In Vitro Antioxidant-Activity Evaluation of Gallic-Acid-Grafted Chitosan

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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

18

3624 Horsebarn Road Extension, U-4017, Storrs, CT 06269-4017, USA.

19

Phone: (860) 486-2186.

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Fax: (860) 486-3674.

21

Email: [email protected] 1

ACS Paragon Plus Environment


22

ABSTRACT

23

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.

37

Keywords: Chitosan, gallic acid, conjugate, free radical induced grafting method, antioxidant

38

activity

39 40 41

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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.

61

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



63

(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

70

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

81

Chitosan (low molecular weight), 2,2'-azino-bis(3-ethylbenzothiazoline-6)-sulphonic acid

82

(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).

86

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.

101

The measurement of GA content in GA-CS was carried out according to the Folin–

102

Ciocalteu method with slight modification.21 Briefly, the freeze-dried GA-CS powder was 5



103

dissolved in deionized water at 1 mg/mL. Then, 0.5 mL of each sample was mixed with 1 mL of

104

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.

110

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.

120

The optimal GA-CS with the highest GA grafting degree was used for the pKa measurement.

121

Briefly, 10 mg of GA-CS powder was dissolved in deionized water at concentration of 1 mg/mL

122

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,

124

Ltd., Worcestershire, UK).

125

2.5 Morphological properties of GA-CS

126

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).

131

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

136

of DPPH solution (0.4 mM DPPH in methanol) was mixed with 50 µL of each sample in a

137

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.

142 7



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.

160

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

162

concentration. 8


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2.6.3. Reducing power assay

164

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



ORAC (µM TE) =

184

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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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203

3. RESULTS AND DISCUSSION

204

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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223

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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243

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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283

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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303

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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323

with polyphenol contents in the chitosan-based conjugates.26 By comparing to other phenolic

324

acid-chitosan conjugate synthesized using the same method, which had 74.54% DPPH

325

scavenging activity with a 45.8 mg GAE/g of grafting degree,28 the GA-CS in the present study

326

showed both greater grafting degree and superior DPPH scavenging activity.

327

3.4.2. ABTS radicals scavenging capacity

328

The ABTS assay has been widely applied for testing the preliminary free radicals

329

scavenging ability of antioxidant compounds. Nevertheless, the scavenging capacity of GA-CS

330

synthesized by the Vc/H2O2 redox pair has not been reported. As shown in Fig. 5B, our results

331

revealed that the scavenging capacity against ABTS radicals of all samples, including chitosan,

332

GA-CS and GA shared similar trends with the results in the DPPH assay. The scavenging activity

333

of chitosan increased slowly with the increase of concentration. At the concentration range from

334

0.05-0.2 mg/mL, the scavenging activity of GA-CS dramatically increased. It is notable that the

335

scavenging activity of GA-CS increased rapidly and reached it plateau at 0.4 mg/mL (99.49%),

336

which was even slightly higher than that of GA control (99.1%), which might be explained by

337

the synergistic reaction between chitosan and GA in the conjugate. The IC50 values for chitosan,

338

GA-CS and GA were 3.526, 0.076 and 0.003 mg/mL, respectively. Yu et al. prepared GA grafted

339

carboxymethyl chitosan conjugate via EDC/NHS modification method and reported that the IC50

340

is 0.291 mg/mL.40 The comparison indicated that the GA-CS prepared by free radical induced

341

grafting method had superior ABTS radical scavenging activity than that synthesized by

342

activated ester-mediated approach. The ABTS radical scavenging activity of as-prepared GA-CS 17



343

is also dramatically higher than other polyphenolic acid-chitosan conjugates, such as ferulic

344

acid41 which was reported to have a IC50 of 0.20 ± 0.02 mg/mL. This may be ascribed to the fact

345

that GA contains more phenolic hydroxyl groups than ferulic acid does, and phenolic hydroxyl

346

groups are known to play an important role in the hydrogen and electron donating ability.

347

3.4.3. Reducing power

Page 18 of 32

348

Reducing power may act as a significant indicator of the antioxidant activity. It is based on

349

the reduction of Fe3+ to Fe2+ by antioxidant agent, followed by the formation of ferric-ferrous

350

complex which has a maximum absorption at 700 nm. As shown in Fig. 5C, native chitosan

351

showed negligible reducing powder at all concentrations tested in this study, while GA-CS

352

showed gradually increased reducing powder as increase of concentration and eventually reached

353

similar capacity with free GA. At the concentration of 1.0 mg/mL, the reducing power of

354

chitosan, GA-CS and GA were 0.009, 2.660 and 2.849, respectively. The IC50 values of chitosan,

355

GA-CS and GA were 79.365, 0.067 and 0.010, respectively. Like the scavenging activity against

356

DPPH and ABTS radicals, it is worth mentioning that the reducing of GA-CS in our study is also

357

much higher that other phenolic acids-chitosan conjugates reported in previous studies.26, 28

358

3.4.4. Oxygen radical antioxidant capacity (ORAC)

359

The ORAC method has been widely adopted to evaluate the antioxidant activity of foods

360

and active compounds.42 However, to our best knowledge, the ORAC assay among modified

361

chitosans, especially the polyphenol-chitosan conjugates, was rarely reported. In fact, the ORAC

362

value of GA-grafted-chitosan has only been published in one study.43 The GA-CS, prepared 18


Page 19 of 32


363

using EDC/NHS chemical cross-linker with a grafting degree of 58.1 ± 1.2 mg GAE/g, exhibited

364

a 19-fold higher ORAC value than native chitosan. In our study, the GA-CS showed an enhanced

365

capacity to scavenge peroxyl radicals with an ORAC value 1.83 folds higher than native chitosan.

366

The ORAC values of chitosan, GA-CS and GA were 1117.2 ± 145.8, 2043.7 ± 352.0, and 2843.8

367

± 335.0, respectively (Fig. 5D). The less increase of ORAC value of GA-CS compared to the

368

previous study might attribute to the different preparation method.

369

3.5. Stability of GA-CS

370

The free radical-initiating reaction is sensitive to oxygen in the condition, resulting in the

371

degradation of GA and the decrease of GA contents in chitosan conjugate.10 Meanwhile, the

372

stability of GA-CS is also greatly affected by temperature that higher temperature may induce

373

faster degradation of GA in the conjugate.44 Therefore, the evaluation of the stability is critical to

374

explore the applications of GA-CS. In the present work, the stability of GA-CS was tested under

375

refrigeration (4 ºC) condition, by monitoring the total phenol content at designated time points

376

(Fig. 6). For both GA-CS and GA, the total phenol content gradually decreased as the extension

377

of storage time and reached to 90.9% and 87.2% at day 14, respectively, owing to the

378

degradation of GA. This result revealed that the GA-CS was able to slightly protect GA from

379

degradation over long period of time due to the chemical conjugation between the chitosan and

380

GA.

381

In summary, in the present study, GA-CS was successfully prepared using free radical

382

induced grafting approach. The grafting of GA was confirmed by FT-IR and UV-vis, suggesting 19



Page 20 of 32

383

that the grafting reactions occurred between amino and hydroxyl groups of chitosan and carboxyl

384

groups of GA. The result of XRD analyses indicated that the enhanced water solubility of

385

GA-CS may be attributed to the decrease in crystallinity, which was further evidenced by the

386

morphological observation. The mass ratio of 0.5:1 between GA and chitosan was considered as

387

the optimal ratio by determining the grafting degree and zeta potential. In addition, GA-CS

388

exhibited excellent reducing power, ORAC value and scavenging capacities against DPPH and

389

ABTS free radicals which were all superior to native chitosan. Therefore, this novel water

390

soluble chitosan-based conjugate prepared by green synthesis method could be explored as a

391

promising antioxidant agent.

392 393

ACKNOLWDGEMENT

394

This work is supported by the Startup funding from University of Connecticut. The SEM study

395

was performed using the facilities in the UConn/FEI Center for Advanced Microscopy and

396

Materials Analysis (CAMMA).

397 398

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Figure captions:

510

Fig. 1. Effects of different mass ratio of gallic (GA) and chitosan (CS) on the (A) grafting degree

511

and (B) zeta potential of gallic acid-chitosan conjugate (GA-CS). The values sharing different

512

superscript letters were statistically different at P

In Vitro Antioxidant-Activity Evaluation of Gallic-Acid-Grafted Chitosan

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