Food Chemistry 118 (2010) 508–511

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Food Chemistry

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Oxidation of (�)-epicatechin is a precursor of litchi pericarp enzymatic browning

Liang Liu a, Shaoqian Cao c, Yujuan Xu a, Mingwei Zhang a, Gengsheng Xiao a, Qianchun Deng d, Bijun Xie b,*

a Sericulture and Farm Produce Processing Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, PR Chinab College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR Chinac College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR Chinad Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, PR China

a r t i c l e i n f o

Article history:Received 9 March 2009Received in revised form 31 March 2009Accepted 7 May 2009

Keywords:FlavanolsEpicatechinPolyphenol oxidaseBrowningOxidation

0308-8146/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodchem.2009.05.019

* Corresponding author. Tel./fax: +86 27 87282966E-mail address: bijunfood@yahoo.com.cn (B. Xie).

a b s t r a c t

The degradation of flavanols had been studied both in the litchi pericarp during the storage and in themodel system containing PPO and flavanols of litchi pericarp. The results showed that (�)-epicatechinwas the optimal endogenous substrate of litchi pericarp PPO, and the procyanidins of litchi pericarp wereoxidised very slowly when incubated alone with PPO. However, (�)-epicatechin could accelerate the oxi-dation of the other flavanols in litchi pericarp through a coupled oxidation pathway. The results obtainedallowed us to draw a conclusion that the oxidation of (�)-epicatechin was a precursor of litchi pericarpbrowning. A pathway of enzymatic browning of litchi pericarp was proposed as follows: with the loss ofcellular compartmentation, the litchi pericarp PPO and flavanols mixed and, then, (�)-epicatechin wasoxidised by the PPO and o-quinones formed. The o-quinones reacted with other flavanols and anthocya-nins, accelerating the oxidation of other polyphenols. Finally, the oxidation of (�)-epicatechin and otherpolyphenols led to the formation of the brown-coloured compounds, resulting in the enzymatic brown-ing of litchi pericarp.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Litchi (Litchi chinensis Sonn.) is a subtropical fruit of high com-mercial value for its white, translucent aril and attractive red col-our. However, the fruit rapidly loses its bright red colour andturns brown once harvested. Postharvest browning of litchi wasthought to be caused by the rapid degradation of the red pigmentand oxidation of phenolic compounds by polyphenol oxidase(PPO), producing brown-coloured products (Akamine, 1960;Huang, Hart, Lee, & Wicker, 1990; Jiang, Zauberman, & Fuchs,1997; Tan & Li, 1984).

Enzymatic browning is caused by the oxidation of phenolic sub-strates by PPO to produce reactive quinones. These quinones arehighly reactive species involved in different reaction pathways.They are powerful electrophiles which may suffer nucleophilic at-tack by other polyphenols, amino acids, proteins to produce dark-brown or black pigment in senescent and postharvested fruits andvegetables (Cabanes, García-Cánovas, & García-Cármona, 1987;Fulcrand, Cheminat, Brouillard, & Cheynier, 1994; García-Carmona,Cabanes, & García-Cármona, 1987; Hurrel & Finot, 1984; Matheis &Whitaker, 1984).

In our previous studies, we found that (�)-epicatechin was themain endogenous substrate of PPO in litchi pericarp (Liu et al.,

ll rights reserved.

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2007a, 2007b), and the enzymically generated (�)-epicatechino-quinone could induce anthocyanins degradation (Liu, Cao, Xie,Sun, & Wu, 2007c). However, those results were obtained in themodel systems, and whether those proposed reactions occurredin the litchi pericarp was ambiguous. Thus, the purpose of thepresent work was to investigate the relationship between the poly-phenols oxidation by PPO and the litchi pericarp browning duringthe storage, so as to confirm our previous conclusions and give afurther understanding for the mechanism of litchi pericarpbrowning.

2. Materials and methods

2.1. Plant material

The fruit of litchi (Litchi chinensis Sonn. cv. Feizixiao) at commer-cial maturation were obtained from Guangdong. The fruit arrivedin the laboratory within 24 h after harvest. Fresh fruits were dis-tributed randomly into groups of 10 fruits, packed in 0.03 mmthick PPE bags (20 � 30 cm) and airproofed with rubber bands,and stored at 3 �C.

2.2. Chemicals

(�)-Epicatechin, (+)-catechin, catechol, 4-methylcatechol andchlorogenic acid were purchased from Sigma (St. Louis, MO),

L. Liu et al. / Food Chemistry 118 (2010) 508–511 509

procyanidin B2 was purchased from Nakahara Science Co. (Ltd).Procyanidin A2 and epicatechin-(4b?8, 2b?O?7)–epicatechin-(4b?8)–epicatechin were obtained and characterised in our previ-ous study (Liu et al., 2007a).

2.3. Colour measurements of litchi pericarp

The Hunter L*, a* and b* values were measured using an Ultra-Scan XE colourimeter (Hunterlab). Hunter L* values show a graduallightness of the pericarp. The Hunter a* values indicate a measureof redness (or �a* of greenness) and b* indicate a measure of yel-lowness (or �b* of blueness) on the hue-circle. Chroma C* gives fur-ther information on the saturation or intensity of colour. Chroma C*

was calculated as follow equation:

C� ¼ ða�2 þ b�2Þ1=2

0

20000

40000

60000

80000

100000

120000

140000

0 10 20 30 40Time (day)

Act

ivity

(U/g

of p

eric

arp)

Fig. 1. Change in the PPO activity during storage at 3 �C. Vertical bars indicate SD.

2.4. Membrane permeability measurement of litchi pericarp

Membrane permeability, expressed by relative leakage rate,was determined according to the method of Jiang and Chen(1995a) with some modifications. Discs were obtained by using acork borer (10 mm in diameter) from 10 fruit pericarps. Twentydiscs were rinsed twice and then incubated in 25 ml of distilledwater at 25 �C, and shaken for 30 min. Electrolyte leakage wasdetermined with a conductivity meter, and then the discs andincubated solution were boiled for 15 min and then cooled to25 �C to assess total electrolytes. The relative leakage was ex-pressed as a percentage of the total electrolytes.

2.5. Extraction of flavanols from litchi pericarp

Fresh litchi pericarp (5 g) from 5 fruits was extracted using acid-ified methanol (methanol:1.5 N HCl (85:15)) in a domestic ultra-sonic bath (Kunshan), at room temperature for 0.5 h. The extractwas filtered and the filter residue re-extracted using the samemethod until a colourless solution was obtained. Filtrates werecombined and the final volume was adjusted to 100 ml in a volu-metric flask. The sample was filtered through a 0.45 lm filter be-fore RP-HPLC analysis.

2.6. Reversed-phase HPLC analysis

The analysis of flavanols of litchi pericarp by reversed-phaseHPLC was performed on a Varian liquid chromatograph, and thedetection was carried out using a photodiode array detector. Thecolumn was a VP-ODS column (150 mm � 4.6 mm ID, 5 lm parti-cle size, SHIMADZU). The method utilised a binary gradient with amobile phase of 2.5% v/v aqueous formic acid (mobile phase A) andacetonitrile/water/formic acid (57.5:40:2.5, v/v/v) (mobile phaseB). A 10-ll sample solution was injected and the elution conditionswere as follow: a linear gradient from 5% to 35% B in 40 min, from35% to 50% B in 5 min, and from 50% to 80% B in 5 min, followed bywashing and reconditioning of the column. Flow rate was 1 ml/min. Calibrations were performed for each compound by injectionof known dilutions. Quantifications were based on peak areas at280 nm for flavanols, at 510 nm for anthocyanins.

2.7. PPO activity analysis

Fresh litchi pericarp (5 g) from 5 fruits were triturated with li-quid nitrogen, and then homogenised with 50 ml of 0.1 M phos-phate buffer (pH 7.5) and 1 g of PVPP for 10 min. Aftercentrifugation at 8000g for 5 min, the supernatant was collectedand the final volume was adjusted to 100 ml in a volumetric flask.

PPO activity was assayed spectrophotometrically at 25 �C using(�)-epicatechin as a substrate by monitoring at 440 nm (Liuet al., 2007b). The reaction medium (3 ml) contained 1 ml of3 mM (�)-epicatechin, 1.98 ml of 50 mM of phosphate buffer (pH7.5), and 0.02 ml of the enzyme solution. One unit of enzymewas defined as the amount of enzyme that caused an increase inabsorbance of 0.001/min at 25 �C.

2.8. Statistical analysis

The Hunter L*, a* and b* values were measured 10 times on eachof the 10 fruits, and the average Hunter values were calculated. Forenzyme activity measurement and quantification of flavanols, eachsample was assayed in triplicate. Means were compared using Tu-key Test with a significance level P < 0.05.

3. Results and discussion

3.1. Change in PPO activity of litchi pericarp during the storage

To evaluate the role of PPO activity played in the browning oflitchi pericarp, we investigated the change in the PPO activity dur-ing the storage. However, there was no significant change(P > 0.05) in the PPO activity of litchi pericarp during the wholestorage (Fig. 1). This suggested that the change in the PPO activityhad no correlation with the browning of litchi pericarp, and thebrowning might be more dependent on whether the PPO mixedwith its phenolic substrates.

3.2. Changes in polyphenols and membrane permeability of litchipericarp during the storage

According to the previous structural identifications (Liu et al.,2007a, 2007c), we found that cyanidin 3-rutinoside was the majoranthocyanins and (�)-epicatechin, procyanidin A2, procyanidin B2and epicatechin-(4b?8, 2b?O?7)–epicatechin-(4b?8)–epicate-chin were the four major flavanols in the litchi pericarp (Fig. 2).These results were in agreement with the previous descriptions(Le Roux, DoCo, Sarni-manchado, Lozano, & Cheynier, 1998; Sar-ni-manchado, Le Roux, Le Guerneve, Lozano, & Cheynier, 2000).

To investigate the relationship between the change of polyphe-nols and litchi pericarp browning, the contents of cyanidin 3-ruti-noside and each major flavanol, the membrane permeability andbrowning of litchi pericarp were measured at intervals of 7 daysduring the storage. Browning of litchi pericarp during the storage

Fig. 2. RP-HPLC chromatographic profile of polyphenols of litchi pericarp, detectedat 280 nm. 1: procyanidin B2; 2: cyanidin 3-rutinoside; 3: (�)-epicatechin; 4:epicatechin-(4b?8, 2b?O?7)–epicatechin-(4b?8)–epicatechin; 5: procyanidinA2.

Table 2Changes in colour and membrane permeability of litchi pericarp during storage at3 �C.

Day Hunter colour parameters Membrane permeability (%)a

L* a* b* C

1 35.61a 15.61a 22.72a 27.57a 17.54 ± 2.87c8 35.7a 15.51a 21.97a 26.89a 17.24 ± 4.38c

15 34.64a 15.64a 21.84a 26.86a 18.74 ± 2.79c22 34.22a 13.54a 21.98a 25.82a 18.42 ± 3.96c29 34.97a 12.60ab 22.44a 25.74a 26.14 ± 6.52b36 29.09b 11.00b 16.38b 19.73b 52.22 ± 14.85a

In each column, values with the same letter are not significantly different; P < 0.05,Tukey comparison.

a Mean ± SD.

2.0

2.2

2.4

2.6

2.8

3.0

3.2

0 1 2 3 4Time (min)

Res

idua

l sub

stra

tes

(mM

)2.980

2.984

2.988

2.992

0 1 2 3 4Time(min)

Res

idua

l sub

stra

tes

(mM

)

Fig. 3. Oxidation rate of (�)-epicatechin (d), procyanidin A2 (N), procyanidin B2(s) and epicatechin-(4b?8, 2b?O?7)–epicatechin-(4b?8)–epicatechin (j) byPPO of litchi pericarp. Inset: amplified plot for the oxidation rate of procyanidin A2(N), procyanidin B2 (s) and epicatechin-(4b?8, 2b?O?7)–epicatechin-(4b?8)–epicatechin (j) by PPO of litchi pericarp.

510 L. Liu et al. / Food Chemistry 118 (2010) 508–511

was assessed by the changes of pericarp colour, expressed as theHunter L*, a*, b* and C* values. The contents of each major flavanolhad no significant change (P > 0.05) in the first 22 days with stor-age at 3 �C (Table 1). Simultaneously, the membrane permeabilitydid not increase significantly (P > 0.05) and litchi pericarp brown-ing did not occur (Table 2).

However, the membrane permeability began to increase andbrowning occurred after 22 days storage (Table 2). This resultagreed with the previous description that the loss of cellular com-partmentation, associated with the enhanced lipid peroxidation,reduced membrane fluidity and increased membrane permeabilitywould occur during the storage of litchi (Jiang & Chen, 1995a,1995b; Lin et al., 1988). Our results also showed that the contentsof major flavanols and anthocyanins began to decline after themembrane permeability increased (Table 1). For example, the con-tent of (�)-epicatechin declined to 71.12% over 29 days, and thenonly 21.39% of the content remained at day 36. This could easilybe explained by the fact that the deterioration in membrane func-tion allows PPO and polyphenols to mix, causing the oxidation offlavanols and the degradation of anthocyanins in the litchipericarp.

3.3. Oxidation of endogenous polyphenols of litchi pericarp by PPO

In our previous study, we found that (�)-epicatechin was themain endogenous substrate of PPO in litchi pericarp (Liu et al.,2007b), while cyanidin 3-rutinoside was not a direct substratefor PPO (Liu et al., 2007c). However, the oxidation of the otherendogenous polyphenols, especially flavanols in litchi pericarp byPPO is unknown. In an attempt to determine the optimal endoge-nous substrate of litchi pericarp PPO, the reaction of litchi pericarpPPO with endogenous flavanols of litchi pericarp had beeninvestigated.

Table 1Changes in the major polyphenols in litchi pericarp during storage at 3 �C.

Polyphenols (mg/g of pericarp)b Day of storage

1 8

Procyanidin B2 1.08 ± 0.05a 1.04 ± 0.03a(�)-Epicatechin 3.74 ± 0.10a 3.68 ± 0.08aProcyanidin A2 1.87 ± 0.08a 1.84 ± 0.06aTrimera 0.82 ± 0.06a 0.80 ± 0.03aCyanidin 3-rutinoside 0.34 ± 0.02a 0.33 ± 0.02a

In each row, values with the same letter are not significantly different; P < 0.05, Tukeya Trimer was epicatechin-(4b?8, 2b?O?7)–epicatechin-(4b?8)–epicatechin.b Mean ± SD.

From Fig. 3, we found that the oxidation rate of (�)-epicatechinby litchi pericarp PPO was much faster than the other flavanols oflitchi pericarp. The low oxidation rates of procyanidin A2, procy-anidin B2 and epicatechin-(4b?8, 2b?O?7)–epicatechin-(4b?8)–epicatechin indicated that procyanidins in litchi pericarpwere poor substrates for PPO (Fig. 3). This result was in agreementwith the previous studies on apple and grape (Cheynier, Owe, & Ri-gaud, 1988; Cheynier & Silva, 1991; Le Bourvellec, Le Quere, San-oner, Drilleau, & Guyot, 2004).

However, those three procyanidins of litchi pericarp disap-peared much faster than expected in the litchi pericarp duringthe storage (Table 1). This was probably due to the fact that those

15 22 29 36

1.03 ± 0.04a 1.01 ± 0.03a 0.53 ± 0.02b 0.15 ± 0.01c3.58 ± 0.06a 3.54 ± 0.07a 2.66 ± 0.03b 0.80 ± 0.02c1.77 ± 0.04a 1.74 ± 0.05a 1.38 ± 0.03b 0.32 ± 0.02c0.80 ± 0.05a 0.73 ± 0.04a 0.58 ± 0.04b 0.28 ± 0.02c0.33 ± 0.03a 0.30 ± 0.02a 0.24 ± 0.02b 0.11 ± 0.01c

comparison.

0

20

40

60

80

100

120

0 50 100 150 200 250

Time (min)

Res

idua

l con

tent

(%)

Fig. 4. Changes of (�)-epicatechin (d), procyanidin A2 (N), procyanidin B2 (s) andepicatechin-(4b?8, 2b?O?7)–epicatechin-(4b?8)–epicatechin (D) during thereaction between PPO and flavanols extract of litchi pericarp. The reaction medium(3 ml) contained 1 ml of 0.5 mg/ml litchi pericarp flavanols, 1.98 ml of 50 mM ofphosphate buffer (pH 7.5), and 0.02 ml of the enzyme solution. Vertical barsindicate SD.

L. Liu et al. / Food Chemistry 118 (2010) 508–511 511

procyanidins were oxidised by the enzymatically generated o-qui-none of (�)-epicatechin. Interestingly, procyanidin B2 disappearedeven faster than (�)-epicatechin in litchi pericarp during the stor-age (Table 1). This was probably attributed to the high antioxidantactivity of procyanidin B2, which could more easily react with theo-quinone of (�)-epicatechin, resulting in the reduction of the o-quinone of (�)-epicatechin back to (�)-epicatechin and the oxida-tion of itself.

In order to confirm the above conclusion that the o-quinone of(�)-epicatechin could accelerate the oxidation of flavanols, thisoxidation had been investigated in the model system which con-tained both flavanols and PPO of litchi pericarp. The rate of thisreaction was very fast and the solution turned dark yellow-brownrapidly. The HPLC analysis showed that the degradation rates ofprocyanidin B2, procyanidin A2 and epicatechin-(4b?8,2b?O?7)–epicatechin-(4b?8)–epicatechin were similar to thedegradation rate of (�)-epicatechin in this model system (Fig. 4),confirming the above conclusion. A similar result had been ob-tained by Cheynier et al. (1988) who suggested that caftaric acidcould induce coupled oxidation of other polyphenols in grapemust-like model solutions containing grape PPO.

The degradation of flavanols could be observed both in the litchipericarp during the browning of litchi pericarp and in the modelsystem containing PPO and flavanols of litchi pericarp. This indi-cated that oxidation of flavanols by PPO played an important rolein litchi pericarp browning. The results presented in this paper fur-ther indicated that the oxidation of flavanols in litchi pericarp wasmainly caused by the couple oxidation of (�)-epicatechin o-qui-none. Moreover, it has been described previously that (�)-epicate-chin o-quinone could induce the degradation of anthocyanins inlitchi pericarp through the couple oxidation pathway (Liu et al.,2007c). Thus, we proposed that the oxidation of (�)-epicatechinwas a precursor of litchi pericarp browning.

In accordance with these results, the pathway of enzymaticbrowning of litchi pericarp was probably as follows: with the lossof cellular compartmentation, the litchi pericarp PPO and flavanolsmixed. Then, (�)-epicatechin was oxidised by PPO and o-quinones

formed. The o-quinones reacted with other flavanols and anthocy-anins, accelerating the oxidation of the other polyphenols. Finally,the oxidation of (�)-epicatechin and other polyphenols led to theformation of the brown-coloured compounds, resulting in theenzymatic browning of litchi pericarp.

Acknowledgement

The financial support provided by the Key Program of UnitedFunds of National Natural Science Foundation of China – NaturalScience Foundation of People’s Government of Guangdong(u0731005) and the Key Program of Natural Science Foundationof Guangdong (07117971) is greatly appreciated.

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