Food

Chemistry
Food Chemistry 98 (2006) 539–544
[Link]/locate/foodchem

Identification of the major flavonoids from pericarp tissues of

lychee fruit in relation to their antioxidant activities
Mouming Zhao a, Bao Yang a, Jinshui Wang a, Baozhen Li a, Yueming Jiang b,*

a
College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640, PeopleÕs Republic of China
b
South China Botanic Garden, The Chinese Academy of Sciences, Guangzhou Le Yi Ju 510650, PeopleÕs Republic of China

Received 14 February 2005; received in revised form 15 June 2005; accepted 15 June 2005

Abstract

Large amount of polyphenolic compounds with strong antioxidant activity was present in the pericarp of harvested lychee
fruits. Flavonoids were extracted with 85% ethanol:15% HCl from lychee fruit pericarp tissues. Most of the lychee flavonoids
were partitioned into the ethyl acetate fraction. Three major components of the ethyl acetate fraction were obtained by reverse
phase high-performance liquid chromatography and determined to be flavanol by their ultraviolet/visible spectra. Furthermore,
these three components were identified as proanthocyanidin B4, proanthocyanidin B2 and epicatechin by nuclear magnetic res-
onance and mass spectrometry. The ethyl acetate fraction, proanthocyanidin B4, proanthocyanidin B2 and epicatechin exhibited
a good antioxidant capability. The hydroxyl radical and superoxide anion scavenging activities of proanthocyanidin B2 was
greater than those of proanthocyanidin B4 and epicatechin, while the epicatechin had the highest DPPH scavenging activity.
 2005 Elsevier Ltd. All rights reserved.

Keywords: Antioxidant activity; Epicatechin; Flavonoids; Litchi pericarp; Proanthocyanidin B4; Proanthocyanidin B2

1. Introduction high-performance liquid chromatography (HPLC) (Lee

& Wicker, 1991). Zhang, Quantick, and Grigor (2000)
Lychee (Litchi chinensis Sonn.) is a tropical and sub- reported that cyanindin-3-glucoside and malvidin-3-
tropical fruit that has high commercial value due in part glucoside may be present in these anthocyanins. Sarni-
to its white and translucent aril and attractive red color Manchado, Roux, Guerneve, Lozano, and Cheynier
(Holcroft & Mitcham, 1996). However, the fruit will (2000) identified the anthocyanins as cyanidin-3-rutino-
rapidly lose its bright color and turn brown once har- side, cyanidin glucoside, quercetin-3-rutinoside, and
vested (Jaiswal, Sah, & Prasad, 1986; Jiang, 2000; Nip, quercetin glucoside, using low-pressure chromatogra-
1988). Lychee fruit pericarp contains a large amount phy, HPLC, UV–visible light spectral analysis, mass
of pigments which are responsible for the red color spectrometry (MS), and nuclear magnetic resonance
(Lee & Wicker, 1991). Prasad and Jha (1978), using (NMR). Recently, Zhang, Pang, Yang, Ji, and Jiang
thin-layer chromatography (TLC), first reported that (2004) reported that the major anthocyanin of lychee
the red color of the lychee fruit was probably due to a fruit pericarp was cyanidin-3-rutinoside. Differences in
mixture of cyanidin and pelargonidin. The red pigments the identification of the anthocyanins may be attributed
were later identified as cyanidin-3-rutiside by TLC and to differences in the extraction and purification proce-
dures (Jiang, Duan, Joyce, Zhang, & Li, 2004). In our
* previous study, most of the red pigments in lychee fruit
Corresponding author. Tel.: +86 20 37252525; fax: +86 20
37252831. pericarp were present in the ethyl acetate fraction and
E-mail address: ymjiang@[Link] (Y. Jiang). the pigments were identified as flavanols. Identification

0308-8146/$ - see front matter  2005 Elsevier Ltd. All rights reserved.
doi:10.1016/[Link].2005.06.028
540 M. Zhao et al. / Food Chemistry 98 (2006) 539–544

of structures of flavanols was needed to explain the loss Table 1

in the skin color of harvested lychee fruit, in relation to Comparison of flavonoid content in hexane, ethyl acetate, butanol and
water fractions
their antioxidant activity.
Recent research has shown that pigments in posthar- Solvent fraction Flavonoid content (mg/g DW) Percent flavonoids
vest fruits exhibit a strong antioxidant activity (Einbond, Hexane 0.04 ± 0.00 0.2 ± 0.01
Reynertson, Luo, Basile, & Kennelly, 2004; Bao, Cai, Ethyl acetate 21.3 ± 0.1 83.1 ± 0.5
Butanol 3.5 ± 0.05 13.6 ± 0.2
Sun, Wang, & Corke, 2005). Antioxidative properties Water 0.8 ± 0.02 3.1 ± 0.06
of pigments resulted from their high reactivity as hydro-
Data were presented as means ± standard deviations of three replica-
gen or electron donors and from their ability to chelate tion determinations.
transition metal ions (termination of the Fenton reac-
tion) (Rice-Evans, Miller, & Paganga, 1997; Wada &
Ou, 2002). It is well established that enzymatic browning of hexane, ethyl acetate, butanol and water were sepa-
of postharvest fruits is related to antioxidant activity rately collected. The flavonoid content in each extract
(Martinez & Whitaker, 1995). Unfortunately, little infor- was determined by aluminum nitrate method (Moreno,
mation on the antioxidant activity of skin pigments from Isla, Sampietro, & Vattuone, 2000). In this study, most
harvested lychee fruit is available. of flavonoids were detected in the ethyl acetate fraction
The objective of this study was to extract and purify (Table 1), and, thus, the ethyl acetate fraction of the
the major flavonoids of lychee fruit pericarp, using 85% flavonoids was used for further purification.
ethanol:15% HCl, hexane, ethyl acetate and butanol and
reverse phase HPLC, and then determine the molecular 2.4. Purification of major flavonoids
weight and chemical structure by MS and NMR analy-
ses. The antioxidant activities of the major flavonoids The ethyl acetate fraction was dried using a rotary
were also evaluated. evaporator at 40 C. The dried extract was re-dissolved
in a small volume of ethanol. A 100 ll of samples was
injected to an AKTATM purifier HPLC system (Amer-
2. Materials and methods sham biotechnology Co., Swiss). The HPLC analysis
was carried out on a reverse phase polystyrene/divinyl
2.1. Materials column (100 · 6.4 mm). Solvent A (water/formic acid =
98:2, v/v) and solvent B (acetonitrile/water/formic
Fresh fruits of lychee (Litchi chinensis Sonn.) cv. acid = [Link], v/v/v) were used as mobile phases. The
Huaizhi at the commercially mature stage were picked elution was allowed to run for 4 min with 3% B and
from a commercial orchard in Guangzhou, China. 97% A, and then solvent B increased from 3% to 50%
Fruits were selected for uniformity of shape and color. B while solvent B decreased from 97% to 50% for
14 min, linearly. The flavonoids were detected at
2.2. Chemicals 280 nm, and three major fractions (P1, P2 and P3 in
Fig. 1) was collected and then freeze–dried separately.
1,1-Diphenyl-2-picryldydrazyl (DPPH), nitro blue
tetrazolium (NBT), phenazine methosulphate (PMS), 2.5. UV–visible spectrophotpmetric analysis
dihydronicotineamidadenine dinucleotide (NADH),
thiobarbituric acid (TBA) and deoxyribose were pur- Each of three major fractions (1 mg) was dissolved in
chased from Sigma Chemical Co. (St Louis, MO, 10 ml of ethanol. The sample solution was scanned at
USA). All other chemicals used were of analytical grade. from 200 to 580 nm, using a UV-2102 PC UV–visible
spectrophotometer (Unico, China) while the spectra
2.3. Extraction of flavonoids were recorded.

Fresh lychee pericarp tissues (5 g) were extracted for 2.6. Molecular weight estimation
2 h at 4 C in 400 ml extraction medium consisting of
85% ethanol:15% HCl by the methods of Lee and Wick- MS system (LCQDECA, Finigan company, USA),
er (1991) and Argolo, AsntÕAna, Pletsch, and Coelho equipped with a Hewlett–Packard 9000 computer sys-
(2004), with a minor modification. After filtering the tem, was used to determine the molecular weight. Sample
extract through Whatman no. 1 paper, the residue was (1 mg) was dissolved in 10 ml of ethanol. A 100 ll of
re-extracted and filtered a second time. Filtrates were sample solution was injected into the MS system. Mass
combined and dried using a rotary evaporator at spectroscopy was recorded with a heat capillary voltage
40 C. The dried extract was re-dissolved in 100 ml of 4.5 kV, a heat capillary temperature of 270 C, sheath
water and then partitioned with 300 ml of hexane, ethyl gas flow rate of 70 units and auxiliary gas flow rate of 10
acetate and butanol squentially. The different fractions units. The scan range of m/z was 200–1200.
 M. Zhao et al. / Food Chemistry 98 (2006) 539–544 541

Fig. 1. Elution profiles of the ethyl acetate fraction of litchi flavonoids separated by reverse phase HPLC.

2.7. NMR spectroscopy 0.5 ml of 1% thiobarbituric acid and 1 ml of 2.8% trichlo-

roacetic acid were added to the mixture. The mixture was
13
C NMR spectra were recorded with a Bruker AC boiled for 10 min and cooled on ice. The absorbance of
300 Instrument at 30 C using 3 mm tubes. Samples the mixture was measured at 532 nm. Percent inhibition
(10 mg) were dissolved in 0.5 ml of MeOH-d4. 13C of deoxyribose degradation was calculated as (1  absor-
chemical shifts were expressed in parts per million bance of sample/absorbance of control) · 100.
(ppm) relative to tetramethyl silane (TMS) as an internal
standard. 2.8.3. Determination of superoxide anion scavenging
activity
2.8. Determination of antioxidant capability The superoxide anion scavenging activity was mea-
sured by the method of Robak and Gryglewski (1988)
2.8.1. DPPH scavenging activity with a minor modification. Samples were dissolved in
The free radical scavenging activity was measured 10 ml ethanol at 0 (control), 20 or 40 lg/ml. A 1 ml ali-
using the method of Shimada, Fujikawa, Yahara, and quot of each sample solution was mixed with 1 ml of
Nakamura (1992) with some modification. Samples were 0.1 M phosphate buffer (pH 7.4) containing 150 lM
dissolved in 10 ml ethanol at 0 (control), 20 or 40 lg/ml. NBT, 60 lM PMS and 468 lM NADH. After 5 min
A 2 ml of 0.2 mM DPPH in ethanol was added to 1 ml of incubation at 25 C the absorbance was measured
of the sample solution. The absorbance at 517 nm was at 560 nm. The superoxide anion scavenging activity
measured after 20 min of incubation at 25 C. The inhi- was calculated as follows: scavenging activity (%) =
bition of DPPH radicals by the samples was calculated (1  absorbance of sample/absorbance of control) ·
according to the following equation: DPPH scavenging 100.
activity (%) = [1  absorbance of sample/absorbance
of control] · 100. 2.9. Data handling

2.8.2. Evaluation of hydroxyl radical scavenging activity All the data were expressed as means ± standard
The hydroxyl radical scavenging activity was mea- deviations of three replication determinations.
sured by the method of Ghiselli, Nardini, Baldi, and
Scaccini (1998). Samples were dissolved in 10 ml ethanol
at 0 (control), 200 or 400 lg/ml. Sample solution (0.1 ml) 3. Results and discussion
was mixed with 0.8 ml of reaction buffer [0.2 M phos-
phate buffer (pH 7.4), 1.75 lmol deoxyribose, 0.1 lmol 3.1. Comparison of flavonoid content in four fractions
iron ammonium sulphate and 0.1 lmol EDTA]. 0.1 ml
of 1.0 mM ascorbic acid and 0.1 ml of 0.01 M H2O2 Hayder et al. (2004) reported that application of
was then added to the reaction solution. The reaction hexane, chloroform and ethyl acetate can partition
solution was incubated for 10 min at 37 C before flavonoids from Myrtus communis. Based on different
542 M. Zhao et al. / Food Chemistry 98 (2006) 539–544

polarities, lychee pericarp flavonoids could be parti- 3.4. Identification of flavonoids

tioned into four fractions of hexane, ethyl acetate, buta-
nol and water. In this study, the ethyl acetate fraction of P1, P2 and P3 were identified as proanthocyanidin
flavonoids accounted for 83.1% of the total quantity B4, proanthocyanidin B2 and epicatechin, respectively,
(Table 1). This study corroborated that of Zhang et al. by UV, MS and NMR analyses, based on following
(2004) who reported that part of lychee anthocyanins characteristics.
had solubility in water. However, the composition of P1: UVmax at 279 nm; [M  H] peak at m/z 577.1; 13C
the butanol fraction of flavonoids is needed to be iden- NMR: 29.6, 38.9, 67.7, 73.7, 79.9, 83.8, 96.3, 97.5, 97.7,
tified further. 99.5, 101.2, 108.2, 114.2–115.9, 119.2, 120.3, 131.2–
132.6, 145.6–146.5 and 155.4–158.7. P1 gave [M  H]
3.2. Separation of ethyl acetate fraction of flavonoids at m/z 577.1 on negative-ion ES–MS, which was consis-
tent with a proanthocyanidin dimmer (Baba, Osakabe,
A reverse phase HPLC gave a successful resolution of Natsume, & Terao, 2002). Its dimeric constitution was
major flavonoids (Robards & Antolovich, 1997; Rohr, apparent from the presence of the four clearly defined car-
Meier, & Sticher, 1999; Merken & Beecher, 2000; Maat- bon signals in the heterocyclic region, with the C-2 carbon
ta, Kamal-Eldin, & Torronen, 2003). In this analysis, signals at d 83.8 and 79.9 ppm being a 2,3-trans flavan unit
the HPLC profiles exhibited three major peaks (P1, P2 with an appended substitute at C-4 and a 2,3-cis terminat-
and P3) of the ethyl acetate fraction (Figs. 1 and 2), with ing unit (Foo, Newman, Waghorn, McNabb, & Ulyatt,
their levels being 3.3, 3.0 and 8.7 mg/g pericarp tissue on 1996). The C-3 carbon signals at d 67.7 and 73.7 ppm were
the basis of dry weight (DW), respectively. Thus, these a flavan terminal unit and an extended unit.
three fractions were collected separately for further P2: UVmax at 279.5 nm; [M  H] peak at m/z 577.3;
13
identification. C NMR: 29.3, 36.9, 66.4, 72.5, 76.3, 79.2, 95.8, 96.3,
97.2, 100.5, 102.1, 107.4, 114.9–115.8, 119.2, 119.3,
3.3. UV–visible spectrophotpmetric analysis 131.4, 131.6, 145.0–146.1 and 155.1–157.9. P2 exhibited
a [M  H] at m/z 577.3 by negative-ion ES–MS, also
Flavanol and flavonol have maximum absorbances at indicating the presence of a proanthocyanidin dimer.
about 280 and 350 nm, respectively, while anthocyanin Its 13C NMR spectrum showed a similar chemical con-
has a maximum absorbance at about 280 and 520 nm stitution to P1. C-2 (d 76.3 and 79.2 ppm) indicated
(Pascale, Erwan, Christine, Yves, & Veronique, 2000). two flavan units which possessed the 2,3-cis configura-
The UV–visible spectrophotometric analysis showed tion. The observation of two upfield methine carbon
that P1, P2 and P3 had the maximum absorbance signals (d 114.9–115.8 ppm), together with those of the
around 280 nm, which suggested that they could be hydroxylated carbon signals (d 145.0–146.1 ppm), was
flavanols. evidence for the presence of a catechol B-ring.

5'
6' 4'
OH
O 1' 3'
8a 2' OH
HO 8
7 2
6 3
5 H
4a 4
OH
OH

Epicatechin

OH OH
O OH O OH
HO HO

OH OH OH OH
OH O OH OH O OH
HO HO

OH OH
OH OH

Proanthocyanidin B4 Proanthocyanidin B2

Fig. 2. Molecular formula of epicatechin, proanthocyanidin B2 and proanthocyanidin B4.

 M. Zhao et al. / Food Chemistry 98 (2006) 539–544 543

Table 2
Comparison of radical scavenging activity (%) of the ethyl acetate fraction, proanthocyanidin B2, proanthocyanidin B4 and epicatechin
Sample Amount (lg) DPPH scavenging activity Hydroxyl radical scavenging activity Superoxide anion scavenging activity
Ethyl acetate fraction 20 50.9 ± 1.0 76.3 ± 0.6 81.4 ± 1.0
40 74.6 ± 1.3 95.7 ± 0.8 91.0 ± 1.1
Proanthocyanidin B4 20 46.1 ± 1.0 42.6 ± 0.7 62.2 ± 0.8
40 54.5 ± 1.0 56.3 ± 1.2 71.3 ± 1.2
Proanthocyanidin B2 20 58.6 ± 0.5 72.9 ± 1.1 79.4 ± 1.6
40 73.8 ± 0.9 96.8 ± 1.3 93.2 ± 1.4
Epicatechin 20 60.4 ± 0.6 38.7 ± 0.3 54.1 ± 1.0
40 76.0 ± 0.8 52.3 ± 0.9 68.4 ± 0.9
Data were presented as means ± standard deviations of three replication determinations.

P3: UVmax at 278 nm; [M  H] peak at m/z 289.1; B2 and epicatechin by UV, NMR and MS analyses. In
13
C NMR: 28.9 (C-4), 66.9 (C-3), 79.4 (C-2), 95.5 (C- addition, proanthocyanidin B2, proanthocyanidin B4
8), 96.2 (C-6), 99.7 (C-4a), 115.3 (C-2 0 ), 115.6 (C-5 0 ), and epicatechin exhibited a strong antioxidant activity.
119.3 (C-6 0 ), 132.1 (C-1 0 ), 145.3 (C-3 0 ), 145.5 (C-4 0 ),
157.0 (C-5), 157.5 (C-7) and 157.6 (C-8a). Identification
of P3 as epicatechin was apparent from the 13C NMR Acknowledgements
spectrum. A methylene carbon in the upfield region (d
28.9 ppm) and two oxygenated methine carbons in the The financial support provided by National Natural
heterocyclic region (d 66.9 and 79.4 ppm) were charac- Science Foundation of China (No. 30425040), the Inter-
teristic of the pyran C-ring of flavanols while the upfield national Foundation for Science (Grant No. E2265/3F)
position of the C-2 carbon (d 79.4 ppm) was character- and Guangdong Provincial Agricultural Research
istic of the epicatechin chemical shift (Lu & Foo, Foundation was greatly appreciated.
1997). The chemical structure of P3 was further corrob-
orated by ES–MS, which gave a [M  H] at m/z 289.1
using a negative-ion probe. References

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