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In Vitro Antioxidant Activity Evaluation of Gallic Acid Grafted Chitosan Conjugate Synthesized by Free Radical Induced Grafting Method Qiaobin Hu, Taoran Wang, Mingyong Zhou, Jingyi Xue, and Yangchao Luo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02255 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 7, 2016
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Journal of Agricultural and Food Chemistry
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In Vitro Antioxidant Activity Evaluation of Gallic Acid Grafted Chitosan
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Conjugate Synthesized by Free Radical Induced Grafting Method
3
Qiaobin Hu, Taoran Wang, Mingyong Zhou, Jingyi Xue, Yangchao Luo*
4
Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269, USA
5 6 7 8 9 10 11 12 13
*Corresponding author.
14 15
Mailing Address:
16
Dr. Yangchao Luo
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Assistant Professor, Department of Nutritional Sciences, University of Connecticut
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3624 Horsebarn Road Extension, U-4017, Storrs, CT 06269-4017, USA.
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Phone: (860) 486-2186.
20
Fax: (860) 486-3674.
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Email:
[email protected] 1
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ABSTRACT
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The major objective of this work was to develop a green and facile process to prepare gallic
24
acid-chitosan conjugate and comprehensively evaluate the physicochemical properties and
25
biological activities of as-prepared water soluble chitosan derivative. A free radical induced
26
grafting approach using ascorbic acid/hydrogen peroxide redox pair was adopted. The obtained
27
conjugate was characterized by FT-IR, UV-vis, XRD, and pKa analysis. The antioxidant
28
activities were evaluated by DPPH, ABTS, reducing power and ORAC assays. The results
29
showed that the mass ratio between gallic acid and chitosan played a vital role in determining the
30
grafting degree and zeta potential of the conjugates, with the ratio of 0.5:1 being the optimal ratio
31
that resulted in the highest grafting degree. The antioxidant assays demonstrated that conjugation
32
significantly improved the antioxidant activities, being dramatically higher than that of free
33
chitosan. It was notable that the DPPH and ABTS scavenging activities of conjugate at 0.4
34
mg/mL reached the same level as the free gallic acid at the equivalent concentration. Our study
35
demonstrated a green and facile synthesis approach to prepare a novel water soluble chitosan
36
derivative which may have promising potentials in the food industry.
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Keywords: Chitosan, gallic acid, conjugate, free radical induced grafting method, antioxidant
38
activity
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1. INTRODUCTION Chitosan
is
a
linear
polysaccharide
comprised
of
D-glucosamine
and
45
N-acetyl-D-glucosamine units linked by β-(1-4) linkages, derived from deacetylation of chitin.
46
Even though chitosan has been proven to possess a lot of excellent properties, including
47
biocompatibility, biodegradability, non-toxic, non-antigenic, available in abundance and low
48
cost,1-4 the poor water solubility is the major obstacle limiting its practical applications.
49
Numerous research works have been carried out to synthesize water soluble chitosan derivatives,
50
such as carboxymethyl chitosan, 5, 6 N-succinyl chitosan,7 and phosphonic chitosan.8
51
Another limitation of chitosan is that it is not a good chain breaking antioxidant due to the
52
lack of H-atom donors. In order to overcome the two problems simultaneously, considerable
53
efforts have been made to modify chitosan. Recently, the polyphenol-chitosan conjugates formed
54
by grafting different polyphenols onto chitosan backbone, have obtained tremendous
55
attention.9-11 Polyphenol is a group of secondary metabolites from plants which play vital roles in
56
growth, reproduction, pigmentation and defense mechanism of ultraviolet radiation and
57
pathogens.12, 13 More importantly, polyphenols are considered as a natural antioxidant and widely
58
employed in foods, drugs, and cosmetics. Therefore, water soluble chitosan-based conjugate with
59
prominent antioxidant activity can be obtained by grafting various polyphenols, for instance,
60
gallic acid,14 caffeic acid,15 ferulic acid,11 salicylic acid,16 and eugenol17 onto chitosan backbone.
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Gallic acid (GA), also known as 3, 4, 5-trihydroxybenzoic acid, is a natural phenolic acid widely
62
distributed in the plants kingdom, especially green tea. The GA grafted chitosan conjugate 3
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(GA-CS) has been synthesized via different methods. For instance, Pasanphan and colleagues
64
prepared water soluble GA-CS and proved that it has increased antioxidant activity against
65
DPPH and hydroxyl radicals.14 However, the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
66
(EDC)/N-hydroxysuccinimide (NHS) and other organic solvents, which could generate toxic
67
reaction products, were used in the fabrication process. Therefore, a safer method named the free
68
radical induced grafting approach has been developed in recent years and adopted to synthesize
69
GA-CS with potential to prevent oxidative stress in cell models.18, 19
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The objective of this study was to prepare, characterize, and investigate the antioxidant
71
activity of GA-CS prepared by free radical induced grafting approach, as a green and facile
72
process using ascorbic acid (Vc)/hydrogen peroxide (H2O2) redox pair. The effects of mass ratio
73
between GA and chitosan on the grafting degree was studied in detail, and the obtained GA-CS
74
was characterized by Fourier transform Infrared (FT-IR), UV-vis, X-ray diffraction (XRD), pKa
75
and scanning electron microscopy (SEM) analyses. Finally, the antioxidant activity of GA-CS
76
was evaluated by various in vitro chemical-based assays and compared with native chitosan and
77
GA.
78 79
2. MATERIALS AND METHODS
80
2.1. Materials
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Chitosan (low molecular weight), 2,2'-azino-bis(3-ethylbenzothiazoline-6)-sulphonic acid
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(ABTS), 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), flourescein sodium salt, and 4
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6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox) were purchased
from
84
Sigma-Aldrich Corp (St. Louis, MO, USA). Folin-Ciocalteau’s phenol reagent and
85
2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from MP Biomedicals (Irvine, CA, USA).
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L-ascorbic acid (Vc) was purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA). GA
87
and all other chemicals were of analytical grade and purchased from Acros Organics (Pittsburgh,
88
PA, USA).
89
2.2. Preparation of (GA-CS)
90
The GA-CS was synthesized according to the ascorbic acid/hydrogen peroxide redox pair
91
method with some modifications.20 Briefly, the stock solution of chitosan (10 mg/mL) was
92
prepared by dissolving in 2% acetic acid (v/v). To prepare GA-CS, 1 mL of 1.0 M H2O2
93
containing 0.054 g of ascorbic acid was added into 50 mL of chitosan solution and stirred for 30
94
min. Then, different amount of GA (the mass ratio of GA to chitosan: 0.1:1, 0.25:1, 0.5:1, 1:1,
95
and 2:1) was introduced into the above mixture and the reaction was carried out at room
96
temperature for 24 h. Afterwards, the mixture was poured into dialysis tubing (MWCO 10,000
97
Da) and dialyzed against deionized water for 48 h with changes of water every 8 h to remove
98
unreacted GA. Finally, the resulting solution was lyophilized using FreeZone Console Freeze
99
Dry System (Labconco, Kansas City, MO, USA) to obtain water soluble GA-CS solid samples.
100
2.3. Determination of grafting degree of GA-CS.
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The measurement of GA content in GA-CS was carried out according to the Folin–
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Ciocalteu method with slight modification.21 Briefly, the freeze-dried GA-CS powder was 5
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dissolved in deionized water at 1 mg/mL. Then, 0.5 mL of each sample was mixed with 1 mL of
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Folin–Ciocalteu reagent (10 times dilution) and reacted for 5 min in the dark, followed by the
105
addition of 2 mL 15% Na2CO3 and deionized water till 10 mL. The mixture was shaken and
106
maintained under room temperature for 2 h and the absorbance of the reaction mixture was read
107
at 750 nm using UV-vis spectrophotometer (Evolution 201, Thermo Fisher Scientific Inc.,
108
Waltham, MA, USA). GA was used as a standard and the grafting degrees of GA-CS were
109
expressed as mg of GA equivalents per g (mg GAE/g) of GA-CS.
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2.4. Characterization of GA-CS
111
The FT-IR spectra of chitosan, GA-CS and GA were recorded on a Fisher Scientific Nicolet
112
Is5 Spectrometer equipped with an iD7 ATR accessory (Thermo Fischer Scientific Inc., Waltham,
113
MA) by scanning from 4000 – 500 cm-1 at a resolution of 4 cm-1 and analyzed using OMNIC
114
software version 8.0.
115 116
The UV-vis spectra were determined by a UV-vis spectrophotometer (Evolution 201, Thermo Fisher Scientific Inc., Waltham, MA, USA) by scanning from 200 to 750 nm.
117
In order to investigate the crystallographic structures of chitosan and GA-CS, the powder
118
XRD spectra was acquired from 2θ = 5-75o on a Rigaku Ultima IV X-ray diffractometer using
119
Cu Kα radiation operating at 40 Kv and 44 Ma.
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The optimal GA-CS with the highest GA grafting degree was used for the pKa measurement.
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Briefly, 10 mg of GA-CS powder was dissolved in deionized water at concentration of 1 mg/mL
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and adjusted into different pH (4.0 – 11.0). The zeta potential of each sample was determined by 6
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laser Doppler microelectrophoresis technology using a Nano Zetasizer ZS (Malvern Instruments,
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Ltd., Worcestershire, UK).
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2.5 Morphological properties of GA-CS
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The morphology of freeze-dried GA-CS powder was observed under scanning electron
127
microscopy (SEM). The control was prepared following the same procedures described in
128
Section 2.2 but in the absence of ascorbic acid, hydrogen peroxide and GA. Samples were
129
deposited onto double adhesive carbon conductive tape before observed under SEM (JSM-6330F,
130
JEOL Ltd., Tokyo, Japan).
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2.6. Assay of antioxidant activity in vitro
132
2.6.1. DPPH radical scavenging assay
133
The DPPH radical scavenging activity was assayed according to the method of Dudonne
134
with slight modifications.22 Chitosan, GA-CS and GA were dissolved in deionized water for
135
preparation of a serious of concentrations (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL). Then, 200 µL
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of DPPH solution (0.4 mM DPPH in methanol) was mixed with 50 µL of each sample in a
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96-well microplate. The mixture was shaken vigorously and allowed to stand in the dark at room
138
temperature for 30 min and the absorbance was measured at 517 nm using a microplate reader
139
(BioTek Instruments, Inc., Winooski, VT, USA). The control was prepared by replacing sample
140
with deionized water, and meanwhile the sample background (sample added in methanol without
141
DPPH) was also measured to avoid any interference of background color.
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DPPH free radical scavenging activity (%) = (1 −
143
As − Ab ) ×100 Ac
144
Where As, Ab, and Ac are the absorbance of the sample, background, and control, respectively.
145
The concentration of the samples that caused 50% inhibition of DPPH radical (IC50) was
146
calculated from the graph of radicals scavenging activity percentage against sample
147
concentration.
148
2.6.2. ABTS radical scavenging assay
149
The spectrophotometric analysis of ABTS radical scavenging activity was carried out in a
150
96-well microplate according to the method of Xie et al23 with modifications. Brifely, 7 mM
151
ABTS aqueous solution was reacted with 4.95 mM potassium persulfate and the mixture was
152
stored in the dark at room temperature for 12 h to generate ABTS free radicals. Then, the ABTS
153
was diluted with PBS (0.2 M, pH 7.4) to a concentration that the absorbance at 734 nm was 0.7 ±
154
0.02 to provide a working solution before usage. The absorbance of a mixture of 200 µL ABTS
155
working solution and 20 µL of each sample was monitored at 734 nm. The control was prepared
156
by replacing sample with deionized water, and meanwhile the sample background (sample added
157
in PBS without ABTS) was also measured to avoid any interference of background color. ABTS free radical scavenging activity (%) = (1 −
158
As − Ab ) × 100 Ac
159
Where As, Ab, and Ac are the absorbance of the sample, background, and control, respectively.
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The concentration of the sample that caused 50% inhibition of ABTS radical (IC50) was
161
calculated from the graph of radicals scavenging activity percentage against sample
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concentration. 8
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Journal of Agricultural and Food Chemistry
2.6.3. Reducing power assay
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The reducing power of chitosan, GA-CS and GA was determined following a previous
165
report with some modifications.24 The reaction was carried out in a mixture containing 50 µL of
166
each sample (1mg/mL), 50 µL of sodium phosphate buffer (PBS, 0.2 M, pH 6.6), and 30 µL of
167
K3Fe(CN)6 (1%, w/v) and was incubated in a 96-well microplate at 50 oC for 20 min. After
168
addition of 30 µL of FeCl3 (0.1%, w/v) and 50 µL of trichloroacetic acid (10%, w/v), the
169
absorbance of the mixture was measured at 700 nm, and the higher absorbance indicated a higher
170
reducing power. The control was prepared by replacing FeCl3 with deionized water.
171
Reducing power = Ac − As
172
Where As and Ac are the absorbance of sample and control, respectively. The concentration of the
173
sample that provided 0.5 of absorbance (IC50) was calculated.
174
2.6.4. Oxygen radical antioxidant capacity assay (ORAC)
175
The antioxidant capacity of chitosan, GA-CS and GA were also determined by the ORAC
176
assay, as reported by Zulueta et al.25 It was evaluated by scavenging peroxide radicals that were
177
generated by AAPH, preventing the loss of fluorescence of the probe. The reaction was carried
178
out in a 96-well microplate and each well contained 20 µL of sample (5 µg/mL)/blank
179
(phosphate buffer solution, pH 7.4)/standard (8 µM), and 120 µL fluorescein (70 nM). The
180
mixture was allowed to stay at 37 oC for 15 min, followed by addition of 60 µL AAPH (12 mM)
181
in PBS solution (pH 7.4). The fluorescence of the mixture was recorded every 1 min for 80 min
182
at excitation and emission wavelengths of 485 and 520 nm, respectively. The ORAC values of
183
samples were expressed as µM Trolox equivalents (µM TE). 9
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ORAC (µM TE) =
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Ct × ( AUCs − AUCb ) × k ( AUCt − AUCb )
185
Where Ct is the concentration of trolox (8 µM), k is the sample dilution factor, and the AUC is the
186
area below the fluorescence decay curve of the sample (AUCs), blank (AUCb), and trolox (AUCt)
187
that were calculated from the following equation: i =80
AUC = 1 + ∑i =1
188
fi f0
189
Where f0 is the initial fluorescence and fi is the fluorescence at time i.
190
2.7. Stability of GA-CS
191
The stability of GA-CS was studied by measuring the total phenol content using UV-vis
192
spectrophotometer (Evolution 201, Thermo Fisher Scientific Inc., Waltham, MA, USA)
193
compared with GA, as a function of storage time. The GA-CS freeze-dried powder and GA were
194
dissolved in deionized water at a concentration of 1.0 mg/mL and stored at refrigeration
195
condition (4 oC) for up to 14 days. At each time point, samples were subjected to the phenolic
196
content measurement following the sample method as described in the Section 2.3.
197
2.8 Statistical Analysis
198
All experiments were conducted in triplicate and data were expressed as mean ± standard
199
deviation. One-way analysis of variance (ANOVA) was performed with the Tukey’s
200
multiple-comparison test to compare the significant difference among samples, using SPSS
201
package (SPSS 13.0 for windows, SPSS Inc., Chicago, IL, USA). The significance level (P) was
202
set as 0.05. 10
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3. RESULTS AND DISCUSSION
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3.1. Optimization of grafting degree
205
The antioxidant activity of chitosan-based conjugates strongly depends on the amount of
206
grafted antioxidant molecules on the chitosan backbone.11, 26 Therefore, in order to synthesize the
207
optimal GA-CS and investigate its physicochemical properties, the conjugates with different
208
mass ratio of chitosan to GA (0.1:1, 0.25:1, 0.5:1, 1:1, and 2:1) were prepared and the grafting
209
degree were evaluated by measuring GA content and zeta potential. As shown in Fig. 1A, the
210
grafting degree significantly increased as the increase of mass ratio up to 0.5:1, followed by a
211
subsequent decrease when the mass ratio reached to 1:1 and 2:1. In this study, the number of
212
macromolecular chitosan radicals induced by the constant concentration of Vc/H2O2 initiator was
213
the same in different samples. The enhancement of grafting degree was possibly due to the
214
increasing of GA ratio within the critical range. However, beyond that range, the excess amount
215
of free GA molecules may inhibit grafting process causing a reduction of grafting degree. This
216
observation was also reported in a previous study,10 showing the highest grafting degree (88.53
217
mg GAE/g GA-g-chitosan) was obtained when the mass ratio of GA to chitosan was 0.5:1.
218
Zeta potential of the conjugates with different mass ratio of GA to chitosan is shown in Fig.
219
1B. It is widely used to quantify the magnitude of the charge in dispersion medium. The change
220
of zeta potential exhibited an opposite trend with grafting degree as affected by the mass ratio of
221
GA to chitosan. Due to the abundant protonated amino groups on its backbone, the native
222
chitosan exhibited a very high magnitude of positive charge (64.2 mV) in acidic condition. On 11
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the contrary, the GA-CS showed much smaller magnitude of positive charge in the range of
224
26.3-33.6 mV, and the higher grafting degree resulted in a less positive charge. This significant
225
reduction in zeta potential may be explained by the less available protonated amino groups due
226
to the formation of peptide bond with GA. The lowest zeta potential was observed when the
227
grafting degree at the GA/chitosan ratio of 0.5:1, confirming the highest grafting degree.
228
Therefore, the GA-CS with a mass ratio of GA to chitosan 0.5:1 was chosen as the optimal
229
sample for subsequent characterizations.
230
3.2. Characterization of GA-CS
231
3.2.1. FT-IR study
232
The FT-IR spectra of chitosan, GA-CS powder, and GA are shown in Fig. 2A to identify
233
their molecular interactions. The spectrum of GA showed strong absorbance bands at 3491 and
234
3269 cm-1 which were attributed to –OH stretching on benzene ring, as well as several peaks
235
within 1450-1600 cm-1 representing the aromatic ring C=C stretching.27 Several typical peaks of
236
amide bonds were observed in the chitosan spectrum, i.e. the absorbance bands at 1651, 1550,
237
and 1316 cm-1, corresponding to C=O stretching (amide I), N–H bending (amide II) and C–N
238
stretching (amide III), respectively.28 There was an absorbance peak at 1589 cm-1, corresponding
239
to N–H bending of the primary amine.29 Besides, the saccharide structure of chitosan was
240
characterized by the asymmetric stretching of C–O–C bridge at 1150 cm-1 and C–O stretching at
241
1060 and 1024 cm-1.30 Compared to chitosan, the N–H bending of the primary amine at 1589
242
cm-1 was not observed in GA-CS, indicating that conjugation reactions occurred at -NH2 sites on 12
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chitosan backbone. Furthermore, the absorbance peaks of C=O stretching and N–H bending
244
shifted from 1651 and 1550 to 1631 and 1531 cm-1, respectively, and became stronger in GA-CS,
245
suggesting the formation of NH–CO group.31 In addition, a new vibration peak at 1731 cm-1 was
246
observed in GA-CS due to the C=C stretching in esters, representing the formation of ester bond
247
between the carboxyl groups of GA and hydroxyl groups of chitosan. The FT-IR results further
248
proved that the conjugation of GA occurred at both amino and hydroxyl groups of chitosan.
249
3.2.2. UV-vis study
250
The UV-vis spectra of chitosan, GA-CS and GA were shown in Fig. 2B. Chitosan showed
251
no absorption peak ranging from 200 to 750 nm, while GA exhibited one characteristic
252
absorption band at 211 nm and another band at 259 nm, which were assigned to the π-system of
253
the benzene ring.23 These two characteristics peaks were observed in GA-CS, confirming the
254
successful conjugation. In addition, a slight red shift of UV-vis absorption peak from 259 nm to
255
261 nm was observed in the spectrum of GA-CS, attributing to the lower energy required for the
256
n–π* and π–π* transition due to the formation of the covalent linkage between chitosan and
257
GA.20
258
3.2.3. Crystallographic structure and thermal property
259
The crystallographic structures of chitosan, GA-CS and GA samples were determined by
260
XRD. As presented in Fig. 2C, two strong peaks were observed in the diffractogram of native
261
chitosan at 2θ = 10.62 o and 20.11 o, pointing out high crystallinity of chitosan.32 However, the
262
peaks of GA-CS were shifted to 12.52 o and 23.29 o, respectively. In addition, the peaks became 13
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broader and weaker, indicating the looseness in packing of chitosan caused by successful
264
introduction of GA. The XRD result revealed that the intensity of inter- and intramolecular
265
hydrogen bonds in GA-CS were significantly reduced, resulting in remarkable decrease in the
266
crystallinity. Therefore, the water solubility of GA-CS was significantly increased compared with
267
native chitosan. Similar results were also reported in previous studies that conjugation of
268
phenolic acids to chitosan induced the increase of amorphous phase and looseness of chain
269
packing.14, 33
270
3.2.4. The pKa of GA-CS
271
The zeta potential and appearance of conjugate with different pH values were shown in Fig.
272
2D. The freeze dried GA-CS was able to directly dissolve in deionized water with a pH of 5.5.
273
The change of zeta potential of GA-CS was inversely proportional to the pH change. At acidic
274
range of pH values (4.0–7.0), the GA-CS exhibited a high transparency and the zeta potential
275
gradually decreased from 29.9 to 5.37 mV as the increase of pH. At pH 8.0, precipitation was
276
observed in GA-CS and the color changed to yellow. Meanwhile, the zeta potential decreased to
277
1.26 mV, which is close to 0 representing the pKa value of GA-CS. Subsequently, with further
278
increase in pH, the GA-CS solution turned more opaque with dark green color and severe
279
precipitation. This phenomenon can be explained by the oxidation of GA and formation of
280
polymerized compounds of o-quinone derivatives which was facilitated by alkalinization,
281
resulting in green coloration of the medium.34
282
To sum up, the GA has been successfully grafted onto CS which was evidenced by the 14
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results mentioned above. In this study, the Vc/H2O2 pair was employed as a redox trigger to
284
initiate the grafting of GA on the chitosan backbone. The possible mechanism for the preparation
285
of GA-CS is illustrated in Fig.3. Based on previous studies, the ascorbic acid is oxidized by H2O2
286
into tricarbonyl ascorbate free radical (AscH•) with the generation of hydroxyl radicals (HO•).35
287
Afterwards, the HO• abstracts hydrogen atom from two major target groups on chitosan,
288
including the amino group at C-2 and hydroxyl group at C-6 position, resulting in the formation
289
of macromolecular chitosan radicals. Curcio and colleagues also suggested that α-methylene is a
290
possible group that the HO• will attack.20 Then, GA can act as an acceptor for macromolecular
291
chitosan radicals, and GA-CS is synthesized by forming ester linkage (carboxyl group of GA and
292
hydroxyl group of chitosan) and peptide linkage (carboxyl group of GA and amino group of
293
chitosan). A different hypothesis has also been proposed that the insertion of GA on chitosan
294
chain occurs at C-2 and C-5 of the aromatic ring of GA.20 The obtained GA-CS was a water
295
soluble conjugate. Compared to the GA-grafted chitosan conjugates that are prepared by other
296
methods
297
enzyme-mediated strategy using tyrosinase or laccase,37 the free radical method showed some
298
advantages, such as no generation of toxic products, low energy consumption and low cost.
299
3.3. Morphological properties of GA-CS
including
activated
ester-mediated
modification
using
EDC/NHS36
and
300
The surface morphology of freeze dried chitosan and GA-CS was observed under SEM,
301
shown in Fig. 4. There were several differences between these two samples. The freeze dried
302
chitosan appeared to be highly packing structural flakes with smooth surface and fiber-like 15
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structure on the edge, owing to the strong inter- and intramolecular hydrogen bonds. Although
304
freeze dried GA-CS also exhibited the flake-like morphology, it was distinct from that of
305
chitosan in the following points. Firstly, the fiberous edge was not observed on freeze dried
306
GA-CS. Secondly, compared to the dense chitosan flake, the surface of freeze dried GA-CS was
307
rough and porous, indicating the greatly decreased hydrogen bonds and the reduction of
308
crystallinity during the grafting process. This result was in accordance to the previous studies.38,
309
39
310
3.4. Antioxidant activity of GA-CS
311
3.4.1. DPPH radicals scavenging capacity
312
DPPH, a stable organic free radical, has been widely used to evaluate the antioxidant
313
activity of various samples. The DPPH scavenging abilities of chitosan, GA-CS and GA are
314
shown in Fig. 5A. With the increase of chitosan concentration in the range of 0.05 to 1.0 mg/mL,
315
chitosan showed a slightly increased trend of scavenging activity but the highest value was only
316
19.63%. As a natural antioxidant molecule, GA showed excellent DPPH free radical scavenging
317
activity at concentrations as low as 0.05 mg/mL which was significantly greater than that of
318
GA-CS and chitosan. The scavenging activity of GA-CS significantly increased as the increase
319
of concentration up to 0.4 mg/mL and reached its plateau at 0.6 mg/mL with the highest
320
scavenging activity of 94.5%, which was the same as the free GA molecule. The IC50 values for
321
chitosan, GA-CS and GA against DPPH radicals were 4.299, 0.196 and 0.045 mg/mL,
322
respectively. It has been reported that the DPPH scavenging activities were positively correlated 16
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with polyphenol contents in the chitosan-based conjugates.26 By comparing to other phenolic
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acid-chitosan conjugate synthesized using the same method, which had 74.54% DPPH
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scavenging activity with a 45.8 mg GAE/g of grafting degree,28 the GA-CS in the present study
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showed both greater grafting degree and superior DPPH scavenging activity.
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3.4.2. ABTS radicals scavenging capacity
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The ABTS assay has been widely applied for testing the preliminary free radicals
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scavenging ability of antioxidant compounds. Nevertheless, the scavenging capacity of GA-CS
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synthesized by the Vc/H2O2 redox pair has not been reported. As shown in Fig. 5B, our results
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revealed that the scavenging capacity against ABTS radicals of all samples, including chitosan,
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GA-CS and GA shared similar trends with the results in the DPPH assay. The scavenging activity
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of chitosan increased slowly with the increase of concentration. At the concentration range from
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0.05-0.2 mg/mL, the scavenging activity of GA-CS dramatically increased. It is notable that the
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scavenging activity of GA-CS increased rapidly and reached it plateau at 0.4 mg/mL (99.49%),
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which was even slightly higher than that of GA control (99.1%), which might be explained by
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the synergistic reaction between chitosan and GA in the conjugate. The IC50 values for chitosan,
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GA-CS and GA were 3.526, 0.076 and 0.003 mg/mL, respectively. Yu et al. prepared GA grafted
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carboxymethyl chitosan conjugate via EDC/NHS modification method and reported that the IC50
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is 0.291 mg/mL.40 The comparison indicated that the GA-CS prepared by free radical induced
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grafting method had superior ABTS radical scavenging activity than that synthesized by
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activated ester-mediated approach. The ABTS radical scavenging activity of as-prepared GA-CS 17
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is also dramatically higher than other polyphenolic acid-chitosan conjugates, such as ferulic
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acid41 which was reported to have a IC50 of 0.20 ± 0.02 mg/mL. This may be ascribed to the fact
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that GA contains more phenolic hydroxyl groups than ferulic acid does, and phenolic hydroxyl
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groups are known to play an important role in the hydrogen and electron donating ability.
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3.4.3. Reducing power
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Reducing power may act as a significant indicator of the antioxidant activity. It is based on
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the reduction of Fe3+ to Fe2+ by antioxidant agent, followed by the formation of ferric-ferrous
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complex which has a maximum absorption at 700 nm. As shown in Fig. 5C, native chitosan
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showed negligible reducing powder at all concentrations tested in this study, while GA-CS
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showed gradually increased reducing powder as increase of concentration and eventually reached
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similar capacity with free GA. At the concentration of 1.0 mg/mL, the reducing power of
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chitosan, GA-CS and GA were 0.009, 2.660 and 2.849, respectively. The IC50 values of chitosan,
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GA-CS and GA were 79.365, 0.067 and 0.010, respectively. Like the scavenging activity against
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DPPH and ABTS radicals, it is worth mentioning that the reducing of GA-CS in our study is also
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much higher that other phenolic acids-chitosan conjugates reported in previous studies.26, 28
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3.4.4. Oxygen radical antioxidant capacity (ORAC)
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The ORAC method has been widely adopted to evaluate the antioxidant activity of foods
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and active compounds.42 However, to our best knowledge, the ORAC assay among modified
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chitosans, especially the polyphenol-chitosan conjugates, was rarely reported. In fact, the ORAC
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value of GA-grafted-chitosan has only been published in one study.43 The GA-CS, prepared 18
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using EDC/NHS chemical cross-linker with a grafting degree of 58.1 ± 1.2 mg GAE/g, exhibited
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a 19-fold higher ORAC value than native chitosan. In our study, the GA-CS showed an enhanced
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capacity to scavenge peroxyl radicals with an ORAC value 1.83 folds higher than native chitosan.
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The ORAC values of chitosan, GA-CS and GA were 1117.2 ± 145.8, 2043.7 ± 352.0, and 2843.8
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± 335.0, respectively (Fig. 5D). The less increase of ORAC value of GA-CS compared to the
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previous study might attribute to the different preparation method.
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3.5. Stability of GA-CS
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The free radical-initiating reaction is sensitive to oxygen in the condition, resulting in the
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degradation of GA and the decrease of GA contents in chitosan conjugate.10 Meanwhile, the
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stability of GA-CS is also greatly affected by temperature that higher temperature may induce
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faster degradation of GA in the conjugate.44 Therefore, the evaluation of the stability is critical to
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explore the applications of GA-CS. In the present work, the stability of GA-CS was tested under
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refrigeration (4 ºC) condition, by monitoring the total phenol content at designated time points
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(Fig. 6). For both GA-CS and GA, the total phenol content gradually decreased as the extension
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of storage time and reached to 90.9% and 87.2% at day 14, respectively, owing to the
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degradation of GA. This result revealed that the GA-CS was able to slightly protect GA from
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degradation over long period of time due to the chemical conjugation between the chitosan and
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GA.
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In summary, in the present study, GA-CS was successfully prepared using free radical
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induced grafting approach. The grafting of GA was confirmed by FT-IR and UV-vis, suggesting 19
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that the grafting reactions occurred between amino and hydroxyl groups of chitosan and carboxyl
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groups of GA. The result of XRD analyses indicated that the enhanced water solubility of
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GA-CS may be attributed to the decrease in crystallinity, which was further evidenced by the
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morphological observation. The mass ratio of 0.5:1 between GA and chitosan was considered as
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the optimal ratio by determining the grafting degree and zeta potential. In addition, GA-CS
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exhibited excellent reducing power, ORAC value and scavenging capacities against DPPH and
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ABTS free radicals which were all superior to native chitosan. Therefore, this novel water
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soluble chitosan-based conjugate prepared by green synthesis method could be explored as a
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promising antioxidant agent.
392 393
ACKNOLWDGEMENT
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This work is supported by the Startup funding from University of Connecticut. The SEM study
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was performed using the facilities in the UConn/FEI Center for Advanced Microscopy and
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Materials Analysis (CAMMA).
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Figure captions:
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Fig. 1. Effects of different mass ratio of gallic (GA) and chitosan (CS) on the (A) grafting degree
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and (B) zeta potential of gallic acid-chitosan conjugate (GA-CS). The values sharing different
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superscript letters were statistically different at P