Antioxidant activities of different fractions of polysaccharide purified from pulp tissue of litchi (Litchi chinensis Sonn.)

Article abstract of DOI:10.1016/j.carbpol.2010.03.021

Three fractions of water-soluble polysaccharide fractions, coded as LFP1, LFP2 and LFP3, were isolated and purified from litchi pulp by DEAE-52 cellulose anion-exchange column and Sephadex G-100 gel-permeation column. Their chemical and physical characteristics were determined by chemical methods, gas chromatography, IR spectrophotometer and gel-permeation chromatography (GPC) with a model 410 refractive index detector. Antioxidant activities of these fractions were investigated using various in vitro assay systems. Results indicated that LFP1 was composed of arabinose, ribose, galactose and glucose, while LFP2 comprised arabinose, rhamnose and glucose. LFP3 was a heteropolysaccharide bounded with protein (2.6%) and composed of arabinose, rhamnose, galactose and glucose. The molecular weights of LFP1, LFP2 and LFP3 were 11.6 × 10⁴, 3.1 × 10⁴ and 2.9 × 10⁴ Da, respectively. LFP3 showed the highest antioxidant activities among the three fractions of polysaccharides, and could be explored as a novel potential antioxidant.

Full text of DOI:10.1016/j.carbpol.2010.03.021

Carbohydrate Polymers 81 (2010) 612–616
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Antioxidant activities of different fractions of polysaccharide purified from pulp
tissue of litchi (Litchi chinensis Sonn.)
Fan-Li Konga,b, Ming-Wei Zhangb , Rui-Bin Kuang , Shu-Juan Yu ,
b,∗
c
a
b
Jian-Wei Chi , Zhen-Cheng Wei
a
College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640, PR China
Key Laboratory of Functional Food, Ministry of Agriculture, Bio-tech Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, PR China
Key Laboratory of Tropical and Sub-tropical Fruit Trees, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Three fractions of water-soluble polysaccharide fractions, coded as LFP1, LFP2 and LFP3, were isolated
and purified from litchi pulp by DEAE-52 cellulose anion-exchange column and Sephadex G-100 gel-
permeation column. Their chemical and physical characteristics were determined by chemical methods,
gas chromatography, IR spectrophotometer and gel-permeation chromatography (GPC) with a model
Received 2 February 2010
Received in revised form 5 March 2010
Accepted 9 March 2010
Available online 16 March 2010
4
10 refractive index detector. Antioxidant activities of these fractions were investigated using various in
vitro assay systems. Results indicated that LFP1 was composed of arabinose, ribose, galactose and glucose,
while LFP2 comprised arabinose, rhamnose and glucose. LFP3 was a heteropolysaccharide bounded with
protein (2.6%) and composed of arabinose, rhamnose, galactose and glucose. The molecular weights of
LFP1, LFP2 and LFP3 were 11.6 × 10 , 3.1 × 10 and 2.9 × 10 Da, respectively. LFP3 showed the highest
antioxidant activities among the three fractions of polysaccharides, and could be explored as a novel
potential antioxidant.
Keywords:
Litchi
Polysaccharide
Purification
Antioxidant activity
4
4
4
©
2010 Elsevier Ltd. All rights reserved.
1
. Introduction
of these substances (Chen, Zhang, Qu, & Xie, 2008; Wang & Luo,
007).
Litchi (Litchi chinensis Sonn.), a tropical to subtropical fruit
2
Oxidation is essential to many organisms for the production of
energy to fuel biological processes. However, many human diseases
such as cancer, rheumatoid arthritis, and atherosclerosis, etc.) are
due to the uncontrolled production of oxygen-derived free radicals
Mau, Lin, & Song, 2002). Since synthetic antioxidants have been
originated from China is cultivated all over the world in warm
climates (Gontier et al., 2000). Litchi fruits have been used to
produce various types of healthy products and foods, e.g., medic-
inal beverages, drinks or soup (Chyau, Ko, Chang, & Mau, 2003;
Jiang, 2000; Jiang & Li, 2003). It may merit investigation as a
potential antioxidant for clinical use. Polysaccharide and major
flavonoids from litchi pericarp tissues has been purified and found
to exhibit beneficial antioxidant activities (Yang et al., 2006). How-
ever, no investigation has been carried out on polysacchrides of
litchi pulp to better effectively exploit the structural and functional
properties of these substances. More detailed work is required
to fully elucidate the antioxidant activities of active polysaccha-
rides.
In this study, the major polysaccharides of litchi fruit pulp
were extracted and purified using a DEAE-52 cellulose column
chromatography and a Sephadex G-100 column chromatogra-
phy. Their chemical and physical characteristics were determined
by chemical methods, gas chromatography, IR spectrophotome-
ter and gel-permeation chromatography (GPC) with a model 410
refractive index detector. In addition, the structure properties and
antioxidant activities of these major polysaccharides were also
investigated.
(
(
suspected of being responsible for liver damage and carcinogen-
esis (Grice, 1988; Qi et al., 2005), it is essential to develop and
utilize effective and naturally derived antioxidants so that they
can protect the human body from free radicals and retard the
progress of many chronic diseases (Kinsella, Frankel, German, &
Kanner, 1993; Nandita & Rajini, 2004). In the past two decades,
many reports have indicated that plant polysaccharides have strong
antioxidant activities and can be explored as novel potential antiox-
idants (Hu, Xu, & Hu, 2003; Jiang, Jiang, Wang, & Hu, 2005; Qi et
al., 2006; Ramarahnam, Osawa, Ochi, & Kawaishi, 1995). Previous
studies indicate that polysacchrides antioxidant activity may be
closely related to chemical properties and structural charateristics
∗ Corresponding author at: Key Laboratory of Functional Food, Ministry of Agri-
culture, Bio-tech Research Institute, Guangdong Academy of Agricultural Sciences,
Guangzhou 510610, PR China.
E-mail address: mwzhh@vip.tom.com (M.-W. Zhang).
0
144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.03.021

F.-L. Kong et al. / Carbohydrate Polymers 81 (2010) 612–616
613
2
. Materials and methods
Bradford’s method, using bovine serum albumin (BSA) as the stan-
dard (Bradford, 1976).
2
.1. Materials and chemicals
Gas chromatography (GC) was used for identication and quan-
tification of monosaccharides in litchi polysaccharide fractions. The
polysaccharide (10 mg) was hydrolyzed with 10 ml of 2 M triflu-
Fresh fruits of litchi (Litchi chinesis Sonn.) cv. Heiye at the
◦
commercially mature stage were offered by the Institute of Fruit
Tree Research, Guangdong Academy of Agricultural Sciences in
Guangzhou, China. Fruits with uniform shape and color were
selected as materials.
DEAE-52 cellulose and Sephadex G-100 were perchased from
Pharmacia Co. (Stockholm, Sweden). d-Arabinose, l-rhamnose, d-
ribose and d-galactose, d-glucose, Nitro blue tetrazolium (NBT),
phenazine methosulfate (PMS), dihydronicotineamidadenine din-
ucleotide (NADH) were purchased from Sigma Chemical Co. (St.
Louis, MO, USA). All other chemicals used were of analytical
grade.
oroacetic acid (TFA) at 120 C for 6 h (Erbing, Jansson, Widmalm,
& Nimmich, 1995). Derivation was then carried out using the
trimethylsilylation reagent according to the method of Guentas et
al. (2001). The trimethylsilylated derivatives were loaded into a
HP5 capillary gas chromatography (GC) column equipped with a
flame-ionization detector (FID), using inositol as the internal stan-
dard. The operation was performed using the following conditions:
H : 16 ml/min; air: 150 ml/min; N : 20 ml/min; injection temper-
2
2
◦
◦
ature: 230 C; detector temperature: 230 C; column temperature
programmed from 130 to 180 C at 5 C/min, holding for 2 min at
180 C, then increasing to 220 C at 5 C/min and finally holding for
min at 220 C.
◦
◦
◦
◦
◦
◦
3
2.2. Extraction of polysaccharides
2.6. Molecular weight determination
Litchi fruit polysaccharide (LFP) was extracted and isolated from
litchi pulp according to the methods of Miyazaki and Nishijima
1981) and Olafsdottir et al. (1999) with minor modifications. 1 kg
The molecular weight of the polysaccharides puried from litchi
pulp was determined by gel-permeation chromatography (GPC)
by the method of Yamamoto, Nunome, Yamauchi, Kato, and Sone
(
litchi fruits pulp were refluxed three times to remove monosaccha-
rides and saccharoses with 80% EtOH. Then it was comminuted and
(
1995), in combination with a high-performance liquid chro-
◦
extracted twice with 1 l portions of distilled water for 4 h at 80 C.
matography instrument (Waters5215, USA) equipped with an
Ultrahydrogel column, a model 410 refractive index detector and a
Millennium 32 Workstation. The column was eluted with distilled
water at a flow rate of 0.6 ml/min. Dextran standards (4400, 9900,
1,400, 43,500, 124,000, 196,000, 277,000 and 845,000 Da) were
used for the calibration curve.
The water extracts were filtered through Whatman No. 1 paper. The
filtrates were combined and concentrated to 250 ml by a rotary
◦
evaporator at 65 C. The proteins in the extracts were removed
using the Sevag reagent, and then the polysaccharides were pre-
2
◦
cipitated with four volumes of ethanol for overnight at 4 C. The
precipitate was collected by centrifugation, washed successively
with ethanol and ether, and dried at reduced pressure, giving LFP
as a crude polysaccharide.
2
.7. Infrared spectral analysis of the polysaccharides
The IR spectrum of the polysaccharides was determined using a
2
.3. Separation and purification of the polysaccharides
Fourier transform infrared spectrophotometer (FTIR, Bruker, Ger-
many) equippment. The purified polysaccharide was ground with
KBr powder and then pressed into pellets for FTIR measurement in
A DEAE-52 cellulose column (2.6 cm × 50 cm) was used to isolate
negatively charged polysaccharides from non-negatively charged
polysaccharides. After loading with sample, the column was eluted
with distilled water for 300 ml at 0.5 ml/min, followed stepwise
by NaCl aqueous solution (0–1 M) for 300 ml, respectively, at
−
1
the frequency range of 4000–500 cm (Kumar, Joo, Choi, Koo, &
Chang, 2004).
2
2
.8. Determination of antioxidant activity
0
.5 ml/min. The major polysaccharide fractions were collected
with a fraction collector and dialyzed against tap water and dis-
tilled water for 48 h, respectively, and concentrated using a rotary
evaporator at 65 C. A 0.5-ml sample of three highest peaks of
.8.1. Superoxide anion-scavenging activity
The superoxide anion-scavenging activity was measured fol-
◦
lowing the method of Qi et al. (2006). Briefly, 1 ml of various
concentrations of sample was mixed with 1 ml of 0.1 M phos-
phate buffer (pH 7.4) containing 150 ␮M NBT, 60 ␮M PMS
and 468 ␮M NADH. The reaction mixture was incubated at
room temperature for 5 min and the absorbance was read at
polysaccharide material was loaded into a Sephadex G-100 gel col-
umn (1.6 cm × 60 cm) and eluted with distilled water at a flow rate
of 0.3 ml/min. The major fractions with the highest polysaccharide
content were collected and then freeze dried.
5
60 nm. The capability of scavenging superoxide radical was
2.4. Determination of the polysaccharides purification
calculated using the following equation: scavenging activity
(
%) = (1 − Asample 560 nm/Acontrol 560 nm) × 100.
The samples were dissolved in distilled water, centrifuged,
and then the filtrate was applied to a Sephadex G-100 col-
umn (1.6 cm × 80 cm), which was eluted with distilled water
at 0.2 ml/min. In addition, the polysaccharides purification
was also identified by cellulose acetate pellicle electrophoresis
2.8.2. Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity was measured by
consulting the method of Wang et al. (2003) with minor mod-
ification. The reaction mixture, total volume 4.0 ml, containing
the sample solution with various concentration, EDTA-Fe2+
(
borax–sodium hydroxide buffer, pH 10) at 40 V for 50 min with
detection using Toluidine Blue.
(
220 ␮M), safranine O (0.23 ␮M), and H O2 (60 ␮M) in potas-
2
2.5. Monosaccharide compositions and properties
sium phosphate buffer (150 mM, pH 7.4), was incubated for
◦
3
0 min at 37 C and the absorbance was read at 520 nm against
The carbohydrate contents of the samples were determined by
a blank. The control contained phosphate buffer instead of the
H O while blanks contained distilled water instead of sam-
ple solution. The capability of scavenging hydroxyl radical was
calculated using the following equation: scavenging activity
the phenol–sulfuric acid method, using glucose as the standard
Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The protein con-
tents in the polysaccharide fractions were measured according to
2
2
(

6
14
F.-L. Kong et al. / Carbohydrate Polymers 81 (2010) 612–616
Table 1
Components of monosaccharide and properties of polysaccharides from fruits of
litchi.
LFP1
LFP2
LFP3
Protein (wt%)
nda
nd
2.6
Carbohydrate (wt%)
Sugar components (mol%)
d-Arabinose
l-Rhamnose
d-Ribose
99.4
99.6
97.4
1.67
nd
2.15
1.54
1.00
1.00
1.37
nd
nd
1.21
2.42
3.44
nd
1.00
1.65
d-Galactose
d-Glucose
a
nd: Not detected.
raphy on Sephadex G-100 column (1.6 cm × 60 cm), respectively
Fig. 2).
To determine the purification of polysaccharides, three fractions
(
Fig. 1. DEAE-52 chromatogram of the crude polysaccharides extracted from litchi
were loaded into Sephadex G-100 column (1.6 cm × 80 cm). Three
fractions were showed both only one symmetrical peak from gel-
filtration chromatography, indicating that no other polysaccharide
was present in the sample. In addtion, only a single spot on cellulose
acetate pellicle electrophoresis was observed using Toluidine Blue.
Based on above results, these three fractions have been purified to
be homogeneous.
fruit.
(
×
%) = [(Asample 520 nm − Ablank 520 nm)/(Acontrol 520 nm − Ablank 520 nm)]
100.
2.8.3. Reducing power
The reducing power was determined according to the method of
Duan, Zhang, Li, and Wang (2006) with some modifications. Briefly,
3
.2. Chemical composition of polysaccharides
1
2
ml of various concentrations of sample solution was mixed with
.5 ml of 0.2 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1%
◦
The polysaccharide content, protein content, and sugar com-
potassium ferricyanide. The mixture was then incubated at 50 C for
positions of LFP1, LFP2 and LFP3 were determined and given in
Table 1. No proteins existed in the LFP1 and LFP2, while LFP3 had
a low protein content of 2.6%. Monose compositions of three frac-
tions were determined by the triuoroacetic acid hydrolysis method.
GC analysis of the trimethylsilyl derivatives showed difference of
these three fractions, with the presence of arabinose, ribose, galac-
tose and glucose in the molar ratio of 1.67:2.15:1.54:1.00 for LFP1
fraction; and arabinose, rhamnose and glucose in the molar ratio of
2
0 min. After 2.5 ml of 10% trichloroacetic acid (w/v) was added, the
mixture was centrifuged at 3000 rpm for 10 min. A 2.5 ml aliquot
of the upper layer was mixed with 2.5 ml of distilled water and
0.5 ml of 0.1% ferric chloride, and the absorbance at 700 nm was
measured. Reducing power was expressed as a percentage of the
activity shown by a 1 mM solution of Vitamin C.
3
. Results and discussion
1
.00:1.37:1.21 for LFP2 fraction, also arabinose, rhamnose, galac-
tose and glucose in the molar ratio of 2.42:3.44:1.00:1.65 for LFP3
fraction.
3.1. Isolation and purification of polysaccharides
An aqueous extract of crude polysaccharides from litchi pulp
tissue was chromatographed on a DEAE-52 anion-exchange col-
umn (2.6 cm × 50 cm) to yield three peaks (Fig. 1), and the yields
of the three fractions were LFP1 2.37%, LFP2 63.92%, LFP3 20.00%.
The polysaccharide absorption peak showed similar time and shape
as the protein absorption peak, suggesting that LFP3 is a likely
a glycoprotein. This finding was similar to previous report that
polysaccharides existing as glycoprotein in grape berries (Saulnier
3.3. Molecular weight determination
The molecular weights of LFP1, LFP2 and LFP3 were determined
by GPC (Yamamoto et al., 1995). The equation of the standard curve
was drawn as log wt = 27.215 − 6.052t + 0.5446t − 0.0167t (where
wt represents molecular weight, while t represents elution time)
using the Millennium32 software. The molecular weights of LFP1,
LFP2 and LFP3 were 116440, 31424 and 28181 Da, respectively. The
2
3
&
Brillouct, 1989). Three fractions were purified by gel chromatog-
Fig. 2. Sephadex G-100 gel chromatogram of the polysaccharides. A 0.5-ml sample of three highest peaks of polysaccharide from DEAE-52 column was loaded into a Sephadex
G-100 gel column (1.6 cm × 60 cm) for further purification.

F.-L. Kong et al. / Carbohydrate Polymers 81 (2010) 612–616
615
Table 2
Molecular weight (MW) and molecular size (MN) of litchi polysaccharides.
Sample
MW
MN
MW/MN
LFP1
LFP2
LFP3
116440
31424
28181
94119
35982
61040
1.24
1.15
4.87
Fig. 5. Scavenging effect of LFPs on hydroxyl radicals. Data represent the
mean ± standard deviation (n = 3).
activity of the polysaccharide fraction (F01) from pericarp tissues
of litchi was about 50.7% and 62.5% at 50 ␮g/ml and 100 ␮g/ml.
In the present study, LFP3 from litchi pulp exhibits a little lower
scavenging activity than those from litchi pericarp by values of
Fig. 3. IR spectra of LFP1, LFP2 and LFP3 in the frequency range 4000–500 cm−1.
4
1.62% and 48.53% at 50 ␮g/ml and 100 ␮g/ml, respectively. Qi et
al. (2006) reported that scavenging activity of Vitamin C for super-
oxide radical was about from 30% at 500 ␮g/ml. Compared to this
result, LFP1, LFP2 and LFP3 had stronger scavenging activity for
superoxide radical than Vitamin C with values of 41.48%, 52.28%
and 66.45% at 500 ␮g/ml respectively (Fig. 4). Our data on the
activities of scavenging superoxides of different contents suggested
that it was likely to contribute towards the observed antioxidant
effect.
MW/MN values of LFP1 and LFP2 were closer to 1 than that of LFP3,
indicating that LFP1 and LFP2 were more homogeneous (Table 2).
3.4. IR spectra of puried LFP fractions
As shown in Fig. 3, the IR spectra of the three LFP fractions
displayed a broad stretching intense characteristic peak at around
−
1
3
419 cm for the hydroxyl group, and a weak C–H stretching band
−1
at around 2937 cm (Santhiya, Subramanian, & Natarajan, 2002).
The two peaks towards 1620 and 1420 cm in the IR spectra of the
three LFP fractions, resulted from the presence of the COO depro-
−1
3.6. Scavenging activity of hydroxyl radical
−
tonated carboxylic group (Manrique & Lajolo, 2002). Two stretching
As shown in Fig. 5, the three polysaccharide fractions were found
to exhibit the ability to scavenge hydroxyl radicals at amounts
between 75 and 450 ␮g. The scavenging effects increased with
increasing concentration. Scavenging effects of three fractions were
37.23%, 42.54% and 55.67% at amount of 450 ␮g/ml, respectively.
Previous report found that scavenging activity of the polysaccha-
ride fraction from pericarp tissues of litchi was about 50.3% at
−1
peaks, at 1144 and 1011 cm in the IR spectra of LFP1, at 1137 and
−
1
−
1
1
022 cm in the IR spectra of LFP2, at 1143 and 1012 cm in the IR
spectra of LFP3, suggested the presence of C–O bonds (Kacuráková,
Capek, Sasinková, Wellner, & Ebringerová, 2000).
3.5. Scavenging activity of superoxide radical
1
00 ␮g/ml (Yang et al., 2006). This value of scavenging activity for
The three polysaccharide fractions were found to have the abil-
hydroxyl radical from litchi pericarp was higher than three polysac-
charide fractions from litchi pulp (Fig. 5). It was reported that
scavenging effect on hydroxyl radical of Vitamin C was about 20%
at 1000 ␮g/ml (Qi et al., 2006). These results proved that polysac-
charides from litchi (pulp and pericarp) had significant effect on
scavenging hydroxyl radical, and some fractions was more pro-
nounced than that of Vitamin C.
ity to scavenge superoxide radical at concentrations between 50
and 500 ␮g/ml (Fig. 4). The scavenging effects of litchi polysac-
charide increased with increasing concentration. The IC50 values
of LFP1, LFP2 and LFP3 for superoxide radical were 960.86 ␮g/ml,
23.54 ␮g/ml and 113 ␮g/ml, respectively. LFP3 exhibited the high-
est scavenging effects. Yang et al. (2006) reported that scavenging
4
3.7. Reducing power
Reducing power of different polysaccharide fractions extracted
and purified from litchi pulp was investigated and showed in Fig. 6.
Values of reducing power of LFP1, LFP2 and LFP3 were 24.09%,
3
2.55% and 45.61% at concentration of 500 ␮g/ml, respectively. The
reducing properties are generally associated with the presence of
reductones, which have been shown to exert antioxidant action
by breaking the free radical chain by donating a hydrogen atom
(Gordon, 1990). Reductones are also reported to react with cer-
tain precursors of peroxide, thus preventing peroxide formation.
The antioxidant activity was concomitant with the reducing power
(Pin-Der-Duh, Pin-Chan-Du, & Gow-Chin-Yen, 1999; Tanaka, Kuie,
Nagashima, & Taguchi, 1988). Our data on reducing power of three
different fractions from litchi pulp indicates that it may likely play
a role in the antioxidation observed.
Fig. 4. Scavenging effect of LFPs on superoxide radicals. Each value is presented as
mean ± standard error of three parallel measurements for each concentration.

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According to the results above, it was concluded that the
water extracting crude polysaccharide of litchi pulp contained pre-
dominantly three polysaccharide fractions (LFP1, LFP2 and LFP3)
purifed by DEAE-52 and Sephadex G-100 column chromatogra-
phy, and purifcation polysaccharide prepared are confirmed of high
purity. Antioxidation test in vitro shows that LFP3 possesses the
strongest scavenging effect of superoxide radical, hydroxyl radical
and reducing power. This evaluation may shed the light on a bet-
ter understanding on the litchi pulp polysaccharide as a potential
functional antioxidant for its high antioxidant activity.
Acknowledgements
This work were supported by the Fund of Natural Science
of Guang Dong province, China (No. 06200586) and National-
Guangdong union fund project (No. U0731005). Authors thanks
Yan Zhang, Ruifen Zhang, Xiaojun Tang and Kennedy Chan for their
great technical supports in study process.
Qi, H. M., Zhang, Q., Zhao, T., Hu, R., Zhang, K., & Li, Z. (2006). In vitro antioxidant
activity of acetylated and benzoylated derivatives of polysaccharide extracted
from Ulva pertusa (Chlorophyta). Bioorganic Medicinal Chemistry Letters, 16,
2441–2445.
Ramarahnam, N., Osawa, T., Ochi, H., & Kawaishi, S. (1995). The contribution of
plant food antioxidants to human health. Trends in Food Science & Technology, 6,
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