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Journal of Food Biochemistry ISSN 1745-4514

EFFECT OF DIFFERENT SOLVENTS ON POLYPHENOLIC CONTENT, ANTIOXIDANT CAPACITY AND ANTIBACTERIAL ACTIVITY OF IRISH YORK CABBAGE
jfbc_545 344..358

AMIT KUMAR JAISWAL, GAURAV RAJAURIA, NISSREEN ABU-GHANNAM1 and SHILPI GUPTA
School of Food Science and Environmental Health, College of Science and Health, Dublin Institute of Technology, Cathal Brugha Street, Dublin 1, Ireland

Corresponding author. TEL: +353-1-402-7570; FAX: +353-1-878-8978; EMAIL: nissreen.abughannam@dit.ie Accepted for Publication December 30, 2010 doi:10.1111/j.1745-4514.2011.00545.x

ABSTRACT
Cabbage is a rich source of a number of bioactive compounds such as avonoids, glucosinolates and their breakdown products, which may have antibacterial, antioxidant and anticancer properties. This investigation was undertaken to estimate the effect of using water and different organic solvents such as ethanol, acetone and methanol at various concentrations on the total polyphenols, antibacterial activity and antioxidant capacity of Irish York cabbage. Water has the highest extraction yield of 3.85%, whereas 60% methanolic extract has the highest content of total polyphenols (33.5 mg gallic acid equivalents per gram [dried weight, dw] of extract) and avonoid (21.9 mg quercetin equivalents per gram [dw] of extract). A concentration-dependent antioxidative capacity was conrmed for different reactive oxygen species, including hydrogen peroxide, metal chelating capacity, reducing power and lipid peroxidation. The crude extracts showed a broad spectrum of antibacterial activity for the different extraction solvents applied; however, 60% methanolic extract (1.4%) exhibited higher antibacterial activity against Listeria monocytogenes (100%).

PRACTICAL APPLICATIONS
The problem of oxidation and microbial contamination are most common aspects of food preservation, especially when the products develop undesirable avors, unpleasant taste, rancid odors, discoloration and other forms of spoilage often responsible for the loss of quality, and safety and shortening of shelf life. Over the last decade, interest in using extracts from plant origin with antibacterial and antioxidant properties to control oxidative deterioration and microbial spoilage has been increasing. Present work demonstrates that conventional organic-solvent extracts of Irish York cabbage are rich in polyphenols and are potential sources for this phytochemical, and have appreciable amount of antioxidant and antimicrobial activity. This extract may be exploited as bio-preservatives in food applications in order to enhance safety and quality of food or neutraceuticals for possible application in food and dietary supplemental products for health promotion.

INTRODUCTION
Polyphenolic compounds from plants have been extensively studied for their antioxidant activity, which is a biological
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function, important in keeping the oxidative stress levels below a critical point in the body (Zhou and Yu 2006; Wijngaard et al.2009;SreeramuluandRaghunath2010).Antioxidants are a special group of compounds that neutralize
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free radicals and other reactive oxygen species (ROS) that are generated in the body (Devasagayam et al. 2004). Free radicals and ROS are highly reactive and unstable chemical species (Wang et al. 2007) which are fundamental to any biochemical process and represent an essential part of aerobic life and our metabolism (Saito et al. 2005). These free radicals and ROS are generated by normal metabolic activity as well as lifestyle factors such as smoking, exercise and diet, and have been associated in the causation and progression of several chronic diseases (Halliwell 1996; Wang et al. 2007). In addition, free radicals can cause lipid peroxidation in foods, thus leading to its deterioration (Halliwell et al. 1995). All aerobic organisms, including human beings, have antioxidant defenses that protect against oxidative damages, and numerous damage removal and repair enzymes are present to remove or repair damaged molecules (Ali et al. 2001). However, this natural antioxidant mechanism can be inefcient; hence, dietary intake of antioxidant compounds becomes important (Halliwell 1994). Besides playing an important role in physiological systems, antioxidants have been applied in the food industry to prolong the shelf life of foods, especially those rich in polyunsaturated fats. These components in food are readily oxidized by molecular oxygen and are major causes of oxidative deterioration, nutritional losses, off-avor development and discoloration. Addition of synthetic antioxidant compounds such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole have been commonly used in processed foods to control lipid oxidation. It has been reported that these compounds have some side effects (Botterweck et al. 2000). In recent times, there has been an increasing tendency toward the use of natural substances instead of the synthetic ones. Also, with the increase in the price of raw materials, the problem of cost benets for chemical production is becoming more considerable. As a result, great deal of efforts have been directed toward identifying low-cost natural products that can replace synthetic chemicals. Plants are a good source of polyphenols. Organic solvents are commonly used for the extraction of polyphenolics from plant material. Factors which determine the recovery of polyphenols from plant materials are inuenced by the solubility of the polyphenols in the solvent used for the extraction process. Water, ethanol, methanol and acetone and their aqueous mixtures are commonly used for extraction purposes (Basile et al. 2005; Abas et al. 2006; Zhou and Yu 2006; Durling et al. 2007; Pantelidis et al. 2007; Wijngaard et al. 2009; Sreeramulu and Raghunath 2010). Cabbage (Brassica oleracea), a member of Brassicaceae (or Cruciferae) family, is a leafy garden plant and is among the most important vegetables consumed worldwide due to its availability in local markets and consumer preference. It is an herbaceous, biennial, dicotyledonous owering plant and thought to have originated in Western Europe. Cabbage is
Journal of Food Biochemistry 36 (2012) 344358 2011 Wiley Periodicals, Inc.

rich in phytochemicals such as avonoids and glucosinolates and their hydrolysis products. It is a good source of healthpromoting compounds that show preventive effects against cancer, atherosclerosis, nephritis and diabetes mellitus (Taveira et al. 2009). Numerous studies have reported on the antioxidant and antibacterial activity of many species of Brassicaceae family (Sherman and Hodge 1936; Ayaz et al. 2008). However, few studies have highlighted the potential importance of cabbage as a source of antibacterial substances (Kyung and Fleming 1994). However, there are no reports available, which compare the level of polyphenols, antioxidant capacity (AOC) and antibacterial potential of Irish York cabbage extracts upon using different extraction solvents. Keeping these facts in mind, the present study investigates the differences in level of polyphenols, AOC and antibacterial potential of Irish York cabbage extracts using water and a range of organic solvents such as ethanol, acetone and methanol.

MATERIALS AND METHODS

Plant Material and Their Preparation
Fresh Irish York cabbage, B. oleracea (seven to eight cabbage heads) were purchased from a local supermarket in Dublin. Immediately, outer leaves and the stem were trimmed off and the heads were then divided into four segments, and the central core removed. The segments were chopped into 0.5 56 cm pieces using a vegetable cutting machine. A 1,000 g of chopped vegetable was crushed with the help of mortar and pestle in the presence of liquid nitrogen and stored at -20C until used.

Preparation of Extracts
Extraction of polyphenols was carried out according to the existing method in our laboratory (Gupta et al. 2010). Briey, 5 g of crushed cabbage leaves were added to three different asks and extracted using 60% ethanol, methanol or acetone with 1 min nitrogen ushing at 20 psi. Flasks were kept in a shaking incubator (Innova 42, Mason Technology, Dublin, Ireland) at 100 rpm and 40C for 2 h. The infusions were ltered with Whatman #1 until a clear extract was obtained. The extracts were evaporated to dryness in a multi-evaporator (Syncore Polyvap, Mason Technology, Dublin, Ireland) at 60C at their respective pressure and were stored at -20C until used. After identifying the proper solvent for extraction, which was methanol in this case, further studies were carried out with different concentrations of methanol (0, 20, 40, 60, 80 and 100%). All of the extractions were carried out in four replicates. All the extracts were dissolved in water, and a concentration of 1 mg/mL was used for phytochemical analysis.
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Phytochemical Analysis
Determination of Total Polyphenolic Content (TPC). TPC of cabbage extracts were determined by the method of Ganesan et al. (2008). Briey, 100 mL aliquot of sample in deionized water were mixed with 2 mL of 2% Na2CO3 and were allowed to stand for 2 min at room temperature. After incubation, 100 mL of 1N FolinCiocalteaus phenol reagent (Sigma-Aldrich, Steinheim, Germany) was added. Reaction mixture was allowed to stand for 30 min at room temperature in the dark. Absorbance of all the sample solutions was measured at 720 nm. Results were expressed as gallic acid (SigmaAldrich) equivalents per gram (GAE/g) of dried weight of extract through the calibration curve of gallic acid. High Performance Liquid Chromatography Coupled with Diode-Array Detector (HPLC-DAD) Analysis of Polyphenolic Compounds. The HPLC system consisted of a reversed-phase HPLC column on an Alliance HPLC (Waters, Milford, MA; e2695 Separations modules) equipped with an auto sampler and controller with dual pump, a 2998 photodiode array detector (PDA) and the Empower software. An Atlantis C18 column (250 mm 4.6 mm, 5 mm particle size) from Waters (Waters) was used for polyphenols separation at 25C. The binary mobile phase according to Tsao and Yang (2003) composed of 6% acetic acid in 2 mM/L sodium acetate (Sigma-Aldrich; Solvent A) and acetronitrile (Fischer Scientic, Leicestershire, U.K.; Solvent B) were used. The system was run with a gradient program. The solvent gradient was as follows: 015% B in 45 min, 1530% B in 15 min, 3050% B in 5 min and 50100% B in 5 min. A ow rate of 1 mL/min was used and total run time for samples was 70 min. Samples and mobile phases were ltered through a 0.22 mm Millipore lter (Millipore, Bedford, MA) prior to HPLC injection and 20 mL of sample were injected. A 20 mL volume of reference compounds solution were injected. The chromatograms were monitored at 280 nm (hydroxybenzoic acid [HBA]), 320 nm (hydroxycinnamic acids [HCA]), 360 nm (avones and avonols) and 520 nm (anthocyanins); complete spectral data were recorded in the range of 220600 nm. Determination of Total Flavonoid Content (TFC). The TFC was determined according to the method of Liu et al. (2009). Briey, 250 mL of extract was mixed with 1.25 mL of deionized water and 75 mL of 5% NaNO2 (SigmaAldrich) solution. After 6 min, 150 mL of 10% AlCl3H2O solution was added. Finally, 0.5 mL of NaOH (1 M) solution was added and the total volume was made up to 2.5 mL with deionized water. Absorbance against blank was determined at
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510 nm. Results were expressed as quercetin (Sigma-Aldrich) equivalents per gram (QE/g) of dried weight (dw) of extract.

AOC Analysis
DPPH Free Radical Scavenging Capacity. The scavenging effect of polyphenols in the York cabbage extracts were monitored as described by Yen and Chen (1995). The assay was performed in a 96-well round-bottom microplate (Sarstedt, Inc., Leicester, U.K.) with 1:1 ratio of 100 mL of 2,2diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich) radical solution (165 mM) and 100 mL of sample. Different concentrations (0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2.0 mg/ mL) were tested for each sample in order to get EC50 value. The DPPH solution was freshly prepared for each experiment in methanol. The reaction mixtures were incubated for 30 min at 25C in dark conditions, and absorbance measured at 517 nm in a microplate reader (Powerwave, Biotek, Winooski, VT). The ability to scavenge the DPPH radical was calculated using the following equation:

Atest A Scavenging capacity (%) = control 100 Acontrol

(1)

where Acontrol is the blank free absorbance of the control (DPPH solution without sample) and Atest is the blank free absorbance of the test sample (DPPH solution plus test sample). Calculated EC50 values indicate the concentration of sample that is required to scavenge 50% DPPH radicals. The lower the EC50 value of the sample, the higher the AOC. Ascorbic acid was used as a reference compound.

Ferric Reducing Antioxidant Potential (FRAP) Capacity. Total antioxidant power of polyphenolic extracts of vegetable samples were measured using FRAP assay, according to the method reported by Benzie and Strain (1996), with some modications. The working FRAP reagent was freshly prepared by mixing 10 volumes of 300 mM acetate buffer, pH 3.6, with one volume of 10 mM 2,4,6-tri[2-pyridyl]-striazine (Sigma-Aldrich) in 40 mM hydrochloric acid and with one volume of 20 mM ferric chloride (Sigma-Aldrich). All solutions were used on the day of preparation. The reaction was performed in a microplate reader with 96-well plates at a temperature of 37C. Preheated 100 mL FRAP reagent at 37C was dispensed in each well with 50 mL of sample or standard. The reagent blank assay was performed by using 100 mL FRAP reagent and 50 mL of water. The AOC of each extract and standard were measured by calculating the increase in absorbance caused by the generated ferrous ions. The absorbance was read after 10 min at 593 nm with the help of a microplate spectrophotometer. Trolox (Sigma-Aldrich)
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was used as a standard, and the results were expressed as milligram trolox equivalents per gram (TE/g) of (dw) of extract. Metal Chelating Ability. The chelating ability of ferrous ions (FIC) by York cabbage extract was estimated by the method of Decker and Welch (1990) with minor modications. Briey, 100 mL of varying concentrations of different samples or standard were mixed with 100 mL of deionized water and 25 mL of ferrous chloride (Sigma-Aldrich, Germany) (0.5 mM) in a microplate. The reaction was initiated by the addition of 25 mL of ferrozine (Sigma-Aldrich, Germany) (2.5 mM) and the reaction mixture was shaken vigorously. The absorbance was recorded at 562 nm with a microplate reader, after 10 min of incubation at the ambient temperature. A reagent blank contained water (25 mL) instead of ferrozine solution while control had 100 mL of water instead of sample. Ethylenediaminetetraacetic acid (EDTA) (5 to 50 mg/mL) was used as a standard compound. The percentage of inhibition of ferrozine-Fe2+ complex formation was calculated using Eq. (1).

metrically (Agilent 8453 with PDA detector, Waldbronn, Germany) by measuring absorption with molar extinction coefcient for H2O2 of 43.6 M-1 cm-1 at 230 nm. Hydrogen peroxide solution (40 mM) was prepared in phosphate buffer (pH 7.4). A total of 3.4 mL of different York cabbage extracts were added to 0.6 mL of hydrogen peroxide solution, and the absorbance was recorded at 230 nm after 10-min incubation, against reagent blank solution (phosphate buffer without hydrogen peroxide solution). BHT was used as a reference compound. The percentage scavenging of hydrogen peroxide was calculated according to Eq. (1).

Antibacterial Activity
Bacterial Strains and Growth Conditions. The bacterial strains used in this study included gram-negative (Salmonella abony NCTC 6017, Pseudomonas aeruginosa ATCC 27853) and gram-positive (Listeria monocytogenes ATCC 19115, Enterococcus faecalis ATCC 7080; Medical Supply Company, Dublin, Ireland). All the cultures were maintained at -70C in 20% glycerol stocks and grown in tryptic soy broth (TSB, pH 7.2, Scharlau Chemie, Barcelona, Spain) at 37C excluding P. aeruginosa, which was grown at 30C for 18 h to obtain subcultures. To obtain a working concentration, a bacterial suspension was prepared in NaCl, 0.85% (BioMerieux, Marcy lEtoile, France) equivalent to McFarland standard of 0.5 with the help of Densimat photometer (BioMerieux). Finally, the suspension was diluted with TSB in order to get a concentration of 1 106 cfu/mL. Antibacterial Activity Assay. The antibacterial assay was carried out through microtiter well method according to the protocol available in our laboratory (Gupta et al. 2010). York cabbage extracts were dissolved in TSB media and 200 mL was added to the rst row of the plate. The rst well of each row had the highest concentration of the tested extracts (2.8%). Serial dilutions along each column were made with the addition of 100 mL TSB. Test bacteria (100 mL) from the 106 cfu/mL suspensions were added to all the wells. Well containing cabbage extracts (100 mL) and sterile TSB (100 mL) considered as negative control (sample blanks) while well containing sterile TSB (100 mL) and bacterial suspension (100 mL) was considered as control. One well of the microtiter plate containing sterile TSB (200 mL) served as a blank to check for contamination. The readings were taken immediately after the plate was inoculated with culture by microtiter plate reader at 600 nm, with 10-s agitation before the measurement of optical density (OD). The microtiter plates were incubated without agitation at 37C for all the test organisms
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Lipid Peroxidation in a Hemoglobin-Induced Linoleic Acid System (LPO). The lipid peroxidation scavenging ability of cabbage extracts was principally determined by a photometry assay reported by Kuda et al. (2005) with slight modications. The test samples (100 mL) or reference compounds were mixed with 25 mL of linoleic acid/ethanol (0.1 M) and 75 mL of 0.2 M phosphate buffer (pH 7.2) in a test tube. The auto-oxidation of linoleic acid (Sigma-Aldrich) in the above reaction mixture was initiated by adding 50 mL hemoglobin (0.08% in water) and was incubated for 60 min at 37C. The peroxidation of linoleic acid was stopped by adding 5 mL of HCl (0.6% in ethanol). The value of peroxidation of samples in the reacted mixture was calculated using the ferric-thiocyanate method, in which color appeared upon the addition of 50 mL of ferrous chloride (20 mM) and ammonium thiocyanate (Sigma-Aldrich; 30%) each, in that order. For the estimation of peroxide value, 200 mL of the above colored reacted mixture was taken in a microplate and absorbance was recorded at 490 nm using a microplate reader. Ascorbic acid was used as standard. The absorbance of sample blank was measured without the addition of hemoglobin in the reaction mixture. The AOC of the samples was calculated using Eq. (1). Hydrogen Peroxide (H2O2) Scavenging Assay. The hydrogen peroxide scavenging capacity of vegetable extracts was measured according to the existing method in our laboratory Rajauria et al. (2010). The concentration of hydrogen peroxide (Sigma-Aldrich) was determined spectrophotoJournal of Food Biochemistry 36 (2012) 344358 2011 Wiley Periodicals, Inc.

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except P. aeruginosa, which was incubated at 30C, and the readings were taken after 24 h. Sodium benzoate (SigmaAldrich) and sodium nitrite (Sigma-Aldrich) were taken as positive controls. Each experiment was done in triplicate and two plates were tested for each organism. The antibacterial activity was determined in terms of percentage of inhibition calculated by the following formula.

I% =

(C24 C0 ) (T24 T0 ) 100 C24 C0

(2)

where I is the percentage inhibition of growth, C24 is the blank compensated OD600 of the control of the organism at 24 h, C0 is the blank compensated OD600 of the control of the organism at 0 h, T24 is the negative control compensated OD600 of the organism in the presence of test extract at 24 h and T0 is the negative control compensated OD600 of the organism in the presence of test sample at 0 h.

Statistical Analysis
All experiments were done in triplicate and replicated at least twice. Results are expressed as mean values standard deviation. Analysis of variance and multiple comparisons (Fishers least-signicant-difference test) were used to evaluate the signicant difference among various treatments using the STATGRAPHICS Centurion XV. Differences at P < 0.05 were considered to be signicant.

RESULTS AND DISCUSSION

Effect of Solvent System
Extraction Yield, Total Phenolics and Flavonoid Contents. For the selection of proper organic solvent for polyphenols extraction, different organic solvents (60% methanol, ethanol or acetone), were examined. The percentage yield of the different solvent extract of York cabbage is shown in Table 1. The extraction yield of these samples varied from 2.47 0.17% to 3.25 0.10%, with a descending order of methanol > ethanol > acetone. These results were in agree-

ment with yield results reported from buckwheat (Sun and Ho 2005) and barley seed extracts (Liu and Yao 2007), which showed the same order of solvents. Polyphenols are plant metabolites characterized by the presence of several phenolic groups (i.e., aromatic rings with hydroxyls). These hydroxyls are very reactive in neutralizing free radicals by donating a hydrogen atom or an electron, chelating metal ions, inactivating lipid free radical chains and preventing hydroperoxide conversions into reactive oxyradicals. Table 1 summarizes the TPC of the different solvent extracts which varied between 25.2 0.38 to 33.5 1.35 mg GAE/g dw of extracts. The methanolic extract exhibited signicantly higher TPC (33.5 1.35 mg GAE/g dw) as compared with the other two solvents (P < 0.05). There have been few studies which evaluated the content of polyphenols in cabbage. Kaur and Kapoor (2002) estimated the TPC of 33 commonly consumed vegetables and reported that the TPC of cabbage was 92.5 mg GAE/100 g (fw). Our results showed that Irish York cabbage had higher TPC, which is 109.1 mg GAE/100 g (fresh weight [fw]). Kim et al. (2004) reported that TPC of cabbage ranged from 110.2 to 153.3 mg/100 g (fw). The results from the present study are in line with the reported results. The TFC of different solvent extracts varied from 16.9 1.12 to 21.7 1.71 mg QE/g of (dw) of extracts (Table 1). It was found that methanolic extract has 22.12 and 11.52% higher avonoid contents as compared with ethanolic and acetone extracts, respectively. Flavonoids are an important part of the diet because of their healthful effect on human nutrition and their antioxidant activity such as free radical scavenging and inhibition of hydrolytic and oxidative enzymes (Siddhuraju and Becker 2003).

HPLC-DAD Quantitative Analysis of Polyphenols. The photodiode array detector allowed recording of UV-visible (UV-vis) spectrum of each peak of the chromatogram and thus allowed explicit attribution of each chromatographic peak to different class of polyphenols, as each class exhibits a characteristic UV-vis spectrum (Fig. 1). Five different groups of polyphenols were identied by comparing their UV-vis spectra with spectra of reference compounds and

Extraction solvent 60% MeOH 60% EtOH 60% Acetone

Extraction yield (%) 3.25 0.10a 3.21 0.18a 2.47 0.17b

TPC GAE/g (dw) 33.5 1.35a 28.1 0.97b 25.2 0.38c

TFC QE/g (dw) 21.7 1.71a 19.2 0.65b 16.9 1.12c

TABLE 1. TOTAL PHENOL, FLAVONOID AND EXTRACTION YIELD OF IRISH YORK CABBAGE EXTRACTS OBTAINED FROM DIFFERENT SOLVENT EXTRACTION SYSTEMS

Data are expressed as mean SD (n = 3). Means not sharing the same letter are signicantly different (LSD) at P < 0.05 probability level in each row. MeOH, methanol; EtOH ethanol; TPC, total phenolic content; TFC, total avonoid content; dw, dried weight; GAE, gallic acid equivaelents; QE, quercetin equivalents.

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271.3
0.08

326.0
0.050

0.040
0.06

238.1

AU

0.04

AU
0.02 0.00 250.00 49.601 Extracted 300.00 nm

0.030

0.020

0.010

364.0
350.00 400.00

0.000 250.00 300.00 nm 350.00 400.00

0.060

254.7 355.9

C
0.20

515.3

0.050

279.6

0.040

0.15

AU

0.030

AU
0.10 0.05 0.00
250.00 300.00 nm 350.00 400.00

0.020

FIG. 1. CHARACTERISTICS UV-VIS SPECTRA FOR DIFFERENT CLASSES OF POLYPHENOLS (A) GALLIC ACID (B) CHLOROGENIC ACID (C) RUTIN AND (D) CYANIDING-3-O-GLUCOSIDE

0.010

300.00

400.00 nm

500.00

reported values (Abad-garca et al. 2009). All the ve polyphenolic groups were quantied using the standard curves of the representative standards. The HBA derivatives (range 255280 nm) were quantied at 280 nm and expressed as gallic acid equivalents (GAE), HCA derivatives (range 310326 nm) at 320 nm and expressed as chlorogenic acidequivalents (CAE), avones (range, 339350 [band I] and 261279 nm [band II]), polymethoxylated avones (PMF) (range 324336 nm), glycosylated avonoid (GSF; range 347370 nm [band I] and 250267 nm [band II]) at 360 nm and expressed as rutin equivalents (RE), and anthocyanins (range 515545 nm [band I] and 275285 nm [band II]) at 520 nm and expressed as cyanidin-3-glucoside equivalents (Cn3GE). A distinctive HPLC chromatogram of the polyphenols extracted from York cabbage at the four different wavelengths are shown in Fig. 2. The chromatogram showed that York cabbage contains a mixture of more than 20 phenolics. Five peaks were identied as HBA derivatives, four peaks as HCA derivatives, six peaks as avones, two as polymethoxy avonoid, and one peak of glycosylated avonols and polymethoxylated avonols. Nielsen et al. 1998 reported that white cabbage leaves contain more than 20 phenolic compounds including glucosides of kaempferol and quercetin with/without further acylation with hydrocinnamic acid. From the results (Table 2), it is clear that there was an effect of different solvents on individual phenolic groups; signicantly higher (P < 0.05) concentration (2.15 mg GAE/g [dw] of
Journal of Food Biochemistry 36 (2012) 344358 2011 Wiley Periodicals, Inc.

extracts and 7.17 mg CAE/g [dw] of extracts) of HBA and HCA, respectively, were found in ethanolic extracts; avones was higher in acetone extracts, whereas the sum of the individual levels of polyphenolic groups was higher (16.96 mg/ mL) (P < 0.05) in methanolic extracts. Ferreres et al. (2007) reported that hydroxycinnamic derivatives are the main polyphenols in Tronchuda cabbage and a similar trend was observed in the present study. No anthocyanin content was found in any of the tested samples. AOC Analysis. Estimation of the total AOC of fruits, vegetables and other plant products cannot be performed accurately by any single method due to the complex nature of phytochemicals (Chu et al. 2000). In the present study, ve different methods were used for the estimation of total AOC of crude extracts. The scavenging of DPPH radical is caused by the donation of hydrogen by phenolics and the scavenging can be quantied by the change in color from purple to yellow. EC50 value is negatively related to the AOC as it expresses the amount of antioxidant required to reduce the radical concentration by 50%. All York cabbage extracts, at the tested concentration, were capable of directly reacting with and quenching DPPH radical (Fig. 3A). The methanolic extract was found to have the highest DPPH radical scavenging capacity with EC50 0.79 0.05 mg/mL of extracts. As compared with methanol, ethanolic and acetone extracts showed 46.83 and 63.29% less scavenging capacity, respectively.
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0.36 0.34 0.32 0.30 0.28 0.26 0.24 0.22 0.20

A
1 1 3

AU

0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes 40.00 45.00 50.00 55.00 60.00 65.00 70.00

2 1 1 1 1 52 3 2 23 3

3 3 3 3 44

0.36 0.34 0.32 0.30 0.28 0.26 0.24 0.22 0.20

B
33

AU

0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes 40.00 45.00 50.00 55.00 60.00 65.00 70.00

1 2 1 1 52 3 2 3

3 3 3 44

0.36 0.34 0.32 0.30 0.28 0.26 0.24 0.22 0.20

AU

0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 5.00 0.36 0.34 0.32 0.30 0.28 0.26 0.24 0.22 0.20 10.00 15.00 20.00 25.00

3 3

2 3 4 4

30.00 35.00 Minutes

40.00

45.00

50.00

55.00

60.00

AU

0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Minutes

FIG. 2. HPLC-DAD PROFILE OF YORK CABBAGE POLYPHENOLS AT FOUR DIFFERENT WAVELENGTHS: (A) 280 NM; (B) 320 NM; (C) 360 NM AND (D) 520 NM. 1-HYDROXYBENZOIC ACID DERIVATIVES; 2-HYDROXYCINNAMIC ACID DERIVATIVES; 3-FLAVONES; 4-METHOXYLATED FLAVONOID; 5-GLYCOSYLATED FLAVONOLS BHT, butylated hydroxytoluene, EDTA, ethylenediaminetetraacetic acid.

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TABLE 2. HYDROXYBENZOIC ACIDS, HYDROXYCINNAMIC ACIDS, FLAVONES, POLYMETHOXYLATED FLAVONES, GLYCOSYLATED FLAVONOID AND ANTHOCYANINS CONTENTS OF IRISH YORK CABBAGE EXTRACTS OBTAINED FROM DIFFERENT SOLVENT EXTRACTION SYSTEMS Extraction solvent 60% MeOH 60% EtOH 60% Acetone HBA GAE/g (dw) 1.67 0.00a 2.15 0.00b 1.89 0.01c HCA CAE/g (dw) 6.96 0.01a 7.17 0.14a 5.92 0.02b Flavones RE/g (dw) 7.84 0.02a 6.42 0.05b 8.39 0.00c PMF RE/g (dw) 0.24 0.01a 0.86 0.01b 0.41 0.01c GSF RE/g (dw) 0.25 0.01a 0.20 0.01b 0.20 0.01b Anthocyanins Cn3GE/g (dw) ND ND ND

Data are expressed as mean SD (n = 2). Means not sharing the same letter are signicantly different (LSD) at P < 0.05 probability level in each row. MeOH, methanol; EtOH, ethanol; CAE, chlorogenic acid equivalents; RE, rutin equivalents; HBA, hydroxybenzoic acid; HCA, hydroxycinnamic acid; PMF, polymethoxylated avones; GSF, glycosylated avonoid.

Peroxyl radicals are formed by a direct reaction of oxygen with alkyl radicals and these radicals attack biomolecules, such as lipids, to initiate free radical chain reactions and cause lipid peroxidation. Methanolic extract showed higher inhibitory ability on lipid oxidation (EC50 4.78 0.53 mg/mL of extract [Fig. 3B]). No signicant difference was found between ethanol and acetone extracts. However, all the extracts showed a rapid and concentration-dependent AOC capacity. In contrast to results reported by Kuda et al. (2005) on edible algae, phenolic compounds from York cabbage showed higher inhibitory capacity for lipid peroxidation.

Iron, in nature, can be found as either ferrous (Fe2+) or ferric ion (Fe3+), with the latter form of ferric ion predominating in foods. The production of highly ROS such as superoxide anion radicals, hydrogen peroxide and hydroxyl radicals are also catalyzed by free iron through HaberWeiss reaction. Among the transition metals, iron is known as the most important lipid oxidation pro-oxidant because of its high reactivity. The ferrous state of iron accelerates lipid oxidation by breaking down hydrogen and lipid peroxides to reactive free radicals via the Fenton reaction. Ferrozine can quantitatively form complexes with Fe2+. In the presence of chelating

2.0 1.6

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FIG. 3. COMPARATIVE STUDY OF ANTIOXIDANT CAPACITY (EXPRESSED AS EC50 VALUES) OF IRISH YORK CABBAGE, EXTRACTED WITH DIFFERENT SOLVENT SYSTEM (METHANOL [M], ETHANOL [E] AND ACETONE [A]). (A) DPPH, (B) LIPID PEROXIDATION, (C) FIC, (D) H2O2 AND (E) FRAP ASSAY All the values are means SD of three parallel experiments in duplicate. DPPH, 2,2-diphenyl-1-picrylhydrazyl; FIC, chelating ability of ferrous ions; FRAP, ferric reducing antioxidant potential.

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agents, the complex formation is disrupted, resulting in a decrease in the red color of the complex. Measurement of color reduction therefore allows estimating the metal chelating activity of the coexisting chelator. Lower absorbance indicates higher metal chelating activity. EDTA (EC50 16.9 mg/mL) is a strong metal chelator; hence, it is used as standard metal chelator agent in this study. Methanolic extracts of York cabbage showed signicantly (P < 0.05) high metal chelating ability compared with the other solvent extracts with a low EC50 value of 1.49 0.03 mg/mL of extract. Signicant difference in the activity, in increasing order, of acetone < ethanol < methanol was found among all the samples (Fig. 3C). Hydrogen peroxide is only mildly reactive by itself but it can sometimes be toxic to cells or food systems because it may give rise to hydroxyl radicals (OH) and singlet oxygen (1O2) by reacting with transition metals ions. Fig. 3D shows the scavenging of exogenously added H2O2 by different solvent extracts from York cabbage. All the three samples and reference compounds potently scavenged H2O2 in a dosedependent manner. It appeared that among the extracts, methanolic extracts had stronger scavenging capacity at low concentrations (1.15 0.06 mg/mL), but it had 6.76 times lower activity than BHT. The reducing power indicates that the antioxidants present in the extracts have a tendency to donate electrons and can reduce the oxidized intermediates of the lipid peroxidation process, thereby acting as primary and secondary antioxidants (Yen and Chen 1995). The reducing power of York cabbage extracts varied markedly with different solvents (Fig. 3E). This work demonstrated that all the extracts had reducing power in the order of methanol > acetone > ethanol. Methanolic extract showed signicantly (P < 0.05) high reducing power (6.0 0.01 mg TE/g) as compared with acetone (5.66 0.08 mg TE/g) and ethanol (5.48 0.64 mg TE/g) extracts. There was no signicant difference observed between acetone and ethanolic extracts. Antibacterial Activity. The selection of the pathogenic bacteria (L. monocytogenes and S. abony) was made after discussions with the Food Safety Authority of Ireland, as these were found to be the most challenging organisms for the safety of food products in Ireland. The other two (E. faecalis and P. aeruginosa) are the most widespread food spoilage microorganisms. Both these organisms are resistant to the commonly used antibiotics and antibacterial agents. Solvents used for extraction had a signicant effect on the antibacterial efcacy of Irish York cabbage. Regardless of the organism, 60% methanol extract exhibited the highest antibacterial activity (Fig. 4) than those of 60% ethanol and 60% acetone extracts. Resistance to extracts was not correlated with taxonomy, as L. monocytogenes (gram-positive)
352

(Fig. 4A) and S. abony (gram-negative) (Fig. 4C) were the most sensitive, followed by P. aeruginosa (Fig. 4B) and E. faecalis (Fig. 4D) as the most resistant. An inhibition of 100% was achieved against L. monocytogenes with an extract concentration of 2.8 and 1.4%, respectively. As the extract concentration was serially diluted, the inactivation effect was seen to reduce. The addition of extract at a concentration of 0.7% showed a reduction of 76% growth. In case of S. abony and P. aeruginosa methanolic extracts showed signicant antibacterial activity in the range of 6075%. Almost 72% S. abony growth was restricted by methanolic extract followed by P. aeruginosa which was inhibited up to 64%. For the above three organisms, ethanol and acetone extracts showed weak inhibitions which were in the range of 2139% only. E. faecalis was the most resistant among all the organisms. An inhibition in the range of 2532% was achieved using any of the three solvents. Antibacterial activities of 60% methanolic extract were also compared with synthetic antibacterial agents such as sodium benzoate and sodium nitrite. A 60% methanolic extract showed signicantly higher (P < 0.05) inhibition against L. monocytogenes (Fig. 4A) as compared with sodium benzoate (16%) and sodium nitrite (4%). S. abony (Fig. 4C) was inhibited to a higher extent (74.7%) with 60% methanol compared with sodium benzoate (68.6%).

Effect of Solvent Concentration

Extraction Yield, Total Phenolics and Flavonoid Contents. After identifying the proper solvent for polyphenols extraction, further studies were carried out using different concentrations of methanol as it could have a bearing on the extraction of polyphenols. Results showed that percentage extraction yields varied considerably as a function of water/ methanol concentration and ranged from 2.24 0.04% to 3.85 0.13%, respectively, for 100 to 0% methanol (Table 3). The extraction yield represents the percentage of material extracted from the cabbage. Thus, components other than polyphenols could be extracted and contributed to yield. The total phenolic contents of different concentrations of methanol and water were examined and presented in Table 3. The phenolic contents of these samples varied from 10.3 0.89 to 33.5 1.34 mg GAE/g (dw) of extracts. Extractions with 60% methanol resulted in the highest amount of total phenolic content, whereas the extraction with water and 100% methanol was signicantly less (P < 0.05). The total avonoid content of different solvent extracts of York cabbage are presented in Table 3. Signicant differences (P < 0.05) were observed in avonoid content of the different extracts.Pure water,20 and 100% methanol produced extracts with the lowest total avonoid content, whereas 60%
Journal of Food Biochemistry 36 (2012) 344358 2011 Wiley Periodicals, Inc.

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PHYTOCHEMICALS AND BIOACTIVITIES OF IRISH YORK CABBAGE

A
100 100

80
Inhibition (%) Inhibition (%)

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60

60

40

40

20

20

0 2.8 1.4 0.7

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Concentration of extract (%)

Concentration of extract (%)

C
100 100

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Inhibition (%) Inhibition (%)

80

60

60

40

40

20

20

0 2.8 1.4 0.7

0 2.8 1.4 0.7

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Concentration of extract (%)

FIG. 4. COMPARATIVE STUDY OF ANTIBACTERIAL ACTIVITIES OF THE FIRST THREE DILUTIONS FOR CABBAGE, EXTRACTED WITH 60% ETHANOL ( ), 60% ACETONE ( ), 60% METHANOL ( ) WITH SODIUM BENZOATE ( ) AND SODIUM NITRITE ( ) AGAINST (A) L. MONOCYTOGENES, (B) P. AERUGINOSA, (C) S. ABONY AND (D) E. FAECALIS All the values are means SD of three parallel experiments in duplicate.

TABLE 3. TOTAL PHENOL, FLAVONOID AND EXTRACTION YIELD OF YORK CABBAGE EXTRACTS OBTAINED UPON USING DIFFERENT CONCENTRATIONS OF METHANOL

Extraction solvent Water 20% MeOH 40% MeOH 60% MeOH 80% MeOH 100% MeOH

Extraction yield (%) 3.85 0.13 3.51 0.15ab 3.32 0.18bc 3.25 0.10bc 2.95 0.13c 2.24 0.04d
a

TPC GAE/g (dw) 10.3 0.89 21.1 0.98b 27.3 0.89c 33.5 1.34d 28.1 0.97c 28.9 0.38e
a

TFC QE/g (dw) 7.5 1.12a 10.8 1.71b 16.9 1.12c 21.9 1.68d 15.0 1.12e 10.0 1.12b

Data are expressed as mean SD (n = 3). Means not sharing the same letter are signicantly different (LSD) at P < 0.05 probability level in each row. GAE, gallic acid equivalents; QE, quercetin equivalents; dw, dried weight; TPC, total phenolic content; TFC, total avonoid content.

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TABLE 4. HYDROXYBENZOIC ACIDS (HBA), HYDROXYCINNAMIC ACIDS (HCA), FLAVONES, POLYMETHOXYLATED FLAVONES, GLYCOSYLATED FLAVONOIDS AND ANTHOCYANINS CONTENTS OF IRISH YORK CABBAGE EXTRACTS OBTAINED UPON USING DIFFERENT CONCENTRATIONS OF METHANOL Extraction solvent Water 20% MeOH 40% MeOH 60% MeOH 80% MeOH 100% MeOH HBA GAE/g (dw) 2.94 0.14a 3.28 0.00b 1.99 0.00c 1.67 0.00d 1.77 0.00d 1.93 0.64c HCA CAE/g (dw) 1.92 0.19a 5.39 0.01b 5.40 0.02b 6.96 1.01c 6.15 0.60d 6.11 0.01d Flavones RE/g (dw) 1.73 0.01a 4.84 0.02b 6.95 0.18c 7.84 0.02d 7.39 0.01e 6.68 0.02f PMF RE/g (dw) ND ND 0.29 0.01a 0.24 0.01b 0.51 0.01c 0.73 0.01d GSF RE/g (dw) ND ND 0.18 0.00a 0.25 0.00b 0.18 0.00a 0.11 0.00c Anthocyanins Cn3GE/g (dw) ND ND ND ND ND ND

Data are expressed as mean SD (n = 2). Means not sharing the same letter are signicantly different (LSD) at P < 0.05 probability level in each row. MeOH, methanol; GAE, gallic acid equivalents; CAE, chlorogenic acid equivalents; RE, rutin equivalents; PMF, polymethoxylated avones; GSF, glycosylated avonoid.

methanolic extracts had the highest avonoid content. The avonoid-rich plants could be a good source of antioxidants that may help to increase the overallAOC of food and protect it against lipid peroxidation (Sharifar et al. 2009). HPLC-DAD Quantitative Analysis of Polyphenols. Different concentrations of methanolic extracts obtained from York cabbage were analyzed for HBA, HCA, Flavones, PMF, GSF and anthocyanins, and the results were presented in Table 4. Signicant difference (P < 0.05) was found in individual groups of polyphenols as the concentration of water/ methanol changes. Twenty percent of methanolic extracts showed a higher concentration of HBA (3.28 mg GAE/g [dw] of extracts); HCA, avones and GSF were higher in 60% of methanolic extracts (6.96 mg CAE/g [dw] of extracts; 7.84 mg RE/g [dw] of extracts; 0.25 mg RE/g [dw] of extracts), whereas PMF was highly extracted with 100% methanolic extracts (0.71 mg RE/g [dw] of extracts). Water and 20% methanolic extracts did not show any PMF and GSP content which could be due to very low contents. A signicantly higher concentration of phenolic content was found in 60% methanolic extracts after summation of individual polyphenols. Previous studies revealed that the major compounds present in cabbage belong to derivatives of quercetin, sinapic acid, p-coumaric acid and kaempferol (Ferreres et al. 2005). Thus, it can be anticipated that these compounds can also be present in methanolic extracts of York cabbage. AOC Analysis. As shown in Fig. 5A, all the methanolic and water extract quenched DPPH radicals to different degrees. Among all the cabbage extracts tested, 60% methanolic extracts showed higher activity than other tested extracts with an EC50 value of 0.79 0.05 mg/mL did, followed by 80% methanolic extract at 0.81 0.04 mg/mL. A relatively low scavenging activity was observed with 100% methanolic extracts with an EC50 value of 1.57 0.14 mg/mL.
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Effect of different York cabbage extracts on oxidation of linoleic acid are presented in Fig. 5B. It was clear that the presence of antioxidants in the York cabbage extracts reduced the oxidation of linoleic acid by hemoglobin. There were signicant differences (P < 0.05) between the different extracts other than 60 and 80% of methanolic extracts. Sixty percent of methanolic extracts was better in their effect on reducing the oxidation of linoleic acid in comparison to other extracts. All the extracts had a lower antioxidant activity than EDTA. The EC50 value of metal chelating capacity of different York cabbage extracts varied from 1.49 0.03 to 8.80 0.20 mg/mL (Fig. 5C). Sixty percent of methanolic extracts were the highest, followed by 40 and 80% methanolic extracts. Pure water, 20 and 100% methanolic extracts were the lowest, and there was no signicant difference observed between 20 and 100% extracts. Figure 5D shows the EC50 value for hydrogen peroxide by different York cabbage extracts and ranges from 1.15 0.06 to 1.68 0.02 mg/mL. Sixty percent of methanolic extract showed signicantly higher activity followed by 80% methanolic extract. In the FRAP assay, the ability of different methanolic extracts of York cabbage to reduce Fe3+ to Fe2+ ranged from 4.27 0.74 to 6.00 0.13 mg TE/g (dw) of extract (Fig. 5E) and 60% methanolic extracts had the highest reducing power.

Antibacterial Activity. Figure 6 illustrates the antibacterial activity of the rst three dilutions of cabbage extracts obtained from different concentrations of methanol (0, 20, 40, 60, 80 and 100%). As can be seen in Fig. 6A, the antibacterial activity of 60% methanolic extract against L. monocytogenes (100%) was signicantly higher among all the concentrations of methanolic extract tested (p < 0.05). A sudden jump (weak to very strong) in the inhibition against L. monocytogenes was seen when extraction was carried out with 60% methanolic extract as compared with the other conJournal of Food Biochemistry 36 (2012) 344358 2011 Wiley Periodicals, Inc.

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10

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EC50 (mg/ml)

0.8

0.4

0.0

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0% M 20% M 40% M 60% M 80% M 100% M

FIG. 5. COMPARATIVE STUDY OF ANTIOXIDANT CAPACITY (EXPRESSED AS EC50 VALUES) OF IRISH YORK CABBAGE, EXTRACTED WITH DIFFERENT CONCENTRATIONS OF METHANOL (M). (A) DPPH, (B) LIPID PEROXIDATION, (C) FIC, (D) H2O2 AND (E) FRAP ASSAY All the values are means SD of three parallel experiments in duplicate. BHT, butylated hydroxytoluene; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EDTA, ethylenediaminetetraacetic acid; FIC, chelating ability of ferrous ions; FRAP, ferric reducing antioxidant potential.

centrations tested, which showed weak activity. Similar patterns of inhibition were found with all the tested bacteria. However, at lower concentrations of extracts tested (0.7%), the activity was recorded to be above 76%. In the case of S. abony, extracts obtained with 60% methanol also exhibited strong inhibition activity (Fig. 6C). This was signicantly higher among all the tested concentrations (P < 0.05). It was observed that there was signicant difference in the activity when the amount of extracts was reduced (P < 0.05). In the case of P. aeruginosa (Fig. 6B), 60% methanolic extract showed moderate activity, but was still signicantly higher among all the concentrations of methanol used for extraction (P < 0.05). An exception to this inhibition pattern was observed with E. faecalis (Fig. 6D), where 80% methanolic extract showed the highest antibacterial activity (62.9%) among all the concentrations tested. Overall, the most susceptible among food pathogenic bacteria tested in this work to the antibacterial activity of cabbage extract was L. monocytogenes, followed by S. abony, and among the food spoilage, P. aeruginosa was more susceptible than E. faecalis.
Journal of Food Biochemistry 36 (2012) 344358 2011 Wiley Periodicals, Inc.

CORRELATION ANALYSIS
A correlation analysis was carried out between phytochemical content and AOC (EC50 value, except FRAP which is mg TE/g [dw] of extract) of York cabbage extracts. In the present study, TPC, TFC, HBA, HCA, Flavones, PMF, GSF were correlated with FRAP, DPPH, LPO, FIC, H2O2 (Table 5). Moderate correlation was seen between TPC and LPO (r2 0.78), but no correlation was found between other antioxidant assays. No correlation between TPC and AOC was found which could be due to the following reasons: the AOC observed was not only from the phenolic contents, but could possibly be due to the presence of some other phytochemicals such as ascorbic acid, tocopherol and pigments, as well as the synergistic effects among them, which also contribute to the total AOC (Sengul et al. 2009). Spectroscopic determination of TPC was positively correlated (r2 0.93) with the summation of the individual polyphenols as examined by HPLC-DAD. TFC correlated with FRAP (r2 0.94) and LPO (r2 0.78); GSF correlated with FRAP (r2 0.81)
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100
Inhibition (%)

100 80
Inhibition (%)

80 60 40 20 0 2.8 1.4 0.7

60 40 20 0 2.8 1.4 0.7

Concentration of extract (%)

C

Concentration of extract (%)

D

100
Inhibition (%)

100 80
Inhibition (%)

80 60 40 20 0 2.8 1.4 0.7

60 40 20 0 2.8 1.4 0.7

Concentration of extract (%)

Concentration of extract (%)

FIG. 6. ANTIBACTERIAL ACTIVITIES OF THE FIRST THREE DILUTIONS FOR CABBAGE, EXTRACTED WITH DIFFERENT CONCENTRATION OF METHANOL (0 [ ], 20 [ ], 40 [ ], 60 [ ], 80 [ ] AND 100% [ ]) AGAINST (A) L. MONOCYTOGENES, (B) P. AERUGINOSA, (C) S. ABONY AND (D) E. FAECALIS All the values are means SD of three parallel experiments in duplicate. TABLE 5. CORRELATION MATRIX SHOWING RELATIONSHIP BETWEEN YORK CABBAGE POLYPHENOLS AND ANTIOXIDANT CAPACITY

and LPO (r2 0.83), whereas HCA (r2 0.80) and avones (r2 0.82) correlated only with LPO. None of the antioxidant assays showed any correlation with HBA and PMF.

DPPH TPC TFC -0.4506 (0.2625) -0.7119 (0.0476) 0.4326 (0.2844) -0.3592 (0.3822) -0.4004 (0.3256) 0.0947 (0.8235) -0.5919 (0.1221)

FIC -0.6893 (0.0586) -0.6479 (0.0823) 0.556 (0.1524) -0.5427 (0.1646) -0.4682 (0.242) -0.225 (0.5921) -0.6386 (0.0883)

FRAP 0.5349 (0.172) 0.9427 (0.0004) -0.5423 (0.1649) 0.5201 (0.1864) 0.6289 (0.0948) 0.0555 (0.8962) 0.8079 (0.0153)

H2O2 -0.5909 (0.123) -0.2389 (0.5688) 0.429 (0.2888) -0.4458 (0.2683) -0.2842 (0.4951) -0.2146 (0.6097) -0.3318 (0.4221)

LPO -0.7842 (0.0212) -0.8717 (0.0048) 0.6035 (0.1132) -0.801 (0.0169) -0.8299 (0.0108) -0.2331 (0.5786) -0.8339 (0.0101)

CONCLUSION
This work demonstrates that conventional organic-solvent extracts of Irish York cabbage is a potential source of polyphenols, and has signicant antioxidant and antibacterial activity. This study also indicates that 60% methanol is the most efcient solvent for the extraction of polyphenolic compounds from York cabbage. The type of extraction solvent had a big inuence on the phytochemical contents, AOC and antibacterial activity. It was found that York cabbage extracts are a mixture of more than 20 phenolic compounds, which belongs to ve different phenolic groups. The present ndings also provided a comprehensive overview to identifying individual polyphenols of Irish York cabbage.
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HBA HCA Flavones PMF GSF

Value in bracket denotes P value. DPPH, 2,2-diphenyl-1-picrylhydrazyl; FIC, chelating ability of ferrous ions; FRAP, ferric reducing antioxidant potential; GSF, glycosylated avonoid; HBA, hydroxybenzoic acid; HCA, hydroxycinnamic acid; PMF, polymethoxylated avones; TFC, total avonoid content; TPC, total phenolic content.

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ACKNOWLEDGMENTS
The authors would like to acknowledge funding from the Irish Government under the Technological Sector Research Scheme (Strand III) of the National Development Plan.

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Journal of Food Biochemistry 36 (2012) 344358 2011 Wiley Periodicals, Inc.