Effects of Polyphenols from Grape Seeds on Oxidative Damage to Cellular DNA
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____________________________________________________________________________http://www.paper.edu.cn Molecular and Cellular Biochemistry 267: 67–74, 2004. c 2004 Kluwer Academic Publishers. Printed in the Netherlands. Effects of polyphenols from grape seeds on oxidative damage to cellular DNA Peihong Fan and Hongxiang Lou School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, P.R. China Received 23 December 2003; accepted 26 May 2004 Abstract Grape seed polyphenols have been reported to exhibit a broad spectrum of biological properties. In this study, eleven phenolic phytochemicals from grape seeds were purified by gel chromatography and high performance liquid chromatography (HPLC). The antioxidant activities of five representative compounds with different structure type were assessed by the free radical- scavenging tests and the effects of the more potent phytochemicals on oxidative damage to DNA in mice spleen cells were investigated. Procyanidin B4, catechin, epicatechin and gallic acid reduced ferricyanide ion and scavenged the stable free radical, α, α-diphenyl-β-picrylhydrazyl (DPPH) much more effectively than the known antioxidant vitamin ascorbic acid, while epicatechin lactone A, an oxidative derivative of epicatechin, did not reduce ferricyanide ion appreciably at concentrations used and was only about half as effective on free radical-scavenging as epicatechin. Mice spleen cells, when pre-incubated with relatively low concentration of procyanidin B4, catechin or gallic acid, were less susceptible to DNA damage induced by hydrogen peroxide (H2O2), as evaluated by the comet assay. In contrast, noticeable DNA damage was induced in mice spleen cells by incubating with higher concentration (150 µM) of catechin. Collectively, these data suggest that procyanidin B4, catechin, gallic acid were good antioxidants, at low concentration they could prevent oxidative damage to cellular DNA. But at higher concentration, these compounds may induce cellular DNA damage, taking catechin for example, which explained the irregularity of dose-effect relationship. (Mol Cell Biochem 267: 67–74, 2004) Key words: antioxidants, comet assay, DNA damage, free radicals, grape seed polyphenols Introduction and influencing gene expression [8] and antiulcer effect [9]. They have also exhibited properties against the oxidation of Grape seed extracts (GSE) have shown vascular effects low-density lipoproteins [10]. The last effect has explained through affecting vascular tone [1], collagen metabolism [2] the ‘French paradox’ [11–13]. In most cases, GSEs activi- and vascular permeability [3]; cytotoxic effects according to ties are related to their antioxidant properties and are mainly its cytotoxicity towards MCF-7, A-427 and CRL 1739 cells attributed to the phenolic compounds. [4]; chemopreventive effects by inhibiting adenomas polypo- Excessive reactive oxygen species (e.g, hydrogen peroxide sis coli (Apc) gene mutation-associated intestinal adenoma or H2O2) may cause irreparable DNA damage, leading to formation [5] and decreasing 12-O-tetradecanoylphorbol-13- mutagenesis and perhaps cancer [14]. Investigation into the acetate (TPA) induced production of reactive oxygen species, nature of DNA damage and repair have provided valuable DNA fragmentation in hepatic and brain tissues and lipid per- insight into aging, human genetics and cancer [15, 16]. Now, oxidation [6]; cytoprotective effects by ameliorating the toxic there is deep interest in identifying free radical scavengers effects of chemotherapeutic agents [7], protecting spermato- or antioxidants that inhibit oxidative DNA damage. We have gonial cells against radiation damage, apoptopic cell death restricted ourselves to the study of cellular DNA damage Address for offprints:H.Lou, School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, 250012, P.R. China (E-mail: [email protected]) 中国科技论文在线___________________________________________________________________________http://www.paper.edu.cn 68 preventing activities of phenolic compounds from grape concentrated until no acetone was left using a rotary evap- seeds using the single-cell gel electrophoresis (comet) as- orator under reduced pressure and a water bath temperature say, in which the cells with damaged DNA display increased <40 ◦. The concentrated solution was extracted four times migration of DNA from the nucleus towards the anode and with 1000 ml of ethyl acetate each time. The ethyl acetate the cell with broken DNA then resembles a “comet” with a extracts were combined, evaporated to remove ethyl acetate brightly fluorescent head and a tail region which was used to and were lyophilized. GSE was obtained. evaluate DNA damage [17–24]. In this paper, eleven compounds were obtained and iden- Gel chromatography fractionation tified from grape seeds adopting modified methods from lit- erature [25–33], including three novel derivative structures, GSE was dissolved in water and was fractionated on a which were not referred in the literature at hand. With rapid Toyopearl TSK HW-40(F) column (70 × 8 cm), eluted screening procedures [34, 35], we determined the antioxi- with methanol in water gradiently. Fraction was detected dant and radical-scavenging activities of five representative by polyamide TLC [CHCl -MeOH-H Oasmobile phase, phenolic phytochemicals with different structure types. The 3 2 stained by 1% K Fe(CN) -FeCl ] and analytic RP-HPLC results were compared with the known antioxidant vitamin 3 6 3 [using a Phenomenex ODS column (5 µm, 4.6 × 250 mm), ascorbic acid. Then using mice spleen cells, we determined 0.2% V/V formate in 20% methanol as mobile phase, flow whether the potent antioxidants could inhibit oxidative dam- rate 1 ml/min, monitored at 270 nm UV absorbance]. age to DNA induced by H2O2. Because phenolic phytochem- icals have pro-oxidant effects in non-cellular systems [36, 37], we also determined if phenolic phytochemicals by them- HPLC separation and purification of individual selves actually induce oxidative DNA damage in mice spleen polyphenols present in fractions from gel chromatography cells, taking catechin as representative. The fraction was separated by semi-preparative HPLC, Material and methods using a Waters ODS semi-preparative column (10 µm, 25 × 100 mm). The HPLC mobile phase contained solvent A(water), solvent B (5% V/V THF in 10% methanol) and Chemicals and equipment solvent C (methanol). The linear gradient system employed, at room temperature, was: 0–60 min 40% solvent A and 60% DPPH, low and normal melting point agarose products were solvent B to 35% solvent A, 55% solvent B and 10% solvent purchased from Sigma (ST. Louis, MO). All other chemi- C, 60–90 min 35% solvent A, 55% solvent B and 10% sol- cals and reagents used in the studies were obtained in the vent C to 100% solvent C. The solvent flow rate throughout purest form available commercially. HPLC was performed the run was 3 ml/min. The column eluate was monitored at using Waters 600–900 Semi-preparative liquid chromatogra- 270 nm UV absorbance in each case and individual polyphe- phy equipped with DAD detector. Lyophilization was done nol peaks were collected, the purity was identified by analytic by an Eyela FD-1000 Freeze-drying meter. Proton nuclear HPLC. If necessary, re-separation was performed using semi- magnetic resonance (1H-NMR) and carbon nuclear mag- preparative HPLC. netic resonance (13C-NMR) were recorded on an Avance 500 Brucker spectrometer. Mass spectra were recorded in an API 4000 mass spectrometer. Infrared spectra were recorded Identification of the individual polyphenols obtained with a Nicolet-Nexus 470 Ft-IR spectrometer. X-ray crystal structure data were collected on a Bruker-P4 Kappa CCD IR, MS, 1H-NMR, 13C-NMR spectra were analyzed and con- diffractometer. UV-VIS spectra were measured with an Agi- trasted with related literature, to identify the structure of the lent 8450 spectrometer. In comet assay, DNA was visualized individual phenols obtained. For certain compound 4, X-ray using Olympus IX-70a inverted fluorescence microscope, the crystal diffraction was used. resulting images were taken with Kodak image system. Initial screening of five representative polyphenols Preparation of grape seed extracts (GSE) from GSE for antioxidant activities Grape seeds were collected from a brewery and milled to In view of the variety and amount of the compounds obtained, powder. The powder (5 kg) was defatted with petroleum catechin, epicatechin, epicatechin lactone A, gallic acid, pro- ether firstly, and then was macerated for 12 h at room tem- cyanidin B4 were selected as representatives for antioxidant perature three times with 2000 ml of water/acetone (30:70, activity assessment. Vit C was used as control. Sample so- V/V) each time. The three macerates were combined and lutions (0–3 mM, dissolved in methanol) were prepared, the 中国科技论文在线___________________________________________________________________________http://www.paper.edu.cn 69 reducing power and free radical-scavenging activity of sam- Following centrifugation, the cells were resuspended in 1ml ple solutions at different concentration were evaluated. The of HBSS. Cells were then challenged at 4 ◦C with 50 µM reducing power of sample solutions were tested by measuring H2O2 for 20 min and immediately analyzed for extent of the absorbance at 700 nm of the resulting solution, after sam- DNA damage. In other experiments, cells were incubated ple solutions were reacted with potassium ferricyanide and with higher concentration (50 µM, 150 µM)