Food Chemistry 114 (2009) 577–581

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Antioxidant properties of various solvent extracts from lychee

(Litchi chinenesis Sonn.) flowers
Shih-Chuan Liu a, Jau-Tien Lin b, Chin-Kun Wang c, Hsin-Yi Chen a,c, Deng-Jye Yang a,*
a
School of Health Diet and Industry Management, Chung Shan Medical University, 110, Jianguo N. Rd. Sec. 1, Taichung 402, Taiwan
b
Department of Applied Chemistry, Chung Shan Medical University, 110, Jianguo N. Rd. Sec. 1, Taichung 402, Taiwan
c
School of Nutrition, Chung Shan Medical University, 110, Jianguo N. Rd. Sec. 1, Taichung 402, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: The antioxidant capacities of the acetone, methanol and water extracts of hot-air dried lychee (Litchi
Received 7 February 2008 chinenesis Sonn.) flowers were estimated with Trolox equivalent antioxidant capacity (TEAC) assay,
Received in revised form 18 August 2008 reducing power and 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) radical-scavenging assay. The con-
Accepted 29 September 2008
tents of antioxidant components in these extracts were also determined. Results showed that the highest
and lowest contents of these components including phenols, flavonoids and condensed tannins were
found in acetone and water extracts, respectively. The antioxidant activities of the lychee flower extracts
Keywords:
for all assays were in the order: acetone extract > methanol extract > water extract. The contents of anti-
Antioxidant
Flavonoid
oxidant components in these extracts were correlated with antioxidant activities.
Lychee flower Ó 2008 Elsevier Ltd. All rights reserved.
Litchi chinenesis Sonn.
Phenol
Tannin

1. Introduction Many reports indicate that flowers contain phenolic com-

Lychee (Litchi chinenesis Sonn.) is a tropical and subtropical fruit
of the Sapindaceae family originating from South-east Asia (Rivera-
López, Ordorica-Falomir, & Wesche-Ebeling, 1999). It is one of the
important commercial crops in Taiwan, which blooms in late
March and matures in late June. The non-climacteric fruit (with a
sweet and pleasant flavour) has a bright red, attractive pericarp
surrounding a white and translucent fleshy aril (Nakasone & Paull,
1998). The juicy fruit can be eaten directly and can also be used to
manufacture juice, vinegar, jelly and wine, or used in ice creams
and sorberts (Salunke & Desai, 1984). Furthermore, lychee has also
been employed in traditional Chinese medicine to promote human
health for a long time.
Many studies have demonstrated that phenolic compounds ex-
ist in the aril and pericarp of lychee fruit (Duan, Jiang, Su, Zhang, &
Shi, 2007; Lee & Wicker, 1991a, 1991b; Rivera-López et al., 1999;
Sarni-Manchado, Le Roux, Le Guernevé, Lozano, & Cheynier,
2000). These compounds can quench reactive free radicals, (Sorata,
Takahama, & Kimura, 1984; Takahama, Youngman, & Elsnert,
1974), and act as antioxidants or agents of other mechanisms,
which contribute to their anti-carcinogenic, anti-inflammatory
and cardio-protective effects, and prevention of degenerative dis-
eases (Biglari, AlKarkhi, & Easa, 2008; Shahidi, 1997).
pounds and have antioxidant action as well (Barreira, Ferreira,
Oliveira, & Pereira, 2008; Elzaawely, Xuan, Koyama, & Tawata,
2007; Ho, Hwang, Shen, & Lin, 2007; Kaur, Alam, Jabbar, Javed,
& Athar, 2006; Susanti et al., 2007). Lychee flower (small, pale
white-light yellow colour) is one of the major nectar sources
in Taiwan and bees could turn the nectar into honey
(Baltrušaitytė, Venskutonis, & Čeksterytė, 2007). This honey also
contains phenolic compounds and exhibits antioxidant activity
(Aljadi & Kamaruddin, 2004). There is, however, no report con-
cerning the antioxidant property of the flower of lychee planted
in Taiwan.
In the present investigation, various solvent (acetone, methanol
and water) extracts of hot-air dried lychee flowers were used to
evaluate their antioxidant capacities (Trolox equivalent antioxi-
dant capacity (TEAC) assay, reducing power and scavenging ability
on 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) radicals). The
contents of total polyphenol, total flavonoid and condensed tannin
in these extracts were also determined.
2. Materials and methods
2.1. Samples

* Corresponding author. Tel.: +886 4 24730022x11867; fax: +886 4 23248188. Fresh lychee (L. chinenesis Sonn.) flowers were obtained from a
E-mail address: djyang@csmu.edu.tw (D.-J. Yang). fruit farm in Taichung County, Taiwan. The flowers were dried in a

0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.09.088
578 S.-C. Liu et al. / Food Chemistry 114 (2009) 577–581
hot-air dryer (Chi-Yeh Electric and Machinery Co., Taipei, Taiwan)
at 40 °C for 16 h before solvent extraction.
2.2. Chemicals
Acetone and methanol (MeOH) were purchased from Merck
(Darmstadt, Germany). Deionised water (dd H2O) was prepared
using an Ultrapure water purification system (Lotun Co., Ltd.
TM
Taipei, Taiwan). Chemicals used for determination of contents of
total phenols, total flavonoids and condensed tannins including
aluminum chloride, (+)-catechin, Folin–Ciocalteu’s phenol re-
agent, gallic acid, sodium carbonate (Na2CO3), sodium hydroxide
(NaOH), sodium nitrite (NaNO2), hydrochloric acid (HCl), and
vanillin were obtained from Sigma (St. Louis, MO), Merck and
Wako Co. (Osaka, Japan). Chemicals employed for antioxidant
capacity assays such as ascorbic acid, 2-20 -azino-bis-(3-ethylbenz-
thiazoline-6-sulfonic acid) (ABTS), 2, 2-diphenyl-2-picrylhydrazyl
hydrate (DPPH), disodium hydrogen phosphate (Na2HPO4),
ferrous chloride (FeCl3), 6-hydroxy-2,5,7,8-tetramethylchromane-
2-carboxylic acid (Trolox), hydrogen peroxide (H2O2), horseradish
peroxidase, potassium ferricyanide (K3Fe(CN)6), sodium dihydro-
gen phosphate (NaH2PO4), and trichloroacetic acid (TCA) were
purchased from Sigma.
2.3. Preparation of lychee flower extracts
Twenty-five grams of dried lychee flowers were extracted with
500 ml of MeOH or acetone at ambient temperature for 24 h fol-
lowed by filtration with an aspirator (Panchun Scientific Co., Kaoh-
siung, Taiwan). The residues were re-extracted with 500 ml of the
same solvent. The same extracts were combined and then concen-
trated to dryness with a rotary evaporator at 35 °C. For water
extraction, 30 g of dried lychee flowers were refluxed with
1500 ml of distilled water at 98 ± 2C for 30 min. After filtration,
the extract was cooled and lyophilised (Vastech Scientific Co.,
Ltd., Taipei, Taiwan). The yield of each extract was calculated. All
of the extractive procedures were performed in dim lighting. The
dried extracts were kept in the dark under nitrogen at 80 °C prior
to analysis.
2.4. Determination of antioxidant contents in lychee flower extracts
All the extracts were re-dissolved in MeOH at a concentration
4 mg/ml for determination of antioxidant contents.
2.4.1. Total phenolic contents
Total phenols were determined according to the method de-
scribed by Julkunen-Titto (1985). An aliquot (50 ll) of each extract
or standard solution was mixed with 1 ml of dd H2O and 0.5 ml of
Folin–Ciocalteu’s phenol reagent. Afterwards 2.5 ml of 20% Na2CO3
solution were added to the mixture followed, by incubating at
ambient temperature in the dark for 20 min. The absorbance
against a blank was measured at 735 nm (Spectro UV–vis auto
spectrophotometer, Labomed Inc., Culver City, CA). Gallic acid
was used to prepare a standard curve (0.025–0.6 mg/ml;
y = 1.3058x + 0.065; r2 = 0.9993; y is the absorbance; x is the solu-
tion concentration). The results were expressed as mg gallic acid
equivalents (GAE)/g extract (dw).
2.4.2. Total flavonoid contents
Flavonoid contents were determined according to the method
of Zhishen, Mengcheng, and Jianming (1999). An aliquot
(250 ll) of each extract or standard solution was mixed with
1.25 ml of dd H2O and 75 ll of 5% NaNO2 solution. After
6 min, 150 ll of 10% AlCl3H2O solution were added. After
5 min, 0.5 ml of 1 M NaOH solution were added and then the
total volume was made up to 2.5 ml with dd H2O. Following
thorough mixing of the solution, the absorbance against blank
was determined at 510 nm. (+)-Catechin was utilised for con-
structing the standard curve (0.05–0.5 mg/ml; y = 0.79x + 0.048;
r2 = 0.9997; y is the absorbance; x is the solution concentration).
The results were expressed as mg catechin equivalents (CE)/g
extract (dw).
2.4.3. Condensed tannin contents
Condensed tannins were determined according to the method
of Julkunen-Titto (1985). An aliquot (50 ll) of each extract or stan-
dard solution was mixed with 1.5 ml of 4% vanillin (prepared with
MeOH) and then 750 ll of conc. HCl were added. The well-mixed
solution was incubated at ambient temperature in the dark for
20 min. The absorbance against blank was read at 500 nm. (+)-Cat-
echin was used to make the standard curve (0.05–1 mg/ml;
y = 0.801x + 0.0555; r2 = 0.9992; y is the absorbance; x is the solu-
tion concentration). The results were expressed as mg catechin
equivalents (CE)/g extract (dw).
2.5. Antioxidant capacity assay of the lychee flower extracts
2.5.1. DPPH radical-scavenging activity
DPPH radical-scavenging activity was determined according to
the method of Shimada, Fujikawa, Yahara, and Nakamura (1992).
An aliquot of each extract or standard solution prepared with
MeOH (200 ll) was mixed with 50 ll of 1 mM DPPH (also prepared
with MeOH). The mixture was shaken followed by incubating at
ambient temperature for 30 min in the dark. The absorbance
against blank was measured at 517 nm. The extract concentration
that could scavenge 50% of the DPPH radicals (EC50) was calculated
from the plot of scavenging activity against the concentration of
extract. The scavenging activity was estimated based on the per-
centage of DPPH radical scavenged compared to a blank sample.
Ascorbic acid and (+)-catechin standards were used for
comparison.
2.5.2. TEAC assay
TEAC assay was carried out according to the method of Arnao,
Casas, Del Río, Acosta, and García-Cánovas. (1990). The absorbance
against blank was determined at 734 nm as a function of extract
concentration, and the scavenging percentage of ABTS+ was
calculated relative to Trolox (a water-soluble analogue of vitamin
E adopted as an antioxidant standard). Antioxidant activity was
expressed as mmol Trolox equivalent (TE)/g extract. The ABTS+
solution (OD734 = 0.65 ± 0.02) was made by mixing ABTS, H2O2
and peroxidase with final concentrations of 100 lM, 50 lM and
4.4 unit/ml, respectively. Trolox (30 ll, 0.05 to 2 mM MeOH),
which was mixed with 970 ll of ABTS+ solution and reacted for
1 min, was used to construct the standard curve (y = 0.7302x
+ 0.6274; r2 = 0.9992; y is the absorbance; x is the solution concen-
tration). (+)-Catechin standard was used for comparison.
2.5.3. Reducing power
Reducing power was determined according to the method of
Oyaizu (1986). An aliquot of each sample or standard solution
prepared with MeOH (250 ll) was mixed with 250 ll of sodium
phosphate buffer (0.2 M, pH 6.6) and 250 ll of 1% K3Fe(CN)6
incubated at 50 °C for 20 min. After adding 250 ll of 10% trichlo-
roacetic acid, the mixture was centrifuged at 3750g for 10 min.
The supernatant (100 ll) was then taken out and immediately
mixed with 100 ll of MeOH and 25 ll of 0.1% ferric chloride.
After incubation for 10 min, the absorbance against blank was
determined at 700 nm. The EC50 value is the concentration at
which the absorbance is 0.5. Ascorbic acid and (+)-catechin stan-
dards were utilised for comparison.
 S.-C. Liu et al. / Food Chemistry 114 (2009) 577–581 579

2.6. Statistical analysis 100

Determination of antioxidant contents and all antioxidant

capacity assays were executed in triplicate and the mean values 80
were calculated. The data were subjected to analysis of variance,

Scavenging activity (%)

and Duncan’s multiple range tests were employed to gauge differ- 60
ences between means. A significant difference was judged to exist
at a level of p < 0.05.
40

3. Results and discussion

20 Ascorbic acid
(+)-Catechin
3.1. Extraction yields and antioxidant contents in the lychee flower
Acetone extract
extracts
0 MeOH extract
Water extract
The highest extraction yield was found in the acetone extract of
the hot-air dried lychee flowers (12.91%). The extract also con- -20
0 50 100 150 200 250 300 350
tained the highest contents of total phenols (316 mg GAE/g ex-
tract), total flavonoids (273 mg CE/g extract) and condensed Concentration(µg/ ml)
tannins (273 mg CE/g extract) (Table 1). Although the extraction
Fig. 1. DPPH radical-scavenging effects of acetone, MeOH and water extracts of
yields between MeOH (9.56%) and water (9.92%) extracts did not lychee flowers, ascorbic acid and (+)-catechin. Each value is expressed as mean ± SD
show a significant difference, the MeOH extract had significantly (n = 3).
higher antioxidant contents (170 mg GAE/g extract for total
phenols, 110 mg CE/g extract for total flavonoids and 45.9 mg
CE/g extract for condensed tannins, Table 1) than the water extract
Table 2
(97.8 mg GAE/g extract for total phenols, 42.8 mg CE/g extract for EC50 values of DPPH radical-quenching activity, reducing power, and antioxidant
total flavonoids and 44.8 mg CE/g extract for condensed tannins, activity (TEAC assay) of lychee flower extracts.
Table 1). Ho et al. (2007) illustrated that rich amounts of total
Sample EC50a of DPPH EC50b of reducing TEAC (mmol
phenols, total flavonoids and condensed tannins existed in longan radical-quenching power TEc/g sample)
(Dimocarpus longan Lour.) flowers. The levels of these components activity (lg sample/ml) (lg sample/ml)
in the various solvent extracts of the flowers also showed differ- Acetone extract 30.1 ± 2.28c 5.27 ± 3.24c 3.46 ± 0.30b
ences. Barreira et al. (2008) also indicated that chestnut flowers MeOH extract 120 ± 8.64b 168 ± 11.56b 1.03 ± 0.01c
contained considerable amounts of polyphenols and flavonoids. Water extract 314 ± 11.23a 199 ± 13.18a 0.59 ± 0.08d
Our results revealed that lychee flower contained remarkable lev- (+)-Catechin 15.7 ± 2.84d 26.9 ± 2.25d 12.6 ± 0.26a
Ascorbic acid 14.7 ± 2.52d 10.8 ± 1.01e –
els of phenols, flavonoids and condensed tannins. Various solvents
would affect extractions of these components. Values (mean ± SD, n = 3) in the same column followed by a different letter are
significantly different (p < 0.05).
a
EC50 means the effective concentration of sample that can decrease DPPH
3.2. Antioxidant capacities of the lychee flower extracts
concentration by 50%.
b
EC50 is the concentration for which the absorbance at 700 nm is 0.5.
With regard to the lychee flower extracts, Fig. 1 illustrates that the c
TE, Trolox equivalents.
sequence for DPPH radical-scavenging ability was acetone extra-
ct > MeOH extract > water extract. At 120 lg/ml, the scavenging abil-
ities on DPPH radicals were 94.9%, 49.2%, 20.9% for the acetone,
Ascorbic acid
MeOH and water extracts, respectively. Table 2 shows that the EC50
values of DPPH radicals scavenging for the acetone, MeOH and water (+)-Catechin

extracts were 30.5, 120 and 314 lg/ml, respectively. For TEAC assay, Acetone extract
the acetone extract showed the highest antioxidant activity, the MeOH extract
MeOH extract next, and the water extract had the lowest activity (Ta- 4.0 water extract
ble 2). The TEAC values for the acetone, MeOH and water extracts
were 3.46, 1.03 and 0.59 mmol TE/g, respectively. Fig. 2 illustrates
that reducing power increased with concentration of each lychee
Absorbance at 700 nm

3.0

Table 1
Extraction yields, and contents of total phenols, total flavonoids and condensed
tannins in lychee flower extracts. 2.0
Sample Extraction Content
yield (%)a
Total phenols Total flavonoids Condensed
(mg GAEb/g (mg CEc/g tannins 1.0
extract) extract) (mg CEc/g extract)
Acetone extract 12.9 ± 1.12a 316 ± 15.34a 273 ± 9.92a 273 ± 10.42a
MeOH extract 9.56 ± 0.73b 170 ± 7.02b 110 ± 1.57b 45.9 ± 4.33b
Water extract 9.92 ± 0.86b 97.8 ± 2.46c 42.8 ± 2.02c 44.8 ± 1.81c 0.0
0 200 400 600 800
Values (mean ± SD, n = 3) in the same column followed by a different letter are
significantly different (p < 0.05). Concentration (µg/ml)
a
Extraction yield (%) = (sample extract weight/sample weight)  100%.
b
GAE, Gallic acid equivalents. Fig. 2. Reducing power of acetone, MeOH and water extracts of lychee flowers,
c
CE, Catechin equivalents. ascorbic acid and (+)-catechin. Each value is expressed as mean ± SD (n = 3).
580 S.-C. Liu et al. / Food Chemistry 114 (2009) 577–581
Table 3
Correlations established between antioxidant activity EC50/TEAC values and contents of antioxidant components.
Value Equation
Total phenols
TEAC Y = 0.0137 X  0.9729
r2 = 0.963, F = 181, p < 0.001
EC50 of DPPH radical-scavenging activity Y = 1.1907 X + 386.11
r2 = 0.834, F = 35.2, p < 0.001
EC50 of reducing power Y = 0.6809 X + 272.45
r2 = 0.946, F = 124, p < 0.001
flower extract. Likewise, the acetone and water extracts exhibited
the highest and lowest reducing power, respectively. Table 2 shows
that the EC50 values of reducing power for the acetone, MeOH and
water extracts were 52.7, 168 and 199 lg/ml, respectively.
Ho et al. (2007) reported that water and ethanol extracts of lon-
gan flowers having high levels of total phenols, total flavonoids and
condensed tannins exhibited good antioxidant ability. For chestnut
water extracts, flower extract had higher polyphenols and flavo-
noids than leaf and fruit extracts; the flower extract also showed
higher antioxidant capacity than the other two extracts (Barreira
et al., 2008). Pérez, Galderón, and Croci (2007) found that rosemary
methanol extract had higher phenolic contents and antioxidant
capacity than its water extract. In our investigation, the highest
amounts of phenols, flavonoids and condensed tannins were found
in the acetone extract of lychee flowers, which had higher activity
in antioxidant assays than MeOH or water extracts.
Correlation analysis (Table 3) showed a strong correlation be-
tween TEAC values and contents of antioxidant components (total
phenols, r2 = 0.963; total flavonoids, r2 = 0.974; condensed tannins,
r2 = 0.976), and so did that between EC50 values of reducing power
and contents of antioxidant components (total phenols, r2 = 0.946;
total flavonoids, r2 = 0.967; condensed tannins, r2 = 0.935). A signif-
icant correlation was also observed between EC50 values of DPPH
radical-scavenging activity and total phenolic contents
(r2 = 0.834), and also the EC50 values and flavonoid contents
(r2 = 0.810). The condensed tannin contents were weakly corre-
lated with EC50 values of DPPH radical-scavenging activity
(r2 = 0.552). Barreira et al. (2008) found that antioxidant activity
EC50 values of DPPH radical-scavenging activity and reducing
power of chestnut water extract correlated with its contents of
phenols and flavonoids. Marimuthu, Wu, Chang, and Chang
(2008) observed that antioxidant activity (TEAC value) of ethanolic
extract from bark of Chamaecyparis obtuse var. Formosan correlated
with its phenolic content. However, some authors (Eberhardt, Liu,
Smith, & Lee, 2001) have mentioned that there is no direct correla-
tion between phenolic content and antioxidant activity. Our results
showed that phenolic components would probably play an impor-
tant role in the antioxidant capacity of the lychee flower extracts.
4. Conclusion
Acetone could extract the highest concentration of antioxidant
compounds (polyphenols, flavonoids and condensed tannins) from
hot-air dried lychee flowers in this investigation; this extract also
showed higher antioxidant activity than those extracted by MeOH
or water. These results reveal that lychee flowers, generally re-
garded as a disposable byproduct, has potential for exploitation
to promote human and animal health.
Acknowledgments
This work was supported by Chun Shan Medical University,
Taichung, Taiwan (Project No. CSMU-96-OM-B-005 and CSMU-
96-OM-A-088).
Total flavonoids Condensed tannins
Y = 0.0130 X  0.1489 Y = 0.0117 X + 0.2758
r2 = 0.974, F = 267, p < 0.001 r2 = 0.976, F = 286, p < 0.001
Y = 1.1046 X + 311.28 Y = 0.8217 X + 254.07
r2 = 0.810, F = 29.9, p < 0.001 r2 = 0.552, F = 8.64, p = 0.022
Y = 0.648 X + 231.98 Y = 0.574 X + 209.55
r2 = 0.967, F = 206, p < 0.001 r2 = 0.935, F = 101, p < 0.001
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