Structure Characterization of a Novel Polysaccharide from

Subscriber access provided by EGE UNIVERSITESI KUTUPHANESI

Article

Structure Characterization of a Novel Polysaccharide from Dictyophora indusiata and its Macrophage Immunomodulatory Activities Wenzhen Liao, Zhen Luo, Dan Liu, Zhengxiang Ning, Jiguo Yang, and Jiaoyan Ren J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504677r • Publication Date (Web): 19 Dec 2014 Downloaded from http://pubs.acs.org on December 23, 2014

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

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

Page 1 of 34

Journal of Agricultural and Food Chemistry

1

Structure Characterization of a Novel Polysaccharide from

2

Dictyophora indusiata and its Macrophage Immunomodulatory

3

Activities

4

Wenzhen Liao†, Zhen Luo‡, Dan Liu†, Zhengxiang Ning†, Jiguo Yang†*, Jiaoyan Ren†*

5 6

(1 College of Light Industry and Food Sciences, South China University of

7

Technology, Guangzhou 510640, China. 2 R&D Center, Infinitus (China) Co,.LTD.

8

Guangzhou 510665, China)

9 10 11 12 13 14 15 16 17 18

Co-corresponding authors:

19

Jiguo Yang, Department of Food Science and Technology, South China

20

University of Technology, Wushan Road 381, Guangzhou, Guangdong, China.

21

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

22

Jiaoyan Ren, Department of Food Science and Technology, South China

23

University of Technology, Wushan Road 381, Guangzhou, Guangdong, China.

24

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

25 1

ACS Paragon Plus Environment


1

Abstract

2

A novel polysaccharide, here named DP1, was isolated from the fruiting body of

3

Dictyophora indusiata using water extraction method. Structure characterization

4

revealed that DP1 had an average molecular weight of 1,132 kDa and was consisted of

5

glucose (56.2%), galactose (14.1%) and mannose (29.7%). The main linkage type of

6

DP1 were proved to be (1→3)-linked α-L-Man, (1→2, 6)-linked α-D-Glc,

7

(1→6)-linked β-D-Glc, (1→6)-linked β-D-Gal and (1→6)-linked β-D-Man by

8

periodate oxidation−Smith degradation and NMR analysis. Immuno-stimulating assay

9

indicated that DP1 could significantly promote macrophage NO, TNF-α and IL-6

10

secretion in murine RAW 264.7 cells involving complement receptor 3 (CR3). The

11

immune activities of DP1 was quite stability under thermal processing (100 °C,

12

121 °C and 145 °C). Besides, DP1 retained stably after acidic/alkline treatment ( pH

13

4.0-10.0), which enable it to be an ideal complementary medicine or functional food

14

for therapeutics of hypoimmunity and immunodeficiency diseases.

15 16

Key words polysaccharide, Dictyophora indusiata, structure characterization, immuno-stimulating, complement receptor 3.

17 18 19

2


Page 2 of 34

Page 3 of 34


1

INTRODUCTION

2

Dictyophora indusiata, a member of the Phallaceae Corda family, is one of the

3

most popular edible mushrooms worldwidely consumed. Due to its attractive

4

appearance, distinctive flavour and high nutritive value, Dictyophora indusiata is also

5

regarded as “queen of the mushrooms” 1. Dictyophora indusiata can also be used as

6

biopharmaceutical material with various therapeutic properties. In recent decades,

7

many active compounds (e.g polysaccharides, flavonoids or proteins) from

8

Dictyophora indusiata have been reported to possess immunoregulation, antioxidant,

9

anti-tyrosinase, anti-inflammatory and neuroprotective activities

2-4

. Thereamong, the

10

polysaccharides of Dictyophora indusiata received much more attention because of

11

their multiple beneficial potency and relatively low toxicity

12

polysaccharides with various bioactivities have been purified from the fruiting body

13

of Dictyophora indusiata, but rare information is available concerning their chemical

14

structure and immunomodulatory activities.

15

5, 6

. To date, a couple of

Macrophage is one kind of the phagocyte which plays pivotal roles in the innate 7, 8

16

immune responses

When the host is invaded by pathogenic organisms,

17

macrophages can serve as antigen-presenting cells (APC) that present antigen to T

18

lymphocytes and thus induce the adaptive immune response

19

macrophages also play important roles in embryogenesis, tumorigenesis, cutaneous

20

wound healing and hematopoiesis

21

cell models to evaluate the immunemodulatory properties of bioactive compounds.

22

Some botanical polysaccharides have been found to enhance the viability of

23

macrophage against pathogenic microorganisms and tumorigenesis by increasing the

24

secretion of nitric oxide (NO) and the production of cytokines including tumor

25

necrosis factor (TNF-a), interleukin (IL-1, IL-2, IL-6, IL-8, IL-10 and IL-12) 16 -18 and

10-15

9

. Furthermore,

. Thus, macrophages are usually used as ideal

3



1

it has been thought that polysaccharides can bind to specific membrane receptors of

2

macrophage and activate the immune response through distinct signal transduction

3

pathways

4

macrophages by polysaccharides are still not clear. For this sake, it is meaningful to

5

explore the structural characterizations, biological functions and molecular

6

mechanisms of the polysaccharides from Dictyophora indusiata.

19, 20

. However, the exact mechanisms involved in the activation of

7

In the present study, a new polysaccharide, here called Dp1, with a purity of >

8

97.5%, was separated from Dictyophora indusiata. The primary chemical structure

9

and conformation of Dp1 were characterized. Also, its immunomodulatory activities

10

and membrane receptors were examined using murine RAW264.7 macrophages

11

model. The thermal stability of the Dp1 bioactivity against heat treatment was

12

investigated and its stability against acidic/alkali treatment at different pH conditions

13

was studied as well. In addition, the possible signal transduction pathways involved in

14

macrophage activation by Dp1 were discussed and the results from this study might

15

supply useful information for better understanding the chemical structure and

16

immunomodulatory activities of botanical polysaccharides.

17

MATREIALS AND METHODS

18

Materials and Reagents

19

Dictyophora indusiata was collected from Zhijin, Guizhou Province, China. The

20

murine macrophage cell line RAW264.7 was obtained from American Type Culture

21

Collection (ATCC, Rockville, MD, USA). Dulbecco’s modified Eagle’s medium

22

(DMEM), Fetal bovine serum (FBS), phosphate-buffered saline (PBS, pH 7.4),

23

penicillin and streptomycin were purchased from Gibco Life Technologies (Grand

24

Island, NY, USA). Anti-TLR2 antibody, anti-CR3 antibody and anti-TLR4 antibody

25

were got from Abcam Inc. (Cambridge, MA). Laminarin and lipopolysaccharide (LPS) 4


Page 4 of 34

Page 5 of 34


1

were

obtained

from

Sigma

Company

(St.

Louis,

MO,

USA).

DEAE

2

(diethylaminoethyl)-52 cellulose and Sephadex G-200 were purchased from GE

3

Healthcare Life Science (Piscataway, NJ, USA). Nitricoxide (NO)-detecting kit was

4

purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China).

5

Mouse IL-6 ELISA kit and Mouse TNF-α ELISA kit were acquired from

6

Neobioscience Technology Co., Ltd. (Shenzhen, China). Standards of dextrans, uronic

7

acid, phycite, glycerol, glycol reference and monosaccharide references (e.g glucose,

8

galactose, xylose, mannose and rhamnose) were purchased from Sigma Company (St.

9

Louis, MO, USA). The ultrapure water was obtained from a Milli-Q water

10

purification system of Millipore (Millipore, Bedford, MA, USA). All the other

11

chemical reagents used in this study were of analytical grade.

12

Extraction and Purification of Polysaccharides from the Fruiting Body of

13

Dictyophora indusiata

14

The fruiting body of Dictyophora indusiata was dried in a hot air-drying oven at

15

40 °C and crushed into powder using a tissue triturator. The powder of fruiting body

16

was extracted with boiling water at a ratio of 1: 30 (w/w) for 2 h and the obtained

17

extract was centrifuged at 4000 × g for 15min. The supernatant was then collected and

18

concentrated at 60 °C. After that, the concentrated supernatant was deproteinated

19

according to the Sevag method

20

deproteination process was repeated 10 times. The resulting solution was mixed with

21

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

22

precipitate were then separated by centrifugation at 5000 × g for 15 min. Finally, the

23

precipitate was dried using vacuum freeze drying and collected as crude

24

polysaccharides.

21

. To remove the protein as much as possible, this

5



1

One hundred miligrams of crude polysaccharides were dissolved in 10 mL of

2

ultrapure water and loaded onto a DEAE-52 anion-exchange chromatography column

3

(2.5 cm × 60 cm). The elution was conducted using ultrapure water first and then by a

4

series of NaCl solution in the sequence of 0.05, 0.1, 0.2, 0.3 and 0.5 M, respectively,

5

at a flow rate of 0.5 mL/min. The eluent fractions were collected and analyzed by the

6

phenol−sulfuric acid method

7

DP2 (eluted by 0.05 M NaCl) were obtained, dialysed at 4°C for 48h and lyophilized.

8

In this study, we mainly focused the investigation of DP1 and the other fraction will

9

be studied later.

22

. Two fractions, DP1 (eluted by ultrapure water) and

10

After that, DP1 fraction was further purified by Sephadex G-200 column

11

chromatography. Twenty milligrams of DP1 was redissolved in 4 mL of ultrapure

12

water and applied to a Sephadex G-200 column (2.5 cm × 60 cm). The sample was

13

eluted with ultrapure water at a speed of 0.5 mL/min, then the eluent was collected

14

and detected using the phenol−sulfuric acid method for the determimation of 97.5%

15

content.

16

Structure Characterization of DP1

17

Molecular Weight Determination of DP1

18

The weight-average molecular weight distribution of DP1 was analyzed by

19

high-performance gel permeation chromatography (HPGPC, Waters, Milford, MA,

20

USA) on a Waters HPLC instrument with a TSK-GEL G-5000 PWXL column (300

21

mm × 7.8 mm i.d., 10 µm) and a TSK-GEL G-3000PWXL column (300 mm × 7.8

22

mm i.d., 6 µm, Tosoh Co., Ltd., Tokyo, Japan), using 0.01 mol/L KH2PO4 as the

23

mobile phase at a flow rate of 0.6 mL/min.

24

Infrared Spectrum Analysis

6


Page 6 of 34

Page 7 of 34


1

The infrared spectra of DP1 was analyzed according to the KBr-disk method

23

2

using a Fourier transform infrared (FT-IR) spectrophotometer (Bruker, Ettlingen,

3

Germany) in the range of 400–4000 cm-1.

4

Chemical Composition of DP1

5

Twenty miligrams of DP1 was hydrolyzed by 4 mL trifluoroacetic acid (TFA) at

6

105 °C for 6 h. Excess of TFA was removed by rotary evaporation. Then the residue

7

was redissolved in methanol and dried by rotary evaporation. Finally, the residue was

8

dissolved in ultrapure water and analyzed by ion exchange chromatography (Dionex

9

ICS-3000) equipped with an integrated amperometric detector and Carbopac PA20

10

column (2 × 250 mm). The elution was conducted at a rate of 0.6 mL/min at column

11

temperature 20 °C. Gradient elution was carried out according to the following

12

process: 0-2 min, 100% 200 mmol/L NaOH; 2.1 - 20 min, 10% 20 mmol/L NaOH and

13

90% ultrapure water; 20.1-40 min, 100% 200 mmol/L NaOH. The glucose, galactose,

14

xylose, mannose and rhamnose standards were derivatised as previously reported.

15

Acetyl inositol was used as the interior reference.

16

Periodate Oxidation-Smith Degradation

17

4

The periodate oxidation-Smith degradation method was taken out according to 24

18

the report of Chi

with some modification. Twenty-five milligrams of DP1 sample

19

was dissolved in 12.5 mL of ultrapure water and incubated with 12.5 mL of NaIO4 (30

20

mmol/L) in the dark at room temperature. During the incubation period, 0.1 mL of the

21

reaction liquid was taken out from the reaction system at different time intervals (0, 6,

22

12, 24, 36, 48 and 60 h), diluted into 100 mL with ultrapure water, and measured at

23

223 nm by a UV-Vis spectrophotometer (Model UV-18000, Shimadzu, Japan) until

24

the absorbance value became invariable. The process of periodate oxidation was

25

stopped by the addition of 2 mL glycol. Then 2 mL of periodate product was titrated 7



1

by 0.01 mol/L NaOH to quantify the production of formic acid. The rest of periodate

2

product was dialyzed in distilled water for 72 h and concentrated by rotary

3

evaporation. Then 70 mg of NaBH4 was added to the residuary solution and kept in

4

the dark for 24 h. The solution was adjusted to pH 5.0 with 0.1 mol/L acetic acid and

5

dialyzed in distilled water for another 72 h. The solution was then concentrated and

6

freeze-dried. Ten milligrams of the residues were hydrolyzed by 4 mL of 2 mol/L TFA

7

at 105 °C for 6 h. The lysate was acetylized with 1 mL of pyridine and 10 mg of

8

hydroxylamine hydrochloride at 90 °C for 30 min. Then 1 mL of acetic anhydride was

9

added to the reaction system and continuously heating at 90 °C for another 30 min.

10

The production of acetate derivative was analyzed by a gas chromatograph system

11

(Aglient, 6890N, Agilent Technologies Inc., Santa Clara, California, USA) with a

12

DB-1701 capillary column (30 m × 0.25 mm × 0.25 µm, J&W Scientific, Fulsom, CA

13

95630, USA) and a flame ionization detector. The linearly heating program is from

14

80 °C to 220 °C at a speed of 2 °C/min, from 220 °C to 250 °C at a speed of 5 °C/min

15

and kept at 250 °C for 5 min. The temperature of detector was set at 300 °C. The

16

phycite, glycerol, glycol, rhamnose, arabinose, xylose, galactose, mannose and

17

glucose were used as standards.

18

Nuclear Magnetic Resonance (NMR)

19

Thirty-five milligrams of DP1 was dissolved in 0.5 mL of D2O in a nuclear

20

magnetic resonance tube and then analyzed by a Bruker 600 MHz Nuclear Magnetic

21

Resonance Apparatus (Bruker Corp., Fallanden, Switzerland) 25.

22

8


Page 8 of 34

Page 9 of 34


1 2

Thermogravimetric (TG) Analysis

3

The thermostability of DP1 was analyzed using a thermogravimetric analyzer

4

(Pyris Diamond TG-DTA, Perkin Elmer Co., Waltham, MA, USA). Five milligrams

5

of DP1 sample was placed in the Al2O3 pan and heated from 60 °C to 600 °C with a

6

heating rate of 10 °C/min. The analysis was conducted under a pure nitrogen gas

7

atmosphere at a flow rate of 30 mL/min.

8

Immunomodulatory Activity and Stability Assay

9

Cell Culture and Immunomodulatory Activity Test

10

RAW264.7 cells were cultured with DMEM plus with 10% of fetal bovine serum

11

(FBS), 100 µg/mL of streptomycin and 100 U/mL of penicillin, and incubated at

12

37 °C with 5% CO2 humidified atmosphere in a carbondioxide cell incubator. Cells

13

were adjusted to a concentration of 1 × 106 cells/mL in the exponential phase, loaded

14

onto the 96-well microplate (100 µL/well) and continuously incubated for 24 h. Then

15

the culture medium was changed to new medium with DP1 samples and incubated for

16

another 24 h. Antibodies (anti-TLR2, anti-CR3 and anti-TLR4, at a concentration of 5

17

µg/mL) and laminarin (500 µg/mL, the inhibitor of dectin-1) were added to cells to

18

investigate the membrane receptors of DP1 on the RAW264.7 cells. The cells were

19

pretreated with antibodies and laminarin for 2 h prior to stimulation with different

20

concentration of DP1 (125, 250, 500, and 1000 µg/mL) and incubated for 24 h. LPS

21

(50 µg/mL) was used as the positive control. After that, the supernatants of cells were

22

collected and the levels of NO, TNF-α and IL-6 were measured using NO-detecting

23

kit, Mouse TNF-α ELISA Kit and Mouse IL-6 ELISA Kit, respectively, according to

24

the manufacturer’s instructions.

25

Immunomodulatory Stability Assay

9



1

Thermal Treatment. DP1 was dissolved in PBS (pH 7.4) to make final

2

concentration of 500 µg/mL and subjected to different heat treatment (100 °C × 30

3

min, 121 °C × 30 min and 145 °C × 30 min, respectively) and then cooled to room

4

temperature. After that, DP1 was sterilized by 0.22 µm microporous filtering film and

5

then incubated with RAW 264.7 cells for 24 h. The changes of NO, TNF-α and IL-6

6

levels were detected as described above.

7

Acidic/alkaline Treatment. DP1 (500 µg/mL) was dissolved in different

8

phosphate buffer to make final pH 2.0, 4.0, 6.0, 7.4, 8.0 and 10.0, respectively, and

9

then kept over night at room temperature. DP1 with different pH value was sterilized

10

by 0.22 µm microporous filtering film and then incubated with RAW 264.7 cells for

11

24 h. The changes of NO, TNF-α and IL-6 levels were detected as described above.

12

Combined Thermal Treatment and Acidic/alkaline Treatment. DP1 (500

13

µg/mL)

was dissolved in different phosphate buffer at pH 2.0, 4.0, 6.0, 7.4, 8.0 and

14

10.0, respectively. Then the solution were heated at 121 °C for 30 min. All samples

15

were sterilized by 0.22 µm microporous filtering film and then incubated with RAW

16

264.7 cells for 24 h. The changes of NO, TNF-α and IL-6 levels were detected as

17

described above.

18

Statistical Analysis

19

Data were presented as mean ± SD for at least three replicates and analyzed by

20

SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Significance was determined at P values

21

below 0.05 by analysis of variance. The difference between three or more groups was

22

analyzed by one-way ANOVA multiple comparisons.

23

10


Page 10 of 34

Page 11 of 34


1

RESULTS AND DISCUSSION

2

Extraction and Purification of Polysaccharides from Dictyophora Indusiata

3

Crude polysaccharides were isolated from the fruiting body of Dictyophora

4

indusiata with a yield of 6.36%. The extracts were first purified by DEAE-52

5

anion-exchange chromatography column (Figure. 1A). Two independent peaks, DP1

6

(eluted by ultrapure water) and DP2 (eluted by 0.05 mol/L of NaCl) were obtained. In

7

the present study, we mainly focused on DP1, and the property of DP2 will be studied

8

in our future work. The DP1 fraction was further purified by Sephadex G-200 column.

9

As shown in Figure. 1B, a single peak of DP1 was observed. Finally, DP1 fraction

10

with a purity of > 97.5% was obtained after dialysis and lyophilization.

11

Characterization of DP1

12

Molecular Weight of DP1

13

The

weight-average

molecular

weight

of

DP1

was

measured

by

26

14

high-performance gel permeation chromatography (HPGPC)

15

1C, a single peak was observed on the chromatogram and the molecular weight of

16

DP1 was determined to be 1,132 kDa. This result showed a great difference from the

17

previous research by Hua et al.

18

polyacchrides from Dictyophora indusiata were 2100.99 and 18.16 kDa. The

19

difference of the extraction method, the habitat and the strain of Dictyophora

20

indusiata may lead to the differences of molecular weights.

21

Monosaccharide Composition Assay

22

The

monosaccharide

1

. As shown in Figure.

which indicated that the molecular weight of two

composition

of

DP1

was

analysed

using

ion

23

chromatography. As shown in Figure. 1D, three monosaccharides including galactose,

24

glucose and mannose were identified according to the elution time of relative

25

monosaccharide standards. The molar percentages of galactose, glucose and mannose 11



Page 12 of 34

1

were 14.1%, 56.2% and 29.7%, respectively. The results were different from that of

2

Hua et al. (2012) who found that water-soluble polysaccharides from Dictyophora

3

indusiata were composed of xylose, galactose, glucose and mannose with the molar

4

percentages of 22.6%, 18.6%, 13.9% and 44.9% 1.

5

Periodate Oxidation-Smith Degradation Analysis

6

The results for the periodate oxidation analysis of DP1 showed that 1.27 mol of

7

periodate was consumed by every molar of sugar residues and 0.63 mol of acid was

8

produced, indicating the existence of (1→) or (1→6)- linked glycosidic bonds. The

9

periodate-oxidized DP1 sample was further analyzed by Smith degradation. Glycerol,

10

erythritol, galactose, glucose and mannose were observed after smith degradation

11

(Figure. 1E). The presence of glycerol indicating the existence of (1→2) or (1→6)-

12

linked glycosidic bonds, while erythritol corresponding to the presence of

13

(1→4)-linked glycosidic bonds. The presence of monosaccharides revealed the

14

appearance of (1→3)-linked glycosidic bonds.

15

Infrared Spectrum Analysis

16

The IR spectra of DP1 is shown in Figure. 2A. The stretching intense

17

characteristic peaks at 3335.69 cm-1 indicated the hydroxyl stretching vibration of the

18

polysaccharide chains. The peak around 2926.33 cm-1 was attributed to the C–H

19

stretching vibration 27. The band at 1648.01 cm-1 may be due to the bound water. The

20

absorption peaks between 1430 and 1200 cm-1 indicated the presence of the O-H

21

deformation vibrations and C-O stretching vibrations, respectively

22

1074.32 and 1040.25 cm-1 suggested that the skeletal modes of DP1 were pyranose

23

rings. The absorption at 866 cm-1 indicated the β- pyranoside linkage in DP1. A

24

characteristic peak at 796.81 cm-1 represented the presence of mannose.

25

NMR Analysis of DP1 12


28

. The bands at

Page 13 of 34


1

The spectra of 1H NMR and 13C NMR of DP1 are shown in Figure. 2B and 2C.

2

The signals of DP1 in 1H NMR and 13C NMR spectra were attributed on the basis of

3

monosaccharide, glycosidic linkages and chemical shifts 29. The resonances signals of

4

13

5

L-mannose, L-arabinose, D-galactose, D-glucose and L-rhamnose

6

95.902 ppm suggested the presence of C-1 of (1→3)-linked α-L-Man unit and the

7

peak at 100.723 ppm indicated the presence of C-1 of (1→3)-linked α-D-Gal unit.

8

Another peak at 104.978 ppm proved the presence of C-1 of (1→2,6)-linked α-D-Glc

9

unit and the peak at 69.469 ppm corresponded to C-6 of (1→6)-linked β-D-Glc unit.

10

Besides, the peat at 68.742 ppm suggested the presence of C-6 of (1→6)-linked

11

β-D-Gal unit and the peak at 66.569 ppm corresponded to the C-6 of (1→6)-linked

12

β-D- Man unit. The peaks in the region of 78.287 ppm to 81.436 ppm （78.287，

13

78.593，78.921，and 81.436 ppm） were attributed to the presence of (1→2), (1→3)

14

or (1→4)-linked glycosidic bonds. The configuration of the glycosidic linkage of DP1

15

was analyzed by 1H NMR spectra. The signals of 1H NMR in the region of

16

5.400-5.843 ppm (5.483, 5.517, 5.569, 5.623, 5.673, 5.678, 5.715 and 5.843 ppm)

17

indicated the presence of α-form furanose. The peaks at 5.000-5.400 ppm (5.005,

18

5.117, 5.131, 5.206, 5.217, 5.244, 5.257, 5.296 and 5.348 ppm) were attributed to the

19

presence of α-form pyranose.

C NMR between 95.0 ppm to 110.0 ppm belonged to the anomeric carbon atoms of

13


29

. The peak at


1

Immunomodulatory Activities of DP1

2

Effects of DP1 on Macrophage NO, TNF-α and IL-6 Production

3

Page 14 of 34

Modulation of the innate immune system can enhance the host’s resistant ability 30

4

to exogenous pathogens threats

. It is thought that the stimulation of the

5

immuno-system might mainly through the activation of macrophage and complement

6

system 31,32. Macrophage activation by immunomodulators is considered to be via the

7

induction of NO expression and enhanced production of cytokines (TNF-a, IL-1, IL-6,

8

IL-12 and IL-10, etc.)

9

IL-6 production were analyzed in this study. Untreated RAW 264.7 cells secreted very

10

little NO, whereas, the addition of DP1 in the medium significantly increased NO

11

secretion in a dose-dependent manner (125−1000 µg/mL) (Figure. 3A). The

12

concentration of NO reached to 11.72 µmol/mL after treatment by 500 µg/mL of DP1,

13

this is higher than a polysaccharide from Lentinula edodes reported by Xu et al. 19. In

14

Xu’s study, the concentration of NO was lower than 4 µmol/mL after treatment by 500

15

µg/mL of Lentinula edodes polysaccharide. Meanwhile, DP1 had similar

16

dose-dependent manner effects on macrophage TNF-α and IL-6 production (Figure.

17

3B and 3C). The macrophage IL-6 levels (eg. 1134 pg/mL of IL-6 at 250 ug/mL of

18

DP1) treated by DP1 is higher than that of some polysaccharide fractions isolated

19

from Opuntia polyacantha 14 (lower than 1000 pg/mL of IL-6 at 250 ug/mL of some

20

Opuntia polyacantha fractions). Whereas, the TNF-α levels (eg. 588 pg/mL of TNF-α

21

at 250ug/mL of DP1) treated by DP1 is lower than that of a polysaccharide from

22

Ganoderma atrum ( over 1400 pg/mL of TNF-α at 160 mg/mL Ganoderma atrum

23

polysaccharide).34

24

immunomodulatory activities by upregulation of NO, TNF-α and IL-6 secretion in

25

RAW 264.7 cells.

33

The

. Thus, the effects of DP1 on macrophage NO, TNF-α and

results

suggested

that

DP1

14


exhibited

remarkable

Page 15 of 34


1 2

Roles of CR3, TLR2, TLR4, and Dectin-1 on DP1-induced Macrophage

3

Immunomodulation

4

It has been proposed that macrophage activation is mediated primarily through

5

the recognition of pathogen-associated molecular patterns (PAMPs) by pattern

6

recognition receptors (PRRs) on the surface of macrophage cells. Stimulation of PRRs

7

leads to the initiation of intracellular signaling pathways and eventually induces

8

transcriptional activation and expression of cytokines. Recent evidence suggests that

9

macrophages might bind polysaccharides and mediate immunomodulating effects

10

mainly through complement receptor 3 (CR3), Toll-like receptors (TLR2 and TLR4)

11

or dectin-135. In the present study, the roles of CR3, TLR2, TLR4 and dectin-1 on

12

DP1-induced

13

including anti-CR3, anti-TLR2, anti-TLR4 and the inhibitor of dectin-1, laminarin,

14

were preincubated with RAW264.7 cells for 2 h and then incubated with DP1 for

15

another 24 h. Laminarin is a β-glucan which can specifically bind to dectin-1 and

16

block the dectin-1-mediated immune responses

17

IL-6 production were determined. As shown in Figure. 4A, 4B and 4C, after treatment

18

of anti-CR3 and DP1, the levels of NO, TNF-α and IL-6 were all visibly decreased

19

comparing to the group treated by DP1 only (p < 0.05). However, no decrease was

20

found in the groups treated by anti-TLR2, anti-TLR4. As for laminarin, it was even

21

enhanced due to its own immuno-stimulating activity. These results indicated that

22

CR3 was one of the receptors of DP1 on RAW264.7. Previous studies have proved

23

that CR3 possessed sugar specificity and the polysaccharides containing

24

N-acetyl-d-glucosamine or mannose in addition to glucose are most likely recognized

25

by CR3 34. Therefore, the mannose and glucose composition of DP1 may contribute to

macrophage

immunomodulation

were

investigated.

Antibodies

36

. The macrophage NO, TNF-α and

15



1

the specific binding of DP1 to CR3 on macrophage. Besides, there might be some

2

other receptors of DP1 on the membrane of macrophage, since the NO level

3

(Figure.4A), TNF-α level (Figure.4B) and IL-6 level (Figure.4C) in treated group by

4

anti-CR3 and DP1 were still higher than those in the group absence of DP1 treatment.

5

CR3 has been proved to be involved in the activation of the phosphoinositide-3-kinase

6

(PI3K), phosphorylation of the downstream serine/threonine kinase Akt and

7

mammalian target of rapamycin (mTOR), activation of the mitogen-activated protein

8

kinase (MAPK), and nuclear factor-κB (NF-κB) signal transduction pathways.

9

Activation of these signaling pathways ultimately leads to gene transcription and

10

production of pro-inflammatory cytokines 16. Therefore, we can infer that the possible

11

molecular mechanism of DP1-induced macrophage immunomodulation was mainly

12

through activation of the PI3K/Akt/MAPK/NF-κB signaling transduction pathways

13

(Figure. 4D), and these pathways will be characterized in our future study.

14

Effects of Thermal Treatment and Acidic/Alkali Treatment on DP1-induced

15

Macrophage Immunomodulation

16

It is well established that severe environmental conditions such as high

17

tempreature, high pressure and extreme pH have great influence on the advanced

18

structure of macromoleculars

19

and acidic/alkali treatments on the immunomodulation activity of DP1 were

20

investigated. It was found that with the increase of temperatures, DP1 exhibited a

21

substantial decrease effects on macrophage NO, TNF-α and IL-6 production, as

22

shown in Figure.5A, 5B and 5C, respectively. It may be due to that high temperature

23

(over 100 °C) can cause the break down of non-covalent bonds such as ionic bonds,

24

hydrogen bonds and hydrophobic effects of DP1, and thus as compared with TNF-α

25

and IL-6, the NO level was more sensitive to heat treatment and the 145 °C

37

. In this study, the effects of different heat treatment

16


Page 16 of 34

Page 17 of 34


1

processing for 30 min destroyed 77.6 % of its native activity. At this point, the

2

immue-sting activities of TNF-α and IL-6 were 24.9% and 57.0% of its native

3

activities, respectively. Although being sensivity to heat treatment, DP1 was still able

4

to upgrade the macrophage NO, TNF-α and IL-6 levels after treatment at 145 °C. The

5

reason for these results may mainly because that thermal treatment just destroyed the

6

higher structure (eg. triple helical) of DP1. Recent evidence confirmed that the

7

helix-coil transition of polysaccharide was induced by heat treatment at about

8

130-145 °C due to the breakage of hydrogen bonds 19. The primary structure may not

9

be damaged under the selected temperatures, although higher structure may likely

10

contributed much to the biological activity of DP1, the primary structure of DP1 also

11

maintained a considerable part of its immue-sting activity. The thermostability of DP1

12

was further analyzed using thermogravimetric analysis (TGA). As shown in Figure

13

5D, neglecting the small weight loss at around 50 °C attributed to the vaporization of

14

retained moisture, the decomposition peak of DP1 was found at 329 °C. This

15

degradation temperature of DP1 was much higher than that of the polysaccharides

16

from haloalkalophilic Bacillus sp. I-450 (290°C) as reported by C. Ganesh Kumar. etc

17

38

18

extreme heat treatments.

. These results further confirmed that DP1exhibited good thermostability under

19

Previous studies have also reported that the non-covalent bonds can also be

20

destroyed in the strong acidic or alkline conditions 39. Thus, the effects of pH changes

21

on DP1 was investigated as well in the present study. The DP1 samples were

22

pretreated by phosphate buffers with various pH (form 2.0 to 10.0) for 12 h. As shown

23

in Figure. 6A, 6B and 6C, it was found that the cells without DP1 protection were

24

very sensitive to different pH treatments, while the addition of DP1 remarkably

25

enhanced the macrophage NO, TNF-α and IL-6 secretion. 17


Especially for IL-6, the


1

protection effect of DP1 was more obvious. It turns out that even at pH 4.0, DP1

2

could still protect the cells to recover 50% of their original NO, TNF-α and IL-6

3

concentration. It demonstrated that the structure of DP1 remained stable in a broad

4

spectrum of pH levels. At alkline conditions (pH 10.0), DP1 showed less protection

5

on the NO level of the cells, but it still contributed to the TNF-α and IL-6 secretion of

6

macrophage.

7

The stability of DP1 against both heat treatment and acidic/alkline treatment

8

were further explored. DP1 samples were dissolved in phosphate buffers with

9

different pH values (form 2.0 to 10.0) and then heated at 121 °C for 30 min. The

10

results demonstrated that DP1 still retained its immunomodulatory activities to

11

enhance macrophage NO (Figure. 6D), TNF-α (Figure. 6E), and IL-6 (Figure. 6F)

12

secretion in RAW 264.7 cells. In this way, DP1 was proved to retain stable

13

immunomodulatory activities even undergoing combined heat treatment and

14

acidic/alkline treatment. It was very interesting that at pH 10.0, the results showed

15

that the contribution of DP1 to the macrophage NO level was even enhanced after

16

combined treatments (Figure. 6D) than single alkline treatment (Figure. 6A) and the

17

reason was not clear yet. With the above results, we suggested the potential of DP1 as

18

a novel immunomodulatory agent.

19

In this study, a new polysaccharide DP1 of 1132 kDa was purified from

20

Dictyophora indusiata. DP1 was consisted of (1→3)-linked α-L-Man, (1→2,

21

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

22

β-D-Man. DP1 possessed significant immunomodulation activities by upregulating

23

macrophage NO, TNF-α and IL-6 secretion. One of the membrane receptors of DP1

24

on RAW264.7 was confirmed to be CR3. The PI3K/Akt/MAPK/NF-κB signaling

25

pathways were thought to be involved in the immunomodulation function of DP1. 18


Page 18 of 34

Page 19 of 34


1

DP1 retained stably across broad spectrum of pH and thermal treatment. Thus, DP1

2

demonstrated

3

hypoimmunity and immunodeficiency population.

4

ACKNOWLEDGEMENTS

the

potential

applications

as

complementary

medicine

for

5

This study was supported by the National Key Technology R&D Program of

6

China in the 12th five-year period (No. 2012BAD33B11), the Fundamental Research

7

Funds for the Central Universities (No. 2014ZZ0063 & 2013ZZ0061) and Guangdong

8

Natural Science Funds for Distinguished Young Scholars (No.S2013050013954).

9

19



1 2

REFERENCES

3

(1) Hua, Y.; Yang, B.; Tang, J.; Ma, Z.; Gao, Q.; Zhao, M., Structural analysis of

4

water-soluble polysaccharides in the fruiting body of Dictyophora indusiata and their

5

in vivo antioxidant activities. Carbohyd Polym. 2012, 87, 343-347.

6

(2) Sharma, V. K.; Choi, J.; Sharma, N.; Choi, M.; Seo, S. Y., In vitro anti-yrosinase

7

activity of 5-(hydroxymethyl)-2-furfural isolated from Dictyophora indusiata.

8

Phytother Res. 2004, 18, 841-844.

9

(3) Geun Goo, B.; Baek, G.; Jin Choi, D.; Il Park, Y.; Synytsya, A.; Bleha, R.; Ho

10

Seong, D.; Lee, C.; Kweon Park, J., Characterization of a renewable extracellular

11

polysaccharide from defatted microalgae Dunaliella tertiolecta. Bioresour Technol.

12

2013, 129, 343-350.

13

(4) Chen, H.; Yang, T.; Chen, M.; Chang, Y.; Lin, C.; Wang, E. I.; Ho, C.; Huang, K.;

14

Yu, C.; Yang, F., Application of power plant flue gas in a photobioreactor to grow

15

Spirulina algae, and a bioactivity analysis of the algal water-soluble polysaccharides.

16

Bioresour Technol. 2012, 120, 256-263.

17

(5) Kanmani, P.; Yuvaraj, N.; Paari, K. A.; Pattukumar, V.; Arul, V., Production and

18

purification of a novel exopolysaccharide from lactic acid bacterium Streptococcus

19

phocae PI80 and its functional characteristics activity in vitro. Bioresour Technol.

20

2011, 102, 4827-4833.

21

(6) Tong, H.; Xia, F.; Feng, K.; Sun, G.; Gao, X.; Sun, L.; Jiang, R.; Tian, D.; Sun,

22

X., Structural characterization and in vitro antitumor activity of a novel

23

polysaccharide isolated from the fruiting bodies of Pleurotus ostreatus. Bioresour

24

Technol. 2009, 100, 1682-1686.

25

(7) Janeway Jr, C. A.; Medzhitov, R., Innate immune recognition. Annu Review of 20


Page 20 of 34

Page 21 of 34


1

Immunol. 2002, 20, 197-216.

2

(8) Uthaisangsook, S.; Day, N. K.; Bahna, S. L.; Good, R. A.; Haraguchi, S., Innate

3

immunity and its role against infections. Ann Allerg, Asthma Im. 2002, 88, 253-265.

4

(9) Birk, R. W.; Gratchev, A.; Hakiy, N.; Politz, O.; Schledzewski, K.; Guillot, P.;

5

Orfanos, C. E.; Goerdt, S., Alternative activation of antigen-presenting cells: concepts

6

and clinical relevance. Hautarzt. 2001, 52, 193-200.

7

(10) Lingen, M. W., Role of leukocytes and endothelial cells in the development of

8

angiogenesis in inflammation and wound healing. Arch Pathol Lab Med. 2001, 125,

9

67-71.

10

(11) Klimp, A. H.; De Vries, E.; Scherphof, G. L.; Daemen, T., A potential role of

11

macrophage activation in the treatment of cancer. Crit Rev Oncol Hemat. 2002, 44,

12

143-161.

13

(12) Foo, N.; Ou Yang, H.; Chiu, H.; Chan, H.; Liao, C.; Yu, C.; Wang, Y., Probiotics

14

prevent the development of 1, 2-dimethylhydrazine (DMH)-induced colonic

15

tumorigenesis through suppressed colonic mucosa cellular proliferation and increased

16

stimulation of macrophages. J Agr Food Chem. 2011, 59, 13337-13345.

17

(13) Pan, M.; Chang, Y.; Tsai, M.; Lai, C.; Ho, S.; Badmaev, V.; Ho, C., Pterostilbene

18

suppressed lipopolysaccharide-induced up-expression of iNOS and COX-2 in murine

19

macrophages. J Agr Food Chem. 2008, 56, 7502-7509.

20

(14) Puangpraphant, S.; de Mejia, E. G., Saponins in yerba mate tea (Ilex

21

paraguariensis A. St.-Hil) and quercetin synergistically inhibit iNOS and COX-2 in

22

lipopolysaccharide-induced macrophages through NFκB pathways. J Agr Food Chem.

23

2009, 57, 8873-8883.

24

(15) Chiu, F.; Lin, J., HPLC analysis of naturally occurring methylated catechins,

25

3''-and 4''-methyl-epigallocatechin gallate, in various fresh tea leaves and commercial 21



1

teas and their potent inhibitory effects on inducible nitric oxide synthase in

2

macrophages. J Agr Food Chem. 2005, 53, 7035-7042.

3

(16) Schepetkin, I. A.; Quinn, M. T., Botanical polysaccharides: macrophage

4

immunomodulation and therapeutic potential. Int Immunopharmacol. 2006, 6,

5

317-333.

6

(17) Lai, P. K. K.; Chan, J. Y. W.; Wu, S. B.; Cheng, L.; Ho, G. K. W.; Lau, C. P.;

7

Kennelly, E. J.; Leung, P. C.; Fung, K. P.; Lau, C. B. S., Anti-inflammatory Activities

8

of an Active Fraction Isolated from the root of Astragalus membranaceus in RAW

9

264.7 Macrophages. Phytother Res. 2014, 28, 395-404.

10

(18) Kulakowski, D. M.; Wu, S.; Balick, M. J.; Kennelly, E. J., Merging bioactivity

11

with liquid chromatography-mass spectrometry-based chemometrics to identify minor

12

immunomodulatory compounds from a Micronesian adaptogen, Phaleria nisidai. J

13

Chromatogr A. 2014, 1364, 74-82.

14

(19) Xu, X.; Yan, H.; Zhang, X., Structure and immuno-stimulating activities of a

15

new heteropolysaccharide from Lentinula edodes. J Agr Food Chem. 2012, 60,

16

11560-11566.

17

(20) Gordon, S., Pattern recognition receptors: doubling up for the innate immune

18

response. Cell. 2002, 111, 927-930.

19

(21) Staob, A. M., Removal of proteins from polysaccharides methods. Carbohydr

20

Chem. 1965, 5, 1-5.

21

(22) Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P.; Smith, F., Colorimetric

22

method for determination of sugars and related substances. Anal Chem. 1956, 28,

23

350-356.

24

(23) Liao, W.; Ning, Z.; Chen, L.; Wei, Q.; Yuan, E.; Yang, J.; Ren, J., Intracellular

25

Antioxidant Detoxifying Effects of Diosmetin on 2,2-Azobis(2-amidinopropane) 22


Page 22 of 34

Page 23 of 34


1

Dihydrochloride (AAPH)-induced Oxidative Stress through Inhibition of Reactive

2

Oxygen Species Generation. J Agr Food Chem. 2014, 62, 8648-8654.

3

(24) Chi, A.; Chen, J.; Wang, Z.; Xiong, Z.; Li, Q., Morphological and structural

4

characterization of a polysaccharide from Gynostemma pentaphyllum Makino and its

5

anti-exercise fatigue activity. Carbohyd Polym. 2008, 74, 868-874.

6

(25) Wu, S.; Bao, Q.; Wang, W.; Zhao, Y.; Xia, G.; Zhao, Z.; Zeng, H.; Hu, J.,

7

Cytotoxic triterpenoids and steroids. Planta Med. 2011, 77, 922-928.

8

(26) Zhao, L.; Dong, Y.; Chen, G.; Hu, Q., Extraction, purification, characterization

9

and antitumor activity of polysaccharides from Ganoderma lucidum. Carbohyd Polym.

10

2010, 80, 783-789.

11

(27) Manrique, G. D.; Lajolo, F. M., FT-IR spectroscopy as a tool for measuring

12

degree of methyl esterification in pectins isolated from ripening papaya fruit.

13

Postharvest Biol Tec. 2002, 25, 99-107.

14

(28) Kacurakova, M.; Capek, P.; Sasinkova, V.; Wellner, N.; Ebringerova, A., FT-IR

15

study of plant cell wall model compounds: pectic polysaccharides and hemicelluloses.

16

Carbohyd Polym. 2000, 43, 195-203.

17

(29) Jing, Y.; Huang, L.; Lv, W.; Tong, H.; Song, L.; Hu, X.; Yu, R., Structural

18

Characterization of a Novel Polysaccharide from Pulp Tissues of Litchi chinensis and

19

Its Immunomodulatory Activity. J Agr Food Chem. 2014, 62, 902-911.

20

(30) Schepetkin, I. A.; Xie, G.; Kirpotina, L. N.; Klein, R. A.; Jutila, M. A.; Quinn, M.

21

T., Macrophage immunomodulatory activity of polysaccharides isolated from Opuntia

22

polyacantha. Int Immunopharmacol. 2008, 8, 1455-1466.

23

(31) Chihara, G., Recent progress in immunopharmacology and therapeutic effects of

24

polysaccharides. Dev Biol Stand.1991, 77, 191-197.

25

(32) Wang, S. Y.; Hsu, M. L.; Hsu, H. C.; Lee, S. S.; Shiao, M. S.; Ho, C. K., The 23



1

anti‐tumor effect of Ganoderma Lucidum is mediated by cytokines released from

2

activated macrophages and T lymphocytes. Int J Cancer. 1997, 70, 699-705.

3

(33) Schepetkin, I. A.; Faulkner, C. L.; Nelson-Overton, L. K.; Wiley, J. A.; Quinn, M.

4

T., Macrophage immunomodulatory activity of polysaccharides isolated from

5

Juniperus scopolorum. Int Immunopharmacol. 2005, 5, 1783-1799.

6

(34) Yu, Q.; Nie, S.; Wang, J.; Yin, P.; Li, W.; Xue, M., Polysaccharide from

7

Ganoderma atrum induces tumor necrosis factor-α secretion via phosphoinositide

8

3-kinase/Akt, mitogen-activated protein kinase and nuclear factor-κB signaling

9

pathways in RAW264.7 cells. Int Immunopharmacol. 2012, 14, 362–368.

10

(35) Bohn, J. A.; BeMiller, J. N., (1→3)-β-D-Glucans as biological response

11

modifiers: a review of structure-functional activity relationships. Carbohyd Polym.

12

1995, 28, 3-14.

13

(36) Gantner, B. N.; Simmons, R. M.; Canavera, S. J.; Akira, S.; Underhill, D. M.,

14

Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor

15

2. J Exp Med. 2003, 197, 1107-1117.

16

(37) Thornton, B. P.; Vĕtvicka, V.; Pitman, M.; Goldman, R. C.; Ross, G. D.,

17

Analysis of the sugar specificity and molecular location of the beta-glucan-binding

18

lectin site of complement receptor type 3 (CD11b/CD18). J Immunol. 1996, 156,

19

1235-1246.

20

(38) Chen, Y.; Chiang, Y.; Hsu, F.; Tsai, L.; Cheng, H., Structural modeling and

21

further improvement in pH stability and activity of a highly-active xylanase from an

22

uncultured rumen fungus. Bioresour Technol. 2012, 123, 125-134.

23

(39) Kumar, C. G.; Joo, H.; Choi, J.; Koo, Y.; Chang, C., Purification and

24

characterization of an extracellular polysaccharide from haloalkalophilic Bacillus sp.

25

I-450. Enzyme Microb Tech. 2004, 34, 673-681. 24


Page 24 of 34

Page 25 of 34


1

(40) Martínez-Felipe, A.; Cook, A. G.; Wallage, M. J.; Imrie, C. T., Hydrogen

2

bonding and liquid crystallinity of low molar mass and polymeric mesogens

3

containing benzoic acids: a variable temperature Fourier transform infrared

4

spectroscopic study. Phase Transit. 2014, (ahead-of-print), 1-20.

5 6

25



Page 26 of 34

1

FIGURE LEGENDS

2

Figure 1. The chromatography of the polysaccharides from Dictyophora indusiata

3

(DP1) by (A) DEAE (diethylaminoethyl)-52 cellulose (DEAE-52), (B) Sephadex

4

G-200, (C) high-performance gel permeation chromatography (HPGPC), (D) Ion

5

exchange chromatography of monosaccharides mixture (D1) and DP1 sample (D2),

6

(E) Gas chromatography of monosaccharides mixture (E1) and DP1 sample (E2).

7

Figure 2. (A) Fourier transform infrared (FT-IR) spectrum, (B)

8

and (C) 1H NMR spectrum of the polysaccharides from Dictyophora indusiata (DP1).

9

Figure

3.

Effects

of

different

concentrations

of

13

C NMR spectrum

Dictyophora

indusiata

10

polysaccharides (DP1) on macrophage (A) Nitric oxide (NO), (B) Tumor necrosis

11

factor (TNF-α), and (C) Interleukin (IL-6) secretion in RAW 264.7 cells. The group

12

without adding DP1 was used as the control and lipopolysaccharide (LPS, 50 µg/mL)

13

was used as the positive control.

14

Figure 4. Roles of CR3, TLR2, TLR4 and Dectin-1 on DP1 (the polysaccharides of

15

Dictyophora indusiata)-induced (A) Nitric oxide (NO), (B) Tumor necrosis factor

16

TNF-α, and (C) Interleukin (IL-6) secretion in RAW 264.7 cells. The cells were

17

incubated with monoclonal antibodies (anti-TLR2, anti-CR3, anti-TLR4) for 2 h and

18

then washed with PBS 3 times before stimulating with DP1. For the dectin-1 group,

19

cells were incubated with laminarin (inhibitor of dectin-1) for 2 h before stimulating

20

with DP1 without washing with PBS. (D) the possible molecular mechanism of

21

DP1-induced macrophage immunomodulation.

22

Figure 5. Effects of different temperature treatment on DP1 (the polysaccharides of

23

Dictyophora indusiata)-induced (A) Nitric oxide (NO), (B) Tumor necrosis factor

24

(TNF-α), and (C) Interleukin (IL-6) secretion in RAW 264.7 cells. DP1 samples were

25

divided into three groups and treated at 100 °C, 121 °C and 145 °C for 30min, 26


Page 27 of 34


1

respectively. Then the cells were incubated with heat treatment DP1 samples for 24 h.

2

(D) The thermogravimetry- differential therogravimetry (TG-DTG) curves of DP1.

3

Figure 6. (A), (B) and (C), effects of different pH treatment on DP1 (the

4

polysaccharides of Dictyophora indusiata)-Induced (A) Nitric oxide (NO), (B) Tumor

5

necrosis factor (TNF-α), and (C) Interleukin (IL-6) secretion in RAW 264.7 cells.

6

Cells were incubated with DP1 at different pH of phosphate buffers for 24 h. The cells

7

of control group were incubated at different pH of phosphate buffers (without DP1)

8

for 24 h. (D), (E) and (F), co-effects of heat (121°C) and different pH treatment on

9

DP1-induced (D) Nitric oxide (NO), (E) Tumor necrosis factor (TNF-α), and (F)

10

Interleukin (IL-6) secretion in RAW 264.7 cells. Cells were incubated with heat

11

treatment (121°C) DP1 at different pH of phosphate buffers for 24 h, versus the

12

control groups (without DP1).

13

27



2.0

B

1.8

Concentration of NaCl (mol/L)

1.6

0.4

1.4 1.2

DP2

1.0

0.3

0.8

0.2

0.6 0.4

0.1

0.2

C

1.0 0.8

0.6 0.4 0.2 0.0

0.0

0.0

0

10

20

300

60

D2

N o rm .

40 35 30

40

E2

25

20

20

40 35

glucose

20

mannos e

45

m in 1 5

25

30

E1 galactose

40

D1

10

xylos e

N orm .

Time (min)

5

16

arabinose

14

Mannose

60

12

Xylose

80

10

Glucose

100

Galactose

Rhamnose

8

rhamnose

0

6

phy cite

0 120

60

45

Mannose

80

50

E

Glucose

100

Galactose

The peak height (nC)

D

40

mannos e

Tube number

120

30

Tube number

glucose

250

phycit e

200

gly col

150

glycerol

100

gly col

50

glyc erol

0

The peak height (nC)

Absorbance (490nm)

1.2

0.5

DP1

Absorbance (490nm)

A

Page 28 of 34

30

20

25

0 6

8

10

12

14

20

16

0

Time (min)

Figure 1 28


5

10

mi n

15

20

25

30

Page 29 of 34


DP1

A

50 40

20 4000

3500

3000

2500

2000

629.53 572.39

890.28 796.81

1547.68

30

1500

1202.53 1074.32 1040.25

60

1374.28

2926.33

1727.93 1648.01

70

1425.46 1319.07 1253.63

80

3335.69

Transmittance (%)

90

2131.46

2348.73

100

1000

Wavenumber (cm-1)

B

C

Figure 2 29


500


35

A Concentration of NO (µmol/L)

a 30

25

20

b 15

bc c

10

d 5

e 0

control

125

250

500

1000

LPS

µg/mL 1000

a

Concentration of TNF-a (pg/mL)

B 800

600

d

cd

125

250

bc

b

500

1000

e 400

200

0

control

LPS

µg/mL

C Concentration of IL-6 (pg/mL)

2000

a b

1500

cd bc

d 1000

500

e 0

control

125

250

500

1000

µg/mL

Figure 3

30


LPS

Page 30 of 34

Page 31 of 34

Journal of Agricultural and Food Chemistry 25

A Concentration of NO (µmol/L)

a 20

15

e

c

ce

e

d 10

b

5

f

-

-

LP S

DP1 (500 µg/mL)

an tiTL R2 an tiTL R4 an tiCR 3 La m in ar in La m in ar in

0

1400

+

+

+ -

-

C

a 1200

a

c e

1000

Concentration of IL-6 (pg/mL)

e

e d

800

b

600

f

200

800

c e

ef

f

600

d 400

200

bg

g 0

D

Figure 4 31


S

in m

in

ar

in ar m in

CR 3

La

4

ti-

-

+

+

La

+

+

+ -

an

-

LR

DP1 (500 µg/mL)

2

-

tiT

-

an

+

LR

+

tiT

+

an

+

+ -

LP S

-

an tiTL R2 an tiTL R4 an tiCR 3 La m in ar in La m in ar in

DP1 (500 µg/mL)

0

LP


+

1000

B

400

+


A

a

1200

B

40

Page 32 of 34

a

30

d

d

25 20

c 15 10

1000


b

5

e

c 600

b 400

200

b 0

untreated

100?

121?

145?

control

2000

a e

untreated

LPS

100?

121?

145?

control

LPS 2

110

D

100

e

DTG

0

90

1500

-2

80

W eight (% )


d

800

0

C

e

d 1000

c

70

-4

60 -6

50 40

500

-8

30 20

b

10

0

untreated

100?

121?

145?

control

-10

TG

LPS

-12

0

Figure 5

0

100

32


200

300

400

Temperature (°C)

500

600

D erivative weight (% m in -1 )

Concentration of NO (µmol/L)

35

A


DP1 control

25

900

B

800

20

c 15

d e

10

A

f 5

bf

C

C BD



a

DP1 control

B

a

700 600

b b

B

AB e 300

A AB

B

400

C

200

0

0

pH2.0

pH4.0

pH6.0

pH10.0

pH8.0

c

1000

100

E

pH6.0

pH4.0

pH8.0

pH10.0

600

d

d

400

200

PBS

pH4.0

pH2.0

a


cd bd

15

b

10

e 5

A

C D

D

CD

800

a

700 600

b b AB

B

e 300

A AB

B

400

C

200 100

B

0

0

pH2.0

pH4.0

pH6.0

pH8.0

500µg/mL

pH10.0

PBS

pH4.0

pH6.0

pH10.0

PBS

pH8.0

pH10.0

PBS

500µg/mL

c

800

b

b 600

d 400

200

0

pH2.0

pH8.0

A

ac

1000

c

500

A

a

DP1 control

1200

a


c

20


D

F

DP1 control

pH6.0

500µg/mL

900

E

A

A

A

A

500µg/mL

DP1 control

b

b 800

0

pH2.0

PBS

a

1200

c

500

DP1 control

1400

C

a


Page 33 of 34

D

pH2.0

C

pH4.0

BC

pH6.0 500µg/mL

Figure 6

33


AB

pH8.0

B

pH10.0

A

PBS


1

Table of Contents Graphic

2

34


Page 34 of 34

Structure Characterization of a Novel Polysaccharide from

Recommend Documents