Plant Foods Hum Nutr (2010) 65:57–63 DOI 10.1007/s11130-010-0154-8
ORIGINAL PAPER
Antioxidant Effect of trans-Resveratrol, Pterostilbene, Quercetin and Their Combinations in Human Erythrocytes In Vitro Renata Mikstacka & Agnes M. Rimando & Ewa Ignatowicz
Published online: 27 January 2010 # Springer Science+Business Media, LLC 2010
Abstract There is evidence that a diet rich in fruit and vegetables may reduce the risk of cancer and other degenerative diseases. However, potential health impact of bioactive phytochemicals is limited by their low amount and relatively poor bioavailability. It has been suggested that the health benefits associated with fruit and red wine consumption could be due to the whole antioxidant pool of the diet microcomponents. In this study, the antioxidant activities of trans-resveratrol, pterostilbene and quercetin, and the effect of their combination were investigated in human erythrocytes in vitro. H2O2-induced lipid peroxidation was assessed by measuring the amount of thiobarbituric acid reactive species. Quercetin and pterostilbene protected erythrocyte membranes against lipid peroxidation (IC50 values = 64±8.7 µM and 44.5±7.8 µM, respectively). Resveratrol was significantly less effective. However, the three compounds protected the erythocytes against hemolysis and GSH (reduced glutathione) depletion to the same extent. Combinations consisting of two compounds (molar ratio 1:1) influenced lipid peroxidation in a concentration–
dependent manner. At lower concentrations, resveratrol with quercetin or pterostilbene inhibited synergistically the oxidative injury of membrane lipids At higher concentrations, an additive effect was observed. These protective effects may partially explain the health benefit of these bioactive microcomponents when together in the diet.
R. Mikstacka (*) Department of Chemical Technology of Drugs, Poznań University of Medical Sciences, Grunwaldzka 6, 60-780 Poznań, Poland e-mail:
[email protected]
Introduction
A. M. Rimando Natural Products Utilization Research Unit, Agricultural Research Service, U.S. Department of Agriculture, PO Box 8048, University, MS 38677, USA E. Ignatowicz Department of Pharmaceutical Biochemistry, Poznań University of Medical Sciences, Swiecickiego 4, 60-781 Poznań, Poland
Keywords Lipid peroxidation . Hemolysis . Combination effect . Resveratrol . Pterostilbene . Quercetin Abbreviations IC50 the concentration required to inhibit hemolysis or lipid peroxidation by 50% DMSO dimethyl sulfoxide AAPH 2,2′-azobis(2-amidinopropane)hydrochloride GSH reduced glutathione TBARS thiobarbituric acid reactive substances CI combination index
Diet rich in fruits and vegetables provides consumers with antioxidant polyphenolic phytochemicals which are supposed to exert positive effect on human health. One of the most abundant beneficial ingredient is quercetin (3,3′,4′,5′,7-pentahydroxyflavone; Fig. 1), a flavonoid occurring in onions, edible fruits, teas and red wine [1]. The other well known molecules: trans-resveratrol (3,4′,5trihydroxystilbene; Fig. 1) and pterostilbene (3,5-dimethoxy4′-hydroxystilbene; Fig. 1) are present mostly in small fruits such as Vaccinium berries and grapes [2, 3]. Natural flavonoids and stilbenes exhibit significant antioxidant activity shown in many experimental models in vitro
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Plant Foods Hum Nutr (2010) 65:57–63
quercetin
trans-resveratrol
pterostilbene Fig. 1 Structures of quercetin, trans-resveratol and pterostilbene
and in vivo, e.g. quercetin was shown to inhibit low density lipoprotein oxidation [4] and resveratrol reduced a phorbolinduced respiratory burst of human neutrophils [5]. A natural analogue of resveratrol, pterostilbene, was shown to be as effective as resveratrol in the assay for peroxyl radicals scavenging activity [6] and protected normal human fibroblasts membrane against lipid peroxidation [7]. In the present study, human erythrocytes have been applied to assess the activity of natural polyphenols considering that red blood cells as oxygen carriers are constantly exposed to oxidative injury. The high oxygen tension, high membrane concentration of polyunsaturated fatty acids and redox active hemoglobin molecules render erythrocytes particularly susceptible to free radical attack. Moreover, human erythrocytes can be obtained during the procedure of relatively low invasiveness. Preventive applications of dietary natural antioxidants in oxidative stress-related diseases should be considered. The crucial role of antioxidants is to counteract overproduction and/or to attenuate functions of cellular reactive oxygen species (ROS) leading to oxidative stress and injuries. Although, the amounts of antioxidant polyphenols that are
ingested by humans may be relatively high, the concentration of active compounds in blood is rather low due to low bioavailability and fast metabolism [8, 9]. Nevertheless, on the basis of epidemiological studies it is suggested that diet rich in fruit and vegetables is associated with reduced risk of cardiovascular disease, cancer, diabetes, Alzheimer’s disease, cataracts, and age-related functional decline. To explain these phenomena the additive and synergistic effects of phytochemicals are proposed as well as modulation of the disease-specific pathways and mechanisms [10]. Combined effects of bioactive food components have been lately the focus of several reports. Ferrer et al. [11] demonstrated that combination of pterostilbene and quercetin inhibited metastatic B16 melanoma cell growth up to ∼56% in vitro and suggested also that pterostilbene and quercetin interfered, in a concentration-dependent manner, with the mechanism of cell division leading to G0/G1 phase arrest. In the study of Mertens-Talcott and Percival [12] an interaction of ellagic acid and quercetin with resveratrol in the induction of apoptosis in human leukemia cells was presented. The enhanced inhibition of adipogenesis and induction of apoptosis in vitro was observed for combination of resveratrol and quercetin [13]. In addition, the combined inhibitory effect of red wine polyphenolic components on cell growth and DNA synthesis was shown in studies on squamous carcinoma cells [14]. The recent studies on the antimutagenic activity of grape extracts suggest that the protective activity may not be attributed to individual polyphenols but rather to synergistic effect of many compounds in the extract [15]. In the present study, we aimed at the assessment of the protective effect of the natural chemopreventive agents, trans-resveratrol, pterostilbene and quercetin individually against AAPH-induced hemolysis and GSH depletion and their combination effect on H2O2-induced lipid peroxidation in human erythrocytes in vitro.
Materials and Methods Materials trans-Resveratrol, quercetin, Trizma Base, Trizma HCl, thiobarbituric acid, sodium azide, butylated hydroxytoluene, dimethyl sulfoxide (DMSO), 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH), 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid) were purchased from Sigma Aldrich Chemical Co., USA. Pterostilbene was synthesized following the previously described method [6]. All other chemicals used were of analytical grade reagents. Peripheral blood was obtained from healthy donors at the Regional Blood Bank of Poznan, Poland. Donors signed informed consent forms.
Plant Foods Hum Nutr (2010) 65:57–63
Methods Preparation of Erythrocytes Blood was collected into heparinized tubes. Erythrocytes and plasma were separated by centrifugation (10 min, 1,000×g). Erythrocytes were washed twice with 0.9% NaCl, once with phosphatebuffered saline (PBS), pH 7.4, and suspended in PBS to obtain approximately 10% hematocrit. Hemoglobin (Hb) was determined with a standard cyanmethemoglobin method. Measurement of Hemolysis Hemolysis of erythrocytes was estimated according to the method described by Tedesco et al. [16] with minor modifications. Erythrocytes in 5% suspension were shaken gently while being incubated with AAPH (final concentration 50 mM) at 37 °C for 3 h. Then the samples were centrifuged (5 min, 1,000×g) and the extent of hemolysis was determined spectrophotometrically by measuring the absorbance of supernatants diluted with PBS (1:9, v/v) at 540 nm. Percentage of hemolysis was calculated in relation to 100% hemolysis caused by deionised water added in nine parts per 1 part of erythrocyte suspension. Glutathione Measurement The concentration of GSH in erythrocytes was determined according to the method of Dise and Goodman [17]. The amount of GSH was expressed as nmol GSH per mg of hemoglobin. TBARS Determination trans-resveratrol, pterostilbene, quercetin or mixtures of two of these compounds in DMSO (at molar ratio 1:1) were added to 0.5 ml of preincubated erythrocyte suspension. Sodium azide (4 mM) was used as a catalase inhibitor. Subsequently, lipid peroxidation was induced by addition of 0.5 ml of 20 mM H2O2 and cells were incubated for 1 h at 37 °C with constant shaking. Erythrocytes incubated in buffer with DMSO (0.5%) were used as controls. The peroxidation of membrane lipids in erythrocytes was measured according to the method described by Stocks and Dormandy [18] with minor modifications. The amount of TBARS, mostly malondialdehyde, was estimated by measuring the absorbance at 532 nm. Lipid peroxidation level was expressed as micromoles of TBARS per g Hb or as % of control value. IC50 values were determined graphically by plotting lipid peroxidation (as percent of control value) versus test compound concentration. Quantification of Combination Effects Effects of combinations of the natural antioxidants on lipid peroxidation in human erythrocytes were assessed using the method of Chou and Talalay [19]. Combination indexes (CI) for every total concentration of mixture were calculated according to . . the following equation CI: ¼ ðDÞ1 ðDxÞ1 þðDÞ2 ðDxÞ2 þ
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. ðDÞ1 ðDÞ2 ðDxÞ1 ðDxÞ2 , where (D)1 and (D)2 are concentrations of compounds 1 and 2 that have x effect when used in combination, and (Dx)1 and (Dx)2 are the concentration of compounds 1 and 2 with the same effect x when used alone. CI1 represent synergism, additivity, or antagonism, respectively. Statistical Analysis Data were expressed as means from three separate experiments performed in triplicate. The tStudent test after the ANOVA procedure was applied to determine the difference among the means, which were considered statistically significant when P
