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Food Chemistry 121 (2010) 132–139

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Antioxidant/prooxidant activity of a polyphenolic grape seed extract

Veronica Sanda Chedea a,*, Cornelia Braicu b, Carmen Socaciu a
a
Department of Chemistry and Biochemistry, University of Agricultural Sciences and Veterinary Medicine, 3-5 Manastur Str., 400372 Cluj-Napoca, Romania
b
Cancer Institute ‘‘I. Chiricuta” Cluj-Napoca, 34-36 Republicii Str., Cluj-Napoca, Romania

a r t i c l e i n f o a b s t r a c t

Article history: The oxidative potential of a polyphenolic grape seed extract, with the idea of using this extract as a nutri-
Received 2 July 2009 tive supplement, was evaluated. Data presented in this work provide in vitro (primary leukocyte culture)
Received in revised form 10 October 2009 UV–Vis spectral evidence, indicating that quinones, as oxidation products, are involved in the modulation
Accepted 7 December 2009
of the antioxidant/prooxidant balance at cellular level in the case of catechin-type compounds (pure cat-
echin (CS) and polyphenolic extract (PE)), in the absence or presence of lipoxygenase (pure (LS) or in raw
soybean extract (LE)) as oxidative stress inducers. The study shows, to some extent, the grape seed
Keywords:
extract tested, considered as an antioxidant nutritive supplement, may have prooxidant activity as well,
Catechin-type compounds
Lipoxygenase
depending on the dose, duration of administration, and other dietary components. The UV–Vis analysis
Polyphenols confirms that the antioxidant activity of this extract might be mediated by prooxidant quinones and oxi-
Quinone dation products of the polyphenols from grape seeds.
UV–Vis spectroscopy Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Oxidative stress, the consequence of an imbalance of prooxi-

Grape seeds are waste products of the winery and grape juice
industry. These seeds contain lipid, protein, carbohydrates, and
5–8% polyphenols, depending on the variety. Grape seed polyphe-
nols contain flavan-3-ols as monomers (catechin, epicatechin, gal-
locatechin, epigallocatechin and epicatechin 3-O-gallate) but also
as procyanidin dimers, trimers, and highly polymerised procyani-
dins, aside from phenolic acid precursors (gallic acid). For this rea-
son, the grape seed extract is considered as a powerful antioxidant
that prevents premature ageing and disease (Schewe, Sadik, Klotz,
Yoshimoto, Kühn & Sies, 2001; Schewe, Kühn, & Sies, 2002).
The most abundant phenolic compounds isolated from grape
seeds are catechin, epicatechin, and procyanidins (as dimers and
trimers) (Shi, Yu, Pohorly, & Kakuda, 2003). Catechin is usually
the most important individual flavanol in both grape skins and
seeds, although epicatechin is also usually well represented. Some
grape varieties display similar levels of both monomers, or an even
higher proportion of epicatechin (Chedea et al., in press; Gonzales-
Manzano, Rivas-Gonzalo, & Santos-Buelga, 2004). Procyanidin B1
has been reported to be the main oligomer in skins (Mateus, Mar-
ques, Gonçalves, Machado, & de Freitas, 2001), whereas all C4–C8
procyanidin dimers (i.e., B1–B4) are usually found in seeds, of
which procyanidin B2 is normally the most abundant (Jordão, Ri-
cardo-da-Silva, & Laureano, 2001). Levels of galloyl flavan-3-ols
are more important in seeds than in skins (Jordão et al., 2001).
* Corresponding author. Tel.: +40 264 595825/596384(5,6,7)x213; fax: +40 264
593792.
E-mail address: cveronica@usamvcluj.ro (V.S. Chedea).
dants and antioxidants in the organism, has rapidly gained recog-
nition as a key phenomenon in chronic diseases: cardiovascular
disease, hypertension, diabetes mellilitus and cancer (Schewe,
2002). The harmful effects of oxidative processes in living organ-
isms can be reduced by the dietary intake of flavan-3-ols and
procyanidins.
Lipoxygenase (LOX, EC 1.13.11.12), a dioxygenase known to be
widely distributed in plants, animals and microorganisms, cataly-
ses the oxidation of polyunsaturated fatty acids to hydroperoxides
(Banerjee, 2006). Peroxyl radical complexes have been reported to
exist during the catalytic cycle of LOX and can serve as sources of
free radicals (Robinson, Wu, Domoney, & Casey, 1995). Thus,
lipoxygenase can be seen as an oxidative stress inducer; also
oxidative stress may favour a concerted package of lipoxygenase-
mediated enzymatic and non-enzymatic lipid peroxidation and
co-oxidative processes (Schewe, 2002). Considering the various
detrimental effects of imbalances or perturbations in fatty acid
oxidation, a considerable interest in the development and charac-
terisation of LOX inhibitors has been reported (Casey & Hughes,
2004; Schneider & Bucar, 2005). Antioxidants such as flavonoids,
which act as free radical quenchers, may act also as LOX inhibitors
(Zhou, Miao, Yang, & Liu, 2005). Schewe et al. (2001), Schewe et al.
(2002) studying the inhibitory effect of () epicatechin and of re-
lated oligomers, procyanidins, towards mammalian lipoxygenase,
suggest general lipoxygenase inhibitory potency of flavanols and
procyanidins that may contribute to their beneficial effects on
the cardiovascular system in man.
Using UV–Vis spectroscopy, the interactions between the
components of the LOX extracted from soybean and of the

0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.12.020
 Author's personal copy
V.S. Chedea et al. / Food Chemistry 121 (2010) 132–139
catechin-type compounds from grape seed extract in leukocyte
culture, were monitored. Leukocytes were taken as a cellular mod-
el for evaluating the oxidative stress caused by LOX in the presence
of catechin-type compounds, according to literature data (Aggar-
wal & Shishodia, 2006; Alvarado et al., 2006; Banerjee, 2006; Casey
& Hughes, 2004; Chedea, Vicasß, & Socaciu, 2008; Yasunari, Maeda,
Nakamura, & Yoshikawa, 2002). Dietary supplementation with
antioxidants improves functions and decreases oxidative stress of
leukocytes (Alvarado et al., 2006). Although the in vitro conditions
do not totally imitate the in vivo ones, the conclusions of this study
aim to provide information that can be related directly to oxidative
processes of cell interaction with dietary components, not only as
pure molecules, but also in raw extract. The dose and time-depen-
dent oxidative degradation of catechin to quinones is involved in
the antioxidant/prooxidant activity balance of these flavonoids.
The data presented here along with those from other laboratories
might be useful information in searching for safe antioxidants
added in appropriate amounts to the human diet as nutritive sup-
plements, and also can constitute a base for the study of lipoxyge-
nase inhibition by catechin-type compounds.
2. Materials and methods
2.1. Chemicals
Catechin standard ((±)-catechin hydrate) and pure soybean
lipoxygenase-1 were purchased from Sigma Chemical Co., St. Louis,
MO. The standard enzyme contained 46.000 units/mg protein.
RPMI, foetal serum, penicillin, streptomycin, L-glutamine, Tri-
ton, Hanks salt containing 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphe-
nyl tetrazolium bromide (MTT) and DMSO were from Sigma, Cluj-
Napoca, Romania.
2.2. Standard solutions preparation
Catechin standard (CS) was solubilised in pure Milli-Q water
and added to the reaction mixture and in cell culture to a final con-
centration of 310 lM (entire dose) and 155 lM (half dose).
Lipoxygenase standard (LS) was solubilised in physiological sal-
ine buffer (PBS; pH 7, as a stock solution of 1 mg protein/1 ml PBS
corresponding to 46.000 enzymatic units/1 ml PBS. For the exper-
iments 575 e.u. LOX/ml reaction mixture or cell culture medium
were taken.
2.3. Extraction of polyphenols from grape seeds and lipoxygenases
from soybeans
The polyphenols from grape seeds (PE) were extracted and
characterised as previously described (Chedea et al., in press).
The total content in catechin-type compounds of this extract was
determined by HPLC as being 1956 mg total catechins/kg dry grape
seeds (Chedea et al., in press).
To obtain the lipoxygenase extract (LE), an aliquot of 5 g soy-
bean meal was mixed with 30 ml of PBS, pH 7.0, and stirred for
1 h at room temperature. It was then filtered through cheesecloth
and centrifuged for 10 min at 16,000 rpm. The supernatant repre-
sents the raw extract LE. The total protein content of LE, deter-
mined by the Gornall method (Gornall, Charles, Bardawill, &
Maxima, 1949), was 27 lg total protein/ml extract.
2.4. Total polyphenol content of PE and LE
The total polyphenol content of the extracts was measured by
the Folin–Ciocalteu method (Singleton, Orthofer, & Lamuela-Rav-
entos, 1999). The grape seed extract contains polyphenols ex-
133
pressed as 3.2 g gallic acid equivalent/kg grape seeds and LE the
equivalent of 1.8 g gallic acid/kg soybeans.
2.5. Leukocytes isolation
Viable leukocytes were readily obtained from sterile whole
(horse) blood drawn with plastic syringe containing cold heparin
solution in physiological saline buffer at a final concentration of
50 IU/ml. When drawn, blood was mixed with heparin and gravita-
tional sedimentation of erythrocytes for 10–30 min (plasma or
‘‘buffy coat” with leukocytes clearly visible above the packet eryth-
rocytes) took place. After centrifugation for 10 min at 1500 rpm (to
precipitate remaining erythrocytes), with a Pasteur pipette the
plasma was transferred and centrifuged again for 10 min at
3500 rpm. The supernatant was discarded and the pellet was
washed with PBS. After a centrifugation of 10 min at 3500 rpm,
the pellet was suspended in media to a concentration of
1  106 cells/ml. Cells were cultured in RPMI medium supple-
mented with 10% foetal serum, 100 U/ml penicillin, 100 mg/ml
streptomycin and 2 mM L-glutamine under standard culture condi-
tion (37 °C, 95% humidified air and 5% CO2).
2.6. Interactions between lipoxygenase (LS or LE) and polyphenols (CS
and PE)
2.6.1. In abiotic conditions
In a volume of 2 ml PBS (pH 7), we added 36 ll CS (to a final
concentration of 310 lM) or 36 ll PE (to a final concentration of
35 lg polyphenols/ml medium). Each experimental variant (CS or
PE in PBS) was mixed with 250 ll LS diluted 1:10 (to a final conc.
of 575 e.u. LOX/ml buffer) and also with 250 ll LE (27 lg total pro-
tein/ml extract).
2.6.2. In biotic conditions
Aliquots of CS or PE (36 ll) were added to the cell culture and
incubated for 3 and 24 h, respectively. After this time 250 ll of
LS or LE were added and after 1 h (at 4 and 25 h, respectively)
the spectra were registered and MTT assay was performed. The
protocol for each experimental variant is presented in Table 1.
2.7. UV–Vis spectroscopy
All the spectra were recorded at room temperature using a
spectrophotometer (JASCO V-500; Cluj-Napoca, Romania) in the
UV–Vis range 200–450 nm.
2.7.1. In abiotic conditions
Spectra were registered for the experimental variants immedi-
ately after mixing the polyphenols with lipoxygenase (t0), and after
1 h incubation. From the spectra registered after 1 h were sub-
tracted the spectra taken at t0. The subtractions were performed
using the software of the spectrophotometer.
2.7.2. In biotic conditions
2.7.2.1. In the extracellular medium. For the experimental variants
where the entire dose (310 lM) of polyphenols was used (experi-
mental variants 1a–8a, 1c–8c) the spectra for the extracellular
medium were registered. The untreated and treated cells were
transferred to Eppendorf tubes and centrifuged for 5 min at
500 rpm. After centrifugation the supernatant absorption spectra
were registered and from the spectra of all the variants were sub-
tracted the spectra of the untreated cells.
2.7.2.2. For the intracellular matrix. The untreated and treated cells
(all experimental variants are indicated in Table 1; entire dose
310 lV and half dose 155 lV) were centrifuged for 5 min at
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134 V.S. Chedea et al. / Food Chemistry 121 (2010) 132–139

Table 1
The protocol for experimental variants of cell co-cultivation with polyphenols and lipoxygenase, standard and in extract.

Exp. var. Legend Exp. protocol

CS or ½CS 1a/1b
PE or ½PE 2a/2b
UV–Vis spectra MTT assay

LS or LE 3a/4a

UV–Vis spectra MTT assay

CS + LS or ½CS + LS 5a/5b
CS + LE or ½CS + LE 6a/6b
UV–Vis spectra MTT assay

PE + LS or ½PE + LS 7a/7b
PE + LE or ½PE + LE 8a/8b
UV–Vis spectra MTT assay

CS or ½CS 1c/1d
PE or ½PE 2c/2d
UV–Vis spectra MTT assay

LS or LE 3c/4d

UV–Vis spectra MTT assay

CS + LS or ½CS + LS 5c/5d
CS + LE or ½CS + LE 6c/6d
UV–Vis spectra MTT assay

PE + LS or ½PE + LS 7c/7d
PE + LE or ½PE + LE 8c/8d
UV–Vis spectra MTT assay

500 rpm; the supernatant was discarded, and the pellet resus- Results were expressed as the percentage (%) of MTT reduction,
pended in 1 ml pure water. A detergent (20 ll of Triton 100) was assuming the absorbance of control cells as 100%.
added to each tube to break the cellular membrane and the mix-
ture was used for spectral analysis. 2.9. Statistical analysis

Data were presented as the mean percentages of control ± stan-

2.8. MTT assay
dard errors of the mean (SEM) from at least three independent
experiments. Experimental data were analysed with the program
The influence of interaction between polyphenols and lipoxyge-
StatView II, performing one-way analysis of variance (ANOVA)
nase on the mitochondrial respiration of leucocytes was evaluated
and Fisher’s protected least significant difference (PLSD) test was
by MTT assay, using a suspension of 2  103 cells per well in 96-
used (a p value of 0.05 was considered significant).
well plates. After 3 and 24 h incubation with polyphenol, respec-
tively, whilst in logarithmic growth cell phase, cells were treated
with lipoxygenase with/without polyphenols. MTT assay was per- 3. Results and discussion
formed at 4 h and 25 h after treatment, respectively.
For the MTT assay, cells were pelleted and washed with PBS, 3.1. Effect of catechin standard (CS) and grape seed polyphenolic
and 150 ll Hanks salt containing MTT (455 lg/ml) were added into extract (PE) on lipoxygenase standard (LS) and extract (LE) in an
each well. After 2-h incubation under standard conditions, the MTT abiotic environment
solution was removed and 200 ll of DMSO were added into each
well. Absorbance was measured at 490 nm using a Biotek Synergy In order to check the interaction between polyphenols and
HT microplate reader (Cluj-Napoca, Romania). lipoxygenase in abiotic conditions, the UV–Vis spectra were
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V.S. Chedea et al. / Food Chemistry 121 (2010) 132–139 135

measured at t0 and at 1 h for the mixtures of CS or PE with LS or LE.
To observe the differences, we subtracted the spectra at t0 from the
ones measured at 1 h; four bands (I–IV) with different absorption
wavelengths were identified.
The first band (I) has absorption maxima at 247 and 252 nm
when PE is present in the reaction mixture, and the second (band
II) at 279 nm when LS was active on CS, inducing a hypsochromic
shift from 280 nm (absorption maximum for CS). The third band
(III) had maxima at 294.5, 296 and 298 nm, indicating the interac-
tion between LE and catechin-rich samples (CS and PE), and the
fourth band (IV) had maxima at 432 and 435 nm, showing the for-
mation of oxidised polyphenolic compounds as quinones and
dimers.
Investigating the oxidation of catechin, as a model of anthocya-
nins and tannins oxidation, Dangles, Fargeix, and Dufour (2000),
have shown that under oxidative conditions catechins dimerize,
absorbing at 400–500 nm, this maximum being influenced by the
pH. An absorption maximum of 420 nm was reported by Mallika
and Dhar (1980) for the polymerised product of tannin, and at
430 nm Brown and Whiteoak (1964) showed the formation of cat-
echin dimer through an o-quinone intermediate. In a model solu-
tion containing cupric ions the autoxidation of catechin was
monitored by HPLC with diode array detection, and numerous
reaction products, colourless compounds and pigments absorbing
at 440, 460 and 550 nm, were detected. Amongst the colourless
derivatives, four dimeric compounds, consisting of two catechin
units linked by a carboxymethine bridge, were present (Es-Safi,
Cheynier, & Moutounet, 2003). Following this idea, as in our case
the absorption maxima of 432 and 435 nm appear after incubation
of LS with CS, and of LS with PE, respectively, we can say that under
the prooxidant environment of lipoxygenase CS and PE undergo
oxidation to quinones and dimerisation.
3.2. Effect of catechin standard (CS) and grape seed polyphenolic
extract (PE) on lipoxygenase standard (LS) and on extract (LE) in cell
culture (biotic environment)
3.2.1. Evaluation of effect in extracellular medium
The oxidative status of cell culture media changes when poly-
phenol antioxidants, and lipoxygenase (prooxidant) are added.
Subtracting the spectra of the untreated cells from those of treated
cells (experimental variants 1a–8a and 1c–8c; Table 1), it can be
seen that oxidation of CS and PE takes place with or without
lipoxygenase added to the media (Table 2). Even though the poly-
phenols are oxidised independently of lipoxygenase, from Table 2
it can be observed that the maximal wavelengths of the oxidation
products are different as a result of the presence or absence of
lipoxygenase. When LS and LE were incubated (1 h) in the cell cul-
ture, the resulting kmax values were attributed to lipoxygenase:
278–279 and 282–283 (exp. var. 3a, 3c, 4a, 4c, 5a, 7a). By co-incu-
bation of LS and LE with cells, treated for 3 h with polyphenols (CS
and PE), only LS kept its lipoxygenase characteristic maxima (5a
and 7a versus 6a and 8a).
When CS was incubated either for 3 or 24 h, autooxidation may
have occurred. As the incubation time increases (3 h versus 24 h)
the absorption maxima increases (var. exp. 1a versus 1c) showing
the tendency of CS to oxidise and form dimers. These observations
seem consistent with the ones of Dangles et al. (2000), indicating
the formation of a catechin o-quinone with an absorption maxi-
mum at 335 nm that then evolves into yellow dimers absorbing
in the region 400–500 nm.
In a study about EGCG (epigallocatechin gallate) stability in cul-
ture media, it was observed that this flavan-3-ol was unstable in
McCoy’s 5A culture medium, forming dimers (Hong et al., 2002).
Several factors, including pH, concentration of proteins, antioxi-
dant levels, and the presence of metal ions, could affect the stabil-
ity of EGCG, of which pH is probably the most critical. The dimers
displayed increased absorbance over a broad range of 350–400 nm,
showing a yellow-brown colour. High quantities of these dimers
are formed under cell culture conditions and the products peaked
at 2–4 h, and significant amounts still remained, even after almost
all of the EGCG disappeared (Hong et al., 2002).
Our results are similar to those of Hong et al. (2002), when
EGCG was oxidised to dimers in mild alkaline conditions. Incubat-
ing CS with the cells (3 h and also 24 h) and adding LS for 1 h, the
oxidation of the flavan-3-ol in the presence of the enzyme is regis-
tered (5a, 5c). In the presence of LE, in the media of cells co-incu-
bated with CS (3 and 24 h), the oxidation of the polyphenols takes
place with quinone formation. In the case of PE incubated with
cells (3 and 24 h) and LE addition (8a, 8c), a major increase of
the absorption maxima attributed to quinone formation and the
dimerization of polyphenols was observed.
3.2.2. Evaluation of effect in intracellular matrix
Fig. 1 presents the effect of cell cocultivation with CS, PE, LS, LE
and combinations of these (see Table 1), as the UV–Vis spectra

Table 2
The absorption maxima registered in the extracellular environment after lipoxygenase addition at different times (1, 3 and 24 h). The absorption maxima were obtained by
subtraction of untreated cells spectra from those of treated cells. For the legend of the experimental variants see Table 1.

1h 3h 24 h Attributed effect
Experimental Absorption Experimental Absorption Experimental Absorption
variant maxima (nm) variant maxima(nm) variant maxima(nm)
1a (CS) 253.5; 297.5 1c (CS) 250.5; 343 Catechin oxidation
2a (PE) 308 2c (PE) 250; 324.5 Polyphenols oxidation
225; 230.5 Oxidation products as result of LOX action
3a, 3c (LS)
278; 282.5 LOX
278; 283 LOX
4a,4c (LE)
217.5; 249.5; 288; 303 Polyphenol oxidation
5a (CS + LS) 279; 283 5c (CS + LS) LOX

217.5; 249.5; 291 249.5; 306 Catechin oxidation

6a (CS + LE) 218.5; 288; 308.5–340 6c (CS + LE) 351 Polyphenol oxidation-quinone formation
278; 282.5 LOX
7a (PE + LS) 7c (PE + LS)
217; 218.5; 249.5; 300.5 307.5 Polyphenol oxidation
8a (PE + LE) 339.5 8c (PE + LE) 351.5 Polyphenol oxidation-quinone formation
506.5 488.5 Polyphenol oxidation and dimerization
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136 V.S. Chedea et al. / Food Chemistry 121 (2010) 132–139

Fig. 1. Overlapped spectral patterns of cell lysate after 3 h (A and B) or 24 h incubation (C, D and E) of cells with polyphenols before lipoxygenase was co-incubated for 1 h.
Detailed explanation of figures (A–E) are made in text. For the experimental variant identification see Table 1. The graph from figure F represents the UV–Vis spectral maxima
as determined for band IV, the quinone formation, with the maxima around 400 nm.

indicate. Analysing the UV–Vis spectra from Fig. 1, as in the case of
LS, LE, CS and PE interactions in abiotic conditions, four absorption
bands were observed.
The spectral patterns presented correspond to the experimental
variants 5a (Fig. 1A) and to 5b, 6a, 6b, 7a, 7b, 8a, 8b (Fig. 1B). The
spectra from Fig. 1C correspond to experimental variant 5c,
Fig. 1D to variants 6c, 6d, 8c and 8d and Fig. 1E to variants 7c, 5d
and 7d. For all the figure 1 represents the cells cocultivated with
CS or PE, 2 represents the cells cocultivated with LS or LE for 1 h
and 3 represents cells cocultivated with CS or PE, where LS or LE
were added and incubated for 1 h before the spectral measure-
ments were done.
From Fig. 1A it can be observed that the peak at 400 nm (band
IV) is higher when both the catechin and lipoxygenase are pure (CS
and LS) and at entire dose (Fig. 1A curve 3, compared with 1 and 2).
From here we can deduce that in these conditions, an enzymatic
oxidation with quinone formation is possible. For all the other
cases at 3 h polyphenols cocultivation with the cells followed by
LS or LE addition, the peak at 400 nm has a lower intensity than
in the case of cells cocultivation with polyphenols or lipoxygenase
alone (Fig. 1B curve 3, compared with 1 and 2). This proves that in
these experimental variants (5b, 6a, 6b, 7a, 7b, 8a, 8b), when at
least a polyphenolic extract was present, the quinone amount low-
ers as result of the interaction between polyphenols and
lipoxygenase.
When the cells were incubated for 24 h with CS and then for 1 h
with LS, at 400 nm, the three curves were nearly the same (Fig. 1C).
This confirms that a longer incubation time (24 h versus 3 h) of CS
lowers the quinone amount formed as a result of LS activity. In the
case of experimental variants 6c, 6d, 8c and 8d, the highest absorp-
tion for band IV is registered by curve 2 followed by 3, and finally 1
(Fig. 1D). In all these cases, lipoxygenase is present as the extract,
LE. This spectral order may result from a polymerisation of poly-
phenols, and also by the inhibitory activity of the polyphenols
against LE, when incubated for 24 h with the cells (curve 2 and 1
versus 3, Fig. 1D). It also might be said that the quinones formed
within the cells in 24 h (maximum at 400 nm for curve 1 versus
3, Fig. 1D) are consumed when LE is added, so these oxidation com-
pounds may have also inhibitory effect against LE activity. Fig. 1E
presents the pattern for variants 5d, 7c and 7d. In all cases, lipoxy-
genase is used as standard (LS), and the absorption maximum at
400 nm is higher in the case of curve 1. This indicates the forma-
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V.S. Chedea et al. / Food Chemistry 121 (2010) 132–139 137

Table 3
The absorption maxima (nm) for the four spectral bands (shown in Fig. 1) in cell culture when the polyphenols (CS and PE) were incubated 3 or 24 h, followed by the addition
(1 h) of LS and LE as Table 1 indicates.

Experimental conditions Absorption band Absorption maxima(nm) Attribution

CS and PE (3 h) Band I 241–244 Conjugated dienes, products of lipoxygenase oxidation
LS and LE (1 h) Band II 282–288 Polyphenols free and oxidised
Band III 307–310
Band IV 402–407 Polyphenol oxidation-quinone formation
CS and PE (24 h) Band I 244–245 Conjugated dienes, products of lipoxygenase oxidation
LS and LE (1 h) Band II 293–299 Polyphenols free and oxidised
Band III 308
Band IV 397–402 Polyphenol oxidation-quinone formation

tion of quinone during polyphenols incubation for 24 h. Curves 2
and 3 have almost the same absorption at 400 nm (Fig. 1E).
Fig. 1E shows that these quinones were ‘‘consumed” when LS
was added, the absorption band IV (400 nm) decreasing (curve 3
and 2 versus 1 Fig. 1E).
Fig. 1F represents the quinone formation, for all the experimen-
tal variants (Table 1) used in this experiment, compared with the
control (100%). The results are explained in correlation with those
of the MTT assay in Section 4.
Table 3 shows the absorption maxima for the four bands in cell
culture when the polyphenols were incubated at 3 and 24 h,
respectively. We give a tentative attribution to these maxima, cor-
relating the results from cell culture with the ones in the abiotic
environment and with literature data.
In abiotic conditions, the oxidation of polyphenols occurs with
dimer formation. In cell culture (Table 3) we suppose that there
is polyphenol oxidation with quinone formation without
dimerization.
3.3. Influence of single or combined lipoxygenase–polyphenol samples
on cell respiration (MTT test)
Fig. 2 presents the percentage of inhibition or stimulation of
respiration (measured by the MTT reduction to formazan) for cells
co-incubated with CS and PE with/without LS or LE (the experi-
Fig. 2. Cellular respiration (as measured by the mitochondrial MTT conversion to
formazan) after co-incubation with different mixtures of polyphenols (CS and PE)
and lipoxygenases (LS and LE) for different periods (3 and 24 h) and different doses
(entire or half dose). Values are expressed as percent of control for the experimental
variants tested and presented as well in Table 1 (for an easier understanding of the
graphic the codification used in Table 1 was not applied for this figure); LS, standard
lipoxygenase; LE, lipoxygenase extract; CS, catechin standard; PE, polyphenolic
extract.
mental variants indicated in Table 1 and also represented in
Fig. 1). The respiration is generally stimulated under oxidative
stress, if a similar number of cells are cultivated (same protein con-
tent). In our experiment, high values of MTT indicate a prooxidant
effect, a lower value, compared with the control (100%), an antiox-
idant action.
According to Fig. 2 the MTT test indicates the fact that LS and LE
added to the cell culture had no effect on the mitochondrial respi-
ration of the cells. MTT test shows that 3 h incubation of CS with
the cells had no significant effect, but at 24 h there is an increase
of the mitochondrial respiration, due to the formation of oxidation
products. At 24 h, for an entire dose of CS incubated with the cells,
the oxidation products are accumulating, significantly increasing
the level of prooxidant effect, compared with at 3 h, registering a
dose/effect relationship. There is no statistically significant effect
at the half dose CS, neither at the 3 h nor at 24 h. In the case of
CS co-incubated with cells and then LS added, the mitochondrial
respiration is slightly increased compared with the control but
not statistically significant. The MTT values decreased most when
at 24 h, CS at entire dose was incubated with the cells before add-
ing LS. There is no dose/effect relationship in this case. Compared
with the case of CS + LS, we can say that LE, added to the cells
co-cultivated with CS (for 3 h or 24 h, at entire or half dose), gave
a stronger prooxidant effect (higher values of MTT). LE + CS had the
strongest prooxidant effect at 3 h for the entire dose, compared
with all the experimental variants tested and presented in Fig. 2.
For the entire dose in the case of CS in the presence of LS or LE low-
er levels of MTT were observed at 24 h than at 3 h. At half dose CS
in the presence of LS or LE compared with the variant when only CS
was incubated with the cells without LS or LE, the MTT values
show an increase in the mitochondrial respiration at 24 h com-
pared with 3 h so there is an intensification of prooxidant effect
with time.
Co-cultivating PE at entire dose with the cells gave an increased
mitochondrial respiration; at half dose the mitochondrial respira-
tion decreases in intensity. In this case both at 3 h, and 24 h, there
was registered a dose/effect relationship; decreasing the dose de-
creases the intensity of mitochondrial respiration. At 3 h MTT has
higher values compared with the control (statistically significant
values) but at 24 h MTT level is almost equal with the control.
Co-incubating PE with the cells for 3 h at entire dose followed by
LS addition activates the respiratory process very fast (in 3 h). In
the case of co-cultivating the cells with PE and adding LE the acti-
vation of respiration is observed much later (at 24 h at entire dose).
There is registered a perturbation of the prooxidant/antioxidant
balance induced differently by LS and LE and related with the incu-
bation time of polyphenols. There was also registered a related
dose effect at 3 h for PE + LS and at 24 h for PE + LE. LS induces a
fast prooxidant effect and LE a slower one.
At entire dose, comparing PE incubated with the cells for 3 h
and LE added, with PE only; the prooxidant effect decreases on
addition of LE so we have an antagonist effect between PE and LE
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138 V.S. Chedea et al. / Food Chemistry 121 (2010) 132–139

(Fig. 2). At 24 h incubation of PE and than LE added, compared with
PE, the prooxidant effect is significantly increased, removing the
antagonistic effect.
4. Conclusions
Data presented in this work provide UV–Vis spectral evidence
indicating the in vitro formation of quinones, oxidation products
of the catechin-type compounds tested. The composition in poly-
phenols of the extracts tested (PE and LE) was evaluated using
the LC–MS technique and it was found that epicatechin and cate-
chin are the major compounds in PE, representing together with
ECG 60% of total polyphenols, followed by procyanidin dimers
(28%) and trimers (12%) (Chedea et al., in press). In LE the main
identified compounds, were the isoflavones, daidzein and geni-
stein, but in very low amounts (Chedea, Echim, & Vicasß, 2009).
The fate of these polyphenolic compounds was checked in abiotic
conditions and in cellular culture, in the extracellular medium as
well as in the intracellular matrix. As a prooxidant inducer we took
the standard soybean lipoxygenase as well as a raw extract from
soy beans containing LOX-1 and LOX-3 isoenzymes (Chedea
et al., 2008) because of the interest in the inhibition of this enzyme
by different classes of polyphenols.
Extensive literature data refer to the antioxidant activity of cat-
echins as one of their health-promoting properties (Ren, Qiao,
Wang, Zhu, & Zhang, 2003; Aggarwal & Shishodia, 2006). It has also
been demonstrated by some studies that the pro-oxidant activity
of flavonoids can contribute to their health-promoting activity,
by inducing important detoxifying enzymes, pointing to a benefi-
cial effect of a supposed toxic chemical reaction (Azam, Hadi, Khan,
& Hadi, 2004; Lee-Hilz et al., 2006).
Our study shows that the oxidation products of polyphenols –
as pure molecules (CS) or in grape seed extract (PE) – are formed
within the cellular matrix and also in the extracellular medium.
Different patterns of oxidation were determined by obtaining dif-
ferent UV–Vis absorption maxima for the reaction products, differ-
ence given by the state of the molecules, pure or in extracts. In
abiotic conditions LS shows a strong prooxidant effect towards
CS and also towards PE. The enzymatic oxidation of catechin and
of polyphenols takes place with the formation of dimers. The situ-
ation is affected by the cellular environment. In the extracellular
medium catechin and polyphenols are oxidised in the presence
of LS but no dimer formation takes place. This shows that the reac-
tivity of polyphenols as well as lipoxygenase activity is modulated
by the cells through other molecules and their enzymatic appara-
tus. Hong et al. (2002) have shown that the stability of EGCG was
increased in the presence of cells; this effect may be caused by sta-
bilizing factors from cells, such as proteins and antioxidants. In our
case, autoxidation of catechin and other polyphenols from the ex-
tract takes place in the extracellular medium. The absence of di-
mers in this case may be due also to the presence of amino acids
and proteins, in cell culture medium, that can form complexes with
quinones, blocking them from further dimerisation.
In the intracellular matrix quinone formation was observed, as a
result of interaction between catechins and lipoxygenase (Table 4).
Our study shows that quinones are involved in the modulation of
the antioxidant/prooxidant balance at cellular level in the case of
catechin-type compounds (CS and PE) in the presence or absence
of lipoxygenase as oxidative stress inducer. In the case of 3 h incu-
bation time, for almost all experimental variants a prooxidant ef-
fect was registered for either low or high quinone formation. No
effect and high levels of quinones were observed in the case of
CS half dose, and PE + LE. Only for pure catechin at entire dose after
3 h incubation time did the measurements show an antioxidant ef-
fect and production of high amounts of quinones.
A longer incubation time (24 h) has an influence on both qui-
none formation and observed effect. Pure catechin gives at entire
dose a prooxidant effect and high quinone formation but at half
dose antioxidant action coupled with low amounts of quinones.
When LOX was added, either pure or in extract, independently of
the catechin dose, a low amount of quinones and a prooxidant ef-
fect resulted, except for the situation when LS acted as prooxidant
in the presence of pure catechin and the prooxidant effect was re-
placed by an antioxidant effect. In case of the extract tested gener-
ally, low quinone formation was registered, a high level being
measured for LE at half dose of PE. At 24 h incubation time the anti-
oxidant action of PE is registered for almost all experimental vari-
ants, excepting the situation of PE at entire dose and of PE + LE,
when the prooxidant environment was observed.
Besides their antioxidant properties, catechins have been de-
scribed as displaying pro-oxidant activity, having the potential to
oxidise to quinones or semiquinones, resulting in redox cycling
and reactive oxygen species production as well as in thiol, DNA
and protein alkylation (Galati & O’Brien, 2004; van der Woude,
Boersma, Alink, Vervoort & Rietjens, 2006). Because flavonoids
tend to act as antioxidants rather than oxidants, at first glance, oxi-
dative stress generated by the production of quinones does not
seem relevant to explain the curative properties of these
compounds.
We have demonstrated by UV–Vis spectroscopy, that the qui-
nones are involved in the modulation of lipoxygenase activity in

Table 4
The quinone formation (expressed from UV–Vis absorption) in cell culture by polyphenol oxidation as determined by UV–Vis spectroscopy and the mitochondrial stress,
determined from MTT assay. The increase ("), decrease (;) or non-changed (NC) conditions are compared with untreated cells, (% of control). See also Fig. 1E and Fig. 2.

Exp. variant 3h 24 h
Quinone formation MTT assay Effect Quinone formation MTT assay Effect
CS "* ; AOX + Hq "* " ProOX + Hq
CS + LS "* " ProOX + Hq ;* ; (very little) AOX + Lq
CS + LE "* "* ProOX + Hq ; " ProOX + Lq
½CS "* ffiwith the control NE + Hq ;* ; AOX + Lq
½CS + LS NC " ProOX + Lq ;* " ProOX + Lq
½CS + LE "* " ProOX + Hq ;* "* ProOX + Lq
PE " "* ProOX + Hq ; "* ProOX + Lq
PE + LS NC "* ProOX + Lq ;* ; AOX + Lq
PE + LE "* ffiwith the control NE + Hq ;* "* ProOX + Lq
½PE "* "* ProOX + Hq NC ;* AOX + Lq
½PE + LS "* " ProOX + Hq ;* ; AOX + Lq
½PE + LE ;* " ProOX + Lq "* ; AOX + Hq

Hq, High quinone; Lq, Low quinone; AOX, Antioxidant; ProOX, Prooxidant; NE, No effect; NC, non-changed.
*
Statistically significant for p < 0.05.
 Author's personal copy
V.S. Chedea et al. / Food Chemistry 121 (2010) 132–139
the presence of polyphenols within the cells. This conclusion is in
agreement with that of Sadik, Sies, and Schewe (2003) and Baner-
jee (2006). An irreversible covalent modification of soybean LOX by
flavonoids has been suggested, whereby during the formation of
fatty acid peroxyl radical in the LOX pathway, the flavonoids are
co-oxidised to a semi-quinone or quinone, which in turn may bind
to sulfhydryl or amino groups of the enzyme causing inhibition.
This study shows that to some extent the grape seed extract
tested, considered as an antioxidant nutritive supplement, might
have prooxidant activity as well, depending on dose, duration of
administration and other dietary components. The extract had an
antioxidant action at a longer incubation time, at half and entire
dose against pure lipoxygenase and at half dose against the lipoxy-
genase extract. The UV–Vis analysis proves that the antioxidant
activity of this extract might be mediated by the prooxidant qui-
nones/oxidation products of the polyphenols from grape seeds.
The eventual prooxidant activity could be due to the redox sys-
tem of phenols–quinones. The so called ‘‘futile redox-cycling of
quinones”, forms reactive oxygen species from the simple flava-
noid o-quinones (o-quinones of catechin in our case) that can act
as prooxidants (Bors, Michel, & Stettmaier, 2000). Procyanidin o-
quinone is capable of producing oligomeric compounds through
various coupling reactions that retain the number of hydroxyl
groups, unlike the simple flavanoid o-quinones that can act as pro-
oxidants by forming reactive oxygen species through futile redox
cycling (Bors et al., 2000). In their study Bors et al. (2000) propose
that proanthocyanidins are superior antioxidants compared to
flavonols proper, whose quinones are more likely to redox-cycle
and act as prooxidants. Following this statement we can say that
the grape seed extract components, catechin and epicatechin on
one side and proanthocyanidins and dimers on the other side,
partially act in an antagonistic way to the procyanidin antioxidant
effect enhancing the prooxidant activity of the o-quinones of
monomeric flavan-3-ols.
It is not surprising that quinones generate an antioxidant effect
because they can be reduced to semiquinones by radicals and then
back to diphenols; also the catechins o-quinones can react via phe-
nolic coupling reactions to form dimers and oligomers, each retain-
ing its original number of reactive hydroxyl groups, i.e. enhancing
its antioxidant capacity until a level is reached when the oligomers
become insoluble and precipitate (Bors, Foo, Hertkorn, Michel, &
Stettmaier, 2001). For this reason the antagonistic effect between
catechin and procyanidins from the grape seed extract is registered
only partially, namely, when the catechin quinones have a prooxi-
dant activity. A synergistic effect is registered when both catechins
and procyanidins give an antioxidant action.
Acknowledgement
This work was supported by a grant from the Romanian Na-
tional University Research Council, project TD 392/2007.
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