Preparation of bioactive polysaccharide nanoparticles with enhanced

Statistical analysis. Each measurement was conducted in at least triplicate samples, and the. 190 results were reported as the mean ± standard deviat...
1 downloads 8 Views 1MB Size
Subscriber access provided by UNIV OF YORK

Bioactive Constituents, Metabolites, and Functions

Preparation of bioactive polysaccharide nanoparticles with enhanced radical scavenging activity and antimicrobial activity Yang Qin, Liu Xiong, Man Li, Jing Liu, Hao Wu, Hongwei Qiu, Hongyan Mu, Xingfeng Xu, and Qingjie Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00388 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43

Journal of Agricultural and Food Chemistry

1

Preparation of bioactive polysaccharide nanoparticles with enhanced radical scavenging activity and antimicrobial activity

2

Yang Qin1†

3

Liu Xiong1† Man Li† Jing Liu‡ Hao Wu† Hongwei Qiu†

Hongyan Mu†

Xingfeng Xu† Qingjie Sun*

4 †

5

College of Food Science and Engineering, ‡Central Laboratory, Qingdao Agricultural University

6

(Qingdao, Shandong Province, 266109, China)

7

1

8

*Correspondence

9

[email protected]), College of Food Science and Engineering, Qingdao Agricultural University,

10

Equally-contributing author author

(Tel:

86-532-88030448,

Fax:

86-532-88030449,

266109, 700 Changcheng Road, Chengyang District, Qingdao, China.

ACS Paragon Plus Environment

e-mail:

Journal of Agricultural and Food Chemistry

11

ABSTRACT: Due to their biocompatibility and biodegradability in vivo, natural polysaccharides

12

are effective nanocarriers for delivery of active ingredients or drugs. Moreover, bioactive

13

polysaccharides, such as tea, Ganoderma lucidum, and Momordica charantia polysaccharides (TP,

14

GLP, and MCP), have antibacterial, antioxidant, antitumor, and antiviral properties. In this study,

15

tea, Ganoderma lucidum, and Momordica charantia polysaccharide nanoparticles (TP-NPs,

16

GLP-NPs, and MCP-NPs) were prepared via the nanoprecipitation approach. When the ethanol to

17

water ratio was 10:1, the diameter of the spherical polysaccharide nanoparticles was the smallest,

18

and the mean particle size of the TP-NPs, GLP-NPs, and MCP-NPs was 99±15, 95±7, and 141±9

19

nm, respectively. When exposed to heat, increased ionic strength and pH levels, the nanoparticles

20

exhibited superior stability and higher activity than the corresponding polysaccharides. In

21

physiological conditions (pH 7.4), the nanoparticles underwent different protein adsorption

22

capacities in the following order: MCP-NPs> TP-NPs> GLP-NPs. Moreover, the 2,

23

2-Diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical, and superoxide anion radical scavenging

24

rates of the nanoparticles were increased by 9%–25%, as compared to the corresponding

25

polysaccharides. Compared to the bioactive polysaccharides, the nanoparticles enhanced

26

antimicrobial efficacy markedly and exhibited long-acting antibacterial activity.

27

KEYWORDS bioactive polysaccharides, nanoprecipitation, free radicals scavenge, protein

28

adsorption, cytotoxicity

ACS Paragon Plus Environment

Page 2 of 43

Page 3 of 43

Journal of Agricultural and Food Chemistry

29

INTRODUCTION

30

Bioactive polysaccharides are natural polymers that exist widely in plants, fungi, animals,

31

and microorganisms. They engage in a broad spectrum of physiological activities, including

32

antioxidation, antimicrobial, antitumor, antiviral, and lipid-lowering.1-4 Moreover, bioactive

33

polysaccharides are biocompatible and biodegradable in vivo and are nontoxic in a wide range of

34

doses. Thus, there is growing interest among researchers regarding the benefits of bioactive

35

polysaccharides for human health and other areas.5,6 However, bioactive polysaccharides belong to

36

Class III of the Biopharmaceutics Classification System, which means that the oral absorption of

37

polysaccharides is poor and erratic. Furthermore, due to their hydrophilic and uncharged nature,

38

neutral polysaccharides demonstrate low bioavailability and clearance by the reticuloendothelial

39

system, which indicates their restricted bioactivity in vivo.7

40

To improve the bioavailability and bioactivity of functional ingredients, various nanoparticle

41

delivery systems have been developed, such as nanomicelle, nanovesicle, nanoliposome, and

42

nanogels.8-11 Because bioactive polysaccharides provide a wide range of benefits for various

43

biological processes,12,13 nanocarriers loaded with bioactive polysaccharides have become a

44

prevalent research topic. For example, Kong et al. prepared silica–chitosan nanoparticles for

45

encapsulation of Antrodia camphorata polysaccharides and demonstrated their increased

46

antitumor effects.14 Sun et al. found that the ophiopogon polysaccharide liposome could activate

47

macrophages, and its efficacy was significantly better than Ophiopogon polysaccharide.15 Liu, et

48

al. reported that Ganoderma lucidum polysaccharides (GLP) encapsulated in liposomes induce

49

more powerful antigen-specific immune responses than GLP alone.16 Wang et al. found that the

50

histidine modified Auricularia auricular polysaccharide nanomicelles with a diameter of 157.2

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

51

nm were more easily ingested by cells than Auricularia auricular polysaccharide.17 Qiu et al.

52

prepared bioactive polysaccharide-loaded maltodextrin nanoparticles with sizes of 80–120 nm,

53

and they found that the stability of polysaccharide-loaded maltodextrin nanoparticles was

54

improved at high salt concentrations.18

55

Due to the small size and high surface/volume ratio, polysaccharide-loaded nanoparticles

56

display specific characteristics that differ from those of the corresponding bulk material, such as

57

better dispersibility, higher stability, and higher penetration rates through biological barriers.

58

However, to the best of our knowledge, there are no studies that report the preparation of bioactive

59

polysaccharide nanoparticles using only bioactive polysaccharides. We hypothesized that

60

polysaccharide nanoparticles may improve bioactivities of polysaccharides. To these this

61

hypothesis, we fabricated three bioactive polysaccharide nanoparticles using tea polysaccharide

62

(TP), Ganoderma lucidum polysaccharide (GLP), and Momordica charantia polysaccharide (MCP)

63

via the nanoprecipitation method. We first investigated the morphological characteristics and size

64

distribution of the bioactive polysaccharide nanoparticles: tea polysaccharide nanoparticles

65

(TP-NPs), Ganoderma lucidum polysaccharide nanoparticles (GLP-NPs), and Momordica

66

charantia polysaccharide nanoparticles (MCP-NPs). Moreover, we explored the stability,

67

cytotoxicity, and protein adsorption of the polysaccharide nanoparticles. We further explored

68

antioxidant and antimicrobial activities of polysaccharide nanoparticles in vitro by comparison

69

with the polysaccharides.

70

MATERIALS AND METHODS

71

Materials. TP, GLP, and MCP were provided by Ci Yuan Biotechnology Co., Ltd. (Shanxi,

72

China). 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and hydrogen peroxide (H2O2) were purchased

ACS Paragon Plus Environment

Page 4 of 43

Page 5 of 43

Journal of Agricultural and Food Chemistry

73

from Sigma Chemical Co. (St. Louis, MO, United States). Mouse embryonic fibroblast (MEF)

74

was provided by the American Type Culture Collection (ATCC). 3-(4, 5-dimethylthiazol-2-yl)-2,

75

5-diphenyltetrazolium bromide (MTT), and Dulbecco’s Modified Eagle’s Medium (DMEM) were

76

obtained from Sigma Chemical Co. (St. Louis, MO, United States). Gram-negative bacteria

77

Escherichia coli (E. coli, ATCC 25922) and Salmonella typhus (S. typhus, ATCC 6897), and

78

Gram-positive bacteria Staphylococcus aureus (S. aureus, ATCC 25923) and bacillus subtilis (B.

79

subtilis, ATCC 60511) were purchased from Nanjing Bianzhen Biological Technology Co., Ltd.

80

Luria-Bertani (LB) broth powder was supplied by Thermo Fisher Scientific Inc. (Beijing, China).

81

All other reagents used were of analytical grade.

82

Preparation of bioactive polysaccharide nanoparticles. Three types of bioactive

83

polysaccharides (TP, GLP, and MCP) were used to prepare nanoparticles using the

84

nanoprecipitation method of Qiu et al.19 with some modifications. In brief, polysaccharide solution

85

(2%, w/v) was prepared by mixing 2 g of polysaccharide powder in 100 mL of deionized water,

86

stirring the solution for 1 h at room temperature (25 °C), and then centrifuging the solution at

87

3,500 g for 5 min to remove any insoluble components. The polysaccharide solution was adjusted

88

to different pH levels using a 0.5 M NaOH solution (pH 5.0 for GLP; pH 6.0 for TP and MCP).

89

Afterwards, a fixed quantity of 95% ethanol (30, 40, 50, 100, and 200 mL) was added drop-wise

90

into 10 mL of polysaccharide solution at room temperature, which was continually stirred using a

91

magnetic stirrer. Furthermore, the solution was kept under continuous mechanical stirring for

92

another 2 h. Afterward, the suspension was centrifuged (10,000 g for 15 min), and the precipitates

93

were rinsed three times with 95% ethanol to remove excess water. Subsequently, the dried TP-NPs,

94

GLP-NPs, and MCP-NPs were obtained by conducting the lyophilization process (-80 °C for 72 h)

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

95

and kept in a plastic bag until further use.

96

Size, ζ-potential, and polydispersity index. The size distributions, average size, and

97

polydispersibility index (PDI) of polysaccharide nanoparticles were determined by the dynamic

98

light scattering (DLS) technique using a Zetasizer Nano ZS90 (Malvern Instruments, U.K.). The

99

electrical characteristic (ζ-potential) was determined by particle electrophoresis using the same

100

instrument. The nanoparticle dispersions were diluted (0.1%) with ultrapure water to avoid

101

multiple scattering effects and were placed into the measurement chamber. Then, the dispersions

102

were equilibrated at 25±1 °C prior to analysis.

103

Transmission electron microscopy (TEM). The morphologies of the TP-NPs, GLP-NPs,

104

and MCP-NPs were analyzed with a Hitachi 7700 TEM (Tokyo, Japan) at an acceleration voltage

105

of 80 kV. A small droplet of nanoparticle suspension with a concentration of 0.1% (w/v) was

106

deposited onto a carbon-coated copper grid (300 meshes) and then lyophilized for more than 6 h to

107

obtain dry samples for further observation.

108

Fourier transform infrared spectroscopy (FTIR). The chemical structures of the TP and

109

TP-NPs were measured using FTIR (Tensor 27, Jasco Inc., Easton, MD, USA). The background

110

obtained from the scan of KBr was automatically subtracted from the sample spectra. The

111

measured spectral region was between 4,000 and 400 cm−1.

112

Temperature, ionic strength, and pH stability. The turbidity, average size, and PDI of the

113

TP-NPs (1 mg/mL) at different temperatures, ionic strength (NaCl) levels, and pH levels were

114

determined using a spectrophotometry (Shimadzu-2600, Kyoto, Japan) and a Zetasizer Nano ZS90

115

(Malvern Instruments, U.K.). The particle suspensions were divided into fifteen groups: four

116

groups were incubated at temperatures of 4, 25, 37, and 65 °C for 2 h and then returned to room

ACS Paragon Plus Environment

Page 6 of 43

Page 7 of 43

Journal of Agricultural and Food Chemistry

117

temperature. Another six groups were dispersed in NaCl solutions (0, 100, 200, 300, 400, and 500

118

mM) at 0.1% (w/v) for 3 h at room temperature. The other groups were adjusted to the desired pH

119

values (2, 5, 7.4, and 9) using 0.5 M HCl/NaOH solution.

120

In vitro cytotoxicity. The in vitro cytotoxicity of the TP-NPs, GLP-NPs, and MCP-NPs were

121

assessed using an MTT assay according to the method used by Carmichael et al.20 with some

122

modifications. MEF cells in DMEM media (8,000 cells/well) were respectively seeded into each

123

well of a 96-well tissue culture plate (Costar, Corning, NY, USA) and cultured until they reached

124

confluence (24–36 h). One hundred microliter aliquots of TP-NP dispersions at concentrations of 0,

125

25, 50, 100, 150, and 200 µg/mL (the concentrations of the GLP-NPs and MCP-NPs were 0; 250;

126

500; 1,000; 1,500; and 2,000 µg/mL) were added to the cell culture wells, respectively. The plate

127

was incubated for 24 h (at 37 °C) in a 5% CO2 humidified atmosphere. Next, 100 µL of MTT

128

solution (5 mg/mL) were added to each well for further incubation for 4 h at 37 °C. The total

129

number of cells was determined by the absorbance at 570 nm. Cell viability was expressed as a

130

percentage of the control absorbance, defined as 100%.

131

Protein adsorption assay. To study the protein adsorption of the TP-NPs, GLP-NPs, and

132

MCP-NPs, bovine serum albumin (BSA) was chosen as a model protein. The polysaccharide

133

nanoparticles (0.15 mg/mL) were coincubated with BSA (0.25 mg/mL) in a phosphate buffer

134

solution (pH 7.4) at 37 °C. At different time points, 1 mL aliquots of each sample were centrifuged

135

(10,000 g, 20 min) to precipitate protein-adsorbed aggregates. Afterward, a BSA standard curve

136

was established using a BCA Protein Assay Kit. Meanwhile, at the same condition, the

137

concentration of the protein that had not been adsorbed was measured using a microplate reader.

138

The ratios of adsorbed protein at different time points were then calculated.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

139

Free radical scavenging activity assays. The DPPH free radical scavenging activity was

140

determined using the standard DPPH assay method with slight modifications. In brief, 2 mL of

141

DPPH solution (0.2 mM in 95% ethanol) mixed with 2 mL of various concentrations of bioactive

142

polysaccharide or nanoparticle solutions (10-300 µg/mL for TP and TP-NPs; 200-2000 µg/mL for

143

GLP and GLP-NPs; 30-2000 µg/mL for MCP and MCP-NPs), respectively. The reaction mixture

144

was shaken well and then incubated in the dark for 30 min at room temperature. Afterward, the

145

absorbance of the resulting solution was read at 517 nm against a blank. DPPH radical scavenging

146

activity was calculated using equation (1):

147

DPPH scavenging rate (%) = [1-(A1- A2)/A0] ×100% (1)

148

A0 is the absorbance of the control (DPPH solution with no sample); A1 is the absorbance of the

149

test sample (DPPH solution with sample or positive control); and A2 is the absorbance of the

150

blank (sample with no DPPH).

151

Hydroxyl radicals (•OH) were generated by the Fenton reaction. Briefly, the reaction system

152

contained 1.0 mL FeSO4 (9.0 mM), 1.0 mL H2O2 (8.8 mM), 1.0 mL salicylic acid (9.0 mM), and

153

1.0 mL of various concentrations of the bioactive polysaccharides or nanoparticles (10-300 µg/mL

154

for TP and TP-NPs; 200-2000 µg/mL for GLP and GLP-NPs; 30-2000 µg/mL for MCP and

155

MCP-NPs). The mixed solution was incubated at 37 °C for 1 h, and then the absorbance of the

156

mixture was recorded at 510 nm against a blank. The •OH scavenging activity was calculated by

157

equation (2):

158

Scavenging rate (%) = [1-(A1-A2)/A0] × 100% (2)

159

A0 is the absorbance of the control (reaction solution with no sample); A1 is the absorbance of the

160

test sample (reaction solution with sample or positive control); and A2 is the absorbance without

ACS Paragon Plus Environment

Page 8 of 43

Page 9 of 43

Journal of Agricultural and Food Chemistry

161

salicylic acid.

162

The superoxide radical scavenging activity was determined using the same method as that

163

used for the free radical scavenging activity. In brief, the system that generates superoxide radicals

164

was based on the autoxidation of the pyrogallol reaction. Five milliliters of 50.0 mM Tris-HCl

165

buffer (pH 8.1) was mixed with 4.0 mL bioactive polysaccharides or nanoparticles at different

166

concentrations (10-300 µg/mL for TP and TP-NPs; 200-2000 µg/mL for GLP and GLP-NPs;

167

30-2000 µg/mL for MCP and MCP-NPs), respectively. After the mixture was incubated for 20 min

168

at 25 °C, 1.0 mL of 3.0 mM pyrogallol was added to the mixture, and the mixture was incubated

169

for 5 min (at 25 °C). Then 1.0 mL HCl (10.0 mM) was added to terminate the reaction, and the

170

absorbance was monitored at 320 nm. The superoxide radical scavenging activity was calculated

171

by equation (3):

172

Scavenging rate (%) = [1-(A1-A2)/A0] × 100% (3)

173

A0 represents the absorbance of the control (reaction solution with no sample); A2 represents the

174

absorbance of the blank (sample with no pyrogallol); and A1 represents the absorbance of the test

175

sample (reaction solution with sample or positive control).

176 177

Additionally, ascorbic acid (Vc) was used as positive control in DPPH, •OH, and superoxide radical scavenging assays.

178

Determination of antibacterial activity. The inhibitory effects of bioactive polysaccharides

179

(GLP and MCP) and nanoparticles (GLP-NPs and MCP-NPs) on the growth rate of four types of

180

bacteria (S. aureus, Salmonella, E. coli, and B. subtilis) were measured using the broth

181

microdilution test recommended by the Clinical and Laboratory Standard Institute.21 Serial

182

doubling dilutions of the GLP and GLP-NP suspensions at concentrations of 0; 250; 500; 1,000;

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

183

1,500; 2,000; and 3,000 µg/mL and the MCP and MCP-NP suspensions at concentrations of 0; 250;

184

500; 1,000; 1,500; 2,000; 3,000; and 4,000 µg/mL in fresh broth were placed in a test tube,

185

receptively. Bacteria suspensions were inoculated to achieve approximately 1 × 106 CFU/mL of

186

bacterial concentration in each tube. Different bacteria suspensions were grown in LB broth

187

supplemented with different concentrations of bioactive polysaccharides or nanoparticles at 37 °C

188

for 24 h. The optical density (OD) of each tube was monitored by a UV–vis spectrometer at 600

189

nm (Shimadzu-2600, Kyoto, Japan).

190

Statistical analysis. Each measurement was conducted in at least triplicate samples, and the

191

results were reported as the mean ± standard deviation. The data were analyzed using SPSS V.17

192

software (SPSS Inc., Chicago, IL). Duncan's multiple range test was also applied to compare the

193

difference of means from the ANOVA, using a significance level of 5% (p < 0.05).

194

RESULTS AND DISCUSSION

195

Morphology and size of bioactive polysaccharide nanoparticles. The particle size and

196

ζ-potential of the TP-NPs prepared at different ethanol to water ratios (3:1, 4:1, 5:1, 10:1, and 20:1)

197

are shown in Figure 1. The ratios of ethanol to water displayed a significant impact on the particle

198

size distribution of the obtained nanoparticles. As the ratio increased from 3:1 to 10:1, the mean

199

particle size of the TP-NPs significantly decreased from 248±11 nm to 99±15 nm, and the PDI

200

value also decreased from 0.45 to 0.33. The results suggested that small and well-distributed

201

TP-NPs could be fabricated at higher ethanol to water ratios. Similarly, Tan et al. obtained starch

202

acetate nanospheres using nanoprecipitation and found that the average particle size can be

203

reduced by increasing the volume of anti-solvent.22 Furthermore, the ζ-potential of the TP-NPs

204

ranged from -24.5 mV to -22 mV.

ACS Paragon Plus Environment

Page 10 of 43

Page 11 of 43

Journal of Agricultural and Food Chemistry

205

The TEM images of the TP-NPs also show a similar trend (Figure 2). As the ratio of ethanol

206

to water increased from 3:1 to 20:1, the size decreased, and the particles were gradually changed

207

from an irregular to a more spherical shape. At low volume ratios (3:1 and 4:1), the nanoparticles

208

coalesced rapidly and formed large aggregates with loose structures in the presence of excess

209

water; therefore, the formed nanoparticles were mostly non-spherical in shape and demonstrated

210

an inhomogeneous size distribution (Figure 2A, B). When a high volume of ethanol (5:1, 10:1,

211

and 20:1) was added to the polysaccharide solution, a high degree of supersaturation probably

212

resulted in a spatially uniform distribution of nuclei and a low crystal growth rate. The decrease of

213

the amount of coalescence leads to a decrease in particle size.23,24

214

It should be pointed out that the TP solution did not show any change when illuminated by a

215

laser beam (Figure 3). After ethanol was added drop-wise into the solution, Tyndall light

216

scattering was observed along the beam path, which indicated the presence of nano-sized

217

particles. Moreover, the beam intensity of the TP-NPs suspension gradually increased as the

218

ethanol to water ratio increased.

219

Furthermore, the mean size and morphologies of the GLP-NPs and MCP-NPs at different

220

ethanol to water ratios are shown in Figures S1, S2, S3, and S4, respectively. When the ethanol to

221

water ratio was high (5:1, 10:1, and 20:1), the PDI values of both the nanoparticles were smaller

222

than 0.50 (ranged from 0.39 to 0.26), indicating that the nanoparticles were monodispersed

223

without obvious aggregation. At an ethanol to water ratio of 10:1, the GLP-NPs and MCP-NPs

224

were spherical, with sizes of 95±7 and 141±9 nm, respectively. Moreover, according to the TEM

225

results, we speculated that the ethanol to water ratios improved the morphologies of the

226

nanoparticles, while the polysaccharide types did not play a role. These results showed that the

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

227

ethanol to water ratio of 10:1 was optimal to prepare the TP-NPs, GLP-NPs, and MCP-NPs, and

228

thus the obtained polysaccharide nanoparticles were used for further investigation of their

229

stability, antioxidant, and antimicrobial activity.

230

Fourier transform infrared spectroscopy (FTIR). The spectra of the TP and TP-NPs are

231

shown in Figure 4. The absorption spectra of the TP and TP-NPs exhibited a broad peak at

232

3,600–3,200 cm–1 corresponding to intra- or inter-molecular hydrogen bonds with O-H stretching

233

vibration and a weak band at around 2,940 cm-1, which is characteristic of weak C-H asymmetric

234

vibration.25 There were characteristic peaks for the asymmetric (1,700–1,600 cm−1) and

235

symmetric (1,400–1,300 cm−1) stretching of carboxylate anions groups, indicating that carboxyl

236

groups existed in the samples.26,27 The stretching vibrations of S=O had an absorption peak of

237

about 1,300–1,200 cm−1, which was evidence of sulfuric radicals and suggested that all the

238

TP-NPs had sulfated groups.28 The spectra also indicated that TP-NPs had similar adsorption

239

peaks, but the intensities of the peaks were different among them. Compared with TP, the

240

characteristic bands at 3,600–3,200 cm−1 in the TP-NPs shifted to a shorter wavelength, which

241

indicated that the hydrogen bonds between molecular chains in the TP-NPs became stronger.

242

Particle stability. Bioactive polysaccharide nanoparticles may be used in foods and

243

beverages with different external environments depending on the nature of the product. Moreover,

244

they may be exposed to considerable variations in temperature, ionic strength, or pH level as they

245

pass through the human gastrointestinal tract after ingestion. Consequently, it is important to

246

understand the influence of temperature, ionic strength, and pH level on the size, charge, and

247

stability of polysaccharide nanoparticles.

248

Figure 5 shows the effect of different temperature treatments (4, 25, 37, and 65 °C) on the

ACS Paragon Plus Environment

Page 12 of 43

Page 13 of 43

Journal of Agricultural and Food Chemistry

249

mean size, PDI, and turbidity of the TP-NPs. As temperature increased from 4 to 65 °C, no

250

significant changes were observed for particle size, PDI, and turbidity, indicating that the TP-NPs

251

were stable and homogeneous. Presumably, the excellent thermal stability of the TP-NPs was

252

probably due to the steric repulsion between the nanoparticles.

253

The effects of salt content (0–500 mM NaCl) on the size distribution, mean size, PDI, and

254

turbidity of the TP-NPs are shown in Figure 6. Compared with the control, the size distribution,

255

the values of mean size, PDI, and turbidity of the TP-NP suspensions did not change markedly

256

with the addition of 100–300 mM NaCl. These results suggested that the TP-NPs exhibited

257

excellent salt stability. When the salt concentration was over 300 mM, there was an increase in

258

particle size and turbidity. This could be because the inter-particle repulsion was reduced by

259

sufficiently high ionic strength through the electro-screening effect, which allowed for potential

260

contact to occur between nanoparticles, and formed agglomerations, thereby increasing the size of

261

the TP-NPs.

262

Variations in the mean size, ζ-potential, PDI, and turbidity of the TP-NPs over a wide pH

263

range (pH 2.0–9.0) are shown in Figure 7. The size distribution of the TP-NPs remained relatively

264

stable under neutral and alkaline conditions, but it increased significantly under acidic conditions.

265

The TP-NPs had a small size of ~ 95 nm in neutral and alkaline conditions, but the size increased

266

remarkably to about 220 nm in acidic conditions. Likewise, the PDI and turbidity of the TP-NPs

267

showed the same trend regarding size. Jahanshahi and Babaei reported that the ζ-potential of

268

protein nanoparticles was around -30 mV, which should be large enough to ensure good stability

269

through electrostatic repulsion.29 Because the ζ-potential of the TP-NPs was nearly zero or slightly

270

negative at lower pH values (2.0), the particles gathered to form large aggregates. These results

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 14 of 43

271

indicated that the stability of the TP-NPs in neutral and alkaline conditions was better than in

272

acidic conditions. Additionally, the PDI values of the TP-NPs were about 0.29–0.36, and TP-NPs

273

appeared relatively stable across the various pH levels (Figure 7C). Tang et al. suggested that good

274

stability of camptothecin nanosuspension provides its ability to target blood fluid, which helps

275

improve bioactivity of camptothecin.30

276

Cell

viability

of

polysaccharide

nanoparticles.

The

cytotoxicity

of

bioactive

277

polysaccharides and the nanoparticles (TP-NPs, GLP-NPs, and MCP-NPs) against MEF cells

278

were estimated by MTT assay. After 24 h of cell exposure in the medium containing various

279

concentrations of samples, the cell viabilities were still higher than 80% (Figure 8), suggesting

280

that the bioactive polysaccharides and nanoparticles exhibited no toxicity. Polysaccharide

281

extracted from tea materials can be classified as very low toxicity polymers or as unclassified with

282

oral administration.31,32 Our results suggested that the TP-NPs, GLP-NPs, and MCP-NPs could be

283

safely used as functional food materials.

284

Protein adsorption assay of polysaccharide nanoparticles. We evaluated the interaction of

285

the TP-NPs, GLP-NPs, and MCP-NPs with proteins, using BSA as a model plasma protein. Figure

286

9 shows that in physiological conditions (pH 7.4), the nanoparticles underwent different protein

287

adsorption capacities in the following order: MCP-NPs> TP-NPs> GLP-NPs. This could be due to

288

the surface charge and size of particles. Compared to the other two nanoparticles, the GLP-NPs

289

showed reduced nonspecific protein adsorption, thereby indicating prolonged blood circulation.

290

Functionality and opsonization of the nanoparticles begins immediately after they are introduced

291

into plasma. Therefore, bioactive polysaccharide nanoparticles, especially MCP-NPs, could be

292

used as a functional ingredient or nanocarrier with improved stability in blood.

ACS Paragon Plus Environment

Page 15 of 43

Journal of Agricultural and Food Chemistry

293

Antioxidant activity assays of polysaccharide nanoparticles

294

DPPH radical scavenging activity. Polysaccharides that can donate hydrogen have been

295

proved to reduce the stable DPPH radical to yellow diphenylpicrylhydrazine.12,33 Total DPPH

296

scavenging effects of bioactive polysaccharides and nanoparticles at varying concentrations were

297

measured (Figure S5). Evidently, of all the test samples, the DPPH radical scavenging activity was

298

displayed in the most concentration-dependent manner. The higher the concentrations of the

299

samples were, the stronger the radical scavenging activity was. Furthermore, all kinds of

300

polysaccharide nanoparticles presented stronger scavenging activities than their corresponding

301

bioactive polysaccharides at every concentration point. The scavenging rates of the TP-NPs,

302

GLP-NPs, and MCP-NPs at the highest test concentrations (300; 2,000; and 2,000 µg/mL)

303

increased by 12.5%, 10.1%, and 11.25%, compared with the TP, GLP, and MCP, respectively

304

(Figure S5). As shown in Table 1, the 50% inhibitory concentration (IC50) values of TPS, GLP,

305

and MCP were 71.70, 870.42, and 746.82 µg/mL, respectively. In contrast, the IC50 values of the

306

TP-NPs, GLP-NPs, and MCP-NPs were 46.49, 687.91, and 485.47 µg/mL. These results indicated

307

that these polysaccharide nanoparticles had a noticeable effect on scavenging free radicals.

308

Previously, Schaffazick et al.34 and Yen et al.35 reported a similar effect, in which the nanoparticle

309

system increased the antioxidant ability of melatonin anti-lipid peroxidation. Similarly, previous

310

studies have suggested that the nanoparticles of natural bioactive components (kaempferol and

311

resveratrol) demonstrate improved antioxidant activity in vitro.36,37

312

The NaCl and thermal treatments showed a decreased antioxidant activity capacity for most

313

of the bioactive polysaccharides (Figure S5 and Table 1). The antioxidant activities of the

314

bioactive polysaccharides especially decreased after heating treatment. However, the TP-NPs,

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

315

GLP-NPs, and MCP-NPs were stable in heating solutions and had higher DPPH radical

316

scavenging activities than the corresponding bioactive polysaccharides did. The ionic strength

317

treatment also inhibited the antioxidant activities of the three bioactive polysaccharides, but when

318

they formed nanoparticles, the polysaccharide nanoparticles exhibited stronger antioxidant activity.

319

At the highest tested concentration, compared with TP, GLP, and MCP in the presence of heating

320

and NaCl treatment, the scavenging rates of the TP-NPs, GLP-NPs, and MCP-NPs were increased

321

by 19.8% and 24.7%; 13.7% and 14.4%; and 17.8% and 14.5%, respectively. These results

322

indicated that nanoparticles had stronger stability against ionic strength and thermal treatment.

323

Hydroxyl radical scavenging activity. The •OH possesses extremely high reactivity and

324

can induce severe damage to functioning biomolecules in living cells and functional ingredients in

325

food systems.38 Though not as strong as that of Vc, all samples exhibited •OH scavenging effects

326

within the test concentrations (Figure S6). Previous studies of the antioxidant activity of

327

polysaccharides have suggested that the OH group, carboxyl group, sulfate group, and

328

monosaccharide constituent may affect the antioxidant activity of polysaccharides.39-42 As shown

329

in Figure S6, polysaccharide nanoparticles possessed higher •OH scavenging activities than the

330

corresponding bioactive polysaccharides. The scavenging rate of the TP-NPs at a concentration of

331

300 µg/mL increased by 9.6%, and the scavenging rates of the GLP-NPs and MCP-NPs at a

332

concentration of 2,000 µg/mL increased by 12.4% and 15.2%, respectively. Under NaCl and

333

thermal treatment, the •OH scavenging activity of the bioactive polysaccharides was decreased

334

obviously, while the polysaccharide nanoparticles only slightly decreased.

335

After heating treatment, the IC50 values of the TP-NPs, GLP-NPs, and MCP-NPs (Table 2)

336

for scavenging •OH were calculated to be 51.26, 820.29, and 579.67 µg/mL, which were smaller

ACS Paragon Plus Environment

Page 16 of 43

Page 17 of 43

Journal of Agricultural and Food Chemistry

337

than those of bioactive polysaccharides (the IC50 values of the TP, GLP, and MCP were 193.75,

338

1209.3, and 1061.54 µg/mL). At the highest concentration, the •OH scavenging rates of the

339

TP-NPs, GLP-NPs, and MCP-NPs were increased by 14.8% and 20.5%; 20.7% and 15.4%; and

340

21.8% and 15.1%, respectively (Figure S6). These results revealed that the polysaccharide

341

nanoparticles exhibited stronger antioxidant activity than the bioactive polysaccharides.

342

Superoxide anion radical scavenging activity. In organisms, superoxide anion radical

343

indirectly initiates lipid peroxidation and can form strong reactive oxidative species, and thus the

344

presence of superoxide anion radicals is harmful for biomolecules.43-45 Therefore, the superoxide

345

anion radical scavenging activities of bioactive polysaccharides and nanoparticles at different

346

concentrations were evaluated, and the results are shown in Figure S7 and Table 3. All samples

347

exhibited scavenging activities for superoxide anion radicals. Zhao et al. speculated that

348

polysaccharides had electron-withdrawing groups (like keto or aldehyde) that could facilitate the

349

liberation of hydrogen from the O-H bond and thus stabilize the superoxide anion.28 The

350

polysaccharide nanoparticles also had higher scavenging activities for superoxide anion radicals

351

than the corresponding bioactive polysaccharides. After NaCl and heating treatment, compared

352

with the bioactive polysaccharides, the scavenging rates of the TP-NPs, GLP-NPs, and MCP-NPs

353

at the highest concentration were increased by 14.9% and 13.7%; 24.1% and 21.2%; and 16.6%

354

and 18.3%, respectively. Raveendran et al. reported that mauran polysaccharide/chitosan

355

nanoparticles could inhibit the oxidative damage caused under natural conditions, and they also

356

presented a prospective incipient polysaccharide nanoparticle made of extremophilic bacterial

357

origin for defending oxidative stress in vitro.46

358

Antibacterial activity in vitro. A normal intestinal microbial environment is important for

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

359

reducing the risk of gut diseases triggered by pathogenic microorganisms.47,48 Therefore, the

360

effects of the GLP-NPs and MCP-NPs on the growth of the Gram-positive bacteria S. aureus and

361

B. subtilis and the Gram-negative bacteria E. coli and Salmonella after an incubation period of 24

362

h were investigated. For comparison, the antibacterial activities of GLP and MCP were also

363

determined. The OD profiles of the bacteria as a function of concentrations are presented in Figure

364

10.

365

For all of the bacteria types, the OD values of all the samples reduced as the concentration

366

increased, indicating that all the tested samples exhibited antimicrobial activity. Li et al. reported

367

that Cyclocarya paliurus polysaccharides at a concentration of 1 mg/mL exhibited considerable

368

inhibitory activities against S. aureus and B. subtilis.49 As shown in Figure 10, GLP and MCP

369

exhibited higher antibacterial activity against the Gram-positive bacteria than against the

370

Gram-negative bacteria. E. coli had a more negatively charged cell surface than S. aureus and B.

371

subtilis.50 The interaction between bacterial cells and sub-micrometric materials depends on the

372

van der Waals and electrostatic interactions. Furthermore, for the same kind of bacteria, the OD

373

values of the GLP-NPs and MCP-NPs were lower than those of GLP and MCP, respectively.

374

Quantitatively, when the concentration of samples was 3,000 µg/mL, the OD values of the

375

GLP-NPs and MCP-NPs against S. aureus were 0.073 and 0.105, respectively, while the OD

376

values of GLP and MCP were 0.142 and 0.162, respectively. The polysaccharide nanoparticles

377

acted as excellent antibacterial agents against both Gram-positive and Gram-negative bacteria

378

when compared to polysaccharides. This was most likely due to the smaller size and higher zeta

379

potential of the polysaccharide nanoparticles. Likewise, Nguyen et al. reported that the

380

chitosan/sodium tripolyphosphate nanoparticles with smaller size and more positive ζ-potential

ACS Paragon Plus Environment

Page 18 of 43

Page 19 of 43

Journal of Agricultural and Food Chemistry

381

showed higher antibacterial activity against Streptococcus pneumoniae.51

382

Additionally, antibacterial activity as a function of the incubation time of bioactive

383

polysaccharide and nanoparticles against bacteria is shown in Figure S8. Antimicrobial activity of

384

the test samples against bacteria displayed a significant increasing trend (p < 0.05) as the

385

incubation time increased from 2 to 24 h. However, when the incubation time exceeded 24 h,

386

especially for 48 h, the OD values of GLP and MCP against bacteria increased markedly, while the

387

OD values of the GLP-NPs and MCP-NPs were almost constant, which indicated that the

388

GLP-NPs and MCP-NPs could effectively limit the increase in viable cell numbers, and exhibited

389

long-acting antibacterial activity. This could be because that the nanoparticles adhered to the

390

bacterial surface, irreversibly disrupted the membrane structure of the bacteria, and subsequently

391

penetrated cells and effectively inhibited protein activity, which ultimately led to bacteria

392

apoptosis.52,53

393

In summary, we successfully prepared the TP-NPs, GLP-NPs, and MCP-NPs using

394

nanoprecipitation. The polysaccharide types did not affect the morphology of the corresponding

395

nanoparticles. The high ethanol to water ratios produced monodispersed spherical nanoparticles.

396

The diameter of polysaccharide nanoparticles decreased as the ethanol/polysaccharide ratio

397

increased from 3:1 to 10:1. The nanoparticles exhibited better antioxidant activity and radical

398

scavenging activity than the bulk polysaccharides. Furthermore, the functional performances of

399

the TP-NPs, GLP-NPs, and MCP-NPs were more stable than those of the bioactive

400

polysaccharides when exposed to heat and increased ionic strength. In comparison to native

401

polysaccharides, GLP-NPs and MCP-NPs exhibited greater antimicrobial activity and prolonged

402

the antibacterial effect of the Gram-positive bacteria, S. aureus and B. subtilis, and the

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

403

Gram-negative bacteria, E. coli and Salmonella. The results suggest that the functional

404

nanoparticles prepared in this study could be applied as powerful antioxidant and antibacterial

405

materials in the food, cosmetics, and biopharmaceutical industries.

406

Supporting Information

407

DLS and TEM analysis of GLP-NPs (Figure S1 and Figure S2) and MCP-NPs (Figure S3

408

and Figure S4); the DPPH, •OH, and superoxide anion radical scavenging effects of TP-NPs,

409

GLP-NPs, and MCP-NPs at different concentrations after NaCl (100 mM) and 65 °C treatment

410

(Figure S5, Figure S6, and Figure S7); the antibacterial activity of GLP and GLP-NPs at the

411

concentration of 1.25 mg/mL, and MCP and MCP-NPs at the concentration of 2.0 mg/mL (Figure

412

S8).

413

Notes

414 415

The authors declare no competing financial interest. ACKNOWLEDGMENTS

416

This work was supported by the Special Funds for Taishan Scholars Project of Shandong

417

Province (No. ts201712058) and the Natural Science Foundation of Shandong Province of China

418

(ZR2017MC044).

ACS Paragon Plus Environment

Page 20 of 43

Page 21 of 43

Journal of Agricultural and Food Chemistry

419

REFERENCE

420

(1) Giavasis, I. Bioactive fungal polysaccharides as potential functional ingredients in food and

421 422 423 424 425 426 427 428 429

nutraceuticals. Curr. Opin. Biotech. 2014, 26, 162–173. (2) Popa, E. G.; Reis, R. L.; Gomes, M. E. Seaweed polysaccharide-based hydrogels used for the regeneration of articular cartilage. Crit. Rev. Biotechnol. 2015, 35(3) 1–14. (3) Ren, L.; Perera, C.; Hemar, Y. Antitumor activity of mushroom polysaccharides: a review. Food Funct. 2012, 3(11), 1118–1130. (4) Freitas, F.; Alves, V. D.; and Reis, M. A. M. Advances in bacterial exopolysaccharides: from production to biotechnological applications. Trends Biotechnol. 2011, 29(8), 388–398. (5) Huang, X.; Nie, S. The structure of mushroom polysaccharides and their beneficial role in health. Food Funct. 2015, 6(10), 3205–3217.

430

(6) Yan, J. K.; Wang, W. Q.; Wu, J. Y. Recent advances in cordyceps sinensis, polysaccharides:

431

mycelial fermentation, isolation, structure, and bioactivities: a review. J. Funct. Foods, 2014,

432

6(1), 33–47.

433

(7) Li, Z.; Wang, L. N.; Lin, X.; Shen, L.; Feng, Y. Drug delivery for bioactive polysaccharides to

434

improve their drug-like properties and curative efficacy. Drug Deliv. 2017, 24(2), 70–80.

435

(8) Mukhopadhyay, P.; Mishra, R.; Rana, D.; Kundu, P. P. Strategies for effective oral insulin

436

delivery with modified chitosan nanoparticles: a review. Prog. Polym. Sci. 2012, 37(11),

437

1457–1475.

438 439 440

(9) Shimanovich, U.; Bernardes, G. J. L.; Knowles, T. P. J.; Cavaco-Paulo, A. Protein micro- and nano-capsules for biomedical applications. Chem. Soc. Rev. 2013, 43(5), 1361-1371. (10) Wang, S.; Su, R.; Nie, S. F.; Sun, M.; Zhang, J.; Wu, D. Y.; Moustaid-Moussa N. Application

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

441

of

442

phytochemicals. J. Nutr. Biochem. 2014, 25(4), 363–376.

443 444 445 446 447 448

nanotechnology

in

improving

bioavailability

and

bioactivity

Page 22 of 43

of

diet-derived

(11) Bhatia, S. Marine polysaccharides based nano-materials and its applications. Nat. Polym. Drug Deliv. Syst. 2016, 185–225. (12) Xiao, J. B.; Jiang, H. A review on the structure-function relationship aspect of polysaccharides from tea materials. Crit. Rev. Food Sci. 2015, 55(7), 930–938. (13) Wang, J. Q.; Hu, S. Z.; Nie, S. P.; Qiang, Y.; Xie, M. Y. Reviews on mechanisms of in vitro antioxidant activity of polysaccharides. Oxid. Med. Cell. Longev. 2016, 2016(64), 5692852.

449

(14) Kong, Z. L.; Chang, J. S.; Chang, K. L. B. Antiproliferative effect of antrodia camphorata

450

polysaccharides encapsulated in chitosan–silica nanoparticles strongly depends on the

451

metabolic activity type of the cell line. J. Nanopart. Res. 2013, 15(9), 1–13.

452

(15) Sun, W. J.; Hu, W. J.; Meng, K.; Yang, L. M.; Zhang, W. M.; Song, X. P.; Qu, X. H.; Zhang, Y.

453

Y.; Ma, L.; Fan, Y. P. Activation of macrophages by the ophiopogon polysaccharide liposome

454

from the root tuber of ophiopogon japonicus. Int. J. Biol. Macromol. 2016, 91, 918–925.

455

(16) Liu, Z. G.; Xing, J.; Zheng, S. S.; Bo, R. N.; Luo, L.; Huang, Y.; Niu, Y. L.; Li, Z. H.; Wang,

456

D. Y.; Hu, Y. L.; Liu, J. G.; Wu, Y. Ganoderma lucidum polysaccharides encapsulated in

457

liposome as an adjuvant to promote Th1-bias immune response. Carbohydr. Polym. 2016,

458

142, 141–148.

459

(17) Wang, Y. Y.; Li, P. F.; Chen, F.; Jia, L. Q.; Xu, Q. H.; Gai, X. M., Yu, Y. B.; Di, Y.; Zhu, Z. H.

460

L.; Liang, Y. Y.; Liu, M. Q.; Pan, W. S.; Yang, X. G. A novel pH-sensitive carrier for the

461

delivery of antitumor drugs: histidine-modified auricularia auricular polysaccharide

462

nano-micelles. Sci. Rep-UK 2017, 7(1), 4751.

ACS Paragon Plus Environment

Page 23 of 43

Journal of Agricultural and Food Chemistry

463

(18) Qiu, C.; Qin, Y.; Jiang, S. S.; Liu, C. Z.; Xiong, L.; Sun, Q. J. Preparation of active

464

polysaccharide-loaded maltodextrin nanoparticles and their stability as a function of ionic

465

strength and pH. LWT-Food Sci. Technol. 2017, 76, 164–171.

466

(19) Qiu, C.; Yang, J.; Ge, S. J.; Chang, R. R.; Xiong, L.; Sun, Q. J. Preparation and

467

characterization of size-controlled starch nanoparticles based on short linear chains from

468

debranched waxy corn starch. LWT-Food Sci. Technol. 2016, 74, 303–310.

469

(20) Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Evaluation of a

470

tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing,

471

Cancer Res. 1987, 47, 936−942.

472 473 474 475

(21) Wikler, M. A. Correction of a reference to clinical laboratory standards institute interpretive criteria. Clin. Infect. Dis. 2008, 46(11), 1798. (22) Tan, Y.; Xu, K.; Li, L.; Liu, C.; Song, C.; Wang, P. Fabrication of size-controlled starch-based nanospheres by nanoprecipitation. ACS Appl. Mater. Interfaces 2009, 1(4), 956–959.

476

(23) Kakran, M.; Sahoo, N. G.; Tan, I. L.; Li, L. Preparation of nanoparticles of poorly

477

water-soluble antioxidant curcumin by antisolvent precipitation methods. J. Nanopart. Res.

478

2012, 14, 757.

479 480

(24) Joye, I. J.; Mcclements, D. J. Production of nanoparticles by anti-solvent precipitation for use in food systems. Trends Food Sci. Tech. 2013, 34(2), 109–123.

481

(25) Fan, J.; Feng, H. B.; Yu, Y.; Sun, M. X.; Liu, Y. R.; Li, T. Z.; Sun, X.; Liu, S. J.; Sun, M. D.

482

Antioxidant activities of the polysaccharides of chuanminshen violaceum. Carbohydr. Polym.

483

2017, 157, 629–636.

484

(26) Zeng, H. L.; Miao, S.; Zhang, Y.; Lin, S.; Jian, Y. Y.; Tian, Y. T.; Zheng, B. D. Isolation,

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

485

preliminary structural characterization and hypolipidemic effect of polysaccharide fractions

486

from fortunella margarita, (lour.) swingle. Food Hydrocolloids 2016, 52, 126–136.

487

(27) Chen, C.; You, L. J.; Abbasi, A. M.; Fu, X.; Liu, R. H.; Li, C. Characterization of

488

polysaccharide fractions in mulberry fruit and assessment of their antioxidant and

489

hypoglycemic activities in vitro. Food Funct. 2016, 7(1), 530–539.

490

(28) Zhao, Z. Y.; Huangfu, L. T.; Dong, L. L.; Liu, S. L. Functional groups and antioxidant

491

activities of polysaccharides from five categories of tea. Ind. Crops Prod. 2014, 58(1), 31–35.

492

(29) Jahanshahi, M.; Babaei, Z. Protein nanoparticle: A unique system as drug delivery vehicles.

493

Afr. J. Biotechnol. 2008, 7, 4926–4934.

494

(30) Tang, X. J.; Han, M.; Yang, B.; Shen, Y.; He, Z. G.; Xu, D. H.; Gao, J. Q. Nanocarrier

495

improves the bioavailability, stability and antitumor activity of camptothecin. Int. J.

496

Pharmaceut. 2014, 477, 536–545.

497

(31) Chen, G. J.; Yuan, Q. X.; Saeeduddin, M.; Ou, S. Y.; Zeng, X. X.; Hong, Y. Recent advances

498

in tea polysaccharides: extraction, purification, physicochemical characterization and

499

bioactivities. Carbohydr. Polym. 2016, 153, 663–678.

500

(32) Guo, M.; Ding, G. B.; Guo, S. J.; Li, Z. Y.; Zhao, L. Q.; Li, K.; Guo, X. R. Isolation and

501

antitumor efficacy evaluation of a polysaccharide from nostoc commune vauch. Food Funct.

502

2015, 6(9), 3035–3046.

503

(33) Granato, D.; Grevink, R.; Zielinski, A. A. F.; Nunes, D. S.; van Ruth, S. M. Analytical

504

strategy coupled with response surface methodology to maximize the extraction of

505

antioxidants from ternary mixtures of green, yellow, and red teas (camellia sinensis var.

506

sinensis). J. Agric. Food Chem. 2014, 62(42), 10283–10296.

ACS Paragon Plus Environment

Page 24 of 43

Page 25 of 43

Journal of Agricultural and Food Chemistry

507

(34) Schaffazick, S. R.; Pohlmann, A. R.; de Cordova, C. A.; Creczynski-Pasa, T. B.; Guterres, S.

508

S. Protective properties of melatonin-loaded nanoparticles against lipid peroxidation. Int. J.

509

Pharmaceut. 2005, 289(1–2), 209–213.

510

(35) Yen, F. L.; Wu, T. H.; Tzeng, C. W.; Lin, L. T.; Lin, C. C. Curcumin nanoparticles improve

511

the physicochemical properties of curcumin and effectively enhance its antioxidant and

512

antihepatoma activities. J. Agric. Food Chem. 2010, 58(12), 7376–7382.

513

(36) Tzeng, C. W.; Yen, F. L.; Wu, T. H.; Ko, H. H.; Lee, C. W.; Tzeng, W. S.; Lin, C. C.

514

Enhancement of dissolution and antioxidant activity of kaempferol using a nanoparticle

515

engineering process. J. Agric. Food Chem. 2011, 59, 5073−5080.

516

(37) Lee, C. W.; Yen, F. L.; Huang, H. W.; Wu, T. H.; Ko, H. H.; Tzeng, W. S.; Lin, C. C.

517

Resveratrol nanoparticle system improves dissolution properties and enhances the

518

hepatoprotective effect of resveratrol through antioxidant and anti-inflammatory pathways. J.

519

Agric. Food Chem. 2012, 60(18), 4662−4671.

520

(38) Jiang, J. Y.; Kong, F. S.; Li, N. S.; Zhang, D. Z.; Yan, C. Y.; Lv, H. Purification, structural

521

characterization and in vitro antioxidant activity of a novel polysaccharide from boshuzhi.

522

Carbohydr. Polym. 2016, 147, 365–371.

523 524

(39) Chen, R. Z. Antioxidant and immunobiological activity of water-soluble polysaccharide fractions purified from Acanthopanax senticosu. Food Chem. 2011, 127, 434–440.

525

(40) Shi, M. J.; Wei, X.; Xu, J.; Chen, B. J.; Zhao, D. Y.; Cui, S.; Zhou, T. Carboxymethylated

526

degraded polysaccharides from enteromorpha prolifera: preparation and in vitro antioxidant

527

activity. Food Chem. 2017, 215, 76–83.

528

(41) Liu, Y.; Zhang, B.; Ibrahim, S. A.; Gao, S. S.; Yang, H.; Huang, W. Purification,

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

529

characterization and antioxidant activity of polysaccharides from flammulina velutipes

530

residue. Carbohydr. Polym. 2016, 145, 71–77.

531

(42) Xie, J. H.; Wang, Z. J.; Shen, M. Y.; Nie, S. P.; Gong, B.; Li, H. S.; Zhao, Q.; Li, W. J.; Xie,

532

M. Y. Sulfated modification, characterization and antioxidant activities of polysaccharide

533

from cyclocarya paliurus. Food Hydrocolloids 2015, 53, 7–15.

534

(43) Xie, M. H.; Hu, B.; Wang, Y.; Zeng, X. X. Grafting of gallic acid onto chitosan enhances

535

antioxidant activities and alters rheological properties of the copolymer. J. Agric. Food Chem.

536

2014, 62(37), 9128–9136.

537

(44) Xie, J. H.; Tang, W.; Jin, M. L.; Li, J. E.; Xie, M. Y. Recent advances in bioactive

538

polysaccharides from Lycium barbarum L., Zizyphus jujuba Mill, Plantago spp., and Morus

539

spp.: Structures and functionalities. Food Hydrocolloids 2016, 60, 148–160.

540

(45) Di, T.; Chen, G. J.; Sun, Y.; Ou, S. Y.; Zeng, X. X.; Ye, H. Antioxidant and

541

immunostimulating activities in vitro of sulfated polysaccharides isolated from gracilaria

542

rubra. J. Funct. Foods 2017, 28, 64–75.

543

(46) Raveendran, S.; Palaninathan, V.; Nagaoka, Y.; Fukuda, T.; Iwai, S.; Higashi, T., Mizuki, T.;

544

Sakamoto, Y.; Mohanan, P. V.; Maekawa, T.; Kumar, D. S. Extremophilic polysaccharide

545

nanoparticles for cancer nanotherapy and evaluation of antioxidant properties. Int. J. Biol.

546

Macromol. 2015, 76, 310–319.

547

(47) Hudson, J. A.; Frewer, L. J.; Jones, G.; Brereton, P. A.; Whittingham, M. J.; Stewart, G. The

548

agri-food chain and antimicrobial resistance: a review. Trends Food Sc. Tech. 2017, 69(2017),

549

131–147.

550

(48) Omar, A.; Nadworny, P. Review: antimicrobial efficacy validation using in vitro, and in vivo,

ACS Paragon Plus Environment

Page 26 of 43

Page 27 of 43

Journal of Agricultural and Food Chemistry

551

testing methods. Adv. Drug Deliver. Rev. 2017, 112, 61–68.

552

(49) Li, Q. Q.; Hu, J. L.; Xie, J. H.; Nie, S. P.; Xie, M. Y. Isolation, structure, and bioactivities of

553

polysaccharides from cyclocarya paliurus (batal.) iljinskaja. Ann. NY. Acad. Sci. 2017, 1398

554

(1), 20–29.

555

(50) Costalat, M.; Alcouffe, P.; David, L.; Delair, T. Controlling the complexation of

556

polysaccharides into multi-functional colloidal assemblies for nanomedicine. J. Colloid Interf.

557

Sci. 2014, 430, 147–156.

558

(51) Nguyen, T. V.; Nguyen, T. T. H.; Wang, S. L.; Vo, T. P. K.; Nguyen, A. D. Preparation of

559

chitosan nanoparticles by TPP ionic gelation combined with spray drying, and the

560

antibacterial activity of chitosan nanoparticles and a chitosan nanoparticle–amoxicillin

561

complex. Res. Chem. Intermediat. 2017, 43, 3527–3537.

562

(52) Dai, X. M. ; Guo, Q. Q.; Zhao, Y.; Zhang, P.; Zhang, T. Q.; Zhang, X. G.; Li, C. X. Functional

563

silver nanoparticle as a benign antimicrobial agent that eradicates antibiotic-resistant bacteria

564

and promotes wound healing. ACS Appl. Mater. Inter. 2016, 8(39), 25798–25807.

565

(53) Ivask, A.; Elbadawy, A.; Kaweeteerawat, C.; Boren, D.; Fischer, H.; Ji, Z.; Chang, C. H.; Liu,

566

R.; Tolaymat, T.; Telesca, D.; Zink, J. I.; Cohen, Y.; Holden, P. A.; Godwin, H. A. Toxicity

567

mechanisms in escherichia coli vary for silver nanoparticles and differ from ionic silver. ACS

568

Nano, 2014, 8(1), 374–386.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

569

FIGURE CAPTION

570

Figure 1 Size distribution (A: Intensity; B: Number) and mean size, PDI, and ζ-positional (C) of

571

TP-NPs prepared at different ethanol to water ratio (3:1, 4:1, 5:1, 10:1, and 20:1) at pH 6.0.

572

Figure 2 TEM images of TP-NPs prepared at different ethanol to water ratio (A: 3:1, B: 4:1, C: 5:1,

573

D: 10:1, and E: 20:1) at pH 6.0, respectively. Scale bar is 200 nm.

574

Figure 3 Photo showing the aspect of TP solution (a), and TP-NP suspensions (0.5%) prepared at

575

different ethanol to water ratio (b: 3:1, c: 4:1, d: 5:1, e: 10:1, and f: 20:1) and the Tyndall effect by

576

a light beam incident from the left side.

577

Figure 4 FTIR images of TP-NPs prepared at different ethanol to water ratio at pH 6.0.

578

Figure 5 Effect of different temperature (4, 25, 37, and 65 °C) on size distribution (A: Intensity; B:

579

Number), mean size, PDI, and turbidity of TP-NPs prepared with ethanol to water ratio of 10:1 at

580

pH 6.0. Data are presented as the average of triplicate measurements with standard deviation (n =

581

3).

582

Figure 6 Effect of various ionic strength (0-500 mM, NaCl) on size distribution (A: Intensity; B:

583

Number), mean size, PDI, and turbidity of TP-NPs prepared with ethanol to water ratio of 10:1 at

584

pH 6.0. Data are presented as the average of triplicate measurements with standard deviation (n =

585

3).

586

Figure 7 Effect of various pH-levels (2, 5, 7.4, and 9) on size distribution (A: Intensity; B:

587

Number), mean size, PDI, ζ-potential, and turbidity of TP-NPs prepared with ethanol to water

588

ratio of 10:1 at pH 6.0. Data are presented as the average of triplicate measurements with standard

589

deviation (n = 3).

590

Figure 8 Cytotoxicity of MEF treated with TP-NPs (A), GLP-NPs (B), and MCP-NPs (C) at

ACS Paragon Plus Environment

Page 28 of 43

Page 29 of 43

Journal of Agricultural and Food Chemistry

591

different concentrations.

592

Figure 9 BSA adsorptions of TP-NPs, GLP-NPs, and MCP-NPs after incubating at 37 °C (pH 7.4)

593

for various time intervals.

594

Figure 10 Antibacterial activity of native polysaccharides and polysaccharide nanoparticles against

595

various microorganisms in LB broth after 24 h incubation at 37 °C. The red arrow represents the

596

concentration of samples from low to high.

597

A: GLP and GLP-NPs at the concentration of 0, 250, 500, 1000, 1500, 2000, and 3000 µg/mL; B:

598

MCP and MCP-NPs at the concentration of 0, 250, 500, 1000, 1500, 2000 3000, and 4000 µg/mL.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 30 of 43

599

Table 1 IC50 values of DPPH radical scavenging activity of bioactive polysaccharides and

600

nanoparticles after NaCl (100 mM) and 65 °C treatment. Sample

IC50 (µg/mL) Control

NaCl

Temperature

TP

71.70±3.28c

80.85±3.01b

123.71±9.81a

TP-NPs

46.49±3.12b

46.95±2.74b

56.26±3.25a

GLP

870.42±19.32c

961.11±23.19b

1180.43±20.15a

GLP-NPs

687.91±20.73b

681.19±22.31b

860.67±19.41a

MCP

746.82±22.80c

921.35±20.88b

1215.38±22.57a

MCP-NPs

485.47±18.33c

525.16±19.43b

643.48±17.20a

601

Values mean ± SD indicates the replicates of three experiments. Different letters in the same row (a-c) indicate

602

significant differences (upper case) (P < 0.05).

ACS Paragon Plus Environment

Page 31 of 43

Journal of Agricultural and Food Chemistry

603

Table 2 IC50 values of hydroxyl radicals scavenging activity of bioactive polysaccharides and

604

nanoparticles after NaCl (100 mM) and 65 °C treatment. Sample

IC50 (µg/mL) Control

NaCl

Temperature

TP

68.49±2.78c

87.61±3.08b

193.75±6.87a

TP-NPs

39.54±2.23c

46.72±2.01b

51.26±2.16a

GLP

854.95±18.92c

961.11±19.90b

1209.33±25.77a

GLP-NPs

481.58±20.71b

427.91±21.59c

820.29±23.81a

MCP

526.55±21.29c

773.28±21.78b

1061.54±22.40a

MCP-NPs

273.50±14.27c

300.61±17.83b

579.67±16.97a

605

Values mean ± SD indicates the replicates of three experiments. Different letters in the same row (a-c) indicate

606

significant differences (upper case) (P < 0.05).

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 32 of 43

607

Table 3 IC50 values of superoxide radical scavenging activity of bioactive polysaccharides and

608

nanoparticles after NaCl (100 mM) and 65 °C treatment. Sample

IC50 (µg/mL) Control

NaCl

Temperature

TP

100.53±4.34c

189.75±3.13b

327.31±5.72a

TP-NPs

78.20±3.31b

68.91±3.39c

109.86±4.75a

GLP

771.27±20.89c

949.12±30.01b

1362.37±29.77a

GLP-NPs

426.18±19.72b

415.29±21.59b

760.67±28.06a

MCP

452.86±19.94b

765.22±20.82b

1320.21±29.79a

MCP-NPs

246.74±19.89b

279.61±16.52b

543.49±30.64a

609

Values mean ± SD indicates the replicates of three experiments. Different letters in the same row (a-c) indicate

610

significant differences (upper case) (P < 0.05).

ACS Paragon Plus Environment

Page 33 of 43

Journal of Agricultural and Food Chemistry

25

A

TP-NPs 1:3 TP-NPs 1:4 TP-NPs 1:5 TP-NPs 1:10 TP-NPs 1:20

20

15

B

TP-NPs 1:3 TP-NPs 1:4 TP-NPs 1:5 TP-NPs 1:10 TP-NPs 1:20

20

N um ber ( % )

Intensity ( % )

25

10

5

15

10

5

0

0

10

100

1000

3000

10

100

1000

3000

Size (nm)

Size (nm)

300

C

0.5

Mean size (nm) PDI ζ -positional (mV) 0.4

250

0 -5 -10

200 0.3

-15

150 0.2

-20

100 0.1

50

-25

0.0 -30

0 3

4

5

10

20

The ratio of ethanol to water

611

Figure 1 Size distribution (A: Intensity; B: Number) and mean size, PDI, and ζ-positional (C) of

612

TP-NPs prepared at different ethanol to water ratio (3:1, 4:1, 5:1, 10:1, and 20:1) at pH 6.0.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

A

Page 34 of 43

C

B

200 nm

200 nm

200 nm

E

D

200 nm

200 nm

613

Figure 2 TEM images of TP-NPs prepared at different ethanol to water ratio (A: 3:1, B: 4:1, C: 5:1,

614

D: 10:1, and E: 20:1) at pH 6.0, respectively. Scale bar is 200 nm.

ACS Paragon Plus Environment

Page 35 of 43

Journal of Agricultural and Food Chemistry

a

b

c

d

e

f

615

Figure 3 Photo showing the aspect of TP solution (a), and TP-NP suspensions (0.5%) prepared at

616

different ethanol to water ratio (b: 3:1, c: 4:1, d: 5:1, e: 10:1, and f: 20:1) and the Tyndall effect by

617

a light beam incident from the left side.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

TP-NPs 1:4 TP-NPs 1:10 TP

Intensity (%)

TP-NPs 1:3 TP-NPs 1:5 TP-NPs 1:20

Page 36 of 43

4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

618

Figure 4 FTIR images of TP-NPs prepared at different ethanol to water ratio at pH 6.0.

ACS Paragon Plus Environment

Page 37 of 43

Journal of Agricultural and Food Chemistry

25

A

4 °C 25 °C 37 °C 65 °C

20

B

4 °C 25 °C 37 °C 65 °C

20

Number ( % )

Intensity ( % )

25

15

10

15

10

5

5

0

0 10

100

1000

10

3000

100

350 300

1000

3000

Size (nm)

Size (nm)

C

Mean size (nm) PDI Turbidity

0.6

0.6

0.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.1

250 200 150 100 50 0 4

25

37

65

Temperature (°C) 619

Figure 5 Effect of different temperature (4, 25, 37, and 65 °C) on size distribution (A: Intensity; B:

620

Number), mean size, PDI, and turbidity of TP-NPs prepared with ethanol to water ratio of 10:1 at

621

pH 6.0. Data are presented as the average of triplicate measurements with standard deviation (n =

622

3)

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

30

A

0 mM NaCl 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl 500 mM NaCl

Intensity ( % )

15

B

25

N u m b e r (% )

20

10

5

Page 38 of 43

0 mM NaCl 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl 500 mM NaCl

20 15 10 5

0

0 10

100

3000

1000

10

100

Size (nm)

300 250

1000

3000

Size (nm)

0.6

Mean size (nm) PDI Turbidity

C

0.5

0.4 0.5

200 0.3 150

0.4 0.2

100 0.3 50 0

0.2 0

100

200

300

400

0.1

0.0

500

NaCl (mM) 623

Figure 6 Effect of various ionic strength (0-500 mM, NaCl) on size distribution (A: Intensity; B:

624

Number), mean size, PDI, and turbidity of TP-NPs prepared with ethanol to water ratio of 10:1 at

625

pH 6.0. Data are presented as the average of triplicate measurements with standard deviation (n =

626

3).

ACS Paragon Plus Environment

Page 39 of 43

Journal of Agricultural and Food Chemistry

A

18 16

Intensity ( % )

40

pH = 2.0 pH = 5.0 pH = 7.4 pH = 9.0

14

B

35

N u m b e r (% )

20

12 10 8 6

pH= 2.0 pH= 5.0 pH= 7.4 pH= 9.0

30 25 20 15 10

4

5

2 0

0

10

100

1000

3000

10

100

10

300

0

250

-10

1000

3000

Size (nm)

Size (nm)

C

Mean size (nm) Turbidity

PDI ζ - potential

0.7 0.6

0.6 0.5

200 0.5 0.4

-20

150 0.4 0.3

-30 100 -40

0.3 0.2

50 -50 -55

0.2 0.1

0 2

5

7.4

9

pH 627

Figure 7 Effect of various pH-levels (2, 5, 7.4, and 9) on size distribution (A: Intensity; B:

628

Number), mean size, PDI, ζ-potential, and turbidity of TP-NPs prepared with ethanol to water

629

ratio of 10:1 at pH 6.0. Data are presented as the average of triplicate measurements with standard

630

deviation (n = 3).

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

120

A

TPS

TP-NPs

Cell viability ( % )

100 80 60 40 20 0 25

0

50

100

200

150

Concentration (µg/mL)

120

B

GLP GLP-NPs

Cell viability ( % )

100 80 60 40 20 0 0

250

500

1000

1500

2000

Concentration (µg/mL)

120

C

MCP MCP-NPs

Cell viability (% )

100 80 60 40 20 0 0

250

500

1000

1500

2000

Concentration (µg/mL) 631

Figure 8 Cytotoxicity of MEF treated with TP-NPs (A), GLP-NPs (B), and MCP-NPs (C) at

632

different concentrations.

ACS Paragon Plus Environment

Page 40 of 43

Journal of Agricultural and Food Chemistry

Protein adsorption ratio ( % )

Page 41 of 43

35

TPS-NPs GLP-NPs MCP-NPs

30 25 20 15 10 5 0

5

10

15

20

25

30

35

40

Time (h) 633

Figure 9 BSA adsorptions of TP-NPs, GLP-NPs, and MCP-NPs after incubating at 37 °C (pH 7.4)

634

for various time intervals.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

1.0

A

0.8

OD 600

0.6

0.4

0.2

0.0

GLP

GLP-NPs

GLP GLP-NPs GLP

S. aureus Bacillus subtilis

1.0

GLP-NPs

E. coil

B

OD 600

0.8

0.6

0.4

0.2

0.0

MCP MCP-NPs MCP MCP-NPs MCP MCP-NPs

S. aureus

E. coil

Salmonella

635

Figure 10 Antibacterial activity of native polysaccharides and polysaccharide nanoparticles against

636

various microorganisms in LB broth after 24 h incubation at 37 °C. The red arrow represents the

637

concentration of samples from low to high.

638

A: GLP and GLP-NPs at the concentration of 0, 250, 500, 1000, 1500, 2000, and 3000 µg/mL; B:

639

MCP and MCP-NPs at the concentration of 0, 250, 500, 1000, 1500, 2000 3000, and 4000 µg/mL.

ACS Paragon Plus Environment

Page 42 of 43

Page 43 of 43

Journal of Agricultural and Food Chemistry

Graphic Abstract Antimicrobial MCP S. aureus MCP-NPs MCP E.coli MCP-NPs MCP Salmonella MCP-NPs

1.0

Light beam

0.8

OD600

Tyndall effect

0.6 0.4 0.2

Add dropwise

0.0

0

5

10 15 20 25 30 35 40 45 50

Time (h)

Stirring

TEM image

Ethanol Polysaccharide Bioactive

polysaccharide

Antioxidant

100

Scavenging ability (%)

640

80 60 40 Vc TP TP/NaCl TP/65 °C

20 0

0

50

100

150

TP-NPs TP-NPs/NaCl TP-NPs/65 °C

200

250

Concentration (µg/mL)

ACS Paragon Plus Environment

300