Enzymatic Browning and Its Prevention

Chapter 11

Evolution of Chlorogenic Acid o-Quinones in Model Solutions 1

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Florence Richard-Forget , Marie Josèphe Amiot , Pascale Goupy , and Jacques Nicolas 2

Downloaded by UNIV OF ARIZONA on December 4, 2012 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0600.ch011

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Biochimie des dégradations, Station de Technologie des Produits Végétaux, Institut National de la Recherche Agronomique, Domaine Saint-Paul, B.P. 91, 84143 Montfavet Cedex, France Chaire de Biochimie Industrielle et Agro-alimentaire, Conservatoire National des Arts et Métiers, 292 rue Saint-Martin, 75141 Paris Cedex 03, France

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The oxidation of phenolic compounds into their corresponding o-quinones catalyzed by polyphenoloxidase (PPO) is now well documented. However, in spite of their considerable significance with regard to browning, few studies of non-enzymatic secondary reactions involving o-quinones are available. The present study describes the evolution of chlorogenic acid o-quinones. Following the oxidation of chlorogenic acid by purified apple PPO, chlorogenic acid o-quinone and some secondary products have been characterized. Two evolution pathways of o-quinones have been suggested depending on the pH conditions. After addition of (-)epicatechin to oxidized solutions of chlorogenic acid, three condensation products have been purified and identified. These products are themselves enzymatically and non-enzymatically (by cooxidation with chlorogenic acid o-quinone) oxidizable. In addition, they exhibit competitive inhibition properties towards polyphenoloxidase. Discoloration phenomena occurring in apple juice, purée and slices are mainly related to enzymatic browning. However, browning can also originate from non-enzymatic reactions such as the Maillard reaction which occurs in heat processed apple products. Basically, enzymatic browning can be defined as an initial enzymic oxidation of phenolic compounds into slightly colored quinones, catalyzed by polyphenoloxidases. The most prevalent substrates in apples for PPO are chlorogenic acid and (-)-epicatechin, chlorogenic acid being more readily oxidized than (-)-epicatechin (1). Moreover, according to Amiot et al. (2), apple browning susceptibility is mainly dependent on the relative proportions of flavan-3-ols and hydroxycinnamic acids derivatives. Apples 0097-6156/95/0600-0144$12.00/0 © 1995 American Chemical Society In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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Chlorogenic Acid o-Quinones

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contain hydroxycinnamic esters, flavanols, anthocyanins, flavonols and dihydrochalcones (2-4) the contribution of which to browning has been reported by numerous authors (5-7). The o-quinones formed are very reactive entities and are subjected to further secondary reactions, both enzymic and non-enzymic, leading to pigments. Depending on the phenolic compound from which they originate and on environmental factors of the oxidation reaction, the o-quinones show great differences in stability and reactivity and the colors of the corresponding pigments differ widely in hue and intensity (8-10). Different evolution pathways of o-quinones involving phenolic or nonphenolic compounds have been described in the literature: i. nucleophilic additions involving sulfites (14,15), thiols or amino groups of aminoacids or peptides have been identified (16,17). Recently, the addition of water to DOPA or 4-methylcatechol o-quinone has been described yielding the triphenols (8,18). The so-formed triphenols are readily oxidized by polyphenoloxidase or by an excess of quinones, leading to the formation of /j-quinones. ii. o-quinone can react with another molecule of the parent phenol, leading to the formation of dimers. These dimers have been suggested to undergo further oxidation, yielding larger oligomers with different color intensities (9,19). Oszmianski and Lee (20) have pointed out that catechin produced mainly dimers and polymers with a low polarity while chlorogenic acid produced mainly polymers. iii. o-quinones are also reported to react with a different phenol molecule, either leading to a copolymer or regenerating the original phenol and giving a different o-quinone by coupled oxidation (11,14). Following this last pathway, chlorogenic acid o-quinones have been postulated to oxidize tyrosine (21), some anthocyanins (22), flavonol and dihydrochalcone glycosides (5,6) and procyanidins (5,6). However, due to the difficulties of discriminating between enzymatic and nonenzymatic mechanisms, these coupled reactions have not been clearly demonstrated by previous authors. o-Quinones are also known to form copolymers with other phenolic compounds. Following the oxidation of (+)-catechin-chlorogenic acid mixture, six copolymers have been observed and spectrophotometrically characterized by Oszmianski and Lee (20). The structures and the susceptibility to oxidation of these compounds remain unknown. The purpose of the present work was to investigate the secondary reactions involving chlorogenic acid o-quinone both in model solutions containing chlorogenic acid alone and in mixtures of chlorogenic acid with a second phenolic compound. Specific attention will be given to the condensation products formed from the oxidation of a (-)-epicatechin-chlorogenic acid mixture.

In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Experimental Materials. PPO was extracted from apple flesh (cv Red Delicious, picked at commercial maturity) and 120-fold purified in two steps: fractional precipitations by ammonium sulphate and hydrophobic chromatography with Phenyl-Sepharose CL4B (Pharmacia), according to the method of JanovitzKlapp et al. (23). Chlorogenic acid, (-)-epicatechin and (+)-catechin, kaempferol, quercetin and its glycosides (quercetin-3-galactoside, -3-glucoside, -3-rhamnoside and -3-rutinoside), phloretin and phlorizin, cyanidin and its glycosides (cyanidin-3-glucoside and -3-galactoside) were from Extrasynthèse (Genay, France); 4-methylcatechol and all other chemicals were of reagent grade from Sigma (St. Louis, MO). Assay of PPO activity. PPO activity was polarographically assayed according to the method of Janovitz-Klapp et al. (23) using 20 mM 4-methylcatechol as substrate. Activity was expressed as nmol. of 0 consumed per second (nkat) under the assay conditions. To study the susceptibility to oxidation of the three copolymers, analyses were carried out by polarography with a reaction mixture containing 30 μΐ of purified copolymer and 10 nkat PPO in a total volume of 3 ml of a Mc Ilvaine's buffer at pH 4.5. For inhibition studies with copolymers, 4-methylcatechol varied between 1 and 20 mM in the control and two concentrations of copolymers. All assays were performed in duplicate and apparent Vm and Km values were determined using a nonlinear regression data analysis program developed for IBM PC by Leatherbarrow (24). 2

Preparation and HPLC analysis of model solutions. All of the enzymatic reactions were carried out with purified apple PPO (5 or 20 nkat. ml* ) in a reaction vessel at pH 4.5 (5 for copolymers analysis) and 30*C, in the presence of vanillic acid (internal standard for HPLC analysis) using air agitation unless otherwise specified. The concentrations of chlorogenic acid and (-)-epicatechin varied between 1 and 10 mM. For incubation period tested, 0.5 ml was withdrawn from the reaction vessel and immediately mixed with an equal amount of stopping solution containing 2 mM NaF (15). The residual phenols and oxidation products were separated by HPLC (9010 pump and 9050 UV detector driven by a 9020 workstation from Varian) on 10 μΐ samples using the isocratic and the gradient conditions described by Richard et al. (15). The spectra were obtained using a diode array detector (Waters 990). 1

Preparation of the (-)-epicatechin- chlorogenic acid copolymers. The (-) epicatechin-chlorogenic acid copolymers were enzymically prepared in a Mc Ilvaine buffer at pH 5 and 3 0 X . Purified apple PPO (250 nkat) was added to 50 ml of buffer solution containing 10 mM chlorogenic acid and 4 mM (-) epicatechin. After 20 minutes oxidation, the reaction was stopped by adding 10 ml of a stopping solution (60% acetonitrile-40 % H 0 at pH 2.6) containing 10 mM NaF. Copolymers were isolated by semi-preparative HPLC using a Lichrosorb column (25 cm χ 1 cm, 10 μπι, Interchrom) at 30*C. Detection 2


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Chlorogenic Acid σ-Quinones

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was monitored at 280 nm. The elution solvent was 10% acetonitrile - 90% H 0 (adjusted to pH 2.6 with H P0 ) delivered at a flow rate of 2.5 ml.min by a Varian 9010 pump. Copolymers were concentrated by adsorption on C Sep Pack cartridges (Millipore UK Ltd) following by elution with 2 ml methanol. 2

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HPTLC Characterization. Copolymers isolated (10 u\ of methanolic solution) were applied to 10 χ 10 cm silica gel TLC plates (DC-Fertigplatten, Merck ref n'5577). The TLC eluent used was 15% HOAc. After developing, the plates were immediately dried under cold air and observed under 366 nm excitation before and after fuming with ammonia and /or spraying the following reagents: vanillin (1% in 11 M HCL) to detect flavan-3-ols, which turn red, and, 2,4 aminoethyldiphenylborate (1% in methanol), revealing caffeoyl derivatives as green fluorescent spots under 366 nm after fuming with ammonia. Fast Atom Bombardment Mass spectrometry (FAB-MS). Positive mode spectra were recorded with a MR-JEOL-DX 300-3 KeV spectrometer using NBA as a matrix. Results and Discussion Oxidation products of chlorogenic acid. The disappearance of chlorogenic acid and the formation of oxidation products were monitored by HPLC at 280 nm. A typical chromatographic profile (corresponding to 15 min oxidation) is shown in Figure 1. The initial reaction products were mainly four peaks more polar than the parent phenol, labelled CGi, CG , CG and QCG. The use of NaF stopping solutions supplemented with ascorbic acid (2 mM) allowed us to identify QCG as the chlorogenic acid o-quinone and also to characterize CG3 as a quinonic structure with CG2 as its corresponding parent phenol. Chlorogenic acid oxidation also produced compounds which were more apolar, the number and the amount of these increased with reaction time so that at least 20 were present after 30 min oxidation. These products have been supposed to be chlorogenic acid oligomers: using different experimental conditions, Oszmianski and Lee (6) reported the presence of 6 condensed products. When the detection was carried out at 400 nm, QCG, CG and the less polar products were still observed. Figure 2 shows the kinetics of the oxidation of chlorogenic acid catalyzed by apple PPO and of the formation of the quinone of chlorogenic acid (QCG) and of CG and CG . Only QCG reached a maximum after 3-5 min and then steadily decreased to almost zero after 30 min. C G increased in the reaction mixture. CG3 increased also, but more slowly. CG3 seemed to be a compound produced from QCG with an important lag time or a secondary product from CG2. When the enzymic oxidation was stopped with NaF after 3 min, a slight decrease in chlorogenic acid concentration was apparent whereas the quinone content decreased rapidly and the areas of CG and C G increased. The same experiment was carried out with reaction 2

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Enzymatic Browning and Its Prevention

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