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Food Research International 38 (2005) 395–402

Oxidation of caffeic acid in the presence of LL-cysteine: isolation of2-S-cysteinylcaffeic acid and evaluation of its antioxidant properties

Daniel Bassil a, Dimitris P. Makris a,b,*, Panagiotis Kefalas a

a Department of Food Quality Management and Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania (M.A.I.Ch.),

P.O. Box 85, 73100 Chania, Greeceb Department of Enology and Beverage Technology Technological Educational Institute (T.E.I.) of Athens, Ag. Spyridona Str.,

12210 Egaleo, Athens, Greece

Received 22 October 2004; accepted 28 October 2004

Abstract

Caffeic acid (3,4-dihydroxycinnamic acid) was oxidized in wine-like model solutions (citrate buffer, pH 3.5) containing LL-cysteine,

by means of sodium periodate that mimics the mechanism of polyphenol oxidase (PPO). The reaction lead to the formation of a

LL-cysteine/caffeic acid adduct, which was isolated and tentatively identified as 2-S-cysteinylcaffeic acid (2-CCA), on the basis of

LC-MS and 1H NMR data. The antioxidant properties of 2-CCA were assessed by employing the DPPH and a ferric-reducing test,

and compared with both caffeic acid and LL-cysteine, but also with gallic acid, which was used as a reference antioxidant. The results

indicated that the adduct exhibits slightly improved antiradical activity in relation with the parent molecule (caffeic acid), but its

reducing capacity was dramatically reduced, a fact that was theoretically ascribed to its strong chelating ability.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Antiradical activity; Caffeic acid; LL-Cysteine; Polyphenol oxidation; Reducing power; Sodium periodate

1. Introduction

Hydroxycinnamates are phenylpropanoid metabo-

lites that occur widely in plants (Herrmann, 1989), and

plant products such as wine, tea and coffee (Clifford,

1999). There is an increasing awareness that hydroxycin-

namates and their conjugates are bioactive plant foodingredients, possessing potent in vitro antioxidant activ-

ity, which might have beneficial health impact in vivo

(Kroon & Williamson, 1999). Caffeic acid (3,4-dihydr-

0963-9969/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodres.2004.10.009

Abbreviations: AAE, ascorbic acid equivalents.; 2-CCA, 2-S-cystei-

nylcaffeic acid; GRP, grape reaction product (2-S-glutathionylcaftaric

acid); TPTZ, 2,4,6-tripyridyl-s-triazine; TRE, Trolox equivalents.* Corresponding author. Tel.: +30 28210 35056; fax: +30 28210

35001.

E-mail address: dimitris@maich.gr (D.P. Makris).

oxycinnamic acid) has been shown to be a a-tocopherolprotectant in LDL (Laranjinha, Vieira, Madeira, & Al-

meida, 1995), while caffeic acid conjugates such as chlor-

ogenic (caffeoylquinic) and caftaric (caffeoyltartaric)

acids were demonstrated to be more powerful antioxi-

dants in a number of different systems (Fukumoto &

Mazza, 2000; Meyer, Donovan, Pearson, Waterhouse,& Frankel, 1998).

Caffeic acid and its derivatives are good substrates of

polyphenol oxidases, and under certain conditions may

undergo oxidation in plant tissues or products of plant

origin. However, caffeic acid, due to its o-diphenol fea-

ture, is also prone to oxidation by O2 (autoxidation)

and other biologically relevant oxidants, such as peroxy-

nitrile (Kerry & Rice-Evans, 1998). The oxidation ofcaffeic acid but also other structurally similar compo-

nents has been shown to proceed via the formation of

396 D. Bassil et al. / Food Research International 38 (2005) 395–402

o-quinones, which may spontaneously react and yield a

spectrum of products (Cilliers & Singleton, 1991; Fulc-

rand, Cheminat, Brouillard, & Cheynier, 1994). These

reactions are considered to be of high significance, since

they are implicated in enzymic browning (Matheis, 1983;

Mathew & Parpia, 1971; Saltveit, 2000), but also in pro-cesses that alter both the aesthetic and nutritional status

of foods, by reacting with pigments (Cheynier, Souquet,

Kontek, & Moutounet, 1994; Kader, Irmouli, Zitouni,

Nicolas, & Metche, 1999; Kader, Nicolas, & Metche,

1999), amino acids (Friedman, 1996; Pierpoint, 1969),

and proteins (Hurrel, Finot, & Cuq, 1982; Kroll & Rawel,

2001; Rawel, Kroll, & Riese, 2000).

Caftaric acid (trans-caffeoyltartaric acid) is the mostabundant caffeic acid derivative in grapes (Singleton,

M, Zaya, & Trousdale, 1986) and wines (Makris, Psarra,

Kallithraka, & Kefalas, 2003), and upon oxidation by

the enzyme polyphenoloxidase (PPO) during grape must

processing, it forms a conjugate with the tripeptide glu-

tathione (2-S-glutathionylcaftaric acid, Fig. 1) known as

‘‘Grape Reaction Product’’ (Singleton, Salgues, Zaya, &

Trousdale, 1985; Singleton, Zaya, Trousdale, & Salgues,1984). A recent analytical report on Hellenic, varietal

red wines showed that its content might represent

7.9% of the hydroxycinnamate fraction (Arnous, Mak-

ris, & Kefalas, 2001), while in Hellenic white wines of

‘‘Appellation of Origin’’, its amount was as high as

13.1% of all hydroxycinnamates (Makris et al., 2003).

In spite of its important contribution to the polypheno-

lic profile, however, its antioxidant properties havenever been investigated, as opposed to a number of

HO

HO

O

OOH

COOH

COOHS

NH

NH2

HO O

O

NH

HO OO

Fig. 1. Chemical structure of 2-S-glutathionylcaftaric acid, an adduct

formed after oxidation of caftaric acid during grape must processing.

other wine constituents, including caffeic acid, catechin,

procyanidins, flavonols (quercetin, rutin, etc.) and

anthocyanins, for which a substantial body of informa-

tion is available. The present investigation was under-

taken in order to synthesise and isolate a structurally

similar product that arises from caffeic acid oxidationin the presence of LL-cysteine, and examine any alteration

to the antioxidant behaviour of the parent molecule

(caffeic acid) provoked by conjugation with the thiol

(LL-cysteine).

2. Materials and methods

2.1. Chemicals

Water for HPLC analyses was nanopure. Acetonitrile

(MeCN) and trifluoroacetic acid (TFA) were of HPLC

grade. Caffeic acid, LL-cysteine, Trolox�, gallic acid,

hydrogen peroxide, 2,4,6-tripyridyl-s-triazine (TPTZ)

and ascorbic acid were from Sigma Chemical Co.

(St. Louis, MO).

2.2. Oxidation with sodium periodate

The oxidation of caffeic acid with sodium periodate

was performed in a citric acid solution (5 g L�1), ad-

justed to pH 3.5 with sodium hydroxide, to simulate

conditions that may be encountered in musts and wines.

Caffeic acid was added in 49 mL of citric acid solution,in a 100-mL, round-bottom flask, and stirred by means

of a magnetic stirrer to form slurry. One milliliter of so-

dium periodate solution (60 mM) was then added drop-

wise over a 10-min period, under continuous stirring.

The final concentrations of caffeic acid and sodium

periodate in the reaction medium were 2 and 3 mM,

respectively. After the addition of sodium periodate

the reaction mixture was left under stirring and sampleswere taken at regular intervals for HPLC analysis. For

the generation of caffeic acid/LL-cysteine adduct, oxida-

tion of caffeic acid was performed as above, but an ex-

cess of LL-cysteine (4 mM) was included in the reaction

mixture. The same reactions were also performed in

MeOH, to ascertain whether reaction medium affects

product formation.

2.3. Isolation of the caffeic acid/LL-cysteine adduct

A generalized scheme of the procedures carried out

may be seen in Fig. 2. Analytically, oxidation of caffeic

acid (2 mM) in the presence of LL-cysteine (4 mM) was

carried out in 50% aqueous MeOH with an excess of so-

dium periodate (5 mM), in a 1000-mL flask (final vol-

ume of the reaction mixture 500 mL). The addition ofsodium periodate was performed as above, and the mix-

Caffeic a cid (2 m M)+ L-Cysteine ( 4 mM) in 50% MeOH

Sodium periodate(5 mM)

20 hours

MeOH removal under vacuum

Centrifugation (6000 rpm)

Clear aqueous solution

n-butanol wash (x 2)

Concentration under vacuum to approx 5 mL

Ion exchange column chromatography

Collection of fractions, monitoring composition by TLC

Fig. 2. Schematic representation of the procedures carried out for the

isolation of 2-CCA.

D. Bassil et al. / Food Research International 38 (2005) 395–402 397

ture was allowed to react for 20 h at room temperature.

MeOH was removed by evaporation in a rotary

evaporator (T 6 40 �C), and the residual aqueous solu-

tion was centrifuged at 6000 rpm to remove undisolved

material. The clear aqueous solution was washed twicewith n-butanol in a separation funnel, and then further

concentrated to approximately 5 mL under vacuum.

The concentrate was applied onto an ion exchange resin

(diethylaminoethyl cellulose) and successively washed

with: (i) 50 mL of phosphate buffer (pH 7), (ii) 50 mL

phosphate buffer (pH 5), (ii) 30 mL distilled water,

and (iv) 50 mL aqueous acetic acid (2%, pH � 3). Mon-

itoring of fraction composition was accomplished withTLC on cellulose plates, using 15% acetic acid as the

development eluent, and ninhydrine and phosphomo-

lybdic acid (5% in ethanol) as visualization agents.

2.4. HPLC analysis

The equipment used was an HP 1090 liquid chromato-

graph, coupled with anHP 1090 diode array detector and

controlled byAgilent ChemStation software. The column

was a LiChrospher RP-18, 5 lm, 250 · 4 mm (Merck),

protected by a guard column packed with the same mate-

rial. Both columns were maintained at 40 �C. Eluent (A)was 0.1% aqueous trifluoroacetic acid (pH 2.3) and (B)

MeCN:water (4:6 v/v), containing 0.1% trifluoroacetic

acid, and the flow rate was 1 mL min�1. The elution

program used was as follows: from 100% to 50% A in

40 min, then to 20% A in 10 min, and finally isocratic

for another 10 min (total run time 60 min). Monitoring

of the eluate was performed simultaneously at 280, 320,and 420 nm.

2.5. Liquid chromatography–mass spectrometry

A Finnigan MAT Spectra System P4000 pump was

used coupled with a UV6000LP diode array detector

and a Finnigan AQA mass spectrometer. Analyses were

carried out on a Superspher RP-18, 125 · 2 mm, 4 lm,column (Macherey-Nagel, Germany), protected by a

guard column packed with the same material, and main-

tained at 40 �C. Samples were analysed employing ESI

at the positive mode, with acquisition set at 12 eV, cap-

illary voltage 4.90 kV, source voltage 50 V, detector

voltage 650 V and probe temperature 450 �C. For the

development of the chromatograms an acetic acid

(2%)-MeOH gradient was used in 45 min.

2.6. 1H NMR

Spectra were recorded on a Bruker DRX, 400 MHz,

using D2O as solvent.

2.7. Measurement of the antiradical activity (AAR)

An aliquot of 0.025 mL of sample was added to 0.975

mL DPPH� solution (60 lM in MeOH), vortexed, and

the absorbance was read at t = 0 and t = 30 min. Results

were expressed as Trolox� equivalents (mM TRE) using

the following equation:

AAR ðmM TREÞ ¼ 0:018�%DA515 þ 0:017;

as determined from linear regression, after plotting

%DA515 of known solutions of Trolox� against concen-

tration, where

%DA515 ¼At¼0515 � At¼30

515

At¼0515

� 100:

2.8. Measurement of the reducing power (PR)

Determination of the reducing ability was performed

as described previously (Arnous et al., 2001). Sample

(0.05 mL) and 0.05 mL of ferric chloride (3 mM in

5 mM citric acid) were mixed well in a 1.5-mL Eppen-

dorf tube, and incubated for 30 min in a water bath at

37 �C. Following this, the mixture was added 0.90 mL

of 1 mM TPTZ solution in 0.05 M HCl, and vortexed.

After exactly 10 min the absorbance was read at620 nm. The PR was calculated from a calibration curve,

established by plotting known amounts of ascorbic acid

398 D. Bassil et al. / Food Research International 38 (2005) 395–402

against A620. Results were expressed as ascorbic acid

equivalents (mM AAE) using the following equation:

A620 ¼ 0:679� PR � 0:008:

For the blanks, distilled water was added instead offerric chloride/citric acid. All measurements were cor-

rected according to dilution factor.

2.9. Statistics

Measurements of both antiradical activity and reduc-

ing power were performed at least in triplicate (n = 3).

The data are given as average values ± standard devia-tion (SD).

3. Results

Caffeic acid oxidation with sodium periodate has

been demonstrated to generate the o-quinone (Fulcrand

et al., 1994), which is very prone to nucleophilic attack,and may readily react with appropriate electroniophilic

compounds, such LL-cysteine (Fig. 3). The reaction was

HO

HO

OS

NH2

OH

O

HO

HO

COOH

NH2

SH

O

OH

L-Cysteine

trans-Caffeic acid

2-S-Cysteinylcaffeic acid

sodium periodate

Fig. 3. Cascade of reactions involving caffeic acid oxidation with sodium peri

acid.

performed both in a model solution at pH 3.5, resem-

bling grape must conditions, and in 50% aqueous

MeOH. In each case the HPLC analyses indicated the

presence of a predominant compound with UV maxima

at approximately 244 and 320 nm (data not shown).

Residual caffeic acid was also detected at levels notexceeding 2%, based on peak area. Upon prolonged

incubation, however, after the addition of sodium perio-

date, no caffeic acid was detected, and the predominant

peak was of over 92% purity, on the basis of peak area

recorded at 320 nm.

This peak was isolated employing ion exchange

raisin and a sequence of solutions with decreasing pH,

and five fractions were collected. Remaining LL-cysteinewould normally be expected to elute in fraction ii (see

Section 2), due to its isoelectric point, which is 5.02.

The product was expected in fraction iv, considering

that it might have an isoelectric point close to 3, since

it could be viewed as an amino acid with two carboxylic

groups, similar to LL-glutamic acid, which has an isoelec-

tric point equal to 3.22. HPLC analysis of the fraction iv

gave a purity of the product of approximately 96%.The LC–ESI-MS examination, which was performed

at positive ion mode, gave as predominant peak a spe-

OH

O

O

COOH

trans-Caffeic acid o-quinone

Nucleophilic attack

odate, and nucleophilic attack of LL-cysteine to form 2-S-cysteinylcaffeic

D. Bassil et al. / Food Research International 38 (2005) 395–402 399

cies with m/z = 331.9. This was tentatively identified as

the reaction product (2-CCA) under the form of an ad-

duct with MeOH, which was used as one of the eluents

[2-CCA + H+ + MeOH = 332], since some compounds

are susceptible to adduct formation if ES ionisation

takes place in the presence of specific solvents. Further-more, a minor peak with m/z = 240 was identified as a

fragment resulting from cleavage of caffeic acid side

chain, as indicated in Fig. 4. However, the peak with

m/z = 354 could not be assigned to any species deriving

from 2-CCA or any adduct thereof, and could presum-

ably represent impurities.

The 1H NMR analysis showed a loss of long-range

secondary splitting and no signal at about 7 ppm witha coupling constant of 1.8 Hz, confirming the absence

of a proton at the 2-position (Table 1). These assign-

ments indicated a substitution of caffeic acid at this po-

sition of the ring. Substitution occurred through the

cysteine sulphur, since the single b 0 was seen as a broad

triplet at 3.05, and the a 0 protons as a doublet. The

trans-configuration was maintained as shown by the

coupling constants of a and b protons at 15.7 Hz. Thesedata are in accordance with previous reports (Cheynier,

Trousdale, Singleton, Salgues, & Wylde, 1988; Cilliers &

Singleton, 1990).

The antioxidant efficiency of 2-CCA was assessed by

two characteristic in vitro tests, which are based on dif-

Fig. 4. LC-ESI analysis of 2-CCA (positive ion mode), showing the principa

Some fragmentation of 2-CCA also occurred, as indicated by the characteri

ferent chemical background. The antiradical activity

was evaluated with the well-established DPPH� test,

and the ferric-reducing capacity with the TPTZ method-

ology (Arnous et al., 2001). For a credible comparison,

the parent molecule (caffeic acid) and LL-cysteine were

also tested. Moreover, gallic acid was used as a referenceantioxidant, since its ability is known and well-studied.

The results from the antioxidant tests are analytically

presented in Table 2. Conjugation of caffeic acid with

LL-cysteine appeared to enhance slightly the antiradical

efficiency, as 2-CCA exhibited an increased value by

6.9%. Contrary to that, however, the reducing power

was greatly affected, and 2-CCA showed an ability

which was almost 7.1-fold lower in relation with caffeicacid. Gallic acid was proven to be of unsurpassed abil-

ity, both in terms of antiradical activity and reducing

power.

4. Discussion

The first step in an o-diphenol oxidation, such ascaffeic acid, is the formation of o-quinone. This reac-

tion may be performed by various ways, including en-

zymes (Matheis & Whitaker, 1984; Pierpoint, 1966),

electroxidation (Giacomelli, Ckless, Galato, Miranda,

& Spinelli, 2002; Hapiot et al., 1996; Hotta, Sakamoto,

l ion detected, an adduct of 2-CCA with MeOH and H+ (m/z = 331.9).

stic ion of m/z = 241.

Table

11H

NMR

shifts

andcouplingconstants

ofcaffeicacidand2-C

CA

protons

12

3

OH

4

OH

5

6

O

HO

a

b

NH

2

S

O

OH

a'b'

Aromatic/vinylprotonshifts

(ppm)

Cysteineprotonshifts

(ppm)

25

6a

ba0

b0

Caffeicacid

7.09(d,J=2.1

Hz)

6.84(d,J=8.2

Hz)

7.02(dd,J=8.2,2.1

Hz)

6.25(d,J=15.7

Hz)

7.50(d,J=15.7

Hz)

2.83(d,J=8.3

Hz)

3.05(t,J=8.3

Hz)

2-C

CA

–7.21(d,J=8.2

Hz)

7.39(d,J=8.2

Hz)

6.30(d,J=15.7

Hz)

7.88(d,J=15.7

Hz)

Spectrawererecorded

inD

2O.

Table 2

Comparative assessment in relation to antioxidant properties of the

parent molecule (caffeic acid), and its adduct with cysteine (2-CCA)

Sample AAR (mM TRE)a PR (mM AAE)b

Caffeic acid 0.67 ± 0.11 26.47 ± 0.31

Cysteine 0.46 ± 0.02 4.33 ± 0.04

2-CCA 0.72 ± 0.03 3.74 ± 0.24

Gallic acid 2.30 ± 0.19 29.30 ± 1.00

a Antiradical activity (DPPH) assay, expressed as Trolox equiva-

lents (mM TRE).b Reducing power (TPTZ assay), expressed as ascorbic acid

equivalents (mM AAE).

400 D. Bassil et al. / Food Research International 38 (2005) 395–402

Nagano, Osakai, & Tsujino, 2001), oxidising agents

(Dangles, Fargeix, & Dufour, 1999; Kerry & Rice-

Evans, 1998; Makris & Rossiter, 2002a, 2002b), and

alkaline conditions (Cilliers & Singleton, 1989; Makris

& Rossiter, 2000). Likewise, caffeic acid o-quinone can

be generated by oxidation with sodium periodate

(Fulcrand et al., 1994), as also demonstrated for simple

phenolic substances bearing an o-diphenol structure,such as dopamine (Jimenez et al., 1984), and 4-methyl-

catechol (Cabanes, Garcıa-Canovas, & Garcıa-Car-

mona, 1987; Kalyanaraman, Premovic, & Sealy,

1987; Valero, Escribano, & Garcia-Carmona, 1988).

For more complex molecules, however, such as flavo-

nols, oxidation with periodic acid has been claimed

to proceed via formation of hemiketals, which rear-

range to yield the more stable ring-chain tautomericbenzofuranone derivatives (Makris & Rossiter, 2002b;

Smith, 1963; Smith, Webb, & Cline, 1965). Once caf-

feic acid o-quinone is formed, it tends to react with

an array of nucleophilic reagents, including those pos-

sessing sulfhydryl (–SH) groups (Pierpoint, 1966). As a

matter of fact, compounds with sulfhydryl features ap-

pear far more reactive towards o-quinones, compared

with amines (Friedman, 1994). For this reason, inthe case of caffeic acid oxidation in the presence of

LL-cysteine, only the thioether (S-adduct) is formed

but no N-adduct is observed, either the reaction is car-

ried out enzymically (Cilliers & Singleton, 1990), or

under the conditions described in this study.

The testing on the antioxidant characteristics of

2-CCA showed that its antiradical activity (AAR) was

somewhat enhanced compared with caffeic acid, suggest-ing that hydrogen-donating ability by the phenolic

hydroxyls increased upon conjugation with LL-cysteine.

This might be ascribed to the positive conjugative effect

exerted by S, which may weaken O–H bonds of the phe-

nolic hydroxyls, thereby decreasing their bond dissocia-

tion energy. Decreased O–H bond-dissociation energy

of phenolic hydroxyl groups was shown to account for

high antioxidant efficiency of several natural polyphe-nols (Kondo, Kurihara, Miyata, Suzuki, & Toyoda,

1999; McPhail, Hartley, Gardner, & Duthie, 2003).

O

COOH

NH2O

O

S3+Fe

O

Fig. 5. Postulated chelate of 2-CCA with Fe3+. Formation of such a

complex would bind Fe3+ more effectively than caffeic acid thus

hindering redox reactions, which might explain the much weaker

reducing effects of 2-CCA.

D. Bassil et al. / Food Research International 38 (2005) 395–402 401

Alternatively, it could be hypothesized that 2-CCA rad-

ical(s) formed after donation of hydrogen(s) are more

stable compared with the corresponding of caffeic acid,

and thus they do not act as pro-oxidants. This might

also hold true for the corresponding quinone (Bors, Mi-

chel, & Stettmeier, 2000). As regards the reducing power

(PR) that was found to be drastically reduced, this israther a consequence of enhanced chelating ability,

which would hinter Fe3+ from freely redox-cycle with

phenolic hydroxyl groups. Upon the action of LL-cysteine

carboxyl group, 2-CCA could presumably form a stable

chelate of the type seen in Fig. 5, and therefore Fe3+

would be prevented not only from reacting with the phe-

nolic hydroxyls, but also generating oxidizing species

(free radicals) in the Fenton system. Similar action hasbeen reported for novel antioxidants produced by con-

densation of the amino acids glycine and LL-serine with

salicylaldehyde, which were demonstrated to suppress

iron-catalyzed free radicals by mimicking the binding

site of iron-sequestering proteins (Kitazawa and Iwa-

saki, 1999).

Although the antioxidant properties of 2-CCA and

similar conjugates of biological relevance are far frombeing characterized, it is claimed that the findings pre-

sented herein illustrated to some extent the effect of con-

jugation, and may serve as a guide for assessing the

antioxidant behaviour of other natural, structurally sim-

ilar derivatives, such as 2-S-glutathionylcaftaric acid,

and the impact of the relevant reactions in food systems,

such as white wines. Furthermore, the cascade of reac-

tions leading in the formation of similar compoundsmay also serve as a means of constructing novel bio-

based antioxidants with improved properties and higher

performance in inhibiting various forms of oxidative

deterioration of foods.

Acknowledgement

The personnel of the Department of Pharmacy, Divi-

sion of Pharmacognosy, University of Athens, is

thanked for performing the NMR analyses.

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