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Structural Characterization of a Polysaccharide Fraction from Platycladus Orientalis (L.) Franco and Its Macrophage Immunomodulatory and Anti-Hepatitis B Virus Activities Zehua Lin, Wenzhen Liao, and Jiaoyan Ren J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01387 • Publication Date (Web): 27 Jun 2016 Downloaded from http://pubs.acs.org on June 27, 2016

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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.

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Physicochemical Characterization of a Polysaccharide Fraction from

2

Platycladus

3

Immunomodulatory and Anti-Hepatitis B Virus Activities

Orientalis

(L.)

Franco

and

Its

Macrophage

Zehua Lina, Wenzhen Liaoa,b*, Jiaoyan Rena*

4 5 6

a

7

Guangzhou, 510641, China

8

b

School of Food Science and Engineering, South China University of Technology,

Institute of Food Safety and Nutrition, Jinan University, Guangzhou, 510632, China.

9 10 11 12 13 14 15

Co-corresponding authors:

16

*Wenzhen Liao, School of Food Science and Engineering, South China University of

17

Technology, Wushan RD., Tianhe District, Guangzhou, China.

18

E-mail: [email protected]; [email protected]

19

*Jiaoyan Ren, School of Food Science and Engineering, South China University of

20

Technology, Wushan RD., Tianhe District, Guangzhou, China.

21

Tel: (+86)20-87112594; Fax: (+86) 20-38897117; E-mail: [email protected]

22


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23

Abstract

24

A polysaccharide fraction, here called POP1, was purified from the leaves of

25

Platycladus Orientalis (L.) Franco by water extraction and alcohol precipitation.

26

Physicochemical indicated that POP1 had a relative molecular weight of 8.10 × 103

27

Da and consisted of rhamnose (5.74%), arabinose (12.58%), mannose (10.97%),

28

glucose (64.96%) and galactose (6.55%). The main linkages type of POP1 were

29

comprised of (1→5)-linked α-L-Ara, (1→3)-linked α-L-Man, (1→6)-linked β-L-Rha,

30

(1→4)-linked

31

(1→3,6)-linked β-D-Gal and terminated with α-L-Man and α-D-Glc residues based on

32

the periodate oxidation, Simith degradation, methylation and NMR analysis. POP1

33

exhibited excellent immunostimulating activity by enhancing macrophage NO, TNF-α,

34

IL-6 and IL-12 secretion and activating related mRNA expression. Besides, POP1

35

showed significant anti-HBV activity through inhibiting the expression of HBsAg

36

(IC50=1.33 ± 0.12 mg/mL) and HBeAg (IC50=1.67 ± 0.13 mg/mL) and interfering the

37

HBV DNA replication (IC50=0.80 ± 0.03 mg/mL). The present study suggested that

38

POP1 could be used as immunoregulatory agent in functional foods for the prevention

39

of HBV infection.

α-D-Glc,

(1→6)-linked

α-D-Glc,

(1→6)-linked

β-D-Gal,

40 41

Keywords:

Platycladus

Orientalis

(L.)

Franco;

Polysaccharide;

42

characterization; Immunostimulating activity; Antiviral activity

43


Structure


44

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INTRODUCTION

45

Platycladus Orientalis (L.) Franco is widely cultivated in Asian countries. The

46

leaves and seeds of Platycladus Orientalis (L.) Franco are used as functional foods for

47

a long history treating for inflammation, gout, asthma, etc.1, 2 It is also safe to be used

48

as functional food materials approved by China Food and Drug Administration

49

(CFDA).3

50

value

51

neuroprotective biological activities.4-7

Previous studies demonstrated that P. orientalis has a high medicinal

due

to

its

antimicrobial,

anti-inflammatory,

antihyperlipidemic

and

52

The studies into P. orientalis were mostly focused on its flavonoids components for

53

antioxidant activity8 and anti-hyperuricemic effect;9 essential oils for antimicrobial

54

activity;4 diterpenes for antifibrotic activity10 and anti-inflammatory activity11.

55

Besides, polysaccharides are considered as one of the major bioactive

56

P. orientalis, however, rare information about their structure and biological activities

57

are available. In recent decades, natural polysaccharides isolated from different

58

sources such as mushrooms and algae have attracted great attentions due to their

59

diverse

60

immunodeficiency virus), hypoglycemic, anti-inflammatory and immunological

61

activities.12-16

biological

activities,

including

anticancer,

compounds of

anti-HIV

(human

62

Evidences indicated that natural polysaccharides can activate immunologic

63

response, enhance the secretion of cytokines including interleukin-6 (IL-6), IL-12,

64

TNF-α, interferon gama (IFN-γ) and nitric oxide (NO).17 Macrophages play important

65

roles on the signal transmission of nonspecific immune response.18, 19 In the present

66

study, murine macrophage RAW 264.7 cells were used as cell model to investigate the

67

immunostimulating activity of polysaccharides from P. orientalis.

68

Human hepatitis B virus (HBV) is known as the major pathogenic factor of


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69

developing chronic liver disease, cirrhosis and hepatocellular carcinoma.20 Traditional

70

anti-viral drugs for chronic HBV infection have their limitations, such as low efficacy,

71

insufficient uptake, and the development of HBV drug resistance.21 Therefore, the

72

exploration of new antiviral agents for the prevention of HBV infection are urgenly

73

desired.22

74

In the present work, the polysaccharide fraction (POP1) was extracted and purified

75

from the leaves of P. orientalis. The structural information including molecular weight,

76

monosaccharide composition, linkage information was analyzed the primary chemical

77

structure of POP1 was characterized. Moreover, its immuomodulatory activity was

78

examined using murine macrophage RAW 264.7 cells, the antiviral activity of POP1

79

against HBV was evaluated using HepG2.2.15 cells model system. The results could

80

provide useful information about the structure and bioactivity of the P. orientalis

81

polysaccharides, which will be helpful for potentially commercial use of the

82

polysaccharides as functional foods ingredients with immunoregulatory or HBV

83

infection prevention activities.

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MATERIALS AND METHODS

85

Platycladus Orientalis (L.) Franco was obtained from Weifang, Shandong Province,

86

China. The murine macrophage cell line RAW 264.7 was purchased from the Chinese

87

commercial agent of ATCC (American Type Culture Collection). The DMEM culture

88

medium (Dulbecco’s modified Eagle’s medium), FBS (fetal bovine serum), PBS

89

(phosphate-buffered saline, pH 7.4) and antibiotics (streptomycin and penicillin) were

90

Gibco® brand from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

91

Lipopolysaccharide (LPS), 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium

92

bromide (MTT), Griess reagent, standards of dextrans, erythritol, glycerol, ethylene

93

glycol and monosaccharide standards were obtained from Sigma-Aldrich Co. LLC.



94

(St. Louis, MO, USA). DEAE Sepharose Fast Flow and Sephadex G-100 were

95

purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Mouse IL-6,

96

IL-12 and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were purchased

97

from Neobioscience Technology Co., Ltd. (Shenzhen, China). First Strand cDNA

98

Synthesis Kit for RT-PCR (AMV), FastStart Universal SYBR Green Master (Rox)

99

was purchased from F. Hoffmann-La Roche Ltd. (Basel, Switzerland). HBeAg and

100

HBsAg detecting kits were obtained from Shanghai Kehua Bio-engineering Co., Ltd.

101

(Shanghai, China).

102

Extraction and purification of polysaccharides from the leaves of P. orientalis

103

The leaves of P. orientalis were dried at 60 °C for 4 h and grounded. The leaves

104

powder was mixed with deionized water at a ratio of 1:20 (w/w) and kept at 100°C for

105

2 h, and centrifuged(4,000g, 20 min). The supernatant was concentrated at 60 °C and

106

deproteinated 10 times using Sevag method.23 The resulting solution was mixed with

107

three volumes of absolute ethanol and kept overnight at 4 °C. The precipitate were

108

collected by centrifugation at 4,000g for 20 min. Finally, the precipitate was

109

lyophilized as the crude polysaccharides.

110

The crude polysaccharides (200 mg) was dissolved in deionized water (10 mL) and

111

fractionated by a DEAE Sepharose Fast Flow anion-exchange chromatography

112

column (1.6 × 20 cm). The elution was firstly performed using deionized water,

113

followed by NaCl step gradient (0.05, 0.10, 0.20, 0.30, and 0.50 M, respectively), the

114

flow rate was set at 1.0 mL/min. The sugar contents of all the fractions were

115

determined by the phenol-sulfuric acid method.24 Two fractions, POP1 (deionized

116

water elution) and POP2 (0.05 M NaCl elution) were collected, dialyzed at 4 °C for

117

48 h, and lyophilized. The present study was mainly focused on POP1, POP2 will be

118

investigated in the future study.


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The POP1 was further purified using a Sephadex G-100 column chromatography.

120

Ten milliliter of POP1 solution (5 mg/mL) was loaded on a Sephadex G-100 column

121

(1.6 × 80 cm) and eluted by deionized water (1.0 mL/min), and the sugar contents was

122

determined as mentioned above.

123

FT-IR spectrum analysis

124

The infrared spectrum of POP1 was recorded on a Fourier transform infrared

125

(FT-IR) spectrometer (VERTEX 33, Bruker, Germany) in the infrared region of

126

4000-400 cm–1 at a resolution of 4 cm–1.

127

Determination of molecular weight

128

High performance gel permeation chromatography (HPGPC) was used to determine

129

the molecular weight of POP1. HPGPC was conducted on a Waters system using TSK

130

gel G5000PWXL-CP column connected with TSK gel G3000PWXL-CP column.

131

Samples were eluted by KH2PO4 (0.02 moL/L, 0.6 mL/min). Eight analytical

132

standards of dextran for GPC (5.0×103, 1.2×104, 2.5×104, 5.0×104, 1.5×105, 2.7×105,

133

4.1×105, 6.7×105 Da) were used to make a standard curve. The sample and standards

134

were both dissolved in 0.02 moL/L KH2PO4.

135

Monosaccharide composition analysis

136

Monosaccharide composition were analyzed as described previously with minor

137

modification.23 Ten milligram of POP1 was hydrolyzed with TFA (2 M, 4.0 mL) at

138

110 °C for 6 h, and then the solution was dried totally.

139

The hydrolysates were mixed with hydroxylamine hydrochloride (10 mg) and

140

pyridine (0.5 mL) at 90 °C for 30 min. After that, the resulting solution was mix with

141

acetic anhydride (0.5 mL) and incubated at 90 °C for half an hour. The resulting

142

products were dissolved in chloroform and analyzed using a Agilent 7890A gas

143

chromatograph system (Agilent Technologies, USA) with a HP-5 capillary column.



144

The carrier gas was N2 with a speed of 25 mL/min. The temperature was 100 °C, a

145

linear increase to 160 °C at 3 °C/min, followed by linear increase at 10 °C/min to

146

250 °C, and kept for 5 min. The injector and detector was 250 °C. A set of

147

monosaccharides(mannose, rhamnose, galactose, fucose, xylose, glucose and

148

arabinose) were used as standards. The inositol was used as the internal standard and

149

it’s concentration was 1.0 mg/mL.

150

Periodate oxidation and Smith degradation analysis

151

The analysis of Periodate oxidation and Smith degradation were performed

152

according to the previously study with some modifications.25 POP1 (25 mg) was

153

mixed with NaIO4 (30 mM, 12.5 mL) in the dark. The absorption at 223 nm was

154

measured at different time intervals (0, 6, 12, 24, 36, 48, 60, and 72 h) until the

155

absorbance became invariable. The reaction was terminated by glycol (2 mL) and

156

titrated by NaOH (0.01 M). The resulting product was used for Smith degradation.

157

The periodate product was dialyzed and the molecular weight cut-off of the dialysis

158

membrane was 3.5 × 103 Da. Then, NaBH4 (70 mg) was mixed and reacted in dark for

159

24 h. The pH of solution was changed to 6.0 with HOAc (0.1 M) and dialyzed for

160

another 72 h. The residues was freeze-dried and hydrolyzed by TFA (2.0 M, 4 mL) for

161

6 h at 110 °C. The hydrolysate product was then acetylazed and analyzed by a Agilent

162

7890A chromatograph system as mentioned above.

163

Linkage analysis by methylation

164

Methylation of POP1 was carried out according to previous study described by Zhu

165

et al. with minor revision.26 Twenty milligram of dry POP1 was dissolved in DMSO

166

(6 mL) and mixed with NaOH (100 mg) and kept at 25 °C for 30 min. The

167

methylation of POP1 was conducted by methyl iodide (2 mL) in the dark. The

168

methylated POP1 was extracted by chloroform (4 mL) and hydrolyzed by TFA (2M,


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169

10 mL) at 110 °C for 2 h. The POP1 hydrolysates were mixed with NaOH and NaBH4

170

and kept at room temperture for 2h. And the reaction was terminated by HOAc. The

171

resulting solution was then dried and acetylated by acetic anhydride and pyridine. The

172

acetic anhydride was decomposed by distilled water, and methylene chloride (4 mL)

173

was used to extract the acetylated derivatives. The resulting partially methylated

174

alditol acetates (PMAAs) were detected by a Agilent 6890-5975I GC-MS instrument

175

(Agilent Technologies, USA) with a HP-5 capillary column. The carrier gas was N2

176

and its flow rate was 25 mL/min. The temperature was programmed from an initial

177

temperature of 100 °C, a linear increase to 160 °C at 3 °C/min, followed by linear

178

increase at 10 °C/min to 250 °C , and kept at 250 °C for 5 min. The temperature of the

179

injector and detector was 250 °C. The temperature of ion source was set at 300 °C.

180

The analysis was performed in the electron impact ionization mode with ionizing

181

voltage of 70 eV. The range of mass charge ratio (m/z) was set at 30 to 450.

182

Nuclear magnetic resonance (NMR) spectroscopy

183

Twenty-five milligram of POP1 was redissolved by D2O (0.5 mL). The

184

one-dimensional NMR spectra of 1H and 13C were analyzed with a Bruker 600 MHz

185

NMR spectrometer (AVANCE III HD 600, Bruker, Germany) at 25 °C. The operating

186

conditions were set as follows. 1H NMR: spin, 15 Hz; relaxation delay, 2 s;

187

acquisition, 1000 scans.

188

33000 scans.27

189

Thermal gravimetric analysis (TGA) and differential scanning calorimetric

190

(DSC) analysis

13

C NMR: spin, 15 Hz; relaxation delay, 2 s; acquisition,

191

The thermostability of POP1 was investigated by DSC-TG analysis using a

192

Simultaneous Thermal Analyzer (STA 449 F3, NETZSCH, Germany). POP1

193

(approximately 5 mg) was heated in Al2O3 crucibles. The heating program was



194

conducted under static conditions (25-600 °C) with a increase rate of 10 K min-1. The

195

analyses were carried out under N2 atmosphere (40 mL/min).

196

Immunomodulatory activities of POP1

197

Cell culture

198

RAW 264.7 cells were incubated in DMEM plus with penicillin (100 U/mL),

199

streptomycin (100 µg/mL), and FBS (10%) and kept at 37 °C in a CO2 incubator (95%

200

air and 5% CO2) . Cells were at a density of 1 × 106 cells/well and seeded onto 96

201

well microplates. After 24 h, cells were treated with different concentrations of POP1

202

sample for another 24 h.

203

Cytotoxicity assays

204

The effect of POP1 on the viability of RAW 264.7 cells was investigated using

205

MTT assay. Cells at a density of 5 × 103 cells/well were seeded in 96-well plates and

206

treated by POP1 (62.5-1000 µg/mL) for 24 h. MTT solution (20 µL, 5 mg/mL) were

207

added as visualization reagent for 4 h and dissoved by 100 µL of DMSO solution.

208

Then the absorbance of cell plate was determined by a VICTOR™ X microplate

209

reader (PerkinElmer Inc., MA, USA) at 570 nm after 10 min. The cell viability was

210

caculated as following:

211

Cell viability (%) = A/B × 100%

212

(A), The absorbance of treated group. (B), The absorbance of control group.

213

Effects of POP1 on macrophage NO, TNF-α, IL-6 and IL-12 secretion

214

Macrophage activation by immunomodulators is believed to be via the induction of

215

NO expression and enhancement of cytokines (TNF-α, IL-6, and IL-12, etc.)

216

secretion.25 The macrophage NO content after treatment by POP1 was determined by

217

Griess method. The levels of on macrophage NO, TNF-α, IL-6 and IL-12 were

218

examined by by commercial ELISA Kits.25 The positive control group was treated by


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219

LPS (50 µg/mL).

220

Inhibition of POP1 on macrophage IL-6, TNF-α and iNOS gene expressions

221

The aqueous POP1 solution was prepared at three treatment concentrations of 62.5,

222

250, 1000 µg/mL. RAW 264.7 cells were cultured in 96-well plates for 24 h to reach

223

an 80% confluence before the addition of POP1-containing media. Macrophage cells

224

were collected and washed 3 times with PBS for RNA exaction. In brief, the total

225

RNA of RAW 264.7 cells was extracted and reversed into cDNA. The reverse

226

transcription was achieved at 65 °C for 10 min, then at 25 °C for 10 min, at 55 °C for

227

30 min, and at 85 °C for 5 min to synthesis cDNA. Reverse transcriptase-generated

228

complementary DNA encoding iNOS, TNF-α, and IL-6 genes was amplified by PCR

229

using specific primers. The sequences of specific primers are listed in Table 1.

230

GAPDH was used as internal standard. For quantitative RT-PCR assay, the reaction

231

mixture included 10 µL FastStart Universal SYBR Green Master, 2 µL of cDNA, 2 µL

232

of forward and reverse primers (IL-6, TNF-α, iNOS and GAPDH), and water (PCR

233

grade) in total volume of 20 µL. Gene amplification was performed using a Applied

234

BiosystemsTM Prism 7500 sequence detection system (Thermo Fisher Scientific Inc.,

235

USA). Amplification included 1 stage of 95 °C for 10 min followed 40 cycles of a

236

2-step loop: 95 °C for 15 s and 60 °C for 1 min.

237

Antiviral activity

238

Cell culture

239

HepG2.2.15 cells derived from the hepatoma cell line HepG2 transfected with the

240

full genome of HBV have been widely used as cell model for the investigation of

241

anti-HBV activity.28 HepG2.2.15 cells were cultured in RPMI-1640 medium

242

containing 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 200 µg/mL

243

G418, and maintained at 37 °C in a moist atmosphere containing 5% CO2.



244

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Cytotoxicity assays

245

The cytotoxic effects of POP1 on HepG2.2.15 cells were evaluated by MTT essay.

246

Cells were seeded in 96-well culture plates at a density of 5 × 103 cells per well and

247

cultured at 37 ºC for 24 h. Then the cells were incubated with different concentrations

248

(62.5-1000 µg/mL) of POP1 for 6 days, and later, the culture medium was removed

249

and replaced with fresh medium plus with different concentrations of POP1 every 48

250

h during the incubation. After six days incubation, the cytotoxic effects of POP1 on

251

HepG2.2.15 cells were investigated by MTT assay as mentioned above.

252

HBsAg and HBeAg analysis

253

The HepG2.2.15 cells were plated at a density of 1 × 106 cell per well on 96-well

254

plates and cultured for 24 h. Then the cells were treated with different concentrations

255

of POP1 (61.25-1000 µg/mL) for six days. The medium was removed and refresh

256

with medium plus with different concentrations of POP1 every 48 h during the

257

incubation. After incubation, the supernatants were collected and centrifugated (5000g,

258

5 min). The levels of HBsAg and HBeAg were detected by comercial ELISA kits

259

according to the protocols provided by the manufacturer. The results were read at 450

260

nm by a microplate reader. The inhibition rate (%) of HBsAg and HBeAg expressions

261

were calculated as following:

262

HBsAg (or HBeAg) inhibition (%) = (Acontrol – Asample)/Acontrol × 100%, and the 50%

263

inhibitory concentration (IC50) on cells was calculated.

264

HBV DNA extraction and real-time polymerase chain reaction (RT-PCR)

265

HepG2.2.15 cells were treated and harvested as mentioned above. Extracellular

266

virion HBV DNA was extracted according to the RT-PCR method as described

267

previously.29 HBV DNA solution (2.5 µL) and primers (0.4 µM) including forward

268

primer

(5′-CAGTTTACTAGTGCCATTTGTTCAGTG-3′)


and

reverse

primer

Page 13 of 43


269

(5′-AAAAGGGACTCAAGATGTTGTACAG-3′) were mixed to a 25 µL reaction

270

system. The PCR procedure was performed on a 7500 Real-Time PCR Systems

271

(Applied Biosystems, CA, USA). The PCR parameters were set as follewing:

272

Initial denaturation: 95 °C for 15 s, Primer annealing: 58 °C for 1 min, Extension:

273

75 °C for 35 s for 45 cycles. HepG2.2.15 cell culture was used quality control for the

274

quantification of HBV DNA and quantified by the branched DNA (bDNA) method to

275

estimate the copy number of virus in each well. The inhibitory rate of POP1 on HBV

276

DNA was calculated by formula as following:

277 278

The inhibitory rate (%) = (Copies control – Copies sample)/Copies control × 100% Statistical analysis

279

All of the assays were performed independently in triplicate. Statistical analysis

280

was carried out using the SPSS 19.0 software (IBM Corporation, NY, USA). One-way

281

analysis of variance (ANOVA) was performed to analyze the difference between two

282

or more groups, and P value below 0.05 was considered as statistically significant.



283

RESULTS AND DISCUSSION

284

Purification of polysaccharides from Platycladus Orientalis (L.) Franco

285

The crude polysaccharides were isolated from the dry leaves of P. orientalis by

286

water extraction and ethanol precipitation method with a yield of 1.61 ± 0.06% (w/w).

287

The crude polysaccharides were purified by DEAE Sepharose Fast Flow column

288

(Figure 1A). Two major peaks, POP1 (eluted by deionized water) and POP2 (eluted

289

by 0.05 M NaCl), were obtained. According to the elution profile of Figure 1A, most

290

of the polysaccharides were eluted by by distilled water (POP1). Because DEAE

291

Sepharose Fast Flow is a weak anion exchanger, so the POP1 may belong to neutral

292

polysaccharide and carry little negative charge. In present study, we mainly focused

293

on POP1, and POP2 will be studied in our future work. The yield of POP1 from the

294

crude polysaccharide was calculated to be 3.95 ± 0.32% (w/w). The POP1 fraction

295

was further purified by a Sephadex G-100 column. As shown in Figure 1B, a single

296

and symmetrical peak of POP1 was observed. After dialysis and lyophilization, the

297

dried POP1 was proved to be composed of 94.50 ± 0.85% (w/w) total carbohydrate.

298

Structural characterization of POP1

299

Molecular weight of POP1

300

The molecular weight distribution of POP1 was determined by HPGPC. POP1 was

301

a homogeneous polysaccharide fraction with a single absorption peak (Figure 1C).

302

The average molecular mass of POP1 was calculated as 8.10 × 103 Da according to

303

the dextran standard curve.

304

Monosaccharide composition of POP1

305

The monosaccharide composition of POP1 was analyzed using GC system. As

306

shown in Figure 2, the monosaccharide composition of POP1 was made up of

307

rhamnose (5.74%), arabinose (12.58%), mannose (10.97%), glucose (64.96%) and


Page 14 of 43

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308

galactose (6.55%). Glucose represented the greatest proportion compared to the other

309

monosaccharide. Thus, glucose may be the main backbone of the structure of POP1.

310

Periodate oxidation and Smith degradation analysis

311

The type of glycosidic linkage of polysaccharide can be preliminarily

312

characterized by the consumption of NaIO4 and the production of HCOOH in

313

periodate oxidation.30 Results from periodate oxidation showed that 0.8 mol of

314

periodate was consumed by 1.0 mol of sugar residue and 0.4 mol HCOOH was

315

produced. The production of HCOOH indicated the existence of T-linked or 1,

316

6-linked sugar residues. The amount of NaIO4 consumption was approximately two

317

times of the production of HCOOH, indicating the existence of 1-; 1, 6-; 1, 3-; 1, 3, 6-;

318

1, 2, 3-; 1, 2, 4-; 1, 3, 4-; 1, 2, 3, 4-linked glycosidic linkages31. The results of Smith

319

degradation process removed most of the rhamnose, arabinose, mannose, glucose and

320

galactose residues, and produced a large amount of glycol, glycerol and erythritol,

321

indicating that these glycosidic residues may exist in the 1-; 1,2-; 1,2,6-; 1,4-; 1,4,6-;

322

1,6-linked forms.31

323

Methylation analysis of POP1

324

Methylation is commonly used to analyze the glucosidic bond types of natural

325

polysaccharides.32 The methylated polysaccharides were hydrolyzed with TFA, and

326

the resulting monosaccharides were converted into partially methylated alditol

327

acetates (PMAAs) and then analyzed via GC-MS. Basing on the mass spectrum

328

patterns and standard data in the CCRC Spectral Database for PMAAs, the

329

methylation analysis of POP1 are shown in Table 2. The following conclusions can be

330

drawn from Table 2: (1) the arabinose residues were present as 1,5-linked

331

Araf residues; (2) the mannose residues were composed of 1,6-linked and terminal

332

Manp residues; (3) the rhamnose residues were made up of 1,6-linked Rhap residues;



333

(4) the glucose residues were present as 1,4-linked; 1,6-linked and terminal

334

Glcp residues; (5) the galactose residues were composed of 1,6-linked and

335

1,3,6-linked Galp residue.

336

FT-IR spectrum of POP1

337

The infrared spectrum reveals the major functional groups and the chemical bounds

338

of the compound investigated. As shown in Figure 3A, the characteristic bands in the

339

regions of 3427.73, 2925.77, and 1639.10 cm-1 belonged to hydroxyl stretching

340

vibration, C-H stretching vibration, and associated water, respectively.33 The peak

341

around 1029.54 cm-1 was attributed to the stretching vibration of C-O. In the region of

342

1200-950 cm-1, several intense signals at 1151.29, 1079.94 and 1029.54 cm-1

343

suggested the existence of C-O stretching vibrations of glycosidic bonds and pyranoid

344

rings.32, 34, 35

345

NMR spectra of POP1

346

The 1H NMR and

13

C NMR spectra of the POP1 were shown in Figure 3B & C.

347

Chemical shifts of resonances in the 1H and 13C NMR spectra of POP1 were exhibited

348

in Table 3. The 1H and

349

results of methylation analysis, monosaccharide composition and previous reports in

350

the literature.

13

C NMR spectra of POP1 were analyzed according to the

351

As shown in Table 3, the main linkages type of POP1 were comprised of

352

(1→5)-linked α-L-Ara, (1→3)-linked α-L-Man, (1→6)-linked β-L-Rha, (1→4)-linked

353

α-D-Glc, (1→6)-linked α-D-Glc, (1→6)-linked β-D-Gal, (1→3,6)-linked β-D-Gal and

354

terminated with α-L-Man and α-D-Glc residues.

355

Thermal stability analysis of POP1

356

The thermal stability of POP1 was analyzed using thermal gravimetric (TG) and

357

differential scanning calorimetric analysis (DSC). Figure 4 shows TG, derivative TG


Page 16 of 43

Page 17 of 43


358

(derivative of the weight loss curve, DTG) and DSC curves for POP1. The peak

359

position and weight loss of POP1 was shown in Table 4. Peak I indicated that the

360

gradual loss of water was accompanied by the breakage of hydrogen bonds and the

361

weight loss of water.36 The second mass loss for POP1, with a weight loss onset of

362

126.9 °C, presented peaks of the DTG at 322.9 °C with a residual mass of 66.95%.

363

The DSC curves revealed that there were two endothermic events around the peak of

364

57.0 °C and 322.9 °C with the value of DSC was 0.736 and 6.215 mW/mg,

365

respectively, may be attributed to water evaporation (Region I) and depolymerization

366

and degradation reaction (Region II), which was in accordance with the TGA

367

results. Thermal gravimetric analysis and differential scanning calorimetric analysis

368

further confirmed that the thermally decomposed temperature of POP1 was around

369

320 °C.

370

Based on the above results,

POP1 polysaccharide degradation was proved to

371

proceeding four distinct phases: (1) desorption of physically absorbed water; (2)

372

removal

373

accompanied by the rupture of C–O and C–C bonds in the ring units resulting in the

374

evolution of CO, CO2 and H2O; (4) formation of polynuclear aromatic and graphitic

375

carbon structures, which was similar with the previous reports on polysaccharide

376

degradation.37

377

Immunostimulating activities of POP1

378

Effect of POP1 on the viability of RAW 264.7 cells

of

structural

water

(dehydration

reactions);

(3)

depolymerization

379

Macrophages play critical roles in the activation of nonspecific defense and specific

380

defense to pathogens.38 Herein, the cytotoxic effect of POP1 on RAW 264.7 cells was

381

evaluated. As shown in Figure 5A, POP1 insignificantly affected the viability of RAW

382

264.7 cells on the selected concentrations (62.5, 125, 250, 500 and 1000 µg/mL),



383

suggested that POP1 exhibited no significant toxicity on murine RAW 264.7 cells.

384

Effect of POP1 on the production of nitric oxide (NO) by RAW 264.7 cells

385

NO is one of the signalling molecules related to macrophage cytolytic function.39

386

As shown in Figure 5B, POP1 (62.5-500 µg/mL) could remarkably enhance the

387

secretion of NO in an obviously dose-dependent manner. The level of NO treated by

388

500 µg/mL of POP1 was 67.72% of LPS (50 µg/mL). Moreover, the content of NO

389

increased to 607.01% compared with the control. The macrophage NO level (e.g.,

390

26.73 ± 1.02 µM) treated by 125 µg/mL of POP1 was much higher than that of other

391

polysaccharides reported by previous studies, for example, an acidic polysaccharide

392

(APS) from Cordyceps militaris (lower than 8.0 µM at 1000 µg/mL APS) and a

393

polysaccharide (L2) from Lentinula edodes (lower than 5.0 µM at 250 µg/mL L2).40, 41

394

It is reported that polysaccharides which including 1,4-α-D-glucosidic and

395

1,4-β-D-glucosidic linkages exhibit more notable positive effect on the macrophage

396

NO production.42 It can be inferred that the glucosidic linkages may contribute to

397

immunomodulatory activities.

398

Effect of POP1 on the production TNF-α by RAW 264.7 cells

399

Tumour necrosis factors (TNF) are a group of cytokines that play an important role

400

to tumor lysis and apoptosis.43 TNF-α can activate macrophages in an autocrine

401

manner to enhance various functional responses and induce the expression of other

402

immune and inflammatory mediators.44 Many polysaccharides can bind to the

403

receptors on the surfaces of macrophages to stimulate the secretion of TNF-α.25

404

As shown in Fig. 5C, POP1 significantly stimulated the secretion of TNF-α at

405

different concentrations from 62.5 µg/mL to 1000 µg/mL in a dose-dependent manner.

406

The enhancement of the TNF-α production was 224.38% of the control group by

407

500 µg/mL of POP1 treatment. The macrophage TNF-α level (e.g. 1807.37 ± 115.59


Page 18 of 43

Page 19 of 43


408

pg/mL at 250 µg/mL POP1) treated by POP1 was relative higher than those from

409

other polysaccharides, such as a polysaccharide (DP1) from Dictyophora indusiata

410

(588 pg/mL at 250 µg/mL DP1) and a polysaccharide (L2) from Lentinula edodes

411

(lower than 1100 pg/mL at 250 µg/mL L2).25, 41 Similarly, the polysaccharides from

412

fruit bodies of Grifola frondosa45 and Dictyophora indusiata (DP1)25 were also

413

reported to stimulate the inducement of cell TNF-α and IL-6 release under specific

414

concentration.

415

Effect of POP1 on the secretion of IL-6 and IL-12 by RAW 264.7 cells

416

Interleukins are secreted proteins and signal molecules secreted by white blood

417

cells (leukocytes). Interleukin 6 (IL-6) is a pleiotropic cytokine which plays a critical

418

role in immunological responses.46 As shown in Figure 5D, cells of control group

419

secreted little IL-6 (8.88 ± 5.42 pg/mL), while the POP1 treatment (62.5-250 µg/mL)

420

significantly increased IL-6 secretion in an obvious dose-dependent manner. The

421

macrophage IL-6 level (e.g. 1050.58 ± 15.75 pg/mL at 250 µg/mL POP1) induced by

422

POP1 was comparable to that by a polysaccharide (DP1) from Dictyophora indusiata

423

(1134 pg/mL at 250 µg/mL DP1).25

424

In immune responses, IL-12 induces T cells to generate IFN-γ and lytic activity.

425

Thus, IL-12 acts a central and important role as a link between the nonspecific and

426

specific immune systems.47 As shown in Figure 5E, the level of IL-12 was

427

significantly enhanced by POP1 treatment. At the dose of 62.5 µg/mL, the amount of

428

IL-12 was enhanced approximately 35 times compared with the control group it,

429

which was comparable to 59.64% of LPS treatment group (50 µg/mL). The function

430

of polysaccharides to induce macrophages cytokines (IL-1α, IL-6, IL-10 and TNF-α)

431

secretion had also been proved by the fractions from Ganoderma lucidum (Reishi)

432

and Cordyceps sinensis fungus.48, 49 As compared to the previous report, we found



433

here that the macrophage IL-12 level (996.79 ± 9.50 pg/mL) induced by 62.5 µg/mL

434

of POP1 is much higher than that by a polysaccharide from Grifola frondosa.50

435

Effect of POP1 on the mRNA expression of iNOS, IL-6 and TNF-α

436

Previous studies indicated that the activation of macrophage is regulated by the

437

immune-related genes.51 To investigate whether the increased secretion of NO, IL-6

438

and TNF-α were due to the increased expression of iNOS, IL-6 and TNF-α mRNA,

439

the mRNA expression of iNOS, IL-6 and TNF-α in RAW 264.7 cells after POP1

440

treatment was investigated using RT-PCR. As shown in Figure 5 (F-H), the mRNA

441

level of iNOS, IL-6 and TNF-α was increased after POP1 stimulation. As estimated

442

by densitometric measurement, the expressions of iNOS, IL-6 and TNF-α treated by

443

62.5 µg/mL of POP1 were significantly higher than those of the control group. These

444

results demonstrated that the immune-stimulating of POP1 on macrophages are

445

mainly through the act related genes expression of the immune cell cytokines.

446

Similarly, other polysaccharides could also increase the mRNA expression of

447

cytokines as observed on RAW 264.7 cell model. For example, Platycodon

448

grandiflorum polysaccharides could enhance iNOS and TNF-α level,52 Talinum

449

triangulare polysaccharides boosted the gene expression levels of iNOS, TLR2, TLR4

450

and IL-1β,51 and Cordyceps militaris polysaccharides contributed to the mRNA

451

expression of IL-1β, IL-6, IL-10, and TNF-α.40 The function of polysaccharides on the

452

expression of cytokines might be related to membrane receptors TLR4, CD14 and

453

SRA, and MAPK signaling pathways.53

454

Base on the above results and pervious study, we speculated that the possible

455

underlying mechanism of the immunostimulating activity of POP1 may mainly

456

through the recognition of membrane receptors, leading to the activation of the

457

transcription factors (e.g. nuclear factor-κB) through specific signaling cascade, with


Page 20 of 43

Page 21 of 43


458

subsequent stimulation of the related mRNA expression. Taken together, the

459

regulation of these transcription pathways induced the expression of cytokines (Figure

460

6).

461

Antiviral activity of POP1

462

Effect of POP1 on cell viability of HepG2.2.15 cells

463

HepG2.2.15 cell, a derivative of HepG2 stably expressing HBV, is usually used as

464

ideal cell mode to evaluate the anti-HBV activity of bioactive compounds.22 It was

465

necessary to evaluate the cytotoxicity of POP1 on HepG2.2.15 cells before investigate

466

the anti-HBV activity of POP1. As shown in Figure 7A, MTT assays indicated that

467

POP1 didn’t show evident cytotoxicity on HepG2.2.15 cells after 6 days of

468

incubation.

469

Effect of POP1 on HBsAg and HBeAg production of HepG2.2.15 cells

470

HBV is a kind of DNA virus belongs to the Hepadnaviridae family.54 The inner

471

core of HBV is consist of viral genome, DNA polymerase, hepatitis B core antigen,

472

hepatitis B e antigen (HBeAg). Hepatitis B surface antigen (HBsAg) is mainly found

473

on the surface of the virus.54 The inhibition effect of POP1 on HBsAg and HbeAg

474

secretions were investigated after six days incubation. As shown in Figure 7 B&C,

475

The levels of HBsAg and HBeAg expression was significantly inhibited by POP1.

476

The calculated 50% inhibitory concentration (IC50) of POP1 for inhibition HBsAg and

477

HBeAg expression were 1.33 ± 0.12 mg/mL and 1.67 ± 0.13 mg/mL, respectively. In

478

addition, when the concentration of POP1 was above 250 µg/mL, the inhibition of

479

HBsAg presented a dose-dependent manner. At POP1 concentration (1000 µg/mL), it

480

demonstrated better HBsAg and HBeAg inhibition, even exceeded that of the positive

481

control group (LAM, 20 µM). These results suggested that POP1 might be a potential

482

functional food ingredient with the feature of reducing the of risk HBV infections.



483

Similarly, a sulfated extracellular polysaccharide (curdlan sulfate) was found to

484

significantly inhibit HBV infection of HepG2.2.15 cells and HepaRG cells.55 The

485

sulfation group was proved to have dominant contribution on this bioactivity and the

486

possible mechanism might be that the polysaccharide acts as an competitor of HBV

487

binding to the receptors of menbrane of host cells.55 Since there is no sulfation group

488

in the POP1, there may be some other mechanism for POP1 to interfere with the HBV

489

life cycle.

490

Effect of POP1 on HBV DNA replication

491

The antiviral activities of POP1 against HBV DNA replication were analyzed using

492

RT-PCR technique. As shown in Figure 7D, the expression of HBV DNA was

493

significantly inhibited by POP1 in an obviously dose-dependent manner. The level of

494

HBV DNA treated by 500 µg/mL of POP1 was equal to that of the positive control

495

treated with Lamivudine (LAM, 20 µM) (P < 0.01). The calculated IC50 of POP1 for

496

inhibition HBV DNA replication was 0.80 ± 0.03 mg/mL. Similary, Li et al. found

497

that the polysaccharides sulfated derivatives of curdlan sulfate (CS3) could effectively

498

inhibit the HBV replication in HepG2 and HepaRG cells. Wu et al. reported that a low

499

molecular-weight sulfated derivative polysaccharide (PGS) could bind and enter into

500

HepG2.2.15 cells to interfere with HBV transcription.55 Besides, PGS effectively

501

inhibited the expression and secretion of HBsAg and HBeAg in HepG2.2.15 cells.

502

These results demonstrated that the antiviral activities of POP1 might mianly through

503

the interference of HBV replication.

504

In summary, a new polysaccharide POP1 with an average molecular weight of 8.10

505

× 103 Da was purified from the leaves of P. orientalis and it primary chemical

506

structure was characterized.

507

and TNF-α secretion through activating of the related mRNA expression in RAW

POP1 can significantly stimulate the NO, IL-6, IL-12


Page 22 of 43

Page 23 of 43


508

264.7 cells. Besides, POP1 exhibited antiviral activity against HBV by inhibiting the

509

expression of HBeAg, HBsAg and HBV DNA replication. These results suggested

510

that POP1 could be used as an ingredient of anti-visus or immunostimulatory

511

functional foods.

512



513

Acknowledgments

514

The authors gratefully acknowledge the Guangdong Natural Science Funds for

515

Distinguished Young Scholars (No. S2013050013954), Program for New Century

516

Excellent Talents in University (NCET-13-0213), Guangdong Province Funded

517

Research Projects (2015A010107003 and 2014TQ01N645), and the Fundamental

518

Research Funds for the Central Universities, SCUT (2014ZZ0058).

519


Page 24 of 43

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520

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TNF-α, IL-12, IFN-γ, IL-2, IL-4, IL-10 and IL-17. New Biotechnology 2015, 32, 85-95.

645

(48) Chen, H.-S.; Tsai, Y.-F.; Lin, S.; Lin, C.-C.; Khoo, K.-H.; Lin, C.-H.; Wong, C.-H. Studies on

646

the immuno-modulating and anti-tumor activities of Ganoderma lucidum (Reishi) polysaccharides.

647

Biorg. Med. Chem. 2004, 12, 5595-5601.

648

(49) Meng, L.-Z.; Feng, K.; Wang, L.-Y.; Cheong, K.-L.; Nie, H.; Zhao, J.; Li, S.-P. Activation of

649

mouse macrophages and dendritic cells induced by polysaccharides from a novel Cordyceps

650

sinensis fungus UM01. Journal of Functional Foods 2014, 9, 242-253.

651

(50) Kodama, N.; Komuta, K.; Sakai, N.; Nanba, H. Effects of D-fraction, a polysaccharide from

652

Grifola frondosa on tumor growth involve activation of NK cells. Biol. Pharm. Bull. 2002, 25,

653

1647-1650.

654

(51) Wang, L.; Nie, Z.-K.; Zhou, Q.; Zhang, J.-L.; Yin, J.-J.; Xu, W.; Qiu, Y.; Ming, Y.-L.; Liang, S.

655

Antitumor efficacy in H22 tumor bearing mice and immunoregulatory activity on RAW 264.7

656

macrophages of polysaccharides from Talinum triangulare. Food & Function 2014, 5, 2183-2193.

657

(52) Yoon, Y. D.; Kang, J. S.; Han, S. B.; Park, S.-K.; Lee, H. S.; Kang, J. S.; Kim, H. M.

658

Activation of mitogen-activated protein kinases and AP-1 by polysaccharide isolated from the

659

radix of Platycodon grandiflorum in RAW 264.7 cells. Int. Immunopharmacol. 2004, 4,

660

1477-1487.

661

(53) Teruya, T.; Tatemoto, H.; Konishi, T.; Tako, M. Structural characteristics and in vitro

662

macrophage activation of acetyl fucoidan from Cladosiphon okamuranus. Glycoconjugate J. 2009,

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26, 1019-1028.

664

(54) Shepard, C. W.; Simard, E. P.; Finelli, L.; Fiore, A. E.; Bell, B. P. Hepatitis B virus infection:



665

epidemiology and vaccination. Epidemiol. Rev. 2006, 28, 112-125.

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(55) Li, P.; Tan, H.; Xu, D.; Yin, F.; Cheng, Y.; Zhang, X.; Liu, Y.; Wang, F. Effect and

667

mechanisms of curdlan sulfate on inhibiting HBV infection and acting as an HB vaccine adjuvant.

668

Carbohydr. Polym. 2014, 110, 446-455.

669

(56) Gong, L.; Zhang, H.; Niu, Y.; Chen, L.; Liu, J.; Alaxi, S.; Shang, P.; Yu, W.; Yu, L. A novel

670

alkali extractable polysaccharide from Plantago asiatic L. seeds and its radical-scavenging and bile

671

acid-binding activities. J. Agric. Food Chem. 2015, 63, 569-577.

672

(57) Jing, Y.; Zhu, J.; Liu, T.; Bi, S.; Hu, X.; Chen, Z.; Song, L.; Lv, W.; Yu, R. Structural

673

characterization and biological activities of a novel polysaccharide from Cultured Cordyceps

674

militaris and its sulfated derivative. J. Agric. Food Chem. 2015, 63, 3464-3471.

675

(58) Senchenkova, S. y. N.; Shashkov, A. S.; Shneider, M. M.; Arbatsky, N. P.; Popova, A. V.;

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Miroshnikov, K. A.; Volozhantsev, N. V.; Knirel, Y. A. Structure of the capsular polysaccharide of

677

Acinetobacter baumannii ACICU containing di-N-acetylpseudaminic acid. Carbohydr. Res. 2014,

678

391, 89-92.

679 680


Page 30 of 43

Page 31 of 43


681

Figure legends

682

Figure 1. Chromatography of the polysaccharides from P. orientalis by (A) DEAE

683

Sepharose Fast Flow, (B) Sephadex G-100 and (C) high-performance gel permeation

684

chromatography (HPGPC) profile of POP1.

685 686

Figure 2. Gas chromatograms of standard monosaccharides (A) and monosaccharide

687

composition of POP1 (B).

688 689

Figure 3. FT-IR (A), 1H NMR (B), 13C NMR (C) spectrums of the POP1.

690 691

Figure 4. Thermal gravimetric analysis (TG) and differential scanning calorimetric

692

(DSC) analysis of POP1.

693 694

Figure 5. Effect of different concentrations of POP1 on RAW 264.7 cells. (A) cell

695

viability of RAW 264.7 cells. (B) macrophage NO secretion. (C) macrophage TNF-α

696

secretion. (D) macrophage IL-6 secretion. (E) macrophage IL-12 secretion. (F) mRNA

697

expression of IL-6. (G) mRNA expression of TNF-α.(H) mRNA expression of iNOS.

698 699

Figure

700

immunomodulation.

6.

Possible

molecular

mechanism

of

POP1-induced

macrophage

701 702

Figure 7. Effect of different concentrations of POP1 on cell viability of HepG2.2.15

703

cells (A) and HBeAg secretion (B), HBsAg secretion (C), HBV DNA replication (D)

704

in HepG2.2.15 cells. Lamivudine (LAM, 20 µM) was used as the positive control.



705

Table 1. Primer sequences of RT-PCR analysis. Primer IL-6

TNF-α

iNOS

GAPDH

706

Sequence (5′-3′) F

TACTCGGCAAACCTAGTGCG

R

GTGTCCCAACATTCATATTGTCAGT

F

GGGGATTATGGCTCAGGGTC

R

CGAGGCTCCAGTGAATTCGG

F

CGGCAAACATGACTTCAGGC

R

GCACATCAAAGCGGCCATAG

F

TTTGTCAAGCTCATTTCCTGGTATG

R

TGGGATAGGGCCTCTCTTGC

F, forward; R, reverse.

707

31


Page 32 of 43

Page 33 of 43

708


Table 2. Glycosidic linkage composition of methylated POP1. Residues

PMAA

Linkage

Molar ratio

L-Araf

2,3-Me2-Araf

1,5-

4.73

D-Manp

2,4,6-Me3- Manp

1,3-

5.54

2,3,4,6-Me4- Manp

T-

16.69

L-Rhap

2,3,4-Me3-Rhap

1,6-

4.46

D-Glcp

2,3,4,6-Me4-Glcp

T-

11.31

2,3,6-Me3-Glcp

1,4-

51.30

2,3,4-Me3-Glcp

1,6-

4.10

2,3,4-Me3-Galp

1,6-

1.00

2,4-Me2-Galp

1,3,6-

0.86

D-Galp

709 710

32



711

Page 34 of 43

Table 3. Chemical Shifts of Resonances in the 1H and 13C NMR Spectra of POP1. chemical shift (ppm) sugar residue C1/H1

C2/H2

C3/H3

C4/H4

C5/H5

C6/H6

reference

→5)-α-L-Ara(1→

100.14/5.21

81.36/4.15

76.55/3.93

84.05/3.63

64.63/4.00

-/-

56

→3)-α-L-Man(1→

95.80/5.12

76.82/4.08

69.72/3.83

67.80/3.63

71.20/3.70

60.46/3.83

33

α-L-Man(1→

100.60/5.09

69.35/4.00

69.96/3.78

66.20/3.93

72.55/3.70

60.51/3.70

57

→6)-β-L-Rha(1→

107.50/4.84

76.55/4.18

81.36/3.63

76.82/4.08

73.34/3.78

-/-

33

α-D-Glc(1→

98.60/5.03

71.54/3.63

73.34/3.70

69.35/3.63

71.06/3.78

61.14/3.83

23

→4)-α-D-Glc(1→

99.64/5.41

76.55/4.62

74.02/4.08

72.90/3.70

71.06/3.83

60.46/3.78

32

→6)-α-D-Glc(1→

108.90/4.96

81.36/4.25

76.82/3.53

71.20/3.63

69.72/3.70

69.96/3.93

32

→6)-β-D-Gal(1→

104.70/4.39

74.56/3.30

76.82/3.43

71.06/3.50

76.18/3.53

63.80/3.70

58

→3,6)-β-D-Gal(1→

105.80/4.50

71.20/3.63

81.36/3.70

69.96/4.15

74.56/3.83

71.06/4.00

58

712 713

33


Page 35 of 43

714


Table 4. Temperature intervals, weight loss and Td of Region I, II and III; and residual weight at 600 °C (Weight loss, %), for POP1. Thermogravimetric parameters

POP1

Region I Temp. interval (°C)

(24.4–126.9)

Td (°C)

57.0

Weight loss (%)

11.41

Region II Temp. interval (°C)

(126.9–341.9)

Td (°C)

322.9

Weight loss (%)

66.95

Region III Temp. interval (°C)

(341.9–599.3)

Td (°C)

352.7

Weight loss (%)

10.28

Residual char 599.3 °C (%)

11.36

715 716 717

34



Page 36 of 43

1.2 0.5

POP1

Absorbance (490 nm)

1.0 0.4

0.8

0.3

0.6

0.2

0.4 POP2 0.2

0.1

0.0

Concentration of NaCl (mol/L)

A

0.0 0

20

40

60

80

100

120

140

160

180

Tube number

718

Absorbance (490 nm)

B

2.0

1.5

1.0

0.5

0.0 0

719

10

20

30

40

50

60

Tube number

C

720 721 722

Figure 1. Chromatography of the polysaccharides from P. orientalis by (A) DEAE

723

Sepharose Fast Flow, (B) Sephadex G-100 and (C) high-performance gel permeation

724

chromatography (HPGPC) profile of POP1.

35


Page 37 of 43


A

250 225 200

Inter standard

B

Glucose

725 726

175

Rhamnose

100 75 50

Arabinose

125

Galactose

Mannose

150

25 0

727 728 729

10

11

12

13

14

15

16

17

18

19

Figure 2. Gas chromatograms of standard monosaccharides (A) and POP1 (B).

730

36


min


A

Page 38 of 43

100

4000

731 732

733 734

735 736 737

3427.73

1029.54

50 40

577.67 1079.94

60

1151.29

70

1382.79

1639.10

80 2925.77

Transmittance (%)

90

3500

3000

2500

2000

1500

1000

-1

Wavenumber (cm )

B

C

Figure 3. FT-IR (A), 1H NMR (B), 13C NMR (C) spectra of the POP1. 37


500


100 322.9 °C, 6.215 mW/mg

Mass/% DSC/(mW/mg) DTG (dm/dt)

80

8 7 6

TG (%)

5 60 4 352.7 °C, 3.835 mW/mg

40

3 2

20

DSC (mW/mg)

Page 39 of 43

57.0 °C, 0.736 mW/mg

1 0

0 100

738 739 740 741

200

300

400

500

600

Temperature (°C) Figure 4. Thermal gravimetric analysis (TG) and differential scanning calorimetric (DSC) analysis of POP1.

38



80

60

40

20

0

50

500

250

125

62.5

c 30

d e

20 10

f

1000

Control 62.5

1500

b b

b

b

b

1000

F Concentration (µ g/mL )

a

1750

1250

125

1000

500

250

750 500 250

1000

a

250

a

a 3500 3000

b b

2500

c

2000

d 1000 500 0

a 2000

1500

125

500

250

Control b

62.5

a

LPS

a

H

b

Control

125

500

1000

LPS

Concentration (µg/mL)

a

250

250

e Control 62.5

a

1000

b

d


G

b

c 500

LPS

1000

b 1000

0 Control 62.5

a

LPS

c

c

1500

LPS

a

62.5

D

4000



742 Concentration of IL-12 (pg/mL)

b

b

40

0 Control

E

C

a

60

Concentration of IL-6 (pg/mL)

B

Concentration (µ g/mL)

ab

Concentration of NO (µ mol/L)

Cell viability (%)

ab

a

ab

b

Concentration of TNF-α (pg/mL)

ab

100

Concentration (µ g/mL)

A

Page 40 of 43

a

1000

250

a

62.5

a

a

LPS

Control b

c 0 Control 62.5

743 744

125

250

500

1000


LPS

0

1

2

3

4

5

6

7

8

9

0

10

log10 Relative mRNA levels (IL-6)

5

10

15

20

25

Relative mRNA levels (TNF-α)

30

35

0

4000

8000

12000

16000

Relative mRNA levels (iNOS)

745

Figure 5. Effect of different concentrations of POP1 on RAW 264.7 cells. (A) cell viability of RAW 264.7 cells. (B) macrophage NO secretion.

746

(C) macrophage TNF-α secretion. (D) macrophage IL-6 secretion. (E) macrophage IL-12 secretion. (F) mRNA expression of IL-6. (G) mRNA

747

expression of TNF-α.(H) mRNA expression of iNOS.

748

39


Page 41 of 43


749 750

Figure 6. Possible molecular mechanism of POP1-induced macrophage immunomodulation.

40



A

B

a 100

b

b

b

b

a

a

a

Page 42 of 43

a

1.6

b

b

OD 450 nm (HBeAg)

Cell viability (%)

1.4 80

60

40

20

c d

1.2 1.0 0.8 0.6 0.4 0.2

0

0.0 Control

125

62.5

500

250

1000

Control 62.5


C

1.8

a

OD 450 nm (HBsAg)

D

a ab

1.6

b

1.4

c

c

1.2

d

1.0 0.8 0.6 0.4 0.2 0.0 Control 62.5

752 753

125

250

500

125

250

500

LAM

1000


1000

LAM

Concentration of HBV DNA (copies/mL)

751

700000

a b

600000

c d

500000

e

400000

e f

300000 200000 100000 0 Control

62.5

125

250

500

1000

LAM



754

Figure 7. Effect of different concentrations of POP1 on cell viability of HepG2.2.15 cells (A) and HBeAg secretion (B), HBsAg secretion (C),

755

HBV DNA replication (D) in HepG2.2.15 cells. Lamivudine (LAM, 20 µM) was used as the positive control.

41


Page 43 of 43


756 757

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42


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