Food Research International 51 (2013) 907913

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Food Research International

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Ascorbic acid modies the free radical scavenging behaviour of catechin: An insight into the mechanism
Mohamed Ahmed Abbas Mahmoud a, b, 1, Veronica Sanda Chedea c, d, 1, Anastasia Detsi e, Panagiotis Kefalas a,
a Department of Food Quality and Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania, Centre International de Hautes Etudes Agronomiques Mditerranennes, Chania, P.O. Box 85, 73100 Chania, Crete, Greece b Agricultural Biochemistry Department, Faculty of Agriculture, Ain Shams University, P.O. Box 68, Hadayek Shobra, 11241 Cairo, Egypt c Laboratory of Animal Biology, National Research Development Institute for Animal Biology and Nutrition Baloteti (IBNA), Calea Bucuresti nr. 1, Balotesti, Ilfov 077015, Romania d Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Japan e Laboratory of Organic Chemistry, Department of Chemical Sciences, School of Chemical Engineering, National Technical University of Athens, Heroon Polytechniou 9, Zografou Campus, 15780 Athens, Greece

a r t i c l e

i n f o

a b s t r a c t
In this study, the behaviour of catechin (Cat) and ascorbic acid (AA) and their mixtures at different ratios was studied in view of elaborating predictions over an eventual pro-oxidant or synergistic antioxidant activity. The Co(II)EDTA luminol chemiluminescence showed that the mixture of Cat:AA (3:1) had the highest antioxidant activity, while the mixture of Cat:AA (1:2) the most pronounced pro-oxidant activity. As the concentration of AA increases, the antioxidant behaviour of the mixture increases strongly as well. The LCMS analysis for the two mixtures (Cat:AA at ratios of 1:2 and 1:3) revealed that the sufcient of AA accounts for the good antioxidant behaviour of catechin (Cat:AA 1:3 with an IC50 of 47.610.00 M and the mixture Cat:AA 1:2 with an IC50 of 75.320.86 M as assessed by chemiluminescence), supported by the formation of structure I (m/z 183). As its concentration decreases, the antioxidant activity drops and this is attributed to the formation of structure II (m/z 255). However, when AA becomes lesser beyond this point, a restrengthening of the antioxidant behaviour has been observed, which may be assigned to the formation of procyanidin structures. Thus, AA may suppress the formation of procyanidin, i.e. creation of C\C bonds between the catechin units. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 29 November 2012 Accepted 9 February 2013 Available online 24 February 2013 Keywords: Catechin Ascorbic acid Antioxidant/pro-oxidant Dimerisation o-Quinone

1. Introduction Flavonoids are polyphenolic compounds ubiquitously distributed in plants and are consumed regularly in the diet in considerable amounts in the form of fruits, vegetables, nuts and derived products such as red wine, tea and chocolate. Based on their chemical structure, avonoids can be classied into several subclasses such as avonols, avones, avanones, avan-3-ols (also referred to as catechins), anthocyanidins, isoavones, dihydroavonols and chalcones (Menendez et al., 2011). Flavan-3-ols and avonols are the most abundant and are widely distributed in food. The average intake of avan-3-ols is estimated to be approximately 1831 mg/day, with catechin and epicatechin being the most abundant (Manach, Scalbert, Morand, Rmsy, & Jimnez, 2004). Due to their ubiquitous distribution, avonoids from different classes are commonly present together in foods and/or are consumed in the same meal. In fact, prototypical avonoid rich foods contain a large number of different avonoids in variable amounts. However, little is known about the interactions between them (Menendez et al.,
Corresponding author. Tel.: +30 28210 35056; fax: +30 28210 35051. E-mail address: panos@maich.gr (P. Kefalas). 1 These two authors contributed equally to the current work. 0963-9969/$ see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.02.023

2011). When in a mixture, interactions between phytochemicals such as avonoids in a particular plant can contribute signicantly to the ability of natural plant extracts to protect human health or mitigate disease damage, because the responsible bioactive compounds seldom work independently (Lila, 2009). Interactions between antioxidative food components are important, and the ultimate results in vivo depend on many factors, including in vitro activities, food processing, and metabolism in human (Wang, Meckling, Marcone, Kakuda, & Tsao, 2011). Catechins are known constituents of green tea and have physiological functions, including antioxidative activity (Huang & Frankel, 1997) and the ability to suppress adipocyte differentiation (Furuyashiki et al., 2004). Flavan-3-ols may have a dual function, both as antioxidants and pro-oxidants, depending on their concentration and exposure time on the cell culture (Braicu, Pilecki, Balacescu, Irimie, & Neagoe, 2011). The stability of tea catechins in aqueous solution has been investigated by many researchers (Chen, Zhu, Wong, Zhang, & Chung, 1998; Friedman & Jrgens, 2000; Tanaka & Kouno, 2003). Aoshima and Ayabe (2007) studied methods to prevent the deterioration of catechin-enriched green tea. They reported that the addition of citric acid to ascorbatecatechin system could decrease the degradation rate of catechin. That attributed to the strong electron-donating capacity of catechin's dissociated phenolic groups. The results of Mahmoud

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(2012) supported this theory when he studied the inuence of citric acid at different concentration on the antioxidant activity of the equimolar mixture of AA and Cat using DPPH radical test. Ortiz, Ferruzzi, Taylor, and Mauer (2008) examined the effects of storage relative humidity and addition of other ingredients, such as citric and ascorbic acids, on catechin stability in green tea powder. Interestingly in their study, ascorbic acid promoted catechin degradation at 75% relative humidity but reduced catechin degradation at 69% relative humidity compared to green tea powder. Only a small amount of the ascorbic acid had dissolved at 69% relative humidity, and at intermediate water activities, moisture may prevent oxidation by hydrating metallic catalysts and forming hydrogen bonds with hydroperoxides. As more water is introduced to the system, the mobility of catalysts is enhanced, thus overcoming the antioxidant potential of ascorbic acid (Ortiz et al., 2008). Biologically, the oxidation of human lipid soluble antioxidants was delayed due to the existence of catechin which is preserved by the presence of ascorbic acid (Lotito & Fraga, 2000). L-Ascorbic acid is a water-soluble vitamin known as vitamin C and is widely used as an additive in foods and beverages due to its strong reducing ability. In this study, the behaviour of catechin (Cat) and ascorbic acid (AA) and their mixtures at different ratios was studied by chemiluminescence. LCMS analyses were performed for the Cat:AA (1:2) and Cat:AA (1:3) mixtures in the view of a mechanistic study on the oxidant status change. A possible involvement of o-quinones as intermediates responsible for the observed activities was also sought. These data were evaluated in view of elaborating considerations over the prediction of an eventual pro-oxidant or synergistic behaviour of mixtures of antioxidants as a continuation of our previous studies (Abou Samra, Chedea, Economou, Calokerinos, & Kefalas, 2011; Chedea, Braicu, & Socaciu, 2010; Choueiri, Chedea, Calokerinos, & Kefalas, 2012). 2. Materials and methods 2.1. Chemicals Boric acid was purchased from Applichem, ethylene diaminetetraacetic acid (EDTA), luminol (3-aminophtalylhydrazide), ascorbic acid (AA), and catechin (Cat), 98%, were purchased from Sigma (Germany). Cobalt(II) chloride hexahydrate, hydrogen peroxide, H2O2 (30%) and methanol were purchased from Merck and ascorbic acid was purchased from Riedel-de-Han (Germany). 2.2. Sample preparation The solutions of Cat and AA and their mixtures were in methanol at total concentrations of 20, 30, 40, 50, 100, 150 and 200 M. The compounds were mixed at molar ratios of 1:1; 1:2; 1:3; 2:1 and 3:1 in a nal volume of 1 ml. 2.3. Co(II)/EDTA-induced luminol chemiluminescence method A chemiluminescence method was used as described by Parejo, Codina, Petrakis, and Kefalas (2000). In this method, H2O2 was used as a radical source which reacts with Co(II) and generates hydroxyl radicals through a Fenton reaction. Then, luminol reacts with a generated hydroxyl radical to form 3-aminophtalate in an excited electronic state which returns to ground state by emission of light. EDTA, as a chelator, is used in order to decrease the speed of the Fenton reaction. The chemiluminescence intensity of this system reaches a plateau which drops in the presence of an antioxidant. Practically, 1 ml of borate buffer solution (50 mM, pH 9.00) containing CoCl26H2O (8.4 mg ml 1) and EDTA (2.63 mg ml 1) was mixed in a test tube with 0.1 ml of luminol solution (0.56 mM in borate buffer 50 mM, pH 9.00), vortexed for 15 s and followed by the addition of 0.025 ml of H2O2 aqueous solution (5.4 mM). After being vortexed for 30 s the mixture was rapidly

transferred into a glass cuvette and the CL intensity (blank) was measured when it reached the plateau (Io). For the measurement of the samples an aliquot of 0.025 ml of sample (pure compound or mixtures) was added into the test tube containing the buffer, luminol and H2O2 solutions as above and the CL intensity (I) was recorded at the plateau. The reactions in the glass cuvette were performed under magnetic stirring. The ratio Io/I was calculated and plotted vs. concentration (mol l 1) of the sample and correlations were established using linear, exponential and polynomial regression analysis. The concentration of sample (IC50), which is required to decrease Io intensity by 50%, was also calculated. For all measurements, a uorimeter (model 6200, Jenway Ltd., Gransmore, Essex, UK) was used, keeping the lamp off and using only the photomultiplier of the apparatus. All the samples were dissolved in methanol, at concentrations of 20, 30, 40, 50, 100, 150 and 200 M. 2.4. LCMS analysis The mixtures Cat:AA (1:3) and Cat:AA (1:2) were chosen for LCMS analysis. These mixtures were prepared by adding in a spherical ask, 100 ml of Co (II) EDTA, 10 ml of borate buffer, 2.5 ml of H2O2, and 2.5 ml of the mixture to be analysed. After 3.30 min, which was the average time for the chemiluminescence signal to reach the plateau, the mixtures were vigorously shaken with ethyl acetate and the organic layer dried on Na2SO4. After vacuum distillation of the solvent on a rotary evaporator, the residues were redissolved in 1 ml of methanol and ltered through 0.45 m syringe lters (Whatman), and the ltrates were used for chromatographic analyses. The analysis was performed using an LC/DAD/MS system comprising a Finnigan MAT Spectra System P4000 pump coupled with a UV6000LP diode array detector and a Finnigan AQA mass spectrometer. The separation was performed on a Superspher, 100 RP-18, endcapped, 125 2 (4 u) (Merck) column at a ow rate of 0.3 ml/min, the column being kept at 40 C. The detection was monitored at 278 nm. For the MSESI (+) conditions the probe temperature was 400 C, the probe voltage was 4 kV and the collision induced fragmentation energy was 20 and 70 eV (CID) in the mass analyser. The mass range was set at 121787 u and the scan rate was at 0.8 scans/s. The following gradient programme was used: (A) AcOH (2.5%) and (B) MeOH; 100% A isocratic elution for 2 min going to 0% after 50 min. The data were processed using the Xcalibur 1.2 software. 2.5. Statistical analysis Data were presented as the mean percentages of control standard errors of the mean (SEM) from at least three independent experiments. Experimental data were analysed with the programme SPSS 16, performing one-way analysis of variance (ANOVA), and the DUNCAN test was used (a p value of 0.05 was considered signicant). 3. Results 3.1. Chemiluminescence The antiradical average IC50 determined by chemiluminescence and the standard deviations of the tested antioxidants and their mixtures are shown in Table 1. In this study, the antioxidant activity of the compounds and their combinations was in the following decreasing order: Cat : AA3 : 1 > Cat : AA2 : 1 > Cat : AA1 : 3 > Cat > Cat : AA1 : 1 > Cat : AA1 : 2 > AA: The results of the study showed that the mixture of Cat:AA (1:2) has the lowest antioxidant tendency of all the mixtures with IC50

M.A.A. Mahmoud et al. / Food Research International 51 (2013) 907913 Table 1 IC50 of the antioxidants (Cat and AA) and their mixtures at different molar ratios (1:1, 1:2, 1:3, 2:1, 3:1) as measured by the chemiluminescence method in triplicate. Samples Cat AA Cat:AA Cat:AA Cat:AA Cat:AA Cat:AA IC50(M) 58.37 0.45d 133.04 0.50g 62.03 0.20e 75.32 0.86f 47.61 0.00c 43.22 0.60b 35.74 1.32a

909

(1:1) (1:2) (1:3) (2:1) (3:1) signicant for p b 0.05.

a, b, c, d, e, f, g

values equal to 75.32 0.86 M. AA has the lowest antioxidant activity the IC50 being 133.04 0.50 M. Cat alone proves to have an IC50 of 58.37 0.45 M less than half value of that of AA. Increasing the amount of Cat determines a lowering of IC50, where the IC50 of the mixture of Cat:AA (3:1) was equal to 35.74 1.32 M and the IC50 of the mixture of Cat:AA (1:2) was equal to 43.22 0.60 M. Interesting for this study is the signicant drop in the IC50 value when comparing the mixtures Cat:AA (3:1) and Cat:AA (1:2), when the IC50 value of 47.610.00 M becomes 75.320.86 M respectively. 3.2. LCMS analysis In order to understand mechanistic interactions and relate them with activity we have selected specic samples for LCDADMS analysis. These were: the mixture Cat:AA (1:3) with an IC50 of 47.610.00 M and the mixture Cat:AA (1:2) with an IC50 of 75.320.86 M as assessed by chemiluminescence. Lasovsky, Hrbac, Sichertova, and Bednar (2007) studied the oxidation of catechol by hydrogen peroxide and Co(II) and proposed nucleophilic addition of hydrogen peroxide onto the p-position of benzoquinone C_O groups formed. The hydroperoxide intermediates then decompose to hydroxylated 1,4-benzoquinones. Based on this approach (Fig. 1), attack para to the carbonyl benzoquinone groups initiates the following proposed mechanisms in view of proposing sustainable justication for the explanation of CL and MS observations. Fig. 2 (A) presents the chromatogram of the mixture Cat:AA (3:1). The peak eluting at tR = 34.58 min was taken in consideration and its UVvis and MS spectra are presented in Fig. 2(B and C). The LCMS data obtained (Fig. 2C) show formation of one major product practically, where fragment m/z 183 is highly abundant.
5' 6'
HO 7 OH

Nucleophilic attack of H2O2 onto position C-1 of the catechin C ring para to carbonyl at C-4, leads to epoxide formation between C-2 and C-1. This step is supported by the fact that position C-2 of the C-ring is easily oxidised (Kefalas & Makris, 2006; Osman, Makris, & Kefalas, 2008). Then a proton shift from the hydroxyl of the semiquinone system results in the opening of the epoxide to yield the C-2 hydroxylated intermediate of the o-quinone of catechin. Opening of the C-ring follows hydroxylation at C-2 (Glen, Turan, Makris, & Kefalas, 2007; Turan, Glen, Makris, & Kefalas, 2007) to yield the tentative structure I (Fig. 3) after reduction by ascorbic acid, which returns ring B to aromaticity. This structure is the only one after other mechanistic assumptions considered to give ground for the explanation of the abundant fragment m/z 183. Compound I still retains an o-hydroxy system, which may account further for the important antioxidant behaviour observed. In the case of the reaction with H2O2, Co(II) and Cat:AA (1:2), structure I is observed together with another compound (structure II) at almost equal areas (278 nm, Fig. 4A). AA being at a lower concentration, the o-quinone is not completely reduced to structure I. Tentatively then, dimerisation is proposed through 4 + 2 cycloaddition between the o-quinone carbonyls and one double bond from the other o-quinone ring (Fig. 5). This way, one of the quinone rings returns to aromaticity and subsequent elimination of 3 water molecules leads to the more stabilised structure (II), which manifests extended -electron delocalisation. This approach is supported by one possible already proposed dimerisation pathway of o-quinone intermediates and their fragmentation thereof (Chedea, Choueiri, Jisaka, & Kefalas, 2012). This hypothesis is propped up by the observed absorbance at max = 386 nm, assigned to the extended conjugated double bond scaffold (Fig. 4B). Also, the highly abundant fragment m/z 255 might be explained after suggested eventual scission of the unstable o-quinone ring as shown in scheme Fig. 5, together with fragment m/z 531 (Fig. 4C).

4. Discussion 4.1. Chemiluminescence The two antioxidants Cat and AA, were tested alone or in combination using luminol chemiluminescence to assess their antioxidant effect as well as to detect synergistic and/or pro-oxidant effects. By plotting Io/I, linear, exponential and polynomial regression analyses were obtained. IC50 was calculated for Io/I = 2 where Io is

8
O

2 3

B
1'
OH

4' 3' OH

A
6 5
OH

C
4

2'

Catechin [O]
O HO O O OH OH HO O O OH O OH

H2 O 2
OH

Products
OH

Fig. 1. A favoured nucleophilic attack of hydrogen peroxide para to the C_O groups formed on the catechol ring of catechin, leading to other products.

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A
RT: 0.00 - 59.99
34.58

18000000 17000000 16000000 15000000 14000000 13000000 12000000 11000000 10000000 9000000 8000000 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 0

NL: 1.84E7 Channel A UV CatAA1

uAU

1.20 42.13 7.84 10.39 13.90 19.98 25.22 26.71 32.92

47.92 52.42 57.11

10

20

30

40

50

Time (min)

B
CatAA1 #4153 RT:34.60 AV:1 SB: 2 33.20, 35.58 NL: 3.23E5 microAU

320000 300000 280000 260000 240000 220000 200000 180000 160000 140000 120000 100000 80000 60000 40000 20000 0

256.0

uAU

330.0

380.0

428.0 454.0 468.0

506.0 522.0 546.0

250

300

350

400

450

500

550

wavelength (nm)

C
100 80 60 40 20 0 53

183.1

Relative Abundance

CatAA1#3004-3026 RT: 34.76-35.00 AV: 11 SB: 52 34.08-34.70 , 35.00-35.61 NL: 3.31E4 F: {0,0} + c ESI sid=20.00 Full ms [ 121.00-787.00]

237.2 264.8 267.1

183.1

432.9 387.4

515.0

573.5

626.1

754.1
CatAA1#3007-3019 RT: 34.80-34.92 AV: 6 SB: 53 34.08-34.67 , 34.96-35.61 NL: 1.74E4 F: {0,1} + c ESI sid=70.00 Full ms [ 121.00-787.00]

40 30 20 10 0 200 300 400 500 600 700

237.2 265.1 301.2 411.6 479.4 541.2 611.3 702.2 767.3

wavelength (nm)
Fig. 2. The chromatogram for the products of the reaction between H2O2, Co(II) and catechin:ascorbic acid (1:3) as determined at = 280 (A). UVvis (B) and MS (C) spectra (20 and 70 eV) of the compound eluting at tR = 34.58 min for the mixture H2O2, Co(II) and catechin:ascorbic acid (1:3).

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911

Catechin
[O]

HO O OH OH

O HO O H2O2 OH

HO

HO HO O O OH

O OH

HO -H2O HO O O

O HO OH OH

HO O OH

O O OH

OH

OH

HO HO O OH OH OH

O O AA HO

HO O OH OH OH

OH OH
m/z 183

I
Fig. 3. Nucleophilic attack of H2O2 onto position C-1 of the catechin skeleton, followed by epoxide formation leading to the opening of ring C, which then after ascorbic acid reduction of the o-quinone system might yield the tentative structure I. This structure is the only one after other mechanistic assumptions considered to give ground for the explanation of the abundant fragment m/z 183 in the case of mixture Cat+AA at 1:3 MS spectrum (see also Fig. 3B).

the intensity without antioxidant and I is the chemiluminescence intensity after the addition of a certain amount of antioxidant. The obtained order of antioxidant tendency was also observed by Abou Samra et al. (2011) when equimolar mixtures of model antioxidants (ascorbic acid (AA), caffeic acid, quercetin, catechin (Cat), and hesperetin) were studied. The oxidation peak potential (Ep) values of Cat and AA show that Cat is a stronger antioxidant than is ascorbic acid; the IC50 values indicate that, when mixed, AA becomes more antioxidant and Cat more pro-oxidant, so we can assume that Cat regenerates AA. As the less efcient antioxidant was regenerated by the better one, we have (in the case of Cat and AA) a decrease in the antioxidant activity of Cat and an increase in that of AA (Abou Samra et al., 2011). AA regenerates Cat from their oxidised forms, o-quinones (Abou Samra et al., 2011) and so as the amount of Cat increases more Cat would be regenerated in order to have a high level of antioxidant propensity. In this idea it can be postulated that by increasing the AA amount from Cat:AA (1:2) to Cat:AA (1:3) a switch from a pro-oxidant to an antioxidant tendency is observed. The chemiluminescence results of mixture Cat:AA (1:2) and Cat:AA (1:3) with an increased amount of catechin would support the idea of a more signicant antioxidant behaviour because of catechin dimerisation through oxidation leading to procyanidin formation. Catechin dimerisation was also proposed as a factor inuencing the antioxidant/pro-oxidant balance in primary leucocyte culture (Chedea et al., 2010). The cyclic voltammetry work of Abou Samra et al. (2011) shows that if (oxidation potential difference between two antioxidants) is large a weak antioxidant behaviour (AA and caffeic acid) was observed because the electrons cannot be efciently transferred from one molecule to the other. When this difference gets smaller, a ne synergy (quercetin and AA) was recorded. If the difference gets smaller further, then antagonism may occur. Thus, antioxidant activity is not only based on intramolecular stabilisation of formed radicals via delocalisation, but

also on intermolecular delocalisation to the extent this one is favoured; in the present study the procyanidin formation by catechin oxidation. 4.2. LCMS analysis According to the redox cycling model of quinones the regeneration of catechin from the corresponding o-quinone would be expected as already shown for quercetin (Choueiri et al., 2012). Catechin, however, disappears according to our data. Quercetin bears an extended -electron system, which is not the case for catechin. This difference may account for a favoured nucleophilic attack of hydrogen peroxide para to the C_O groups formed on the catechol ring of catechin, leading to other products. Catechin at the absence of AA was also tested by LCMS (data not shown). From the reaction of catechin with Co(II) and H2O2, the presence of dimers of catechin (M+ 1 = 579) becomes conspicuous. The catechin dimers (procyanidins), not missing the structural characteristics of catechin, consequently contribute strongly to the antioxidant prole of catechin. 5. Conclusions We may claim that the presence of AA accounts for the good antioxidant behaviour of catechin, supported by the formation of structure I. The antioxidant activity of Cat decreased with the decrease of AA contents and this is attributed to formation of structure II. Then, when AA becomes lesser beyond this point, we observe a re-strengthening of the antioxidant behaviour, which now may be assigned to the formation of procyanidin structures. From these observations we may also suggest that under the conditions of our experiments ascorbic acid suppresses procyanidin formation, i.e. creation of C\C bonds between the catechin units.

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A
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8000000 7500000 7000000 6500000 6000000 5500000 5000000 4500000

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Structure II Structure I

uAU

4000000 3500000 3000000 2500000 2000000 1500000 1000000 500000 0 -500000 0 5 10 15 20 25 30 35 40 45 50 55

10.24 13.45 21.95 26.02 32.00 30.59 36.99 41.91 50.06 33.98 45.36 57.03

Time (min)

B
CatAA2 #5446 RT:45.38 AV:1 SB:2 44.85, 46.28 NL:6.82E4 microAU
260.0

65000 60000 55000 50000 45000 40000

uAU

35000 30000 25000 20000 15000 10000 5000 0 250 300 350 400 450 500
520.0 540.0 378.0 386.0

550

wavelength (nm)

C
100
255.0

Relative Abundance

80
308.9

CatAA2#4100-4124 RT: 45.50-45.76 AV: 12 SB: 99 44.10-45.32 , 45.85-46.92 NL: 1.26E4 F: {0,0} + c ESI sid=20.00 Full ms [ 121.00-787.00]

60 40 20
237.3 309.6 351.0 411.4 496.7 531.0 531.8 532.7 635.1 667.2 756.8 CatAA2#4100-4124 RT: 45.49-45.77 AV: 13 SB: 99 44.10-45.32 , 45.85-46.92 NL: 3.28E3 F: {0,1} + c ESI sid=70.00 Full ms [ 121.00-787.00]

0 26 20 15 10 5 0 200
183.8

255.0

214.4 309.0

309.6 363.0 425.4 492.8 531.3

648.5

731.8

300

400

500

600

700

m/z
Fig. 4. The chromatogram for the products of the reaction between H2O2, Co(II) and catechin:ascorbic acid (1:2) at 280 nm (A). UVvis (B) and MS (C) spectra of the compound eluting at tR = 45.36 min (structure II) for the mixture H2O2, Co(II) and catechin:ascorbic acid (1:2) at 20 and 70 eV.

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HO HO O OH OH OH O O

913

Dimerisation

OH OH HO OH O HO O HO O O HO OH

OH

O O

HO OH O HO O

O m/z 232+Na+=255 O

O O HO

-3 H2O

m/z 530+H+=531

OH HO HO

OH

II
Fig. 5. Proposed dimerisation through 4 + 2 cycloaddition between the o-quinone carbonyls and one double bond from the other o-quinone ring in case of Cat + AA mixture at 1:2 ratio. This way, one of the quinone rings returns to aromaticity and subsequent elimination of 3 water molecules leads to the more stabilised structure (II), which manifests extended -electron delocalisation.

Acknowledgements V.S. Chedea is a Japan Society for the Promotion of Science (JSPS) postdoctoral fellow. References
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