Cent. Eur. J. Biol.• 6(4) • 2011 • 565-574 DOI: 10.2478/s11535-011-0037-5

Central European Journal of Biology

Red may protect blood plasma proteins and lipids

Research Article

Joanna Kolodziejczyk1,*, Joanna Saluk-Juszczak1, Malgorzata M. Posmyk2, Krystyna M. Janas2, Barbara Wachowicz1

1Department of General Biochemistry, University of Lodz, 90-236 Lodz, Poland 2Department of Ecophysiology and Development, University of Lodz, 90-237 Lodz, Poland

Received 11 January 2011; Accepted 31 March 2011

Abstract: The present in vitro study was designed to examine the antioxidative activity of red cabbage anthocyanins (ATH) in the protection of blood plasma proteins and lipids against damage induced by oxidative stress. Fresh leaves of red cabbage were extracted with

a mixture of methanol/distilled water/0.01% HCl (MeOH/H2O/HCl, 50/50/1, v/v/w). Total ATH concentration [µM] was determined with cyanidin 3-glucoside as a standard. Phenolic profiles in the crude red cabbage extract were determined using theHPLC – method. Plasma samples were exposed to 100 µM peroxynitrite (ONOO ) or 2 mM hydrogen peroxide (H2O2) in the presence/ absence of ATH extract (5-15 µM); oxidative alterations were then assessed. Pre-incubation of plasma with ATH extract partly – reduced oxidative stress in plasma proteins and lipids. Dose-dependent reduction of both ONOO and H2O2-mediated plasma protein carbonylation was observed. ATH extract partly inhibited the nitrative action of ONOO–, and significantly decreased plasma – lipid peroxidation caused by ONOO or H2O2. Our results demonstrate that anthocyanins present in red cabbage have inhibitory – effects on ONOO and H2O2-induced oxidative stress in blood plasma components. We suggest that red cabbage ATH, as dietary antioxidants, should be considered as potentially usable nutraceuticals in the prevention of oxidative stress-related diseases.

Keywords: Anthocyanins • Antioxidant • Peroxynitrite • Oxidative stress • Plasma • Red cabbage © Versita Sp. z o.o.

responsible for the red, blue and purple colours of 1. Introduction various fruits and such as berries, grapes, There has been increasing interest in dietary , apples and red cabbage [3]. The polyphenolic structure and their health-promoting properties were originally of ATH determines their antioxidative properties, such attributed to various naturally occurring . as ability to scavenge various reactive oxygen species •- 1 However, studies focused on phytonutrients, such as (ROS): superoxide anions (O2 ), singlet oxygen ( O2), • antioxidant vitamins, have not shown a clear correlation peroxides (ROO ), hydrogen peroxide (H2O2) and the between the consumption of these vitamins and hydroxyl radical •OH [4]. significant cardioprotective effect. These observations Red cabbage ( oleracea var. capitata shifted scientific attention to other compounds present rubra) belongs to cruciferous crops, which in plants, such as polyphenols, which are secondary include , , Chinese cabbage, radish plant metabolites with many beneficial effects [1,2]. and Brussels sprouts. The cruciferous vegetables The most abundant flavonoid constituents of fruits are recognized as possessing a wide range of health and vegetables are anthocyanins (ATH), occurring benefits [5], partly due to the high level of polyphenols, ubiquitously in the plant kingdom. They are water- including ATH. Currently, red cabbage serves as soluble, glycosylated, nonacetylated pigments, a functional food; however, it was also valued for

* E-mail: [email protected] 565 Red cabbage anthocyanins may protect blood plasma proteins and lipids

medicinal purposes in treating headaches, gout, such as anticancer, antilipidemic, antinflammatory and diarrhea and peptic ulcers as early as the 1950s [6]. antioxidant activities [14,15]. Although the ATH uptake Red cabbage is a rich source of polyphenols, with into blood serum is rather low (aproximately 1%) [16], the most abundant class of ATH present as the main the supplementation of ATH or an ATH-rich diet have polyphenolic components in extracts of fresh and been reported to significantly increase serum antioxidant pickled red cabbage. Over 30 compounds potential [17]. The direct antioxidative effect of ATH on have been identified in this plant; approximately 322 mg plasma components remains largely unknown. of ATH per 100 g fresh weight of red cabbage may be Since red cabbage is considered to be promising obtained [3]. The chemical structure of red cabbage ATH functional food in the prevention of cardiovascular is based on cyanidin as aglycone, glycosylated mainly disorders, it is also important to examine its ability to with glucose and/or sophorose (diglucoside), which are break the toxic action of various cardiovascular disease- acylated with different aromatic and aliphatic acids [7]. contributing agents, such as peroxynitrite. The purpose In contrast to the other flavonoids, anthocyanins carry of our study was to assess, in vitro, the antioxidative a positive charge in the ring structure of carbon, and effects of ATH from red cabbage () thus are cations [8]. It is known that the flavylium cation on plasma protein and lipid changes induced by strong form predominantes under acidic conditions (pH 2 or biological oxidative agents: peroxynitrite (ONOO-) and

below), but when pH is raised towards 7, the colourless hydrogen peroxide (H2O2). chalcone pseudobase dominates. McDougall et al. [9] reported that anthocyanins are effectively stable under the acidic gastric digestion conditions, but are unstable 2. Experimental Procedures under small intestine conditions. The total recovery after simulated pancreatic digestion was around 2.1 Materials 25%, compared to around 100% recovery of phenol Human blood was obtained from healthy young (20–25 content. Since anthocyanins make up the majority of years) non-smoking individuals who denied taking any red cabbage polyphenols, these results suggest that medication for the preceding 14 days and had fasted anthocyanins may break down to form new phenolic for at least 12 h. Blood was collected separately into components [9]. ACD solution (citric acid/citrate/dextrose; 5:1 v/v) and Dietary intake of ATH, mainly from red fruits, centrifuged (3000 × g, 15 min) to yield plasma. Plasma certain vegetables (such as red cabbage), and red samples were preincubated (2 min, 37°C) with ATH wine, can reach 200 mg/day [10]. ATH are absorbed in red cabbage extract, (final concentration of 5–15 mM), the glycosylated form from the human gastrointestinal and then the ONOO- solution (final concentration of

tract after consumption [11]; however, subsequent 100 mM) or H2O2 (final concentration of 2 mM) was acylation lowers their absorption. Anthocyanidin added. Peroxynitrite was synthesized according to diglycosides, in the form of sambubioside or rutinoside, the method of Pryor et al. [18]. The concentration of impart increased stability to the anthocyanin molecule ONOO- used in our experiments (100 µM) corresponds and the quantities excreted in urine are less than 0.1% to the concentrations reported to occur in vivo, when · ·- of intake. However, 60-90% of the anthocyanins may the production of NO and O2 is locally elevated disappear from the gastrointestinal tract within 4 h of (inflammation, oxidative stress, ischaemia and

a meal; what happens to the bulk of the anthocyanins reperfusion) [19-21]. The concentration of H2O2 (2 mM) that vanish is unclear. This disappearance may be has been chosen on the base of the available data on the

partially accounted for by degradation; however, it range of H2O2 concentrations used in various bioassays differs for the various aglycones, and may be modified and our previous laboratory protocols [22-24]. further by the nature of the aglycone glycosylation, Anti-3-nitrotyrosine polyclonal antibodies were which further complicates the understanding of this obtained from Abcam (Cambridge, UK). StreptABComplex/ process [12]. HRP Polyclonal Swine Anti-Goat, Mouse, Rabbit The main difficulty in determining the human health Immunoglobulins, Multi-Link were from DAKO (Glostrup, implications of consuming ATH-rich food is that ATH Denmark). Bicinchoninic Acid Protein Assay Kit and are mainly found as a component of heterogenous o-phenylenediamine dihydrochloride (OPD peroxidase mixtures rather than isolated compounds. This has led substrate), rabbit anti-dinitrophenylhydrazine antibodies, some authors to suggest that the potential pro-oxidative anti-rabbit antibodies, and anti-goat/sheep antibodies action of these compounds or extracts should also be coupled with peroxidase were obtained from Sigma (St. taken into consideration [13]. Available data indicates Louis, MO, USA). All other reagents were of analytical that ATH have a wide range of therapeutic advantages, grade and were provided by commercial suppliers.

566 J. Kolodziejczyk et al.

The protocol was accepted by Committee for at 360 nm as rutin equivalents; and anthocyanins at Bioethics in Scientific Research and Rector of University 520 nm as cyanidin 3,5-diglucoside equivalents [27]. of Lodz, decision on 17th June 2010. 2.4 Immunodetection of 3-nitrotyrosine by a 2.2 ATH extraction and purification competition-ELISA test Fresh leaves (5 g) of red cabbage (Brassica oleracea Immunodetection of 3-nitrotyrosine in plasma var. capitata rubra) were homogenised and extracted proteins by a c-ELISA (competition Enzyme-Linked with a 30 ml mixture of methanol/distilled water/ Immunosorbent Assay) test was performed according

HCl (EMix: MeOH/H2O/HCl, 50/50/1, v/v/w) and to the method described by Olas et al. [28]. A 96-well centrifuged [25]. Supernatant was collected as a microtiter dish was coated with 100 µl of nitro-fibrinogen crude red cabbage extract. The extract was then dried (nitro-Fg) at a concentration of 1 µg/ml and 10 mol in a vacuum rotary evaporator in a water bath at 40°C. 3-nitrotyrosine/mol protein and left overnight at 4°C. The condensed solution (30 mg × ml-1) with acidified The concentration of nitrated proteins was estimated

10% MeOH (MeOH/H2O/HCl, 10/90/1, v/v/w) was using a standard curve, expressed as nitro-Fg prepared and then purified on a high load C18 SPE equivalents. mini-column (Alltech Assoc.) previously activated with MeOH. Using this method, 1.5 ml samples were first 2.5 Estimation of carbonyl groups in plasma desalted with 0.01% aqueous HCl before removal proteins by ELISA test of small phenolics by rinsing with ethyl acetate. The Detection of carbonyl groups by ELISA (using column then was eluted with acidified MeOH gradients antidinitrophenylhydrazine antibodies) in plasma - in the following order: 10, 20, 50 and 80%. Solvent (untreated, ATH and ONOO or H2O2-treated plasma) was volume was always 5 ml per column (≈3 × sample carried out according to the procedure of Buss et al. [29]. volume). Colourful fractions in 50% MeOH were The amount of carbonyl groups present in fibrinogen after collected and analysed by HPLC (High-Performance treatment with peroxynitrite (at the final concentration of Liquid Chromatography). For our experiments, ATH of 100 mM) was determined spectrophotometrically as this fraction was once again dried in a vacuum rotary described by Levine et al. [30]. evaporator in a water bath at 40°C, and then the condensed aqueous solution of 130 mM ATH extract 2.6 Production of thiobarbituric acid reactive (84% of ATH) was prepared. The aqueous solution substances (TBARS) in human plasma of ATH was added to plasma samples for the final Incubation of plasma with tested anthocyanin extract concentration range of 5–15 µM. or ONOO- was stopped by cooling the samples in an ATH concentration (mM) in the extracts was determined ice-bath. Plasma samples were then transferred to an and verified spectophotometrically, with the use of cyanidin equal volume of 20% (v/v) cold trichloroacetic acid in 3,5-diglucoside as a standard, and calculated using 0.6 M HCl and centrifuged at 1200 × g for 15 min. One e -1 -1 absorption coefficient 525 = 30 mM × cm [25]. volume of clear supernatant was mixed with 0.2 volume of 0.12 M thiobarbituric acid in 0.26 M Tris at pH 7.0 and 2.3 HPLC analysis of the extract immersed in a boiling water bath for 15 min. Absorbance Phenolic profiles in purified red cabbage extracts were at 532 nm was measured and results were expressed determined using a HPLC Knauer system equipped with as nmoles of TBARS per ml of plasma [31]. a UV–Vis detector and a Eurospher-100 C-18 column (25 cm × 4.6 mm; 5 ml). The binary mobile phase, 2.7 Data analysis according to Dyrby et al. [26], consisted of water/ formic To eliminate uncertain data, the Q-Dixon test was acid (90:10, v/v) (solvent A) and water/acetonitrile/formic performed. All values in this study were expressed as acid (40:50:10, v/v/v) (solvent B). Flow rate was 1 ml/min, mean ± SEM. The statistical analysis was performed and total run time was 50 min. The system was run with a one-way ANOVA for repeated measurements. The with a gradient programme: 0 min: 88% A + 12% B; statistically significant differences were also assessed 26 min: 70% A + 30%B; 40–43 min: 0% A + 100% B; by the paired Student’s t-test. 48–50 min: 88% A + 12% B. Phenolics were divided into four subclasses and quantified by the maximum UV–Vis absorption of each group. The hydroxybenzoic acid 3. Results derivatives were quantified at 280 nm and expressed as gallic acid equivalents; hydroxycinnamic acid derivatives HPLC analysis of the methanolic extracts from red at 320 nm as chlorogenic acid equivalents; flavonols cabbage leaves showed the following polyphenol

567 Red cabbage anthocyanins may protect blood plasma proteins and lipids

groups: hydroxybenzoic acids, hydroxycinnamic acids, found when the detector was set at 520 nm (Figure and anthocyanins (Figure 1). ATH predominated in both 1D). The chromatogram was characterized by peaks at crude and semi-purified extracts, constituting 64% (data retention times of 9.05, 12.47, 25.02, 28.77, 29.6, 32.72, not shown) and 84% of phenolics (Table 1) respectively. and 33.73 mAU. The peaks were also recorded at the The crude extract also contained hydroxycinnamic wavelengths of 280, 320, and 360 nm (Figure 1ABC). The (21%) and hydroxybenzoic (14%) acid derivatives (data situation was similar with PhC defined as hydrocinnamic not shown). However, after extract semi-purification, acid derivatives, whose peaks were maximal at 320 nm the hydroxybenzoic acids were no longer present, (Figure 1B) but whose echo could also be seen at 280 and the hydroxycinnamic acids were limited to 16% of and 360 nm (Figure 1AC). the total PhC (phenolic compounds) pool (Table 1). In The exposure of blood plasma samples to 100 mM

the semi-purified extract, seven ATH-derivatives were peroxynitrite or 2 mM H2O2 resulted in enhanced levels

Figure 1. HPLC profiles of phenolic compounds (PhC) determined in the semi-purified red cabbage extract (84% of ATH). Eachsample was analyzed at four different wavelengths: (A) 280 nm; (B) 320 nm; (C) 360 nm; (D) 520 nm. 1 - hydroxybenzoic acid derivatives; 2 - hydroxycinnamic acid derivatives; 3 – flavonols; 4 – anthocyanins (ATH).

568 J. Kolodziejczyk et al.

PhC in semi-purified extract Classes of phenolic compounds (PhC) Wavelength in red cabbage semi-purified extract (84% of ATH) -1 -1 [mg × ml ] [mg × gDM ] [%]

1 Hydroxybenzoic acids 280 nm <0.001 - -

2 Hydroxycinnamic acids 320 nm 0.018 11 16

3 Flavonols 360 nm <0.001 - -

4 Anthocyanins 520 nm 0.099 58 84

Table 1. Content of major classes of phenolic compounds (PhC) in red cabbage semi-purified extract (84% of ATH). Data determined by HPLC analysis; DM = extract dry matter.

- Figure 2. Effects of ATH extract (5–15 µM) on carbonyl group formation of blood plasma proteins, induced by ONOO (100 µM) and H2O2 (2 mM). - Results are expressed as the percentage of 100 µM ONOO and 2 mM H2O2-induced plasma carbonyl group formation. The results are representative of five independent experiments done in triplicate, and are expressed as means ± SEM. The effects were significant - according to one-way ANOVA test: ATH extract + ONOO - or H2O2-treated plasma vs. appropriate controls: *P<0.05, **P<0.02, ***P<0.01.

- of plasma protein carbonyl groups (protein oxidation) Exposure of plasma to ONOO or H2O2 led to plasma (Figure 2). The carbonylation levels obtained for plasma lipid peroxidation, estimated by the level of thiobarbituric - samples treated with both ONOO and H2O2 were treated acid reactive substances present after exposure. - as 100% for comparison. A significant increase in 3-NT Treatment of plasma with 100 mM ONOO or 2 mM H2O2 generation (protein nitration) in plasma samples treated caused similar effects on plasma lipid peroxidation; with peroxynitrite was also observed (Figure 3). The approximately twofold increase of TBARS was recorded results from the present in vitro study demonstrated that (Figure 4). In the presence of red cabbage ATH extract, - ATH extract from red cabbage reduced the oxidation a significant decrease in both ONOO and H2O2-induced and nitration of blood plasma proteins - dose-dependent lipid peroxidation was observed. The antioxidative - reduction of both ONOO and H2O2-induced plasma action of the extract in protecting against the action of protein carbonylation was observed. In plasma samples ONOO- was more effective; the highest concentration treated with ONOO-, the protective effect of the tested of red cabbage ATH extract (15 µM) diminished the extract against carbonyl group generation was stronger level of TBARS considerably. While the ATH extract (at than in samples treated with H2O2 (Figure 2). The the concentration range of 5–15 mM) was also able to antioxidative action of the highest dose of ATH extract reduce H2O2-induced TBARS levels, its inhibitory effect - (15 mM) against ONOO and H2O2-induced plasma on lipid peroxidation was dose-independent (Figure 4). protein oxidation was similar, and the level of carbonyl groups was diminished by about 30% (Figure 2). ATH extract partly inhibited the nitrative action of peroxynitrite 4. Disscusion - as measured by the c-ELISA test, the level of ONOO-- induced 3-NT was reduced by ATH in a dose-dependent Red cabbage (Brassica oleracea) is a rich source of manner (Figure 3). flavonoids, especially anthocyanins; however, some

569 Red cabbage anthocyanins may protect blood plasma proteins and lipids

Figure 3. Effects of ATH extract (5–15 µM) on nitration of tyrosine residues in plasma proteins induced by ONOO– (100 µM) measured by c-ELISA method. The results are representative of five independent experiments (performed in triplicate) and expressed as means ± SEM of plasma 3-nitrotyrosine Fg equivalents. The effects of different ATH concentrations were statistically significant according to one-way ANOVA test: untreated plasma vs. plasma + ONOO– ****P<0.0001, ATH extract + ONOO– vs. ONOO–-treated plasma: **P<0.02, ***P<0.001.

– Figure 4. Effects of ATH extract on ONOO - and H2O2-induced blood plasma lipid peroxidation. The effects of ATH extract (5–15 µM) on the level – of TBARS in plasma samples treated with H2O2 (2 mM) or ONOO (100 µM) was measured. The results are represented as means ± SEM of five independent experiments done in triplicate. The effects were statistically significant according to one-way ANOVA test: – – untreated plasma vs. plasma + ONOO or H2O2 P<0.01; ATH extract + ONOO or H2O2-treated plasma vs. appropriate controls: **P<0.002, ***P<0.001.

derivatives of hydroxybenzoic and hydroxycinnamic per 100 g fresh cabbage leaves (the average range of acids are also present in this plant [32]. Mazza and Miniati efficacy for this ATH extraction procedure). The phenolic [8] reported the wide range of ATH quantities found in profiles (Figure 1) were determined using the HPLC red cabbage (25-495 mg per 100 g FW). Concerning method, based on the maximum absorption wavelength ATH quality, red cabbage contains 23 different ATH, for different groups of phenolics (i.e. ATH (520 nm), all cyanidin derivatives highly conjugated with flavonols (360 nm), hydroxycinnamic acids (320 nm) (glucose and xylose) and acylated groups (caffeoyl, and hydroxybenzoic acids (280 nm)). The crude extract p-coumarolyl, feruloyl, p-hydroxybenzoyl, sinapoyl and analysed in our experiments contained ATH (64% of oxaoyl) [33]. The determination of a phenolic profile all phenolic compounds), but hydroxycinnamic acid in Brassica vegetables is difficult, as the majority of derivatives (21%) and some hydroxybenzoic acid reference compounds are not commercially available. derivatives (14%) were also observed (data not shown). Our extraction procedure yielded 122–142 mg ATH After semi-purification the extract contained mostly ATH

570 J. Kolodziejczyk et al.

(84%) and a small amount of hydroxycinnamic acid and pathological processes, including atherosclerosis, derivatives (16%) (Figure 1). hypertension, mutagenesis, and cancer [40]. H2O2 may The major ATH of red cabbage are based on a alter protein conformation by oxidation of cysteine and core of cyanidin 3,5-diglucoside, which can be non- methionine residues and, as a secondary messenger acylated, mono-acylated or di-acylated with p-coumaric, and modulator of gene expression, H2O2 can influence caffeic, ferulic and sinapic acids [8,9]. The o-dihydroxy signal transduction pathways, leading to the synthesis structure of the B-ring is an important determinant for of cytokines and growth factors, such as interleukin 6 the antioxidative potential of anthocyanins, as it confers (IL-6) [41] or TGF-b [42]. a higher degree of stability to the flavonoid phenoxyl The results from these experiments indicate that radicals by participating in electron delocalization [34]. the red cabbage extract had an inhibitory effect on According to Li et al. [35], the number of -OH in total peroxynitrite-induced oxidation - we found a significant or in the B-ring, 3’,4’-ortho-dihydroxyl and 3-hydroxyl reduction of plasma protein carbonylation and lipid are the main structural requirements of anthocyanins in peroxidation. Since peroxynitrite is both an oxidative the of oxidative stress-induced endothelial injury. The and nitrative agent, we were also able to estimate inhibitory effect on oxidative stress was significantly the antinitrative properites of red cabbage ATH. The correlated with the intracellular radical scavenging measurements of 3-NT, performed with the use of activity of the tested anthocyanins [35]. the c-ELISA test, showed that ATH extract protected The antioxidative properties of ATH are believed plasma proteins against the nitrative action of ONOO-. to be partly responsible for the beneficial effects of Our findings confirm the antioxidative properties of these compounds on human health. Existing evidence anthocyanins and provide some information about the suggests that ATH may protect the circulatory system and protective effect of ATH on plasma components. These prevent cardiovascular diseases. They display a variety results are consistent with other studies on the role of of effects on blood vessels, platelets, and lipoproteins, anthocyanins and their derivatives in protecting against and may reduce the risk of coronary heart diseases oxidative stress caused by peroxynitrite. According to [36]. Despite these findings, the antioxidative action of Serraino et al. [43], ATH-rich blackberry juice, containing ATH in the protection of plasma components, such as cyanidin-3-O-glucoside, is a scavenger of peroxynitrite, proteins and lipids, remains only partly identified. Our exerting a protective effect against endothelial work was designed to estimate, in vitro, the antioxidative dysfunction and vascular failure. Ramirez-Tortosa effects of ATH from red cabbage on plasma protein and et al. showed that ATH-rich extracts decreased hepatic lipid changes, induced by two physiological oxidants: lipid peroxidation under oxidative stress conditions - peroxynitrite (ONOO ) and hydrogen peroxide (H2O2). in rats [44]. The studies on an anthocyanin aglycone, ONOO- is formed in a rapid reaction between NO• and pelargonidin, performed by Tsuda et al. [45] showed •- - O2 . The generation of ONOO was confirmedin vivo and that this compound protects tyrosine from undergoing a wide range of its pathological implications have been nitration via peroxynitrite scavenging. In the first step reported, which include cardiovascular impacts [37]. of the reaction between ONOO– and pelargonidin, 3-nitrotyrosine, the biological marker of peroxynitrite, p-hydroxybenzoic acid is formed; subsequently, this is related to the formation of atherosclerotic lesions, acid might react with ONOO–, leading to the generation endothelial cell dysfunction, ischaemia/reperfusion of 4-hydroxy-3-nitrobenzoic acid. injury, myocardial infarction, and heart failure [38]. We Some findings suggest that red cabbage ATH used ONOO- at the concentration of 100 µM. Beckmann may have pro-oxidative properties. Philpott et al. [46] et al. indicated that the bolus addition of 250 µM ONOO- underlined the dangers of generalizing to potential health is roughly equivalent to a steady-state level of 1 µM benefits based solely on identification of high ATH content maintained for 7 min [39]. It has been established that in plants, or other similar approaches. Their observations these concentrations are likely to occur in blood vessels, indicated that the red cabbage extracts might not engage • •- where the production of NO and O2 is elevated in antioxidative protection against H2O2 action, and might [19-21,39]. The enhanced generation of superoxide instead behave as pro-oxidants. Contrary to the results anions may also be a source of other ROS (Reactive of Philpott et al. [46], we found the ATH extract from Oxygen Species), e.g. hydrogen peroxide, formed in vivo red cabbage demonstrated antioxidative properties and as a product of two general mechanisms: enzymatic partly protected blood plasma proteins, as well as lipids, or non-enzymatic dismutation of superoxide anions. against H2O2-induced damage. To exclude the possibility

H2O2 is a physiological bacteriostatic agent, and a key of direct interactions between ATH extract and plasma element in the defence mechanisms of inflammatory components, control experiments using blood plasma cells; however, it is also involved in multiple physiological and the tested extract only (no addition of ONOO- or

571 Red cabbage anthocyanins may protect blood plasma proteins and lipids

H2O2) were performed; no pro-oxidative effects of the efficacy on one or more target functions in the body tested ATH were found (data not presented). in a quantifiable way. Beyond adequate nutritional Inflammatory processes - where rapid formation qualities, functional foods should either improve the of peroxynitrite (and other ROS) leads to plasma state of health and well-being and/or reduce the risk component damage - are among the major risk factors of disease. Our studies demonstrate that anthocyanins of cardiovascular diseases, including myocardial present in red cabbage have inhibitory effects on ischemia/reperfusion injury, heart failure, circulatory oxidative stress in human plasma and, as dietary shock, and atherosclerosis. From a health standpoint, antioxidants, may be potentially usable nutraceuticals food is considered functional if it can reveal a beneficial in the prevention of cardiovascular diseases.

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