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Article
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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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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Abstract
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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
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INTRODUCTION
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Platycladus Orientalis (L.) Franco is widely cultivated in Asian countries. The
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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.
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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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developing chronic liver disease, cirrhosis and hepatocellular carcinoma.20 Traditional
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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
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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
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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
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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
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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
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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
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bromide (MTT), Griess reagent, standards of dextrans, erythritol, glycerol, ethylene
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glycol and monosaccharide standards were obtained from Sigma-Aldrich Co. LLC.
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(St. Louis, MO, USA). DEAE Sepharose Fast Flow and Sephadex G-100 were
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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).
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Extraction and purification of polysaccharides from the leaves of P. orientalis
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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.
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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.
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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.
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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.
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Samples were eluted by KH2PO4 (0.02 moL/L, 0.6 mL/min). Eight analytical
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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.
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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
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chromatograph system (Agilent Technologies, USA) with a HP-5 capillary column.
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The carrier gas was N2 with a speed of 25 mL/min. The temperature was 100 °C, a
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linear increase to 160 °C at 3 °C/min, followed by linear increase at 10 °C/min to
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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.
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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
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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.
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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.
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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
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methylation of POP1 was conducted by methyl iodide (2 mL) in the dark. The
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methylated POP1 was extracted by chloroform (4 mL) and hydrolyzed by TFA (2M,
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10 mL) at 110 °C for 2 h. The POP1 hydrolysates were mixed with NaOH and NaBH4
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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
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acetic anhydride was decomposed by distilled water, and methylene chloride (4 mL)
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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.
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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.
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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.
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33000 scans.27
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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
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(approximately 5 mg) was heated in Al2O3 crucibles. The heating program was
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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).
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Immunomodulatory activities of POP1
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Cell culture
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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
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well microplates. After 24 h, cells were treated with different concentrations of POP1
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sample for another 24 h.
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Cytotoxicity assays
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The effect of POP1 on the viability of RAW 264.7 cells was investigated using
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MTT assay. Cells at a density of 5 × 103 cells/well were seeded in 96-well plates and
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treated by POP1 (62.5-1000 µg/mL) for 24 h. MTT solution (20 µL, 5 mg/mL) were
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added as visualization reagent for 4 h and dissoved by 100 µL of DMSO solution.
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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
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caculated as following:
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Cell viability (%) = A/B × 100%
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(A), The absorbance of treated group. (B), The absorbance of control group.
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Effects of POP1 on macrophage NO, TNF-α, IL-6 and IL-12 secretion
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Macrophage activation by immunomodulators is believed to be via the induction of
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NO expression and enhancement of cytokines (TNF-α, IL-6, and IL-12, etc.)
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secretion.25 The macrophage NO content after treatment by POP1 was determined by
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Griess method. The levels of on macrophage NO, TNF-α, IL-6 and IL-12 were
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examined by by commercial ELISA Kits.25 The positive control group was treated by
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LPS (50 µg/mL).
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Inhibition of POP1 on macrophage IL-6, TNF-α and iNOS gene expressions
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The aqueous POP1 solution was prepared at three treatment concentrations of 62.5,
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250, 1000 µg/mL. RAW 264.7 cells were cultured in 96-well plates for 24 h to reach
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an 80% confluence before the addition of POP1-containing media. Macrophage cells
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were collected and washed 3 times with PBS for RNA exaction. In brief, the total
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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.
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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.
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Antiviral activity
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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
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containing 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 200 µg/mL
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G418, and maintained at 37 °C in a moist atmosphere containing 5% CO2.
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Cytotoxicity assays
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The cytotoxic effects of POP1 on HepG2.2.15 cells were evaluated by MTT essay.
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Cells were seeded in 96-well culture plates at a density of 5 × 103 cells per well and
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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.
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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′)
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reverse
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(5′-AAAAGGGACTCAAGATGTTGTACAG-3′) were mixed to a 25 µL reaction
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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
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DNA was calculated by formula as following:
277 278
The inhibitory rate (%) = (Copies control – Copies sample)/Copies control × 100% Statistical analysis
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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.
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RESULTS AND DISCUSSION
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Purification of polysaccharides from Platycladus Orientalis (L.) Franco
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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.
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Structural characterization of POP1
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Molecular weight of POP1
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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
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galactose (6.55%). Glucose represented the greatest proportion compared to the other
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monosaccharide. Thus, glucose may be the main backbone of the structure of POP1.
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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
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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;
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(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
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(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),
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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
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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
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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
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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.
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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
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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.
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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
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(43) Balkwill, F. Tumour necrosis factor and cancer. Nat. Rev. Cancer 2009, 9, 361-371.
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(44) Baugh, J. A.; Bucala, R. Mechanisms for modulating TNF alpha in immune and inflammatory
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disease. Curr Opin Drug Discov Devel 2001, 4, 635-650.
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(45) Wang, Y.; Fang, J.; Ni, X.; Li, J.; Liu, Q.; Dong, Q.; Duan, J.; Ding, K. Inducement of
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cytokine release by GFPBW2, a novel polysaccharide from fruit bodies of Grifola frondosa,
639
through Dectin-1 in macrophages. J. Agric. Food Chem. 2013, 61, 11400-11409.
640
(46) Heikkilä, K.; Ebrahim, S.; Lawlor, D. A. Systematic review of the association between
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circulating interleukin-6 (IL-6) and cancer. Eur. J. Cancer 2008, 44, 937-945.
642
(47) Habijanic, J.; Berovic, M.; Boh, B.; Plankl, M.; Wraber, B. Submerged cultivation of
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Ganoderma lucidum and the effects of its polysaccharides on the production of human cytokines
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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.
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(48) Chen, H.-S.; Tsai, Y.-F.; Lin, S.; Lin, C.-C.; Khoo, K.-H.; Lin, C.-H.; Wong, C.-H. Studies on
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the immuno-modulating and anti-tumor activities of Ganoderma lucidum (Reishi) polysaccharides.
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Biorg. Med. Chem. 2004, 12, 5595-5601.
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(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.
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(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.
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(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:
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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
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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
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Acinetobacter baumannii ACICU containing di-N-acetylpseudaminic acid. Carbohydr. Res. 2014,
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391, 89-92.
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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.
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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
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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
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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
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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
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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.
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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
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A
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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
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Journal of Agricultural and Food Chemistry
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)
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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.
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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
Concentration (µg/mL)
G
b
c 500
LPS
1000
b 1000
0 Control 62.5
a
LPS
c
c
1500
LPS
a
62.5
D
4000
Concentration (µg/mL)
Concentration (µg/mL)
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
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a
1000
250
a
62.5
a
a
LPS
Control b
c 0 Control 62.5
743 744
125
250
500
1000
Concentration (µg/mL)
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
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749 750
Figure 6. Possible molecular mechanism of POP1-induced macrophage immunomodulation.
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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
Concentration (µg/mL)
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
Concentration (µg/mL)
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
Concentration (µg/mL)
Concentration (µg/mL)
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.
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