Antioxidant Nanocomplexes for Delivery of Epigallocatechin-3-gallate

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Antioxidant Nanocomplexes for Delivery of Epigallocatechin-3-gallate Bing Hu, Fengguang Ma , Yingkang Yang, Minhao Xie, Chen Zhang, Ye Xu, and Xiaoxiong Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00931 • Publication Date (Web): 11 Apr 2016 Downloaded from http://pubs.acs.org on April 17, 2016

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Journal of Agricultural and Food Chemistry

Antioxidant Nanocomplexes for Delivery of Epigallocatechin-3-gallate Bing Hu*, Fengguang Ma, Yingkang Yang, Minhao Xie, Chen Zhang, Ye Xu, Xiaoxiong Zeng* College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

∗

To whom correspondence should be addressed. Fax (Tel.): 86-13915983201; Email: [email protected] (B. Hu)；Fax (Tel.):86-25-84396791; Email: [email protected] (X. Zeng) 1

ACS Paragon Plus Environment


1

ABSTRACT

2

Modification of chitosan (CS) through grafting with caffeic acid (CA, CA-g-CS) and

3

ferulic acid (FA, FA-g-CS) significantly improved the solubility of CS under neutral

4

and alkaline environments. Spherical and physicochemical stable nanocomplexes

5

assembled from the phenolic acid grafting CS and caseinophosphopeptide (CPP)

6

were obtained with particle size less than 300 nm and zeta potential of less than +30

7

mV. The net polymer nanocomplexes composed with the phenolic acid grafting CS

8

and CPP showed strong antioxidant activity. The encapsulation efficiencies of

9

epigallocatechin-3-gallate (EGCG) in the CA-g-CS-CPP nanocomplexes and

10

FA-g-CS-CPP nanocomplexes were 88.1 ± 6.7% and 90.4 ± 4.2%, respectively.

11

Improved delivery properties of EGCG were achieved after loading with the

12

antioxidant nanocomplexes, including controlling release of EGCG under simulated

13

gastric environments and preventing its degradation under neutral and alkaline

14

environments.

15

Keywords: Phenolic acid grafting chitosan, Nanocomplexes, EGCG, Antioxidant

16

activity, Controlled release

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18

INTRODUCTION

Chitosan (CS), the deacetylated form of chitin, is mainly originated from

19

shrimp.

It

is

composed

of

glucosamine

known

as

20

2-amino-2-deoxy-(1-4)-β-D-glucopyranan. CS is considered to be the most widely

21

distributed biopolymer and is the only one naturally occurred positively charged

22

polyelectrolyte. Furthermore, it is generally considered biodegradable and nontoxic

23

with an oral LD50 in mice of over 16 g/kg1, and has been approved for dietary

24

applications in Japan, Italy and Finland. Therefore, CS has been extensively

25

investigated for its potential applications in the food, cosmetics, biomedical, and

26

pharmaceutical fields.2 CS and its derivatives are widely used in fabrication of

27

promising nanocomplexes vehicle for oral delivery of therapeutics or nutraceuticals

28

to increase their bioavailability due to its excellent mucoadhesive and

29

absorption-enhancing properties.3, 4

30

The encapsulation and delivery of certain labile drugs and functional dietary

31

ingredients is potential in solving the problem of their poor bioavailability for which

32

the oxidation damage accounts at least partly.5 Many of the labile drug or

33

nutraceutical molecules are oxidation-sensitive, which is the most common cause for

34

their deterioration during storage and/or during their transport to the required target

35

site in the body.6 Therefore, the polymer conjugates with antioxidant activities have

36

been paid a great attention as novel delivery systems for prevention of the drugs or

37

nutraceuticals from oxidation damage.7, 8 In addition, the carrier matrix materials are

38

the major content compared with their payload content in most delivery systems. 3



39

Phenolic compounds are attractive due to their multifunctional properties, such

40

as combined antimicrobial and antioxidant activities. Caffeic acid (CA) and ferulic

41

acid (FA) are two antioxidant phenolic acids extracted from plants. Despite there

42

have been researches focused on grafting CA and/or FA to CS9, 10, the potential of

43

these polysaccharide-phenolic conjugates in the development of nano-delivery

44

systems for labile molecules has been scarcely investigated.

45

Epigallocatechin-3-gallate (EGCG) is the most abundant and active catechin in

46

green tea. It is well known as a strong natural antioxidant in our diet, which can

47

reduce the risk of cancer as well as cardiovascular, neurodegenerative and other

48

diseases in a lot of epidemiological and preclinical studies.11 Considerable evidences

49

indicate that EGCG can suppress cell proliferation, enhance apoptosis, and inhibit

50

cell invasion, angiogenesis and metastasis through inhibiting enzyme activities and

51

signal transduction pathways.12 However, the oral bioavailability of EGCG is quite

52

low13, which is mainly due to its poor stability in physiological neutral environments

53

(plasma and intestinal juice) and poor intestinal absorption.14 Furthermore, the quick

54

degradation of EGCG in plasma and intestinal juice neutral and alkaline

55

environments was mainly caused by the auto-oxidation of EGCG, forming its

56

homodimers.15

57

Therefore, CA and FA were grafted to CS respectively based on an one-pot

58

method reported in our previous study16, which was further confirmed and

59

characterized by the 1H NMR, UV-vis spectra and FT-IR. The solubility of the

60

CA-g-CS and FA-g-CS under pH 7.0 and pH 8.4 was characterized and compared

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with that of CS. Novel polymer nanocomplexes composed with the phenolic acid

62

grafted CS and CPP were prepared and characterized using transmission electron

63

microscopy (TEM) and dynamic light scattering (DLS). CPP is a group of anionic

64

polypeptides, which are released from the N-terminus polar region during the tryptic

65

digestion of milk casein proteins. The antioxidant activity of the nanocomplexes

66

composed with the phenolic acid grafted CS and CPP was evaluated using DPPH

67

radical assay. The oral delivery properties of the nanocomplexes for phytochemicals

68

were characterized through determining their encapsulation efficiency and release

69

profile of EGCG, and the stabilization effects on EGCG in simulated GI

70

environment.

71

MATERIALS AND METHODS

72

Materials. CS (Average molecular weight: ~ 1.5 × 105, Degree of deacetylation ≥

73

90.0%), 1-ethyl-3-(3’-dimethylaminopropyl-carbodiimide) hydrochloride (EDC-HCl)

74

and Folin-Ciocalteau reagent were purchased from Kayon Biological Technology

75

Co., Ltd. (Shanghai, China). CA (purity > 98%), FA (purity > 98%), Gallic acid (GA,

76

purity > 98%), EGCG (purity > 98%), 2,2-Diphenyl-1-picrylhydrazyl (DPPH),

77

β-carotene and linoleic acid were purchased from Sigma Chemical Co. (St. Louis,

78

MO, USA). 1-hydroxybenzotriazole monohydrate (HOBt·H2O) was obtained from

79

Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). CPP was prepared and

80

identified with HPLC-MS-MS as described in our previous study.17 All of other

81

reagents were of analytical grade.

5



82

Synthesis of CA-, FA-grafting CS (CA-g-CS, FA-g-CS respectively). The

83

synthesis was performed based on the one-pot method reported in our previous

84

study16 with some modifications. CS (0.303 g, 1.85 mmol) was stirred in deionized

85

water (30 mL) with HOBt (0.282 g, 1.85 mmol) overnight until a clear solution was

86

obtained. CA or FA (0.311 g, 1.85 mmol) was introduced into the CS solution

87

followed by the dropwise addition of an alcoholic solution of EDC (0.355 g, 1.85

88

mmol, 2 mL). CA-g-CS or FA-g-CS with different substitutions of CA or FA was

89

prepared by changing the ratio between the phenolic acid and glucosamine in CS

90

(1:2, 1:1). The reaction was carried out for 24 h in ambient temperature and

91

atmosphere in dark. The resultant liquid was poured into dialysis bags (MWCO

92

8,000-14,000 Da), dialyzed against deionized water for 6 days with four changes of

93

water each day. Then, the synthesized CA-g-CS or FA-g-CS sample was frozen and

94

dried by lyophilizer to obtain solid conjugate of CA-g-CS or FA-g-CS. Blank CS,

95

acting as a control, was prepared in the same conditions but in the absence of

96

phenolic acids.

97

Characterization of the phenolic acid grafting CS. Characterizations of CA-g-CS

98

and FA-g-CS conjugations were determined by UV-vis spectra, FT-IR and 1H NMR

99

spectra. The UV-vis spectra were determined by a UV-2600 spectrophotometer

100

(Shimazu, Japan) by scanning from 200 to 800 nm. The FT-IR spectra were collected

101

under ambient conditions, using a Nicolet iS10 FT-IR spectrometer (Thermo

102

Electron Corp., Madison, WI). Each spectrum was averaged over 256 scans with 4

103

cm-1 resolution in the range of 500-4000 cm-1. Proton nuclear magnetic resonance

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(1H NMR) spectra were recorded at 25

o

105

CD3COOD/D2O (1%, v/v, J&K Scientific, China) using AVANCE Ⅲ 500 MHz NMR

106

Spectrometer (Bruker, Switzerland).

107

Evaluation of phenolic groups by Folin-Ciocalteu reagent. Phenolic acid (GA, FA)

108

contents were determined according to the previous method.18 Briefly, 0.5 mL of

109

CA-g-CS or FA-g-CS was mixed with 1 mL of Folin-Ciocalteau reagent for 5 min in

110

dark, followed by addition of 2 mL 20% sodium carbonate (Na2CO3). The mixture

111

was shaken and kept at 30 Ⅲ for 1 h, absorbance (Abs) of which was measured at

112

747 nm using a vis-spectrophotometer (Jinghua Instrument 722, Shanghai, China).

113

Gallic acid was used as a standard. The grafting contents of CA-g-CS and FA-g-CS

114

were expressed as mg of GA equivalent per g of copolymer.

115

Characterization of solubility of CA-g-CS or FA-g-CS in neutral and alkaline

116

pH. Equal amounts of CA-g-CS or FA-g-CS with different phenolic acid contents

117

and CS were dissolved with the same concentration. Equal volume of the polymer

118

solutions were adjusted to pH 7.0 and pH 8.4, respectively. After 2 hours, the

119

polymer solutions were centrifuged under 5000 rpm for 30 min. After centrifuge, the

120

suspensions were removed and the precipitation of each sample was lyophilized and

121

weighted. The ratio between the amount of dried precipitate and the amount of

122

original sample were calculated and compared.

123

General procedure for preparation of the nanocomplexes. CA-g-CS-CPP,

124

FA-g-CS-CPP and CS-CPP nanocomplexes were prepared according to our

125

previously reported procedure with modification.17 CA-g-CS or FA-g-CS was

C with samples dissolved in

7



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126

dissolved in deionized water. CS was dissolved in 1% (w/v) acetic acid solution with

127

sonication until the solution was transparent. The aqueous solution of CPP was

128

obtained at a suitable concentration. CA-g-CS, FA-g-CS, CS and CPP solutions were

129

adjusted to pH 6.2 with 1.0 N HCl or NaOH solution. Subsequently, CPP solution

130

was added to the CA-g-CS, FA-g-CS and CS solution, respectively, under stirring at

131

room temperature. For the preparation of EGCG-loaded nanocomplexes, aqueous

132

solution of EGCG was added to the CA-g-CS, FA-g-CS and CS solution before the

133

addition of CPP solution. The formation of CA-g-CS-CPP, FA-g-CS-CPP and

134

CS-CPP nanocomplexes started spontaneously via the CPP initiated ionic gelation

135

mechanism.

136

Characterization of nanocomplexes. The measurements of particle size,

137

polydispersity index (PDI) and zeta potential of the nanocomplexes were performed

138

on a Zetasizer Nano-ZS (Malvern Instruments) on the basis of DLS techniques. All

139

measurements were made in triplicate at 25 ± 1 Ⅲ.

140

The morphological characteristics of the nanocomplexes were examined by a high

141

performance

digital

imaging

TEM

machine

(JEOL

142

High-Technologies Corp., Japan). One drop of the suspension was placed on a

143

copper grid and allowed to evaporate in air. Once evaporated the samples were

144

placed in a TEM for imaging. The accelerating voltage used was 100 kV and the

145

images were taken on a Gatan electron energy loss spectrometry system using 6 eV

146

energy slit.

147

Determination of antioxidant activity of CA-g-CS-CPP and FA-g-CS-CPP

8


H-7650,

Hitachi

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nanocomplexes by in vitro DPPH radicals scavenging assay. DPPH radicals

149

scavenging assay was performed in the slightly modified reported procedure19. CA,

150

FA, CS, CA-g-CS, GA-g-CS, the CS-CPP nanocomplexes, the CA-g-CS-CPP

151

nanocomplexes, and FA-g-CS-CPP nanocomplexes were homogenously dispersed in

152

deionized water of a series of concentrations (0.05, 0.1, 0.25, 0.5, 0.75, 1.0 mg/ml,

153

respectively). A 50 µL sample was added to each well of 96-well microplate, mixed

154

with 200 µL methanolic DPPH· solution (0.4 mM). The reactions were carried out in

155

dark at room temperature for 30 min. Abs was measured at 517 nm by a microplate

156

reader (Bio-Tek µquant, USA).

157

Scavenging Activity = (1 −

A1 − A2 ) × 100% A0

158

Where A0 is the Abs of the control (water instead of sample), A1 is the Abs of the

159

sample, and A2 is the Abs of the sample only (methanol instead of DPPH).

160

Encapsulation efficiency of EGCG and in vitro release profile of EGCG from the

161

nanocomplexes in simulated gastric environments. The encapsulation efficiency

162

of EGCG in the CA-g-CS-CPP, FA-g-CS-CPP and CS-CPP nanocomplexes was

163

determined according to our previously reported method with minor modification.20

164

Briefly, the EGCG-loaded nanocomplexes were carefully transferred into an Amicon

165

Ultra-15 centrifugal filter device (Millipore Co., Billerica, MA, USA) made up of a

166

centrifuge tube and a filter unit with low-binding Ultracel membrane (MWCO 1000).

167

After centrifugation at 4500 g for 75 min, free EGCG penetrated through the

168

Ultracel membrane into the centrifuge tube, and the EGCG-loaded nanocomplexes in

169

the filter unit were obtained for the determination of the in vitro release profile. The 9



170

amount of EGCG in ultrafiltrate was determined by HPLC according to our reported

171

method.21 The encapsulation efficiency of EGCG was calculated using the formula

172

below:

173 174 175

Total amount of EGCG – amount of EGCG in ultrafiltrate

Encapsulation efficiency (%) = ————————————————————————— × 100

Total amount of EGCG

176

The release profiles of EGCG from the CA-g-CS-CPP, FA-g-CS-CPP and

177

CS-CPP nanocomplexes in simulated gastric environments were analyzed by dialysis

178

method. The EGCG loaded nanocomplexes, obtained from the centrifugation in

179

determination of the encapsulation efficiency, were dispersed in the simulated gastric

180

fluid (SGF) solution, which was further immediately placed in the dialysis bags

181

(MWCO 3.5 kDa) (MYM Biological Technology Company, USA). SGF was 0.1 N

182

HCl (pH 1.2) containing 0.1% pepsin. The dialysis bags containing SGF were

183

immersed in acid release medium (0.1 N HCl, pH 1.2). Aliquots of dissolution media

184

(0.5 mL) were withdrawn, and the concentration of EGCG was determined by HPLC

185

after appropriate dilution with water. And, the same volume of buffer (0.5 mL) was

186

fed back to release medium. The percent cumulative amount of EGCG released from

187

the nanocomplexes was calculated as a function of time.

188

Determination of the stability of EGCG loaded in the CA-g-CS-CPP and

189

FA-g-CS-CPP nanocomplexes under neutral and alkaline environments. Equal

190

volume (0.5 mL) of the EGCG and EGCG loaded CA-g-CS-CPP or FA-g-CS-CPP

191

nanocomplexes with the same concentration of EGCG were dispersed in 9.5 mL, pH

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7.4 PBS buffer. At the time intervals of 0, 1, 3, 6h, 0.5 mL sample was withdrawn

193

from the EGCG and

194

FA-g-CS-CPP nanocomplexes solution, respectively, and extracted with same

195

volume (0.5 mL) of ethyl acetate twice.22 Blank PBS buffer (pH7.4, 0.5 mL) was

196

feed back. After extraction, the ethyl acetate phase was rotary evaporated,

197

re-dissolved in water, and analyzed using HPLC according to our reported method.21

198

Statistical analysis. Data were expressed as mean ± standard deviation (SD) of

199

triplicates. The least significant difference (LSD) test and one-way analysis of

200

variance (ANOVA) were used for multiple comparisons by SPSS 16.0. Difference

201

was considered statistically significant if p < 0.05, and very significant if p < 0.01.

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RESULTS AND DISCUSSION

203

Synthesis and characterization of the phenolic acid grafting CS. The phenolic

204

acid grafting CS (CA-g-CS and FA-g-CS) were prepared through conjugating the

205

amino-groups in CS with the carboxyl group in CA and FA, which was mediated via

206

a water-soluble carbodiimides conjugating agent, EDC, according to our previous

207

study.16 The UV-vis spectra of CA-g-CS and FA-g-CS, as well as the corresponding

208

CS, are shown in Figure 1A. Compared with net CS, new absorption peak around

209

300 nm appears for the samples of CA-g-CS and FA-g-CS, which mainly originates

210

from the π-system of the benzene ring in the phenolic acid. The absorption peak can

211

also be observed in the UV-vis spectra of free CA and FA (data not shown).

EGCG loaded CA-g-CS-CPP nanocomplexes and

212

The phenolic acid grafting CS were then characterized by FT-IR, compared with

213

the net CS. The result is shown in Figure 1B. The main characteristic absorption

11



214

bands of CS appear at 1640, 1557, 1374 and 1150-1040 cm-1, which correspond to

215

amide I, amide II, amide Ⅲ groups, and glycosidic linkage (C-O-C), respectively.23

216

After grafting CS with the phenolic acids (CA and FA), the peak height ratio

217

between the absorption bands of amide II and amide I increases. In addition, certain

218

changes in the bands of amide Ⅲ group around 1374 cm-1 could be observed. The

219

amide II bands around 1557 cm-1 arise from the N-H bending vibrations coupled to

220

C-N stretching vibrations. Amide Ⅲ bands arise from the C-N stretching vibration.

221

This phenomenon is caused by the grafting of the phenolic acids on the amino

222

groups in the CS chain. Similar result was also reported in previous study.24

223

Furthermore, the phenolic acids grafting CS were characterized by 1H NMR, and

224

the net CS was used as the control. Figure 1 C and D reveal the 1H NMR spectrum

225

of the CS derivatives grafted with CA (C) and FA (D). In the spectrum of both

226

CA-g-CS and FA-g-CS, a single peak at 2.9 ppm (H-2), multiple peaks at 3.3–3.7

227

ppm (H-3, H-4, H-5, H-6) and a small single peak at 4.4 ppm (H-1) are exhibited,

228

which originate from CS. The protons of N-acetyl glucosamine units exhibit a single

229

peak at 1.8 ppm. Compared with net CS (data not shown), the phenolic acid grafted

230

products show new peaks at 6.2–8.0 ppm (methine protons of phenolic acids),

231

indicating the successful grafting of phenolic acids onto CS. This result is consistent

232

with that of previous studies. However, the signals of the peaks at 6.2–8.0 ppm are

233

comparatively weak, which was caused by the strong signal of the peak representing

234

the D2O at 4.8 ppm.

235

The substitution of the phenolic acids (CA and FA) on CS was determined by

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236

Folin-Ciocalteu reaction, in which, the phenolic compounds took part in a complex

237

redox reaction with phosphotungstic and phosphomolybdic acids.19 The color was

238

developed from

239

phosphomolybdic/phosphotungstic acid complexes caused by the transfer of

240

electrons at basic pH. Calculated from the results of the Folin-Ciocalteu reaction, the

241

substitutions of CA and FA on the dry CA-g-CS and FA-g-CS copolymers with the

242

molar ratios between phenolic acid to glucosamine in CS of 1:2 and 1:1 are shown in

243

Table 1. For CA-g-CS, the grafting ratio of the phenolic acid to CS increases

244

significantly (p < 0.05) with the elevation of the molar ratios between phenolic acid

245

to glucosamine in CS, which might be attributed to higher quantity of phenolic acid

246

available for the reaction. However, an opposite change trend appears for the

247

FA-g-CS, which might be related to the poor solubility of FA. In addition, at the

248

same molar ratio between phenolic acid to glucosamine in CS, the substitution of CA

249

on CS was significantly (p < 0.05) higher than that of FA on CS, which might also be

250

related to the higher solubility of CA compared to FA.

the chromogens formed through

the reduction

of the

251

Figure 2 shows the solubility of the CA-g-CS, FA-g-CS and CS in neutral and

252

alkaline environments. The CA-g-CS and FA-g-CS were synthesized with the same

253

molar ratio between phenolic acid to glucosamine in CS of 1:1. It can be found from

254

Figure 2 that, compared with the plain CS, the precipitation of the copolymers under

255

pH 7.0 and pH 8.4 is very significantly (p < 0.01) reduced after grafting with CA.

256

And grafting with FA significantly (p < 0.05) decreased the precipitation of the

257

copolymers under pH 7.0 and pH 8.4 compared with that of net CS. These results

13



258

indicated that grafting with phenolic acid significantly increased the solubility of CS

259

in neutral and alkaline pH conditions. The solubility of CA-g-CS was higher than

260

that of FA-g-CS, which might be due to the higher substitution of the CA in the

261

copolymer than that of FA. The general pH value in human body, such as the

262

digestive tract, blood vessel, et al, is neutral and alkalescent. However, CS

263

precipitates at neutral and alkaline pH conditions due to deprotonation of amine

264

groups. The pKa value of the amine groups on CS is approximately pH 6.4. This

265

property of CS means that the CS and the CS based biomaterials can be effective

266

only in a limited area of the human body, such as in stomach and duodenum, where

267

the pH values are below or close to its pKa. Therefore, grafting with phenolic acids

268

successfully extends the soluble pH scope of CS, which is the pre-requirement for

269

intestinal mucosal delivery and vascular delivery of the nutraceuticals using CS

270

based biomaterials.

271

Physicochemical properties of the nanocomplexes. The particle sizes of the

272

CS-CPP, CA-g-CS-CPP and FA-g-CS-CPP nanocomplexes are shown in Table 2.

273

The particle size of the CS-CPP nanocomplexes determined by DLS at room

274

temperature is 199.1 ± 3.0 nm (n = 3), with the PDI around 0.250. With the grafting

275

of the phenolic acids, either CA or FA, the particle size of the nanocomplexes

276

increases significantly compared with that of the CS-CPP nanocomplexes. The

277

particle size of the CA-g-CS-CPP nanocomplexes with the CA grafting ratio of

278

108.02±1.08 mg/g is as large as 273.8 ± 1.7 nm, with the PDI around 0.268, which

279

indicates that the obtained nanocomplexes dispersion is highly heterogeneous. The

14


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280

size of the nanocomplexes composed with the FA-g-CS and CPP is 251.3 ± 3.6 nm,

281

which is smaller than that of the CA-g-CS-CPP nanocomplexes due to the lower

282

grafting ratio of FA than that of CA. However, the PDI of the nanocomplexes

283

suspensions increases from around 0.268 and 0.386, indicating the increased

284

heterogeneity of the nanocomplexes dispersion. It might be caused by the

285

heterogeneous distribution of FA that is grafted in the CS molecular chain. Figure 3A

286

and B show the structure and morphology of the CA-g-CS-CPP nanocomplexes and

287

the FA-g-CS-CPP nanocomplexes with CA grafting ratios of 108.02±1.08 (A)

288

FA grafting ratios of 39.71±0.10 mg/g (B), which were characterized using TEM. It

289

could be seen that the nanocomplexes are spherical in shape, dispersed

290

homogenously, which is in consistence with the DLS results.

291

The antioxidant activity of the nanocomplexes. Despite various chemical-based

292

assays can be used to determine antioxidant activities, such as oxygen radical

293

absorbance capacity (ORAC), DPPH• scavenging method, ferric reducing capacity,

294

β-carotene-linoleic acid system etc, they can generally be classified into two types

295

according to the reactions involved, the assays based on hydrogen atom transfer

296

(HAT) reactions and the ones based on electron transfer (ET). The DPPH assay is

297

one representive of ET reactions. As polysaccharides are soluble in water

298

environment rather than the oil conditions, the DPPH assay was selected to measure

299

the antioxidant activity of the phenolic acid grafting CS.

and

300

It can be found from Figure 4 that both the CA-g-CS-CPP and FA-g-CS-CPP

301

nanocomplexes show significantly higher (p

Antioxidant Nanocomplexes for Delivery of Epigallocatechin-3-gallate

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