International Journal of Biological Macromolecules 70 (2014) 150–155

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Preparation and characterization of catechin-grafted chitosan with

antioxidant and antidiabetic potential
Weili Zhu a,∗ , Zhanjun Zhang b
a
Department of Blood Transfusion, Subei People’s Hospital of Jiangsu Province, Yangzhou 225001, Jiangsu, China
b
College of Biological and Chemical Engineering, Yangzhou Vocational University, Yangzhou 225009, Jiangsu, China

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

Article history: In the present study, the preparation, characterization, antioxidant and antidiabetic activities of catechin-
Received 26 April 2014 grafted chitosan (catechin-g-chitosan) were investigated. The graft of catechin onto chitosan was
Received in revised form 6 June 2014 achieved by redox system and confirmed using various instrumental methods. Proton nuclear mag-
Accepted 19 June 2014
netic resonance spectroscopy indicates that catechin has been successfully grafted onto chitosan. The
Available online 1 July 2014
morphology observation shows that chitosan changes to a softened nature with porous surface after
grafting. Catechin-g-chitosan also exhibits reduced thermal stability and enhanced crystallinity com-
Keywords:
pared to chitosan. Moreover, catechin-g-chitosan shows 0.51 of reducing power, 46.81% of hydroxyl
Conjugate
Chitosan
radical-scavenging activity and 67.08% of DPPH radical-scavenging activity at 1 mg/ml, which are much
Catechin higher than that of chitosan. The antidiabetic activity in vitro assays shows that the ␣-glucosidase
Antioxidant inhibitory effect decreases in the order of catechin-g-chitosan > catechin > acarbose > chitosan, and the ␣-
Antidiabetic amylase inhibitory effect decreases in the order of acarbose > catechin-g-chitosan > catechin > chitosan.
The improved antioxidant and antidiabetic activities of catechin-g-chitosan are attributed to the phenolic
groups in the catechin residues.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction Graft copolymerization of phenolic compounds onto chitosan

Chitosan, the cationic (1-4)-2-amino-2-deoxy-␤-d-glucan
partly acetylated to the typical extent close to 0.25, is industrially
produced from marine chitin [1]. Due to its nontoxicity, good
biocompatibility and susceptibility to chemical modification,
chitosan has been shown to be a reactive and functional polymer
with a wide range of applications in biomedicine, pharmacol-
ogy and agriculture [2,3]. The cationic character, along with the
presence of reactive functional groups in chitosan, has given
it particular properties for incorporation with many phenolic
compounds. Previous study has shown that encapsulation of
(−)-epigallocatechin-3-gallate (EGCG) using caseinophosphopep-
tide and chitosan nanoparticles could be a potential approach to
enhance its antioxidant activity in biological systems [4]. Francesko
et al. [5] reported that collagen, collagen/hyaluronic acid (HA) and
collagen/HA/chitosan sponges loaded with EGCG, catechin and
gallic acid efficiently inhibited the myeloperoxidase activity.
∗ Corresponding author. Tel.: +86 514 87373646.
E-mail address: zhuwlyz@126.com (W. Zhu).
can introduce desired properties, such as antioxidant, antimicrobial
and antidiabetic activity, as well as enlarge the field of the potential
applications of chitosan [6–9]. In recent years, a number of initiator
systems have been developed to initiate grafting copolymerization.
Redox systems, such as ceric ammonium nitrate, potassium persul-
fate, ascorbic acid (Vc) and hydrogen peroxide (H2 O2 ), have been
frequently used to produce free radical sites on chitosan backbones
[9–11]. Till now, many studies have been carried out on the graft
copolymerization of phenolic acids, including gallic acid [6,9,12],
ferulic acid [7,13] and caffeic acid [6,8] onto chitosan. However,
only few studies have been focused on the graft copolymerization
of other phenolic compounds, such as flavonoids [14] and tannins
[15] onto chitosan.
Catechin is one of the biologically effective flavonoids present
in the human diet, particularly in wine and tea [16]. Various bio-
logical activities of catechin, including antioxidant, antimutagenic,
anticarcinogenic, antidiabetic, antiinflammatory and antimicrobial
properties, have been reported [17–19]. In order to improve the
antioxidant and antidiabetic activities of chitosan, the grafting of
catechin onto chitosan using Vc and H2 O2 as redox initiator in acetic
acid solution was investigated in this study. The obtained catechin-
grafted chitosan (catechin-g-chitosan) was characterized by many

http://dx.doi.org/10.1016/j.ijbiomac.2014.06.047
0141-8130/© 2014 Elsevier B.V. All rights reserved.
 W. Zhu, Z. Zhang / International Journal of Biological Macromolecules 70 (2014) 150–155
instrumental methods to confirm the conjugation. The antioxidant
and antidiabetic activities of catechin-g-chitosan were also deter-
mined. This study provides novel information on the structure and
bioactivities of catechin-g-chitosan.
2. Materials and methods
2.1. Reagents and chemicals
Chitosan with an average molecular weight (Mw ) of 250 kDa
was purchased from Shanghai Sangon Biotechnology Co. Ltd.
(Shanghai, China). The degree of deacetylation of chitosan
was determined to be 72% by proton nuclear magnetic reso-
nance (1 H NMR). (+)-Catechin, deuterium oxide (D2 O), ascorbic
acid (Vc), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 4-nitrophenyl-
␣-d-glucopyranoside (pNPG), ␣-glucosidase from Saccharomyces
cerevisiae (EC 3.2.1.20), porcine pancreatic ␣-amylase (PPA; EC
3.2.1.1) and azure starch were purchased from Sigma Chemical Co.
(St. Louis, MO, USA). Acarbose was obtained from Bayer HealthCare
Co. Ltd. (Bejing, China). All other reagents were of analytical grade.
2.2. Preparation of catechin-g-chitosan
Catechin was grafted onto chitosan by redox pair system accord-
ing to the method of Liu et al. [20] with some modifications. Firstly,
chitosan (0.5 g) was dissolved in 50 ml of 2% acetic acid solution
(v/v) in a two-necked round-bottom flask. The flask was then placed
in a water bath (20 ◦ C) and bubbled by a slow stream of nitrogen for
30 min to remove the dissolved oxygen. Then, 0.1 g of Vc, 0.5 g of
catechin and 2 ml of 5 M H2 O2 were added into the flask to ini-
tiate the reaction. The grafting process was carried out at 20 ◦ C
for 24 h. The nitrogen atmosphere was maintained throughout the
reaction period. Finally, the reaction mixture was dialyzed against
distilled water for 3 days and lyophilized to obtain catechin-g-
chitosan. The grafting ratio of catechin-g-chitosan was determined
by the Folin–Ciocalteu method and expressed as mg of catechin
equivalents per g (mgCAE/g) of the conjugate [21].
2.3. Characterization of catechin-g-chitosan
To verify the conjugation, catechin-g-chitosan was character-
ized by 1 H NMR, field-emission scanning electron microscopy
(FESEM), thermogravimetric analysis (TGA) and X-ray diffraction
(XRD). 1 H NMR spectra were obtained at 298 K on AVANCE-600
spectrometer (Bruker Inc., Germany). The morphology observation
was performed on S-4800 FESEM (Hitachi Ltd., Japan) at an accel-
erating voltage of 15 kV. Samples were mounted on a metal stub
and sputter-coated with gold. Thermal gravimetric analysis was
conducted with Pyris 1 TGA (Perkin-Elmer Ltd., USA). Each sample
(2.0 mg) was heated from 30 to 800 ◦ C in a platinum pan at a heat-
ing rate of 10 ◦ C/min under nitrogen flow of 20 ml/min. Powder XRD
measurements were performed on D8 Advance X-ray diffractome-
ter (Bruker AXS, Germany). The powder samples were placed on
low-background quartz sample holders. XRD patterns from 10◦ to
80◦ (2) were recorded at room temperature using Cu K␣ radiation.
2.4. Determination of antioxidant activity in vitro
2.4.1. Reducing power assay
Reducing power assay was carried out according to the
method of Oyaizu [22]. Various concentrations of sample solutions
(0.025–1 mg/ml, 2.5 ml) were mixed with 2.5 ml of 0.2 M sodium
phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide
(w/v). The mixture was incubated at 50 ◦ C for 20 min. After 2.5 ml
of 10% trichloroacetic acid (w/v) was added, the mixture was cen-
trifuged at 5000 rpm for 10 min. The upper layer (5 ml) was mixed
151
with 5 ml of deionized water and 1 ml of 0.1% ferric chloride (w/v),
and the absorbance was measured at 700 nm. Higher absorbance
indicates higher reducing power.
2.4.2. Hydroxyl radical-scavenging activity assay
The hydroxyl radical-scavenging activity was determined
according to the method of Jen with some modifications [23]. Vari-
ous concentrations of sample solutions (0.025–1 mg/ml, 1 ml) were
mixed with 1 ml of 9 mM FeSO4 solution and 1 ml of 9 mM salicylic
acid–ethanol solution. The reaction was initiated by the addition
of 1 ml H2 O2 (8.8 mM) to the mixture and carried out at 37 ◦ C for
1 h. Then the absorbance was determined at 510 nm. The hydroxyl
radical-scavenging activity was calculated by the following equa-
tion:
 A1 − A2

Hydroxyl radical scavenging activity (%) = 1 − × 100
A0
(1)
where A0 is the absorbance of the control (water instead of sam-
ple), A1 is the absorbance of the sample and A2 is the absorbance
of the sample only (salicylic acid–ethanol solution instead of FeSO4
and H2 O2 solutions).
2.4.3. DPPH radical-scavenging activity assay
The DPPH radical-scavenging activity was assayed according to
the method of Qiao with some modifications [24]. Briefly, 0.2 ml of
DPPH solution (0.4 mM DPPH in dehydrated alcohol) was mixed
with 1.0 ml of the sample (0.1–4 mg/ml) and 2.8 ml of distilled
water. The mixture was shaken vigorously and allowed to stand
at room temperature for 30 min. The absorbance of the mixture
was measured at 517 nm. The DPPH radical-scavenging activity was
calculated by the following equation:
 A1 − A2

DPPH radical scavenging activity (%) = 1 − × 100 (2)
A0
where A0 is the absorbance of the control (water instead of the
sample), A1 is the absorbance of the sample and A2 is the absorbance
of the sample only (water instead of DPPH).
2.5. Determination of antidiabetic activity in vitro
2.5.1. ˛-Glucosidase inhibitory effect assay
␣-Glucosidase inhibitory effect assay was performed according
to the method of Kim with some modifications [25]. The reaction
mixture contained 1 ml of 0.1 M potassium phosphate buffer (pH
6.8), 1 ml of substrate solution (2.5 mM pNPG in 0.1 M potassium
phosphate buffer), 1 ml of sample (0.05–1 mg/ml) and 1 ml of ␣-
glucosidase (0.2 U/ml in 0.1 M potassium phosphate buffer). After
incubation at 37 ◦ C for 15 min, 1 ml of 0.2 M Na2 CO3 was added to
stop the reaction. Then, the absorbance of the reaction mixture was
determined at 405 nm. ␣-Glucosidase inhibitory effect was calcu-
lated by the following equation:
 A1 − A2

␣-Glucosidase inhibitory effect (%) = 1 − × 100 (3)
A0
where A0 is the absorbance of the control (phosphate buffer instead
of the sample), A1 is the absorbance of the sample and A2 is the
absorbance of the blank (phosphate buffer instead of pNPG).
2.5.2. ˛-Amylase inhibitory effect assay
␣-Amylase inhibitory effect assay was carried out using the
method of Wang with some modifications [26]. Starch azure
(10 mg) as the substrate was suspended in 1 ml of 0.1 M Tris–HCl
buffer (pH 6.9) containing 0.01 M CaCl2 and incubated at 95 ◦ C
for 20 min. The reaction mixture contained 1 ml of the sample
152 W. Zhu, Z. Zhang / International Journal of Biological Macromolecules 70 (2014) 150–155

(0.5–10 mg/ml), 1 ml of PPA solution (1.4 U/ml in Tris–HCl buffer)

and 1 ml of cooled starch azure solution. After incubation at 37 ◦ C
for 10 min, 1 ml of 50% acetic acid (v/v) was added to stop the
reaction. The mixture was centrifuged at 5000 rpm for 30 min,
and the absorbance of the supernatant was determined at 595 nm.
␣-Amylase inhibitory activity was calculated by the following
equation:
 A1 − A2
 Chitosan
␣-Amylase inhibitory effect (%) = 1 − × 100 (4)
A0
where A0 is the absorbance of the control (Tris–HCl buffer instead
of the sample), A1 is the absorbance of the sample and A2 is the
absorbance of the blank (Tris–HCl buffer instead of starch azure).

2.6. Statistical analysis

Catechin-g-chitosan
Data were expressed as mean ± standard deviation (SD) and
evaluated by one-way analysis of variance (ANOVA) followed by 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0
Duncan’s multiple-range tests. All statistical analyses were carried ppm
out by using SAS for Windows. Difference was considered to be
1
significant if p < 0.05. Fig. 1. H spectra of chitosan and catechin-g-chitosan.

3. Results and discussion

3.1. Characterization of catechin-g-chitosan
In order to confirm the conjugation, the product was char-
acterized by chemical and instrumental methods. The grafting
ratio of catechin-g-chitosan is 65.89 mgCAE/g determined by
Folin–Ciocalteu method. 1 H NMR spectra of chitosan and catechin-
g-chitosan are shown in Fig. 1. Chitosan shows a peak at 1.9 ppm
for the methyl protons of acetylated glucosamine residues. Peaks
between 3.3 and 3.7 ppm are attributed to the protons of C–3,
C–4, C–5 and C–6 of the pyranose ring. And peaks at 2.9 and
4.4 ppm are assigned to protons of C-2 and C-1 of the glucosamine
residues, respectively. In the case of catechin-g-chitosan, a new
peak appeared at 4.6 ppm is attributed to the protons of C-2 of
catechin residues. This result confirms the successful grafting of
catechin with chitosan. However, no other proton peaks for cat-
echin is observed in the conjugate. Vittorio et al. found peaks at
6.6–6.9 and 8.8–9.2 ppm in the 1 H NMR spectra of dextran–catechin
conjugate, assigning to the aromatic and phenolic protons of cate-
chin residues, respectively. They indicated that H-6/H-8 of catechin
(A ring) and H-2 /H-5 of catechin (B ring) were the potential graft-
ing positions [27]. Liu et al. [28] suggested that H-6/H-8 of catechin
(A ring) were the grafting positions in inulin–catechin conjugate.
Our results show that the linkage occurred between amino groups
of chitosan and protons of catechin without any site specificity.
These further suggest that the grafting positions between cate-
chin and polysaccharides are mainly depended on the property of
polysaccharides rather than catechin.

Fig. 2. FESEM micrographs of chitosan (a, b) and catechin-g-chitosan (c, d) at different magnifications.
 W. Zhu, Z. Zhang / International Journal of Biological Macromolecules 70 (2014) 150–155
(a)
100
80
Weight (%)
60
40
20
Catechin-g-chitosan
0
0 100 200 300 400 500
Temperature (°C)
(b)
DTG(%/min)
Catechin-g-chitosan
0 200 400
Temperature (°C)
Fig. 3. TGA (a) and DTG (b) curves of chitosan and catechin-g-chitosan.
The morphology of chitosan and catechin-g-chitosan are
observed by FESEM. As shown in Fig. 2, the FESEM image of chitosan
shows a flaky nature with smooth surface due to stronger inter-
action between the chitosan molecules. However, a distinguished
change is observed in the surface morphology after grafting.
Catechin-g-chitosan exhibits a softened nature with porous sur-
face. In addition, the needle-like crystals observed in the conjugate
should be attributed to catechin residues [28]. The change in
morphology of the conjugate indicates that intermolecular and
intramolecular hydrogen bonds of chitosan have been greatly
decreased during the grafting process.
The thermograms of chitosan and catechin-g-chitosan are
shown in Fig. 3. The thermogram of chitosan exhibits two stages.
One in the range of 30–180 ◦ C with maximum decomposition
rate at 170 ◦ C is associated with loss water, the other in the
range of 200–420 ◦ C with maximum decomposition rate at 310 ◦ C
is ascribed to a complex process including dehydration of the
saccharide rings, depolymerization and decomposition of the poly-
mer [29]. However, the thermogram of catechin-g-chitosan shows
three degradation stages. The first stage ranges between 80 and
200 ◦ C with maximum decomposition rate at 140 ◦ C is due to the
weight loss of adsorbed and bound water. The second and third
stages are contributed to the decomposition of catechin-g-chitosan.
Notably, chitosan degrades more slowly than catechin-g-chitosan,
indicating that the thermal stability of chitosan is higher than
catechin-g-chitosan.
The powder X-ray diffractograms of chitosan and catechin-g-
chitosan are shown in Fig. 4. Chitosan exhibits a major peak at
around 20◦ due to 100 and 110 reflections [30]. However, XRD spec-
tra of catechin-g-chitosan show a crystalline area in the region of
10–50◦ due to the grafting of catechin onto chitosan backbones.
This indicates that the introduction of catechin onto chitosan has
increased the crystallinity of chitosan.
153
Intensity
Catechin-g-chitosan
Chitosan
Chitosan
600 700 800
0 20 40 60 80
2 (deg)
Fig. 4. XRD spectra of chitosan and catechin-g-chitosan.
Chitosan
3.2. Antioxidant activity in vitro of catechin-g-chitosan
As shown in Fig. 5a, the reducing power of all samples increases
with increasing concentrations. At the concentration of 0.1 mg/ml,
the reducing power of chitosan, catechin-g-chitosan, catechin and
Vc are 0.10, 0.37, 1.56 and 1.60, respectively. It has been reported
that the reducing power is generally associated with the pres-
600 800 ence of reductones because of their hydrogen-donating ability [31].
Among all samples tested, chitosan has the lowest reducing power
because the strong intramolecular hydrogen bonds weakened the
hydrogen-donating ability of hydroxyl and amino groups. Catechin-
g-chitosan exhibits an excellent reducing power than chitosan. This
is probably due to the grafted catechin moieties destroys the hydro-
gen bonds of chitosan and increases the hydrogen-donating ability
of the conjugate.
As shown in Fig. 5b, the hydroxyl radical-scavenging activities
of chitosan, catechin-g-chitosan, catechin and Vc at the concen-
tration of 1 mg/ml are 30.74, 46.81, 62.02 and 100%, respectively.
Compared with chitosan, catechin-g-chitosan exhibits a higher
hydroxyl radical-scavenging activity. This suggests that the scav-
enging ability of chitosan was enhanced after grafting by inhibiting
the generation of hydroxyl radical via a Fenton-like reaction.
DPPH radical-scavenging activities of each sample are presented
in Fig. 5c. At a concentration of 1 mg/ml, the DPPH-scavenging activ-
ities of chitosan, catechin-g-chitosan, catechin and Vc are 36.67,
67.08, 72.50 and 99.58%, respectively. The DPPH radical-scavenging
activity of catechin-g-chitosan is a little lower than that of cate-
chin. It is well known that catechin is a potent antioxidant, which
acts by scavenging free radicals and ROS [32]. In this study, we
attempt the conjugation of catechin with chitosan in order to
improve the antioxidant ability of chitosan. The above results show
that the conjugate exhibited stronger antioxidant activities than
the unmodified chitosan. Therefore, it suggests that conjugation of
antioxidant phenolics onto chitosan is a useful approach for gener-
ating of novel range of polymeric antioxidants.
3.3. Antidiabetic activity in vitro of catechin-g-chitosan
The ␣-glucosidase inhibitory effects of chitosan and catechin-g-
chitosan are shown in Fig. 6a. At the concentration of 1 mg/ml, the
␣-glucosidase inhibitory effects of chitosan, catechin-g-chitosan,
catechin and acarbose are 27.06, 72.45, 58.86 and 36.65%, respec-
tively. Acarbose, the drug frequently used for the treatment of
type 2 diabetes, exhibits lower ␣-glucosidase inhibitory effect than
catechin. This is consistent with previous study that flavonoids
are effective ␣-glucosidase inhibitors [33]. In addition, the ␣-
glucosidase inhibitory effect of catechin-g-chitosan is much higher
154 W. Zhu, Z. Zhang / International Journal of Biological Macromolecules 70 (2014) 150–155

(a) (a)
1.8
100
1.5
Absorbance at 700 nm

Inhibitory effect (%)

80
1.2
60
0.9
40
0.6
20
0.3

0 0
0 0.2 0.4 0.6 0.8 1
0 0.2 0.4 0.6 0.8 1
Concentration (mg/ml)
Concentration (mg/ml)
(b) 100
(b) 120

Inhibitory effect (%)

80
Scavenging activity (%)

90 60

40
60
20
30
0
0 2 4 6 8 10
0 Concentration (mg/ml)
0 0.2 0.4 0.6 0.8 1
Fig. 6. ␣-Glucosidase (a) and ␣-amylase (b) inhibitory effects of chitosan (--),
Concentration (mg/ml) catechin-g-chitosan (--), catechin (--) and acarbose (-♦-). Data are presented
as means ± SD of triplicates.
(c) 120
the treatment of type 2 diabetes, because of its potent inhibitory
Scavenging activity (%)

effect against ␣-glucosidase and mild inhibitory effect against ␣-

90
amylase.

60 4. Conclusions

30 Our results suggest that catechin can be successfully grafted

0
0 0.2 0.4 0.6
Concentration (mg/ml)
Fig. 5. The reducing power (a), hydroxyl radical (b) and DPPH radical-scavenging
activities (c) of chitosan (--), catechin-g-chitosan (--), catechin (--) and Vc (-♦-).
Data are presented as means ± SD of triplicates.
than catechin and chitosan, indicating that conjugation of catechin
with chitosan can enhance the ␣-glucosidase inhibitory effect of
chitosan.
The ␣-amylase inhibitory effects of chitosan and catechin-g-
chitosan are shown in Fig. 6b. At the concentration of 10 mg/ml,
the ␣-amylase inhibitory effects of chitosan, catechin-g-chitosan,
catechin and acarbose are 17.65, 36.47, 32.35 and 62.94%, respec-
tively. Among all samples tested, acarbose exhibits the highest
␣-amylase inhibitory effect. Catechin shows much lower ␣-
amylase inhibitory effect than acarbose. This result is in agreement
with that of previous study, which has demonstrated that plant-
derived phenolics have low inhibitory activity on ␣-amylase and
high inhibition potential against ␣-glucosidase [34]. Moreover,
catechin-g-chitosan shows higher ␣-glucosidase and ␣-amylase
inhibitory effects than catechin and chitosan. This suggests that
some synergistic action exists between catechin and chitosan on ␣-
glucosidase and ␣-amylase inhibitory effect. Therefore, our results
indicate that catechin-g-chitosan has the potential to contribute to
with chitosan by redox system. Antioxidant and antidiabetic activ-
ities in vitro of chitosan can be greatly enhanced by grafting
with catechin. Catechin-g-chitosan can be explored as a promising
0.8 1 antioxidant and antidiabetic agent in pharmaceutical industry.
References
[1] R.A.A. Muzzarelli, J. Boudrant, D. Meyer, N. Manno, M. DeMarchis, M.G. Paoletti,
Carbohydr. Polym. 87 (2012) 995–1012.
[2] M. Rinaudo, Prog. Polym. Sci. 31 (2006) 603–632.
[3] R. Jayakumar, D. Menon, K. Manzoor, S.V. Nair, H. Tamura, Carbohydr. Polym.
82 (2010) 227–232.
[4] B. Hu, Y. Ting, X. Zeng, Q. Huang, J. Agric. Food Chem. 61 (2012) 875–881.
[5] A. Francesko, D.S. da Costa, R.L. Reis, I. Pashkuleva, T. Tzanov, Acta Biomater. 9
(2013) 5216–5225.
[6] M. Bozic, S. Gorgieva, V. Kokol, Carbohydr. Polym. 87 (2012) 2388–2398.
[7] A. Aljawish, I. Chevalot, B. Piffaut, C. Rondeau-Mouro, M. Girardin, J. Jasniewski,
J. Scher, L. Muniglia, Carbohydr. Polym. 87 (2012) 537–544.
[8] A.O. Aytekin, S. Morimura, K. Kida, J. Biosci. Bioeng. 111 (2011) 212–216.
[9] J. Liu, J. Lu, J. Kan, C. Jin, Int. J. Biol. Macromol. 62 (2013) 321–329.
[10] A. Pourjavadi, G.R. Mahdavinia, M.J. Zohuriaan-Mehr, H. Omidian, J. Appl.
Polym. Sci. 88 (2003) 2048–2054.
[11] A.M.K. Najjar, W.M.Z.W. Yunus, M.B. Ahmad, M.Z.A. Rahman, J. Appl. Polym. Sci.
77 (2000) 2314–2318.
[12] W. Pasanphan, G.R. Buettner, S. Chirachanchai, Carbohydr. Res. 345 (2010)
132–140.
[13] S. Woranuch, R. Yoksan, Carbohydr. Polym. 96 (2013) 495–502.
[14] F. Sousa, G.M. Guebitz, V. Kokol, Process Biochem. 44 (2009) 749–756.
[15] M. Bozic, S. Gorgieva, V. Kokol, Carbohydr. Polym. 89 (2012) 854–864.
[16] S. Mandel, M.B. Youdim, Free Radic. Biol. Med. 37 (2004) 304–317.
[17] B. Yang, A. Kotani, K. Arai, F. Kusu, Chem. Pharm. Bull. 49 (2001) 747–751.
[18] M. Sabu, K. Smitha, R. Kultan, J. Ethnopharmacol. 83 (2002) 109–116.
[19] G.L. Tipoe, T.M. Leung, M.W. Hung, M.L. Fung, Cardiovasc. Hematol. Disord. Drug
Targets 7 (2007) 135–144.
 W. Zhu, Z. Zhang / International Journal of Biological Macromolecules 70 (2014) 150–155
[20] J. Liu, J. Lu, J. Kan, Y. Tang, C. Jin, Int. J. Biol. Macromol. 62 (2013) 85–93.
[21] V.L. Singleton, R. Orthofer, R.M. Lamuela-Raventos, Methods Enzymol. 299
(1999) 152–178.
[22] M. Oyaizu, Jpn. J. Nutr. 44 (1986) 307–315.
[23] J.F. Jen, M.F. Leu, T.C. Yang, J. Chromatogr. A 796 (1998) 283–288.
[24] D. Qiao, C. Ke, B. Hu, J. Luo, H. Ye, X. Zeng, Carbohydr. Polym. 78 (2009) 199–204.
[25] Y.M. Kim, Y.K. Jeong, M.H. Wang, W.Y. Lee, H.I. Rhee, Nutrition 21 (2005)
756–761.
[26] Y. Wang, Z. Yang, X. Wei, Int. J. Biol. Macromol. 47 (2010) 534–539.
[27] O. Vittorio, G. Cirillo, F. Iemma, G.D. Turi, E. Jacchetti, M. Curcio, S. Barbuti, N.
Funel, O.I. Parisi, F. Puoci, N. Picci, Pharm. Res. 29 (2012) 2601–2614.
155
[28] J. Liu, J.F. Lu, J. Kan, X.Y. Wen, C.H. Jin, Int. J. Biol. Macromol. 64 (2014)
76–83.
[29] J. Zhang, Y. Yuan, J. Shen, S. Lin, Eur. Polym. J. 39 (2003) 847–850.
[30] G.E. Luckachan, C.K.S. Pillai, Carbohydr. Polym. 64 (2006) 254–266.
[31] M.N. Siddaraju, S.M. Dharmesh, Mol. Nutr. Food Res. 51 (2007) 324–332.
[32] B.C. Scott, J. Butler, B. Halliwell, O.I. Aruoma, Free Radic. Res. Commun. 19 (1993)
241–253.
[33] M. Shibano, K. Kakutani, M. Taniguchi, M. Yasuda, K. Baba, J. Nat. Med. 62 (2008)
349–353.
[34] E. Apostolidis, C.M. Lee, J. Food Sci. 75 (2010) H97–H102.