Journal of the Science of Food and Agriculture J Sci Food Agric 79 :517–522 (1999)

Degradation of pelargonidin 3-glucoside in thepresence of chlorogenic acid and blueberrypolyphenol oxidase

Farid Kader,1 Jean-Pierre Nicolas 2 and Maurice Metche1,2*1 Ecole Nationale Supe� rieure d’Agronomie et des Indus tries Alimentaires , Laboratoire de Biochimie Applique� e, 2 Avenue de la Foreü t deHaye, BP 172-54 505, Vandoeuvre-les -Nancy Cedex, France

2 Laboratoire de Biochimie Nutritionnelle, Unite� INSERM 308, Faculte� de Me� decine de Nancy, 9 Avenue de la Foreü t de Haye, BP 184-54500, Vandoeuvre-les -Nancy Cedex, France

Abstract : Oxidation of both chlorogenic acid (CG) and pelargonidin 3-glucoside (Pg 3-glc) as well as

their mixture, in the presence of blueberry polyphenol oxidase (PPO) was studied in model solutions.

The reactions were monitored by spectrophotometric, polarographic and HPLC methods. In the

absence of CG, Pg 3-glc is not oxidised by blueberry PPO, but pigment degradation is induced by the

presence of chlorogenic acid. Kinetic studies showed that Pg 3-glc was degraded by a mechanism

involving a reaction between chlorogenoquinone and/or secondary products of oxidation formed from

the quinone and the pigment. Therefore, no coupled oxidation mechanisms occurred with Pg 3-glc, as

expected of its structure. The oxidation of CG by PPO in the presence of Pg 3-glc involved the con-

sumption of 0.6 lmol of oxygen per lmol of CG oxidised, which is close to the stoichiometry calculated

from the oxidation of CG alone (0.63 lmol of oxygen per lmol). The initial phase of the pigment

degradation showed a delay which did not correspond to cresolase activity. This phase could be

explained by a quinone/anthocyanin reaction which is dependent on the chlorogenoquinone concentra-

tion and/or a reaction involving secondary products of oxidation (formed from the

chlorogenoquinone) and Pg 3-glc.

1999 Society of Chemical Industry(

Keywords: pelargonidin 3-glucoside; blueberry polyphenol oxidase; chlorogenic acid; degradation; oxidation;Vaccinium corymbosum

INTRODUCTION

Fruit colour is aþected by several factors which havebeen reviewed by numerous authors. Highbush blue-berry colour is an important quality factor inýu-encing the suitability of the berries for processing.Fresh Highbush blueberry fruits develop an intensebrowning after crushing. Chlorogenic acid was themajor hydroxycinnamic derivative found in blue-berries.1 The mechanisms responsible for browninghave been studied by Kader et al.2 Polyphenol com-pounds and PPO come into contact through decom-partmentation, and enzymatic oxidation of CG cantake place rapidly. The products of oxidation are ableto oxidise anthocyanins, leading to the formation ofcondensation products.

The degradation of non-o-diphenolic anthocyaninsin the presence of both o-diphenolic cosubstrates andPPO have been studied by several authors. Sakamuraand Obata3 and Sakamura et al4 showed that Pg3-glc is not oxidised by eggplant PPO. However, a

degradation is observed in the presence of both CGand eggplant PPO. The degradation of non-o-diphe-nolic anthocyanins has been regarded as a two-stepprocess involving ürst the enzymatic oxidation of theo-diphenolic cosubstrate into the corresponding o-quinone, followed by the reaction of the enzymati-cally generated o-quinone with the anthocyanins.This latter reaction leads to the formation of adductswhich can be oxidised either by enzymatic oxidationor by reaction with the o-quinone.5 Consequently, nocoupled oxidation occurred with non-o-diphenolicanthocyanins as expected of their structure.5,6 Chey-nier et al6 reported that o-diphenolic anthocyaninswere degraded more rapidly than non-o-diphenolicanthocyanins, which favours the proposed mecha-nism. The degradation of non-o-diphenolic antho-cyanins, however, shows an initial lag phase whichdoes not correspond to the cresolase activity since nomolecular oxygen is consumed in the presence ofnon-o-diphenolic anthocyanins ] PPO. According to

* Corres pondence to : Maurice Metche, Ecole Nationale

Supe� rieure d’Agronomie et des Indus tries Alimentaires ,

Laboratoire de Biochimie Applique� e, 2 Avenue de la Foreü t de

Haye, BP 172-54505, Vandoeuvre-leü s -Nancy Cedex, France(Received 22 October 1997; revis ed vers ion received 6 May 1998;

accepted 2 July 1998)

( 1999 Society of Chemical Industry. J Sci Food Agric 0022–5142/99/$17.50 517

F Kader, J-P Nicolas, M Metche

the authors, non-o-diphenolic anthocyanins mayreact with caþeoyltartaric o-quinone or secondaryquinones to form copolymers.

Wesche-Ebeling and Montgomery7 studied thedegradation of Pg 3-glc by strawberry PPO. Modelsystems containing Pg 3-glc ] PPO show a slightloss of pigment (5%), but in the presence of catechin50% of Pg 3-glc was lost after 24 h. The authorsproposed a mechanism of degradation by incorpor-ation of anthocyanins into condensation products ofcatechin by quinone–phenol reactions. According tothe authors, this mechanism also takes place with o-diphenolic anthocyanins such as derivatives of cya-nidin.

The degradation of malvidin 3-glucoside in thepresence of PPO and caftaric acid has been studiedby Sarni et al.5 Kinetic studies showed that malvidin3-glucoside reacted with the enzymatically generatedcaþeoyltartaric o-quinone by a mechanism of con-densation. The oxidation products were monitoredby HPLC, and the UV-Vis spectrum of the com-pound eluted at 23.1 min shows two absorptionmaxima at 290 and 320 nm in the UV region and athird absorption maximum in the Visible region at530–532 nm. The data suggested that the product ofmalvidin 3-glucoside degradation contained both caf-taric acid and anthocyanin moities.

The purpose of this work was to study the degra-dation of Pg 3-glc in the presence of both CG andblueberry PPO. The use of model systems could beuseful in understanding the mechanisms involved inthe degradation of non-o-diphenolic anthocyanins.The reaction mixtures were analysed by spectro-photometric, polarographic and HPLC methods.

MATERIALS AND METHODS

Fruit sampling and storage

Approximately 3.0 kg of ripe blueberries of the‘Coville’ variety were harvested during July 1996 in acommercial plant in north-eastern France (Vosges).Sample ripeness was judged on the basis of skincolour of representative berries. The berries werecleaned, packaged in polyethylene containers (]4¡C)and transported to our laboratory. The fruits werefrozen and stored in the dark at [25¡C under nitro-gen.

Reagents

Pg 3-glc (Callistephin) was purchased from Extra-synthese (Genay, France), CG and the BCA proteinassay reagent from Sigma Chemical Co (St Louis,MO, USA). Methanol (HPLC grade) and formicacid (99% of purity) were obtained from Merck(Darmstadt, Germany) and all other chemicals wereof reagent grade from Merck. Pg 3-glc (1 mM) andCG (1 mM) were dissolved individually in McIlvainebuþer pH 3.5. This buþer was prepared from 0.1 M

citric acid adjusted to the correct pH by addition of0.2 M dibasic potassium phosphate.

Enzyme extraction and PPO activity measurement

PPO was extracted according to the proceduredescribed by Kader et al.8 PPO activity was deter-mined by a polarographic method using a GilsonOxygraph (Villiers-le-Bel, France) equipped with aClark electrode ütted in a 3.1 ml jacketed cell at25¡C. The oxygen uptake was measured in the pres-ence of CG at a ünal concentration of 0.1 mM inMcIlvaine buþer pH 3.5. PPO activity wasexpressed in nanomoles of oxygen consumed persecond in the assay conditions (nanokatals, nkat).The reaction was started by 15.5 ll of blueberryPPO (0.6 nkat, 12.1 nkat mg~1 of protein). Beforeadding the enzyme extract, the solution (CG plusMcIlvaine buþer pH 3.5) was stirred for 20 min tosaturate the solution with oxygen.

Protein concentration (3.2 mg ml~1) was deter-mined according to the method using bicinchoninicacid (BCA) as a speciüc chromogenic reagent.9Bovine serum albumin was used as a standard.

Preparation of model solutions

The assays were performed using three experimentalmethods. The reaction mixtures had ünal volumes of1.0 ml for the spectrophotometric and HPLC assays,and 3.1 ml for the polarographic assays containingMcIlvaine buþer (pH 3.5), PPO (0.194 nkat ml~1),CG (0.1 mM) alone or in combination with Pg 3-glc(0.1 mM) (ünal concentrations). All the reactionswere carried out in air-saturated solutions at 25¡C.Control solutions (ie PPO-free, CG-free solutions)were prepared and incubated under the same tem-perature and stirring conditions.

Analytical methods

The spectrophotometric assays were performedusing a Shimadzu UV 260 spectrophotometer. CGoxidation was recorded at 400 nm and pigment deg-radation was monitored across a spectrum between370 and 600 nm recorded every minute.

The polarographic assays were carried out using aGilson oxygraph equipped with a Clark electrode.

For the HPLC analysis, the reaction mixture wasthe same as described for the spectrophotometricanalysis, but the reaction was time monitored andstopped by adding 100 ll of 20% (w/v) trichloro-acetic acid. All samples were analysed immediatelyby HPLC (Merck-Hitachi L-6200 Intelligent pumpequipped with a diode array detector Merck-HitachiL-3000 connected to a Chromojet integrator). Theconstituents of the medium were separated on aLichrosorb 100 RP-18 reversed phase column(250] 4 mm id, 5 lm) (Merck) using a mobile phaseconsisting of water–formic acid (90 :10, v/v), (solventA) and methanol (solvent B). The conditions were10% to 20% B in A in 5 min followed by 20% to50% B in A in 20 min. Elution was performed at aýow rate of 1.0 ml min~1 and 100 ll of the reactionmixture was injected using a Basic` Marathon auto-matic injector (Spark Holland). Pg 3-glc and CG

518 J Sci Food Agric 79 :517–522 (1999)

Oxidative degradation of pelargonidin 3-glucoside in model solutions

were detected at 495 and 325 nm, respectively. Theresults were expressed as a percentage of the initialconcentration of each substrate. Three analyses wereconducted on two experiments. Each data point isthe mean of six measurements.

RESULTS and DISCUSSION

Degradation of CG alone by the enzymatic extract

CG oxidation was monitored using spectro-photometric, HPLC and polarographic methods. Fig1 shows the eþect of enzyme extract on CG at pH3.5. CG is oxidized by the enzymatic extract into thecorresponding quinone which dispays maximumabsorption at 395–400 nm (Fig 1, curve a). Theabsorbance increased sharply, reached a maximumafter 10 min and then decreased due to the poly-merization of the quinone.10 No degradationoccurred when the crude enzyme was heated at100¡C for 10 min. Adding ascorbic acid to the reac-tion mixture after 2 min of reaction induced aninstantaneous bleaching. In the presence of excessreducing agent, o-quinones formed from CG werereduced to the original o-diphenol.

The HPLC analysis of the reaction mixture con-taining CG ] PPO conducted at various time inter-vals shows that CG oxidation was very fast duringthe ürst 2 min. After 20 min of reaction 29% of theCG remained in the reaction mixture (Fig 1, curveb). This result indicates that PPO is probably inhib-ited by the quinone or by secondary oxidation pro-ducts.11,12 Cheynier et al,13 however, have shownthat PPO is not inactivated by the quinone or thecondensation products. According to those authors,the oxidation of caftaric acid catalysed by grape PPOis protected by condensation products either becausethey compete as substrates for PPO or because caf-taric acid is regenerated from its quinone in theircoupled oxidation.

The oxidation of CG was also monitored by mea-suring oxygen uptake (Fig 2, curve a). CG degrada-

Figure 1. Chlorogenic acid dis appearance by HPLC and(K) (L)chlorogenoquinone formation by s pectrophotometric analys is at

400 nm during the oxidation of 0.1 mM CG in McIlvaine buffer at

pH 3.5 with blueberry PPO (0.194 nkat mlÉ1) at 25¡C. Each datapoint is the mean of s ix determinations . Bars indicate s tandard

deviation.

Figure 2. Kinetic of oxygen uptake in the different model

s ys tems : chlorogenic acid]PPO (0.194 nkat mlÉ1) ;K, |,pelargonidin 3-glucos ide] chlorogenic acid]PPO (0.194 nkatmlÉ1). All the s ubs trates were at a final concentration of 0.1 mM.

Oxygen cons umption was monitored us ing an Oxygraph K-IC

(Gils on) equipped with a Clark electrode at 25¡C. Each data pointis the mean of s ix determinations . Bars indicate s tandard

deviation.

tion consumed molecular oxygen for 10 min, andthen no oxygen was consumed, whereas the HPLCcurve shows that a small amount of CG continued todisappear (Fig 1, curve b). The observed decrease inCG under anaerobic condition has been reported byRichard-Forget et al.14 This is due to a non-enzymatic degradation of CG by reactions involvingo-quinone and the corresponding phenol. Theimportance of this reaction probably varied from onephenol to another depending on the instability of theo-quinone and the pH of the reaction mixture. Suchreactions involving o-quinones and their originatingphenols have already been described by Cheynierand Moutounet15 and Fulcrand et al16 for the oxida-tive degradation of caþeic acid. The products havebeen characterized by Fulcrand et al16 using 1H and13C NMR and MS studies.

The amount of oxygen consumed per lmol of CGoxidised was calculated after 20 min of reaction.Under these conditions, 0.63 lmol of oxygen wasconsumed per lmol of CG oxidised. This is higherthan the theoretical 0.5 value expected according toreaction (1):

chlorogenic acid ] 0.5 O2 ——— ÕPPO

chlorogenoquinone ] H2O (1)

This consumption was lower than the oxygen uptakemeasured by Pierpoint17 for the PPO (tobacco) oxi-dation of CG (0.8 lmol of consumed per lmol ofO2CG oxidised). On the other hand, in the case of CGoxidation by apple PPO, Richard-Forget et al14found a stoichiometry of 0.5 lmol of per lmol ofO2phenol during the initial phase of the reaction (0–3min) which is close to the theoretical value. Accord-ing to these authors, the stoichiometry O2/phenoldepends on the phenol structure, and the conditions

J Sci Food Agric 79 :517–522 (1999) 519

F Kader, J-P Nicolas, M Metche

of oxidation (pH, enzyme activity, phenolconcentration) which inýuenced the relative ratesamong enzymatic and non-enzymatic reactions.Meanwhile, Cheynier et al18 have measured a stoi-chiometry of 0.7 mol of consumed per mol of caf-O2taric acid oxidised, which is close to our value for theoxidative degradation of CG alone. The additionaloxygen uptake observed in the case of CG oxidationis probably necessary to oxidise intermediate pro-ducts in the process of oxidation.

In conclusion, HPLC in combination with polaro-graphic analysis appear to be the most sensitive andaccurate methods to study the oxidation process.

Degradation of Pg 3-glc by blueberry PPO

We studied the degradation of Pg 3-glc (non-o-dip-henolic anthocyanin) in the presence of both PPOand CG. The degradation of this anthocyanin wascarried out by incubating equimolar concentrationsof CG and anthocyanin (ünal concentration 0.1 mM)with blueberry PPO (0.194 nkat ml~1).

It is important to note that Pg 3-glc is not a sub-strate for the PPO since no molecular oxygen wasconsumed (Pg 3-glc ] PPO). Such a result has beendescribed by Sarni et al5 and Cheynier et al.6 Thedegradation of Pg 3-glc occurred only when both CGand blueberry PPO were present (Pg 3-glc ] CG ] PPO) (Fig 3, curve a). This result wouldsuggest that the chlorogenoquinone generated duringthe oxidation of CG by PPO plays an important rolein the mechanism of Pg 3-glc degradation.

In the model system containing PPO ] CG ] Pg3-glc, the presence of the pigment results inincreased oxygen uptake (]0.018 lmol) (Fig 2,curve b) and enzymatic oxidation of CG (]11%)(Fig 3, curve b) compared to the oxidation of CGalone (in the model system PPO ] CG) (Fig 3, curve

Figure 3. Relative degradation kinetics of : pelargonidin|,3-glucos ide (0.1 mM) in pres ence of chlorogenic acid (0.1 mM) ;L,chlorogenic acid (0.1 mM) in pres ence of pelargonidin 3-glucos ide

(0.1 mM) ; and chlorogenic acid alone (0.1 mM) in modelK,s olutions containing blueberry PPO (0.194 nkat mlÉ1) at pH 3.5 at

25¡C. Pelargonidin 3-glucos ide was detected at 495 nm andchlorogenic acid at 325 nm. Each data point is the mean of s ix

determinations . Bars indicate s tandard deviation.

c). If the curves of CG oxidation alone and in thepresence of Pg 3-glc (Fig 3, curves c–b) are com-pared, it can be seen that the oxidative degradationof CG displays the same kinetic behaviour during theürst three minutes, and that the Pg 3-glc degradationis lower (Fig 3, curve a) than the enzymatic oxidationof CG (Fig 3, curve b). After 3 min, 50% of CG and15% of Pg 3-glc are respectively degraded. Duringthe same period of time the amount of oxygen con-sumed is the same (0.070 lmol) for the two modelsystems (PPO ] CG ] Pg 3-glc) and (PPO ] CG)(Fig 2). After 3 min, the rate of Pg 3-glc degradationincreases, the reaction then continues in the sameway as that of CG. After 20 min, 70% of the pigmentand 84% of CG are degraded (Pg 3-glc ] CG ] PPO) even though 73% of CG is oxi-dised in the model system PPO ] CG. After 20 minthe amount of oxygen consumed in comparison tothe enzymatic oxidation of CG alone is higher(]0.018 lmol). A simple calculation shows that0.018 lmol represents the amount of oxygen neces-sary to oxidise 11% of CG, corresponding to the dif-ference between the two model systems.

The oxidative degradation of Pg 3-glc in the pres-ence of CG and blueberry PPO involves a mecha-nism which is somewhat diþerent from that alreadydescribed for derivatives of cyanidin.5,6,12,19h21 Thereaction process between chlorogenoquinone or itsdegradation products and Pg 3-glc can not, however,involve partial regeneration of CG, which can be oxi-dised by PPO. In the case of Pg 3-glc, there is noo-diphenolic function which can justify coupled oxi-dation mechanisms, nevertheless we observed a deg-radation of the pigment.

The simplest hypothesis to explain this behaviouris the concept of a condensation between the chloro-genoquinone or its degradation products and Pg3-glc. In terms of stoichiometry, no signiücant dif-ference in oxygen consumption was observed,between the oxidation of 1 lmol of CG in the pres-ence of Pg 3-glc (0.6 lmol of oxygen per lmol of CGoxidised) and the oxidation of 1 lmol of CG in themodel system CG plus PPO (0.63 lmol of oxygenper lmol of CG oxidised).

The question can then be asked whether it is thequinone or another derivative from the last onereacting with the pigment. During the ürst step ofthe reaction (0 to 4 min), the pigment degradationshows a delay that can be assimilated to a lag phaseobserved in the monophenol monooxygenase activity(cresolase). In addition Pg 3-glc possesses a phenolmoity that can be hydroxylated by PPO, and thismay explain the observed delay. Nevertheless, nooxygen was consumed in the presence of Pg 3-glcplus PPO, thus this hypothesis was abandoned. Inthe case of non-o-diphenolic anthocyanins such aspeonidin 3-glucoside, malvidin 3-glucoside and otherderivatives of malvidin, Cheynier et al6 also observeda delay in their kinetic degradation. The delay can beexplained either by a quinone/anthocyanin reaction

520 J Sci Food Agric 79 :517–522 (1999)

Oxidative degradation of pelargonidin 3-glucoside in model solutions

which can take place at an appreciable rate since thequinone has reached a high level, or by a reactioninvolving secondary products (formed from thechlorogenoquinone) and Pg 3-glc. The formation ofthe secondary products may correspond to theobserved delay in the degradation of Pg 3-glc.

Cheynier et al6 reported that non-o-diphenolicanthocyanins may react with caþeoyltartaric o-quinone or secondary quinones formed from delp-hinidin 3-glucoside and petunidin 3-glucosideleading to the formation of copolymers. These reac-tions have also been mentioned by Sarni et al5 andcan only take place in a mixture of o-diphenolic andnon-o-diphenolic anthocyanins. In our case,however, these reactions cannot be considered sincewe used a puriüed pigment. Wesche-Ebeling andMontgomery7 proposed a mechanism of anthocyanindegradation by strawberry PPO in the presence ofcatechin. According to these authors, the degradationof Pg 3-glc may be due to copolymerisation of antho-cyanins into condensation products formed byquinone–phenol reactions. More recently Sarni et al5demonstrated that Mv 3-glc reacted with the enzy-matically generated caþeoyltartaric o-quinone toform an adduct with a stoichiometry of 1 :1. TheUV-Vis spectrum of the new pigment detected byHPLC suggests that it contains both caþeoyltartaricacid and anthocyanin moities.

Our experimental results are consistent with thosepublished by Sarni et al.5 Moreover, the resultsobtained from Fig 3 enable us to propose additionalremarks such as : (a) blueberry PPO is speciüc forCG oxidation as grape PPO is for caþeoyltartaricacid ; (b) the curves of Fig 2 and 3 both show thatCG oxidation in presence of Pg 3-glc is signiücantlyhigher than that of CG alone. To explain this behav-iour it is necessary to advance the inhibition eþect ofthe oxidation products of CG. The products formedduring the oxidation of CG ] Pg 3-glc ] PPO haveless inhibition eþect on PPO than the productsformed during the oxidation of CG alone. It is prob-able that to that formed during the oxidation of theCG alone, which may explain that more CG is oxi-dised in the presence of Pg 3-glc. During the reac-tion, the ratio of degraded Pg 3-glc to oxidised CGincreased non-linearly. We can also note a discolour-ation of the reaction mixture. The initial diþerencebetween the rate of CG oxidation and the rate of Pg3-glc degradation decreased with time. We can makethe hypothesis that the reaction between the chloro-genoquinone or secondary products of oxidationformed from the chlorogenoquinone and the Pg 3-glcdepends on the concentration of the quinone.

The reaction PPO ] CG ] Pg 3-glc was alsostudied spectrophotometrically across spectrabetween 350 to 600 nm (Fig 4). The decrease inabsorbance at 495 nm indicates the disappearance ofthe Pg 3-glc. The absorbance at 420 nm increasedduring the ürst 3 min of the reaction, reached amaximum and then decreased. The increase in

Figure 4. Abs orbance s pectra (600–350 nm) of the model s ys tem

containing pelargonidin 3-glucos ide (0.1 mM)] chlorogenic acid(0.1 mM)]PPO (0.194 nkat mlÉ1). All s cans were at intervals of 1

min.

absorbance at 420 nm during the ürst three minutesmight be due to the chlorogenoquinone, but after 3min the quinone formed was lower than the degrada-tion of the Pg 3-glc which also absorbed at 420 nm.The UV-Vis spectrum recorded after 20 min of reac-tion is complex and does not provide any informationon the nature of the products formed.

In conclusion, from all these observations it isclear that if it is required to understand the mecha-nism of Pg 3-glc, the chlorogenoquinone must beprepared by chemical oxidation. The reactionbetween o-quinone and anthocyanin allows both theisolation of the reaction products and their structureto be studied. This study is currently being carriedout.

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