Selenylation of Polysaccharide from the Sweet Potato and Evaluation

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES

Article

Selenylation of polysaccharide from the sweet potato and evaluation of anti-oxidant and anti-diabetic activities Bo Yuan, Xu-qin Yang, Meng Kou, Chang-Yan Lu, Yuan-yuan Wang, Jun Peng, Ping Chen, and Ji-hong Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04788 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 10, 2017

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 53

Journal of Agricultural and Food Chemistry

Table of Contents 399x443mm (300 x 300 DPI)

ACS Paragon Plus Environment


1

Selenylation of polysaccharide from the sweet potato and

2

evaluation of anti-oxidant and anti-diabetic activities

3

Bo Yuan, †,ǁ Xu-qin Yang, †,ǁ Meng Kou, †,‡ Chang-yan Lu, # Yuan-yuan Wang, §Jun

4

Peng, † Ping Chen, †, ‡ Ji-hong Jiang†*

5

†

The Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province &

6

School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116, PR

7

China

8 9 10 11

#

§

He Fei First People Hospital, He fei, An-Hui 2183114, China. College of Biomedical Sciences, Xuzhou Medical University, Xuzhou, Jiangsu

221004, China. ‡

Jiangsu Xuzhou Sweetpotato Research Center, Xuzhou, Jiangsu 221131, China

12 13

1


Page 2 of 53

Page 3 of 53


14

ABSTRACT

15

Interest in sweet potato as a functional food is growing. A polysaccharide (SWP)

16

was isolated from the sweet potato tuber and elucidation of its structure as composed

17

of rhamnose, glucose, and galactose undertaken. To improve its activity, selenylation

18

of this novel polysaccharide (Se-SWP) was undertaken by using microwave synthesis.

19

In vitro evaluation showed that the Se-SWP have excellent anti-oxidant activity on

20

scavenging free radical and reducing capacity. In vivo anti-tumour evaluation showed

21

selenylation polysaccharide could effectively inhibit tumour growth (>50%) and

22

adjust immune factors levels in the mice (IL-2, TNF-α, and VEGF). The anti-diabetic

23

potential of Se-SWP was tested in STZ-induced diabetic rats. The results indicated

24

that the Se-SWP treatment significantly reduced the levels of malondialdehyde and

25

other disadvantageous factors that were increased by the STZ-induced treatment.

26

Meanwhile, the Se-SWP treatment caused a significant increase in the activities of

27

enzymatic antioxidants and the levels of non-enzymatic antioxidants in the organs of

28

diabetic rats. All of the activity evaluations indicated that the selenylation method

29

could improve the activity of sweet potato polysaccharide. And its efficacy as a

30

potential therapeutic, which will be the focus of further study.

31 32

KEY

WORDS:

33

anti-diabetic.

Sweet

potato

polysaccharide;

selenylation;

2


anti-oxidant;


34

Introduction

35

In modern medical research, some diseases are caused by metabolic disturbance,

36

such as diabetes mellitus. Diabetes mellitus is a serious chronic metabolic disease and

37

there are about 0.2 billion patients in China alone. Insulin resistance, or impaired

38

synthesis in peripheral tissues of the pancreas, means that the stubbornly high blood

39

glucose level leads to the morbidity rates observed in patients with diabetes mellitus. 1

40

Meanwhile, the abnormalities in the metabolism of lipids and proteins are also factors

41

that cause diabetes mellitus.2 For the treatment of diabetes mellitus, injectable

42

hormones and drugs are the main methods used. In the conventional treatment of

43

those diseases, these medications are the most commonly prescribed to help control

44

their development, however, their effectiveness has been proven and they are known

45

for their untoward side effects. Some researchers hope to develop drugs from

46

medicinal plants because they may pose a less toxic effect. Sweet potato (Ipomoea

47

batatas) is among the most nutritious sub-tropical/tropical foodstuffs. Some studies

48

showed that the sweet potato is also used in traditional medicine practices for type 2

49

diabetes mellitus.3-5

50

Sweet potato is not popular in cooking in many areas in Asia and the Pacific,

51

Africa, and America, but is also used in traditional medicine for the treatment of

52

diabetes mellitus. Meanwhile, it is worth noting the high content, and low toxicity, of

53

polysaccharides in sweet potato tubers: sweet potato polysaccharides (SWP) have

54

attracted more research attention recently and most studies focus on their anti-oxidant

55

and anti-tumour activities.6-9 Because of the hydroxyl radical, the polysaccharide is 3


Page 4 of 53

Page 5 of 53


56

considered a powerful oxidant.10 The formation and propagation of lipid radicals

57

could rearranged the double bonds in unsaturated lipids and destroyed the membrane

58

lipids, even lead to disease. Meanwhile, the polysaccharide can activate the

59

macrophage11 and lymphocyte.12 To a certain extent, the polysaccharide also

60

increasing the secretion of cytokines, such as IL-2, IL-6 and TNF.13 Therefore,

61

research on antioxidant and antitumor, especially exploration of potent natural

62

polysaccharide with low cytotoxicity from sweet potato, has become a hot research

63

field. Identification, or modification, of the SWP is necessary to exploit better the

64

structural and functional properties of these substances.

65

Selenium (Se) is an important trace elements for human health.14 It is a key

66

constituent of Se-dependent enzymes including glutathione peroxidase, types I, II, and

67

III iodothyronine deiodinase, and thioredoxin reductase in the human body.15-18 Se

68

also displays an insulin-mimetic activity both in vitro and in vivo.19 Some researches

69

indicate that the Se participates in synthesis of enzymes and protects the structure and

70

function of biomembrane from over-oxidation and cell damage.20 A lack of selenium

71

could lead to the development and progression of chronic diseases.21 There were

72

about 700 million people with no, or lowered, selenium levels in China22 and this

73

phenomenon is also global.

74

It is generally believed that organic selenium compounds are better and safer

75

than inorganic selenium as medicine.23 There are several reports focused on selenium

76

polysaccharides that have reported that they play an important role in increasing

77

anti-proliferative, antioxidant, and anti-diabetic activity.24, 25 The content of natural 4



Page 6 of 53

78

selenium in polysaccharides is low, so more attention was focused on its artificial

79

synthesis.26, 27

80

In present study, we extracted crude SWP and the activity test showed crude

81

SWP was an anti-oxidant. Meanwhile, isolation of crude SWP and its purification (No.

82

SWPYB01) with a molecular weight of 2.4 × 103 Da was undertaken. Meanwhile, the

83

selenylation derivatives of SWP (Se-SWP) synthesised using the microwave radiation.

84

Characterisation of the structure of Se-SWP by UV, FT-IR spectra, and

85

spectroscopy followed. Then in vivo evaluation of anti-liver tumour and anti-diabetic

86

activity was investigated. In addition, the antioxidant activity in vitro was also

87

established and assayed.

88

Materials and methods

13

C-NMR

89

Materials. The sweet potato tubers were collected from Tongshan, Xuzhou,

90

Jiangsu Province, China. DEAE-52 cellulose was purchased from Pharmacia

91

(Sweden). DEAE–sepharose fast flow and Sephacryl S-300 HR were purchased from

92

Amersham Biosciences (Sweden). Other chemical agents were purchased from

93

Sigma–Aldrich (USA) and Guo-yao Group China (Shanghai, China).

94

Purification of sweet potato polysaccharide. 1kg of dried sweet potato powder

95

was dispersed by stirring for 30 min in 500 mL of deionized water. Then transferred

96

the mixture to an ultrasonic cleaner (KQ-500, Kunshan Ultrasonic Cleaner company)

97

and extracted at a power for 200 W for 30 min. Using the α-amylase

98

(enzyme-substrate, 100 U/g) to hydrolyse the mixture at 60oC with stirring. After

99

hydroxylation, the suspension was centrifuged at 3000 rpm for 10 min. The 5


Page 7 of 53


100

supernatant was concentrated to one fifth of the original volume and then precipitated

101

with 5 volume of 95% ethanol (v/v). The protein removed and combined with a

102

neutral enzyme (papain, 200 U/mL).26, 28 Then, after centrifugation, the supernatant

103

was dialysed with distilled water. About 48 h later, the sample was passed through a

104

DEAE-cellulose column (2.6 × 40 cm). The sample was eluted with distilled water,

105

then with a linear gradient of NaCl at a flow rate of 0.40 mL/min. The main fraction

106

was retained using the automatic collector. Then the sample was dialysed, lyophilised,

107

and further fractionated on a DEAE-sepharose fast column (2.6 × 40 cm), eluted with

108

a linear gradient of NaCl (0 to 0.3 M) at a flow rate of 1 mL/min. The main fraction

109

was collected, dialysed, and lyophilised to obtain the final polysaccharide (No.

110

SWPYB01, hereinafter referred to as SWP). The molecular weight of SWPYB01 was

111

evaluated by gel permeation chromatography on a Sephacryl S-300 HR column

112

(2.6 × 40 cm, Amersham Pharmacia Biotech Ltd, USA) with standard dextrans

113

(dextran Mw: 5 × 106, 2 × 105, 7 × 104, 1 × 104, and 3 × 103; Pharmacia) and glucose.

114

Synthesis of the selenylation modification of SWPYB01 (Se-SWP). We

115

dissolved 1 g SWP powder in 0.6% (v/v) of HNO3 (50 mL) at room temperature with

116

stirring for 30 to 40 min. Then, we added the Na2SeO3 and BaCl2 to the HNO3-SWP

117

solvent and then transferred the mixture to a reflux. The household microwave oven

118

was made over into experimental device that the reflux could be used inside. Stirred

119

the mixture for 5 min at a low power (remained the temperature at about 100 oC) and

120

did six to ten cycles of this. After the reaction, the mixture was cooled with ice to

121

room temperature and the pH adjusted to 7 to 8. Na2SO4 was added to the mixture to 6



122

remove the Ba2+. Then, after centrifugation, the supernatant was dialysed using

123

distilled water overnight. The polysaccharide solutions were concentrated and

124

precipitated with ethanol. After lyophilising in a vacuum freeze-dying machine, the

125

Se-SWP, with different Se contents, was obtained and stored at 4 oC.

126

Evaluation of the absorbance and measurements were carried out using the

127

Thermo Fisher E-300 UV-VIS spectrophotometer (Thermo Fisher, USA) and FT-IR

128

spectra. A Bruker Avance 400 MHz spectrometer (Germany) was used to record the

129

13

CNMR spectra of SWP and Se-SWP (50 mg/mL).

130

The selenium content was determined by the atomic fluorescence spectrometry

131

(AFS-2100, Peking Hai-guang instrument Co., Ltd). The working conditions of the

132

spectrometer were as follows according to the reference with subtle differences: 270

133

V of the negative high voltage, lamp current was 100 mA, the atomization

134

temperature was 200 oC, the height of atomization gas was 8 mm, the flow of shield

135

gas and carrier gas were 400 mL/min and 800 mL/min, respectively. The injection

136

volume was 1mL and the concentrations of standard (selenium standard sample,

137

Shenzhen Pufen Technology Co.) curve were set at 0, 4, 8, 12, 16 and 20 ng/mL.

138

50 mg of Se-SWP weighed accurately was dissolved in 25 mL of ultrapure water，

139

1.0 mL of Se-SWP solution was accurately measured and added into triangular flask

140

with cork and added into 10 mL of HClO4-HNO3(v/v, 1:1). Then the mixture was

141

mixed acid solution and digest for 8 h at 4oC and heat at 120oC replenishing the mixed

142

acid solution timely. Cooled the solution to room temperature after the reaction and

143

diluted accurately into 30 mL with 5% HCl solution. Setting a blank sample solution 7


Page 8 of 53

Page 9 of 53


144

and prepared by the same method above. Detected the fluorescence intensities of the

145

sample solution by the spectrometer.

146

Evaluation of the structure of SWP and Se-SWP. (1) Monosaccharide

147

composition. Some 10 mg of SWP was hydrolysed with 2M TFA at 100 oC in a sealed

148

tube for 12 h. We removed the excess TFA using a water bath at a temperature of 50

149

o

C and co-distilled it with methanol. The hydrolysate was dissolved in distilled water

150

and then analysed using High Performance Liquid Chromatography (HPLC, 1260

151

System, Ageilent. USA) coupled with Evaporative Light-scattering Detector (ELSD,

152

UM5000, Unimicro Technologies, Inc, China) as the detector. All standard and

153

fortified sample solutions were passed through a 0.45 µm filter before use. The

154

mobile-phase solution was degassed in an ultrasonic bath for 20 min before use. The

155

detection conditions were as follows: isocratic elution (acetonitrile and water, 72:28,

156

v/v) with a C18 column chromatography (2.1×50mm, packed with 2.7-µm C18

157

stationary phase). The flow rate of the pump was 0.4 mL/min. The column was

158

equilibrated for about 30 min after the mobile phase was changed and the temperature

159

of the column was kept at room temperature. The ELSD conditions were: gas flow is

160

5.0L/min and evaporating temperature is 80oC.

161

(2) Partial acid hydrolysis. To ensure the quality of the backbone and

162

branched-chains of SWPs, gradual hydrolysis was used: firstly, we hydrolysed the

163

SWP in 0.05 M TFA and then dialysed it. We separated the solution outside the

164

dialysis bag and dried it by rotary evaporations. We dissolved the residue with water

165

and determined the composition by HPLC-ELSD. We collected the solution retained 8



166

in the dialysis bag and dried it by rotary evaporation. Then, we added 0.5 M TFA and

167

continued its hydrolysis and dialysis. The solution outside the dialysis bag was

168

separated and managed as above. The residue inside the bag was dried out and we

169

continued hydrolysis by 2 M TFA and determined the composition of the hydrolysate

170

outside the bag.

171

(3) Smith degradation and methylation analysis. The Smith degradation method

172

is essentially the Fei method with no modifications:28 20 mg of SWP was swollen in

173

10 mL of distilled water, and then 25 mL of 15 nM NaIPO4 was added to the solution.

174

The solution was drawn at 6 h intervals in a dark environment at 4 oC and diluted with

175

distilled water. We determined the consumption of HIO4 and formic acid production

176

with NaOH. The non-dialysate was concentrated and reduced with NaBH4 (40 mg)

177

for 24 h at room temperature and then neutralised to pH 5.0 with 0.1 M HOAc. The

178

mixture product was hydrolysed with 2 M TFA (3 mL) after dialysis and

179

concentration before being tested by HPLC-ELSD.

180

Some 50 mg of SWP was methylated twice according to the method of Ciucanu

181

and Kerek method.29 The methylated products were extracted by CHCl3, and assayed

182

by FI-IR. There was no absorption peak within 3600–3300 cm−1 in the IR spectrum

183

analysis, which indicated complete methylation. Hydrolysis of the product with

184

formic acid and 2 M TFA, and removal of excess acid by co-distillation was followed

185

by reduction with NaBH4 for 24 h, and acetylation with acetic anhydride-pyridine (1:1)

186

at 100 oC for 2 h. The alditol acetates of the methylated sugars were analysed by

187

GC-MS. The gas chromatography (GC) system controlled by the Chemstation 9


Page 10 of 53

Page 11 of 53


188

software and was equipped with a Trace 1300 gas chromatograph (Thermo Fisher,

189

USA) coupled to an ISQ quadrupole mass detector.

190

(4) IR and

13

C-NMR spectroscopy. IR spectra were recorded with a Tensor 27 13

191

Bruker instrument with KBr pellets.

192

Bruker AVANCE AV 400 Spectrometer at 55 oC. The sample was dissolved in D2O

193

and dioxane was used as an internal standard.

194

Assay

of

anti-oxidant

C NMR spectroscopy was measured with a

activity

of

SWP

and

Se-SWP.

The

195

2,2-diphenyl-1-picrylhydrazyl (DPPH, methanolic) radical scavenging capacity of

196

SWP and Se-SWP, was evaluated by the Blois method with no modifications.30

197

Ascorbic acid and BHT were used as standards, and were subjected to the same

198

procedure for comparison. The antioxidant activity was calculated using equation 1:

199

IC(%)=[(A0-AS)/A0]×100%……………………(1)

200

Where As is the absorbance value (517 nm) of the sample and A0 is the

201

absorbance value of the blank (methanol). The concentration of samples that reduced

202

the absorption of DPPH solution by 50% (IC50) was calculated from the calibration

203

curve.

204

The ABTS radical scavenging activity of the SWP and Se-SWP were determined

205

as described by Re.31 The solvent for this method was the menthol. The radical

206

scavenging activities of essential oils and standards (ascorbic acid and BHT) were

207

expressed as the inhibition percentage (IC%), calculated using equation 2:

208

% scavenging (IC%)=[(1-As)/A0]×100%……………………(2)

209

Where As is the absorbance of the sample and A0 is the absorbance (737 nm) of 10



210

the control. The IC50 value was determined as the concentration of the sample

211

required to cause 50% inhibition of the ABTS reaction.

212

The iron (III)-reducing capacity of the SWP and Se-SWP were determined as

213

described by Ud:32 The solvent for this method was the menthol. Determined the

214

absorbance value at 700 nm against blanks and the higher values meant a stronger

215

reducing power. Ascorbic acid and BHT were used as positive controls (+). The

216

results are expressed as the effective concentration at which 0.5 absorbance units

217

(EC50) were obtained from a linear regression analysis.

218

The ferric-reducing ability power (FRAP) of the SWP and Se-SWP were carried

219

out as reported elsewhere.33 The value was expressed as micromoles of Fe2+

220

equivalent per 100 g of sample using the calibration cure of Fe2+.

221

Evaluation of the anti-tumour activity of SWP and Se-SWP. The

222

lipopolysaccharides (LPS) test for the SWP and Se-SWP was in processing with a

223

ELISA kit for LPS (96Tests, DRG, German) and the feminine results showed there is

224

no LPS in the SWP and Se-SWP. The murine H22 hepatoma cell line was purchased

225

from the Key Laboratory of Medical Plant Biotechnology and maintained in

226

Dulbecco’s modified eagle medium supplemented with 10% foetal calf serum and

227

then cultured at 37 °C in 5% CO2. Female Kunming mice (aged 6 weeks) were

228

purchased from the Animal Centre of Xuzhou Medicinal University (Xuzhou, China).

229

All animal procedures were conducted in compliance with the guidelines approved by

230

the Ethics Committee of the School of Life Science of Jiangsu Normal University.

231

The cells were made into the tumour cell suspension with physiological saline at 11


Page 12 of 53

Page 13 of 53


232

a concentration 5 × 108 /mL. The tumour-bearing mice model was established through

233

inoculation of the tumour cell suspension into mice (0.2 mL per mouse) with

234

continued culturing for 24 h. Then, two groups were randomly divided with 40 mice

235

in each group. We continued to feed them under a normal diet for a week, each day

236

five mice were treated and we obtained their peripheral blood. On day 6, the

237

experimental group were subjected to intraperitoneal injected with different Se-SWP

238

solutions once per day for 28 days. The control group were injected with equal

239

volumes of physiological saline. Each week, peripheral blood of five mice was

240

obtained and the serum interleukin-2 (IL-2), tumour necrosis factor-α (TNF-α), and

241

vascular endothelial growth factor (VEGF) levels of the mice determined using the

242

indirect ELISA (kit:48T/96T, Beijing Jianping Jiuxing Bio-medical Co.) assay couple

243

with the procedures described in the commercial kit instructions. We used different

244

concentrations of Se-SWP to evaluate the effect thereof. The same treatment was used

245

in the SWP experiment.

246

After treatment, peripheral blood was obtained from the mice. The tumour was

247

separated and weighed. Tumour inhibition rate (TIR) was calculated using equation

248

334:

249

TIR=[(Tc-Tt)/ Tc] ×100%…………………… (3)

250

Here, Tc denotes the average tumour weight in the control group and the Tt

251

denotes the average tumour weight in the treatment group.

252

Evaluation of the in vivo anti-diabetic and anti-oxidant activity of SWP and

253

Se-SWP. Male SD rats (320 ± 5 g) were also obtained from the Animal Centre of 12



254

Xuzhou Medical University, and maintained in a single air-conditioned room at 25 °C

255

(12 h light:12 h dark cycle). They were fed ad libitum with a standard laboratory diet

256

and had free access to water. The protocol was approved by Institutional Animal

257

Ethics Committee of Jiangsu Normal University (Xuzhou, China).

258

The diabetes rat model was carried out of the described by the method of

259

Fujita.35 The rats were injected intraperitoneally with the streptozocin (STZ) solution,

260

in a citrated buffer (0.1 mol/L sodium citrate and 0.1 mol/L citric acid, pH 4.2–4.5)

261

for over 72 h, we then determined the level of blood glucose and considered a

262

specimen diabetic at, and above, 16.8 mmol/L. There were five treatment groups with

263

five rats each after establishment of the diabetic rat model. The groups were designed

264

as follows, group 1: normal rat as a control group (item number is C); 2: STZ-induced

265

diabetic rat (DR) as a negative control; 3: DR were administered glibenclamide at 30

266

mg/kg/d by oral gavage, as a positive control (PC); 4, STZ-IDM fed 100 mg/kg/d

267

Se-SWP (Se-SWP); 5: STZ-IDM fed 100 mg/kg/d SWP (SWP). To ensure the best

268

dose, different doses of Se-SWP and SWP were selected for evaluation (results not

269

shown) and 100 mg/kg as the best dose was to choose. Glucose levels in tail blood

270

were determined using a glucose meter each day. Insulin, serum total cholesterol(TC),

271

triglyceride(TG), high-density lipoprotein (HDL) and low-density lipoprotein LDL

272

concentrations in serum were determined using commercial kits. Liver, heart,

273

pancreas, and kidney were homogenised. After centrifugation for 10 min at 3000 rpm

274

and 4 °C, the supernatant was collected for determination of hepatic glycogen,

275

catalase (CAT), malonaldehyde (MDA), superoxide Dismutase (SOD), and 13


Page 14 of 53

Page 15 of 53


276

glutathione peroxidase (GSH-Px) using commercial kits.24 To improve the accuracy,

277

solid phase extraction (SPE) technology, as an accumulation technique, was used.

278

The blood of rats was collected for determination of the amount of thiobarbituric

279

acid reactive substances (TBARS), serum glutamate pyruvate transaminase (SGPT),

280

and serum glutamate oxaloacetate transaminase (SGOT) using the standard protocols.

281

The activity evaluation of alkaline phosphatases (ALP), aspartate and alanine

282

transaminases (AST and ALT), total bilirubin (T- bilirubin), creatinine and urea were

283

measured using commercial kits from Biomaghreb, Tunis, Tunisia and Biomerieux,

284

Lyon, France.

285

Statistical analysis. The experiments were conducted in triplicate. Data were

286

reported as mean ± SD for triplicate determinations. ANOVA and Tukey’s tests

287

performed (Minitab for Windows, Version 14, Minitab Inc., PA) to identify

288

differences among means. Statistical significance was set at P < 0.05.

289

Results and discussion

290

Evaluation of the structure of SWP and Se-SWP. (1) Mono-saccharide

291

composition of SWP. A HPLC-ELSD was used as an efficient detection technology to

292

study the mono-saccharide composition of SWP. Compared with the standard

293

mono-saccharide, there were three mono-saccharides identified as rhamnose (Rha),

294

glucose (Glc), and galactose (Gal) after SWP hydrolysis (2 M TFA) (Figure 1A). The

295

mole ratio of the mono-saccharides was 3.2:1.6:6.5 (Rha, Gal, Glc). The Glc was the

296

major ingredient in this polysaccharide.

297

The results of partial acid hydrolysis are shown in Figures 1B, 1C, and 1D: each 14



298

fraction was subjected to HPLC analysis. Figure 1B shows that the mole ratio of Gal

299

and Glc was 0.09:1.2, which indicated that glucose might be the backbone of the

300

structure of SWP. The hydrolysis data showed that the branched structure of SWP was

301

composed of Rha, Gal, and Glc (Figure 1C). Meanwhile, it also indicated that the

302

terminal of each branched structure might be Rha (Figure 1D)28.

303

(2) The type of mono-saccharide of SWP. The periodate-oxidised products were

304

fully hydrolysed and analysed by HPLC after periodate oxidation. Based on the

305

standard data in the CCRS Spectral Database for PMMA’s. The results demonstrated

306

that there was nothing in the oxidation products showing up under HPLC analysis

307

which suggested the following linkages of the mono-saccharides: (1→), (1→2),

308

(1→6), (1→2, 6), (1→4), and (1→4, 6). We extracted and demined the oxidation

309

products using GC-MS: the oxidised products were erythritol, glycerol, glycerimum,

310

and no formic acid was produced. The mole ratio of them was 2:6:7 and the linkages

311

of the mono-saccharides were (1→6) and (1→4). Then, the fully methylated SWP

312

was hydrolysed with acid and analysed by GC-MS. The results showed the presence

313

of three components (Fig. 1E). Compared with the NIST database, those three

314

components were 2, 3, 4-Me3-Glc, 2, 3, 4-Me3-Rha, and 2, 3, 6-Me3- Gal in molar

315

ratio of 5.2:2.7:1.5. Based on the results of periodate-oxidation, the linkages of Glace,

316

Gal, and Rha were deduced as (1→6), (1→4), and (1→6). This result showed a good

317

correlation between terminal and branched residues. In addition, these molar ratios

318

also agreed with the overall mono-saccharide composition of SWP as described

319

above. 15


Page 16 of 53

Page 17 of 53


320

(3) Molecular weight determination of SWP. The SWP was light, freely soluble

321

in water, and was a grey, loose powder once purified by NaCl elution (Figure 2A).

322

The average molecular weight of the polysaccharide was measured with dextran

323

molecular weight standards and determined to have been 2.4 × 103 Da.

324

(4) The spectrum of the structure of SWP and Se-SWP. The UV-vis spectroscopy

325

analysis result of SWP is shown in Figure 2B. The SWP had no adsorption from 250

326

to 600 nm, and especially, none at 280 to 260 nm which indicated the absence of

327

proteins and nucleic acid in the purified SWP.26 The FT-IR spectra of the native SWP

328

is shown in Figure 2C: from the spectrum, 3482 cm-1, 2922 cm-1, and 1130 cm-1 were

329

the characteristic absorption bands of the polysaccharide. There were some peaks

330

between 900 and 800 cm-1 indicating that the structure might contain both α- and

331

β-configurations. The IR spectrum of the SWP showed a strong band at 3482 cm-1,

332

which is a characteristic absorption band of the hydroxyl stretching vibration for the

333

polysaccharide. The bands in the region of 2915 cm-1 and 1585 cm-1 were due to C-H

334

stretching vibration and –OH bending vibrations.

335

The spectrum of 13C-NMR for SWP is shown in Figures 3A and 3B: according to 35

and,28 the resonances in the region of 98-106 ppm in 13C NMR were

336

the literature

337

attributed to the anomeric carbon atoms of Glcp, Galp, and Rhap. The peaks at 103.56

338

ppm corresponded to C-1 of β-L- Rha residues. That at 99.8 ppm corresponded to C-1

339

of the (1→4)-Gal units and indicated that the backbone was composed of

340

(1→4)-linked α-D-Glc residues, which branched at O-3. From the carbon atoms

341

signals, it was suggested the branches might contain (1→3, 4)-linked-Gal, 16



Page 18 of 53

342

(1→4)-linked-Rha, and (1→)-linked-Glc, respectively. Determined the hydrolyzate

343

using GC-MS and inferred the members of branch-structure might be β-L-Rha, D-Glc

344

and D-Gla.

345

The backbone of SWP should contain the repeating units composed of (1→

346

6)-linked-Glc basing on the results of HPLC, GC-MS and13C-NMR. And the branches

347

might contain (1 → 3,4)-linked-Gal. And the branches might contain (1 →

348

3,4)-linked-Gal, (1→4)-linked-Rha, Rha-linked-Gal (or Gla) through placement of (1

349

→6) and linked with backbone.

350

Compare with references and database,28 the composition and link-type of

351

branch-structure might be showed in the figure 3 couple with the data of methylation

352

and NMR.

353

(5) The characterisation of Se-SWP. The selenium content in the Se-SWP was

354

12.74 mg/g through the determination. The result of UV-vis scanning is shown in

355

Figure 2B for comparison with the original SWP. The Se-SWP spectra showed an

356

absorption peak at 338 nm which indicated the presence of selenium and the

357

formation of Se-SWP.26,

358

Figure 2C. Compared with the original SWP, there were two new strong absorption

359

peaks at 916 cm-1 and 781 cm-1 which were attributed to the C-O-Se and Se=O

360

stretching vibration and asymmetric stretching, respectively. This was proof that the

361

selenylation modification of the samples had actually occurred.

362 363

36

Similarly, FT-IR scanning of the Se-SWP is shown in

The selenyl position of polysaccharide was usually determined by

13

C-NMR

spectra.26 The 13CNMR spectra of the native SWP and Se-SWP are shown in Figures 17


Page 19 of 53


364

4C and 4D. Considering that the Rha could not improve the site for the Se, so the

365

salinised region might occur in the Gal. The signals of SWE from 60.25 ppm to 96.98

366

ppm were assigned to C1 to C6 of the unit form of this mono-saccharide. Compared to

367

the original SWP, a new peak at 62.93 in the Se-SWP was assigned to the O-6

368

substituted carbons. It suggested that the Se was a reaction of O-6 form. For single

369

displacement, C6 peaks still remained at 60.25 ppm for Se-SWP, suggesting the

370

primary –OH on the internal side was not salinized.

371

Some references indicated the forms of polysaccharides attaching Se are mainly

372

found to be C2-Se(-O)(=O), C-Se-H, and C-Se(=O)2.24 Meanwhile, the FT-IR could

373

indicate the presence of the β-glucan structure. Some research indicated that the

374

β-glucan structure can help the polysaccharides hold potential health benefits in

375

anti-diabetic, anti-tumour, and immunoregulatory activity.37 As a novel health food in

376

China, the sweet potato was more popular than previously. Most research into sweet

377

potato mainly focused on anthocyanin acid and other ingredients, while the sweet

378

potato polysaccharide was rarely investigated. A few studies concerning its isolation

379

and identification from sweet potato vines, and determination of its molecular weight

380

are available, however, no structural evaluation of them can be found.38 Most studies

381

of sweet potato polysaccharides mainly focus on bio-activity evaluation, such as

382

anti-tumour and anti-oxidant functions.39 In a previous study, a polysaccharide was

383

isolated and purified from the sweet potato tuber. In the present study, an evaluation

384

of the structure through spectroscopy and chromatography found a novel

385

polysaccharide, in which glucose may be the backbone thereof and the branches were 18



386

composed of rhamnose, galactose, and glucose. NMR technology showed that the

387

polysaccharide was a β-type variant. Selenium polysaccharide was synthesised using

388

microwave irradiation and the NMR data evinced its structure. To the best of our

389

knowledge, no information has been reported about the selenylation modification of

390

sweet potato to date.

391

In vitro antioxidant activity analysis. The radical scavenging potential of SWP,

392

Se-SWP, and the control ingredients (ascorbic acid) using a DPPH assay is shown in

393

Figure 5, with their 50% scavenging capacity (IC50 values) of 2.57, 0.72, and 0.67

394

mg/mL, respectively. The radical scavenging capacity of the Se-SWP increased in a

395

concentration-dependent manner (Fig. 5A). The Se-SWP showed the higher

396

scavenging potential than SWP and was closest to the ascorbic acid, a well-known

397

antioxidant reagent.

398

In the ABTS assay, the radical scavenging activity, from strongest to weakest,

399

was as follows: ascorbic acid (IC50 = 0.35 mg/mL), Se-SWP (IC50 = 0.41 mg/mL), and

400

SWP (IC50 = 2.23 mg/mL). Figure 5B shows that all the above quenched ABTS

401

radicals behaved in a dose-dependent manner and the Se-SWP showed nearly the

402

same effect as the ascorbic acid.

403

In the reducing power assay, the Se-SWP was able to reduce ferric ions (Fe3+) to

404

ferrous ions (Fe2+) and the reducing ability increased with increasing concentration

405

(Figure 5C). However, the activity effect of SWP was not obvious, even no effect.

406

While, the Se-SWP exhibited higher activity after selenyl reaction and the reducing

407

power was also closer to that of the control ingredient. In the FRAP assay (Figure 5D), 19


Page 20 of 53

Page 21 of 53


408

which measures the reducing ability of a compound, the Se-SWP was showed better

409

activity than the original SWP and concentration-dependent activity was close to that

410

of ascorbic acid.

411

From the above results, the origin SWP seemed have low or no obviously

412

anti-oxidant activity. However, the selenyl SWP could improve the antioxidant

413

activity. As we known, the polysaccharides are one of the best antioxidants in the

414

natural products.40, 41 There are some reports revealing the antioxidant activities of

415

polysaccharides,42-44 and the mechanisms of the polysaccharides come from the

416

capacity of the free radical scavenging, metal ion, and reducing.45 In a recently reports,

417

three polysaccharides were isolated from the sweet potato and showed better

418

antioxidant activities on DPPH radical, Fe2+ and reducing power.7 Compare with our

419

study, the SWP showed poor activity on the anti-oxidant activity. The structure might

420

be the reasons of the poor activity. Therefore, chemical modification is a good way to

421

improve the activity. In present results, the selenyl reaction might be a good choice for

422

improving the activity of polysaccharides.

423

Meanwhile, there are several mechanisms of antioxidant activity, such as the

424

prevention of hydrogen abstraction, free radical scavenging, prevention of chain

425

initiation, decomposition of peroxides, reducing capacity, and binding of transition

426

metal ion catalysts. The selenium is the important composition of the glutathione

427

peroxidase, which could transfer the peroxide to related alcohol or water and

428

scavenging free radical. This might be the first report of the antioxidant activity of the

429

selenyl sweet potato polysaccharide. 20



430

In vivo anti-tumour activity of SWP and Se-SWP. The effects of SWP and

431

Se-SWP treatment on the expression of IL-2, TNF-α, and VEGF are listed in Table 1.

432

The dynamic change was plotted in Figure 6 where it is seen that the IL-2 and TNF-α

433

levels increased after drug administration. Treatment with Se-SWP increased the

434

serum IL-2 and TNF-α levels but decreased the serum VEGF level of mice in a

435

dose-department manner. Excess Se-SWP (above 100 mg/kg) did not increase the

436

serum levels of IL-2 and TNF-α. However, excess Se-SWP might further decrease the

437

serum VEGF level of the mice (Table 1). The 5-fluorouracil, as a common

438

chemotherapeutic, was widely used in chemotherapy of tumours of the liver. However,

439

its efficacy was worse than that of Se-SWP. A possible reason might be that the

440

Se-SWP was better at raising the immune response than the 5-flurouracil. However,

441

the SWP showed poorer activity than Se-SWP (similarly, it underwent little change at

442

higher concentrations), More remarkably, the effect of Se-SWP was much better than

443

that of the SWP. Figure 6 shows the variation of IL-2, TNF-α, and VEGF levels

444

during the administration period. Increased levels of IL-2 and TNF-α, and decreased

445

levels of VEGF, were seen after dosing. From the results, the SWP also could increase

446

the TNF, however, the immune system might be destroyed and the SWP could repair

447

the system in a certain extent (from 32.97 to 37.06).

448

The correlation of the Se-SWP and tumour weight is listed in Table 2 and

449

treatment with SWP/Se-SWP decreased the tumour weight of the mice and increased

450

the tumour inhibition ratio (TIR) in a dose-dependent manner. When the concentration

451

exceeded 100 mg/kg, there was no effect on the decrease in the tumour weight in mice, 21


Page 22 of 53

Page 23 of 53


452

or any further increase in TIR. The 5-fluorouraci showed better effects than both SWP

453

and Se-SWP, however, it also has many side-effects.

454

IL-2 is an important member of the cytokine network and exhibits several

455

biological activities in the animal body.36 Meanwhile, the TNF-α regulates many

456

cellular functions, including apoptosis, inflammation, immune response, and cell

457

growth and differentiation.46 Those two factors were important elements in the

458

research into the anti-tumour effect. The Se-SWP could increase the content of these

459

factors means that the selenation could improve the efficacy of anti-tumour, especially

460

in the case of the TNF-α level (by about two times). Meanwhile, the Se-SWP could be

461

used as a potential antineoplastic drug in the further cancer therapy. The role of VEGF

462

in tumour growth is well established and inhibition of the VEGF level in the blood is

463

also important during oncotherapy.47 In this in vivo study, Se-SWP could reduce the

464

VEGF content of mice blood. In combination with the result of IL-2 and TNF-α, the

465

selenium polysaccharide increased anti-tumour activity.

466

Recently, the role of dietary carbohydrates has been much more appreciated than

467

just the traditionally known antioxidant from plant sources such as sweet potato.

468

Those carbohydrates, represented by polysaccharides, which exhibit evidence of the

469

prevention of cancer progression.48 In some studies, the polysaccharides were

470

garnered more interesting in diverse biological such as anti-oxidant, antimicrobial,

471

anticancer and anti-inflammatory activities.49-52 In this study, were isolated from

472

sweet potato, and, when selenium-modified, showed favourable activity in prevention

473

of tumour progression: Se-SWP was much better than the original SWP. As we known, 22



474

a reported study which the polysaccharides isolated from the sweet potato showed

475

good anti-tumour activity.7

476

Evaluation of in vivo anti-oxidant and anti-diabetic activity. The results of

477

toxicity testing indicated there was no lethal effect under SWP and Se-SWP when

478

orally fed for 30 days in groups of 10 rats. After the above treatment schedules, the

479

various indicators of experimental rats were similarly as the control specimens. They

480

maintained body weight, organ weight, and underwent no abnormal behaviour.

481

(1) Effect of SWP and Se-SWP on body and organ weight. The body and organ

482

weights in different treatment groups are shown in Figure 7: there was significant

483

intra-group variation in the basal body weight of the DR group compared with the C

484

group. SWP and Se-SWP could help to keep the body weight within a normal range,

485

i.e., 242.37 g and 259.12, respectively. Meanwhile, no differences were observed in

486

the kidney and heart/body weight ratios. The only difference arose in the liver and

487

pancreas, the weights of which were significantly decreased in the DR groups

488

compared with the C group. SWP and Se-SWP could help to maintain normal organ

489

weight ratios with respect to the diabetic control, and positive, groups. Meanwhile,

490

there was no significant difference between SWP and Se-SWP.

491

(2) Hepatic function and plasma lipoprotein analysis. The levels of SGOP and

492

SGPT were all reduce (7.23 U/L and 27.87 U/L, respectively) after administration of

493

streptozocin (p

Selenylation of Polysaccharide from the Sweet Potato and Evaluation

Recommend Documents