]. Wang and Russell [29] suggested contradictory epidemiological study results on the effectiveness of ß-carotene intake to prevent oxidative stress-induced chronic disease are due to the prooxidant property of ß-carotene. Further studies on the metabolism and interactions with other nutrients are required to elucidate their precise physiological roles.

Phenol-rich diets also contained higher levels of vitamin C (84 mg/day) compared to 37 mg/day of vitamin C consumed during phenol-depleted period. Since vitamin C is another potent dietary antioxidant, we cannot rule out its possible confounding effects. Since oxidative stress is an important cause of many chronic diseases, there are suggestions that vitamin C supplementation may lower the risk of chronic diseases. However, clinical trials of vitamin C supplementation with blood oxidative stress markers, including LDL oxidation and lymphocyte DNA damage, showed mixed results [30,31], and Carr and Frei [30] suggested that an intake of 90–100 mg/day vitamin C is required for optimum reduction of chronic disease risk by decreasing oxidative stress. In the present study, high-phenol diets contained 84 mg/day of vitamin C, which may not be enough to reduce oxidative stress. Jacob et al. [32] also showed no difference in lymphocyte oxidative damage between low (5 mg/day) and high (250 mg/day) vitamin C interventions indicating low vitamin C intake did not cause increases in oxidative damage. More studies are required to fully understand the level of dietary vitamin C supplementation to control oxidative stress under different circumstances.

The present study also showed changes in erythrocyte SOD activity. The phenol-depleted diets slightly decreased erythrocyte SOD activity, while phenol-rich diets significantly increased the SOD activity by 41%. Since polyphenols effectively remove oxygen radicals, diminished enzyme degradation partly explains increased SOD activity during phenol-rich intervention [28]. Similar results were observed with parsley and fruit juice intakes [9,10], while lowered intake of fruits and vegetables may lead to an overall decreasing trend in antioxidant enzymes [9]. Since ß-carotene supplementation studies did not show changes in enzyme activities [28], phenols may be a good candidate to reduce radical attack by increasing antioxidant enzyme activities.

Results from this study also indicate that phenol-rich diets reduce oxidative DNA damage, although the effects are not large. At present, a number of in vitro studies have confirmed the antioxidant effects of different polyphenols. Only recently, Boyle et al. [33] showed the decreased DNA damage after 100 g of fried onion consumption in human subjects. Also, three weeks of brussels sprouts consumption reduced the level of oxidized guanosine [34]. The phenolic hydroxyl group is shown to donate electrons to oxygen radicals and also to reduce ferrous ion to ferric ion, thereby suppressing oxidation [35].

The present study indicates phenol-rich diets decrease oxidative stress as assessed by different blood markers. Since phenol-rich diets also contain high levels of antioxidant vitamins, we cannot exclude the possible role of the combination of phenolic and vitamin antioxidants in the observed decrease in oxidative stress. However, positive relationships between urinary excretion of phenols and plasma radical scavenging SOD activity indicates phenols may act as radical scavengers.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
REFERENCES
 
This work was supported by Korean Ministry of Health and Welfare Grant HMP-00-B 22000-0155.

Received June 14, 2002. Accepted October 7, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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