Structural characterization and immune-enhancing activity of a novel high-molecular-weight polysaccharide from Cordyceps militaris

Journal Pre-proof Structural characterization and immune-enhancing activity of a novel high-molecular-weight polysaccharide from Cordyceps militaris

Bao-Lin He, Qian-Wang Zheng, Li-Qiong Guo, Jen-Yi Huang, Fan Yun, Shi-Shi Huang, Jun-Fang Lin PII:

S0141-8130(19)37304-0

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.115

Reference:

BIOMAC 14142

To appear in:

International Journal of Biological Macromolecules

Received date:

10 September 2019

Revised date:

4 December 2019

Accepted date:

14 December 2019

Please cite this article as: B.-L. He, Q.-W. Zheng, L.-Q. Guo, et al., Structural characterization and immune-enhancing activity of a novel high-molecular-weight polysaccharide from Cordyceps militaris, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.12.115

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© 2019 Published by Elsevier.

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Structural characterization and immune-enhancing activity of a novel high-molecular-weight polysaccharide from Cordyceps militaris Bao-Lin Hea,b,1, Qian-Wang Zhenga,b,1,*, Li-Qiong Guoa,b, Jen-Yi Huangc, Fan Yund, Shi-Shi Huanga,b, Jun-Fang Lin a,b,*

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College of Food Science & Institute of Food Biotechnology, South China Agriculture

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a

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University, Guangzhou 510640, China b

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Research Center for Micro-Ecological Agent Engineering and Technology of

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Guangdong Province, Guangzhou 510640, China c

d

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Lafayette, IN 47907, USA

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Department of Food Science, Purdue University, 745 Agriculture Mall Drive, West

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Guangzhou Alchemy Biotechnology Co., Ltd, 139 Hongming Road, Guangzhou

Economic Technology Zone, Guangzhou City, 510760, China

* Corresponding authors Qian-Wang Zheng (Q.W. Zheng) and Jun-Fang Lin (J.F. Lin).

E-mail addresses: [email protected] (Q.W. Zheng) and [email protected] (J.F. Lin). Tel.: +862087570302; Fax: +862085280270.

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Co-first authors Bao-Lin He and Qian-Wang Zheng contributed equally to this work.

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1

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Abstract A novel homogeneous polysaccharide (CMP-Ⅲ) was extracted and purified from C. militaris. Structural characterization revealed that CMP-Ⅲ had an average molecular weight of 4.796 × 104 kDa and consisted of glucose, mannose and galactose with the molar ratio of 8.09:1.00:0.25. The main linkage types of CMP-Ⅲ consisted of

1→2,6)-α-D-Gal

(3.93%)

based

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1→4)-α-D-Glc (70.08%), 1→4,6)-α-D-Man (9.59%), 1→)-α-D-Man (10.79%) and

on

methylation

and

NMR

analysis.

The

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immunomodulatory assay indicated that CMP-Ⅲ significantly promoted macrophage

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phagocytosis and secretion of NO, TNF-α and IL-6. Further study suggested that

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macrophage activated by CMP-Ⅲ involved mitogen-activated protein kinases (MAPKs) and nuclear factor kappa-B (NF-κB) signaling pathways. Overall, these

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results suggested that CMP-Ⅲ could be developed as a potent immunomodulatory

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agent for use in functional foods and dietary supplements.

Key words: Cordyceps militaris; high-molecular-weight polysaccharide; structural characterization; immune-enhancing activity

1. Introduction

Cordyceps militaris, that belongs to the class Ascomycetes, is a well-known traditional Chinese edible and medicinal fungus [1]. It has been used extensively as a restorative drug and folk tonic food in China and other Asian countries, because it contains many active components such as polysaccharides, cordycepin, cordycepic

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acid and carotenoids [2,3]. Among them, polysaccharides are one of the main pharmacological components in C. militaris that has been identified to possess antitumor [4], immunomodulation [5], anti-oxidation [6] and anti-hypolipidemic [7] effects.

Although structural features and pharmacological activities of C. militaris

molecular-weight

polysaccharides.

The

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f

polysaccharides have been studied, the majority of these studies focused on lowmolecular

weights

of

C.

militaris

exhibited

immuno-modulating

activity

[10,11].

The

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polysaccharides

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polysaccharides range from 1.273×103 to 4.36×104 Da [8,9], and these

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high-molecular-weight polysaccharides extracted from C. sinensis, another species belonging to the Cordyceps genus with a high similarity to C. militaris, have been

cell

growth

[12–14]. However,

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tumor

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identified and confirmed to be effective in regulating high-fat diet and inhibiting there

is

still

no

study on the

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high-molecular-weight fraction of C. militaris polysaccharides. In this study, a novel high-molecular-weight polysaccharide named CMP-Ⅲ, was extracted and purified from C. militaris. The physicochemical properties and structural conformation of CMP-Ⅲ were characterized by optical and chemical analyses. Then, its immuno-modulatory effect was investigated using RAW 264.7 cells, and the underlying molecular mechanisms were explored through MAPKs and NF-κB signaling pathways. The ultimate goal of this research is to provide scientific insights into the relationship between the structure and function of C. militaris polysaccharides with respect to human health.

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2. Materials and methods

2.1. Materials and chemicals

The cultured fruiting bodies of C. militaris were obtained from Jiangmen Honghao Biotechnology Co., Ltd. (Guangdong, China) and Liaoning Hongqiao

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Biotechnology Co., Ltd. (Shenyang, China). DEAE-52 and Sephadex G-200 were

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purchased from Solarbio Co. (Beijing, China). Dulbecco’s modified Eagle’s medium

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(DMEM), fetal bovine serum (FBS), streptomycin and penicillin were purchased from Gibco Co. (Grand Island, NY, USA). NO detecting kit was purchased from Nanjing

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Jiancheng Institute of Biotechnology (Nanjing, China). E.Z.N.A. Total RNA Kit Ⅰ was

al

purchased from OMEGA Bio-Tek (Norcross, GA, USA), Fast King RT Kit (With

(Beijing,

China).

Anti-phospho-NF-κB

p65,

anti-NF-κB

p65,

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biotech Co.

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gDNase) and Talent qPCR PreMix (SYBR Green) were obtained from TIANGEN

anti-phospho-IKBα, anti-ERK (ERK), anti-phospho-ERK (p-ERK), antil-JNK (JNK), anti-phospho-JNK (p-JNK), anti-p38 (p38), and antiphospho-p38 (p-p38) primary antibodies (rabbit monoclonal antibodies) were provided by Cell Signaling Technology (Beverly, MA, U.S.A.). All other chemicals and reagents were analytical grade.

2.2. Extraction, isolation and purification of CMP-Ⅲ

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The dried fruiting bodies of C. militaris were pulverized and passed through a 60 mesh sieve. The powder was defatted with 95% ethanol for 24 h, subsequently extracted three times with ultrasonic-assisted hot water (90ºC) at the ratio of 1:20 (w/v) for 2 h each time. The mixture was centrifuged at 5000 × g for 10 min to remove the residue. The supernatant was collected and concentrated by a vacuum rotary

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evaporator (IKA RV8, IKA Instrument Equipment Co. Ltd., Guangzhou, China) at

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55ºC, then precipitated by adding 4 times the volume of 95% ethanol and stored at

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4ºC for 12 h. Brown precipitate was collected by centrifugation (Eppendorf 5810R,

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Germany) at 8000 × g for 10 min and washed twice with absolute ethanol. The

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resulting sample was decolored and deproteinized simultaneously with NKA-9 macroporous resins [15], then dialyzed (3500 Da, 48 h) and lyophilized to obtain C.

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militaris polysaccharide (CMP).

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The CMP (500 mg) was dissolved in 10 mL of ultrapure water and loaded onto a

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pre-equilibrated DEAE-52 column (2.6 cm × 70 cm). The column was eluted with distilled water, 0.1, 0.3 and 0.5 M NaCl solutions at 0.5 mL/min, respectively. The eluents were collected into centrifuge tubes (10 mL/tube) and monitored for polysaccharide content using the phenol-sulfuric acid method [16]. Samples showing the same elution peak were combined, concentrated, dialyzed and lyophilized, here named Fraction Ⅰ (distilled water), Ⅱ and Ⅲ (0.1 and 0.3 M NaCl). About 50 mg of each fraction was dissolved in 4 mL distilled water and loaded onto a Sephadex column (2.6 cm × 80 cm) for further purification. The sample was eluted with ultrapure water at 0.25 mL/min. The eluate (5 mL/tube) was collected and detected as

Journal Pre-proof described above. The main fraction (CMP-Ⅰ, Ⅱ and Ⅲ) was collected, concentrated, dialyzed and lyophilized for the subsequent assays. In this study, focus was put on the structure characterization and

immunomodulatory activity analysis

of the

polysaccharide fraction CMP-Ⅲ.

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2.3. Structure characterization of CMP-Ⅲ

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2.3.1 General physicochemical properties of CMP-Ⅲ

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The total carbohydrate content of CMP-Ⅲ was determined by the

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phenol-sulfuric acid method using glucose as the standard [16]. Total uronic acid content was determined by the m-hydroxydiphenyl method described by

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Blumenkrantz and Asboe-Hansen [17] with glucuronic acid as the standard. The

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protein content was determined by the Bradford’s method [18] using bovine serum

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albumin as the standard. The total phenolic content was determined by the Folin-Ciocalteu assay using gallic acid as the reference [19]. The UV-vis spectra of the CMP-Ⅲ (1.0 mg/mL) were recorded using a spectrophotometer (UV-4802S, unico, Shanghai) in the wavelength range of 190−800 nm at room temperature.

2.3.2 Monosaccharide composition analysis CMP-Ⅲ (10 mg) was hydrolyzed with 4 mL of trifluoroacetic acid (2 M) at 110ºC for 6 h. The acetylation of CMP-Ⅲ was carried out by reacting with 10 mg of

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hydroxylamine hydrochloride and 0.5 mL of pyridine at 90ºC for 30 min. After cooling, 1 mL of acetic anhydride was added to the sample for reaction for another 30 min at 90ºC [20].

The acetate derivatives were analyzed by gas chromatography (GC) using a Shimadzu GCMS-QP2010 Ultra instrument equipped with a DB-1701 capillary

oo

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column (30 m × 0.32 mm × 0.25 µm) and a flame ionization detector (FID). The initial column temperature was set at 70ºC and maintained for 2 min, then linearly

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increased to 180ºC at 20ºC /min, before further increased to 250ºC at 3ºC /min and

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held at 250ºC for 20 min. The temperatures of both injector and detector were set at

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250ºC. A set of monosaccharides (rhamnose, arabinose, fucose, xylose, mannose, glucose, and galactose) were used as the standards, and 1.0 mg/mL of inositol was

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used as the internal standard.

2.3.3 Molecular weight determination and chain conformation analysis

The weight average molecular weight (Mw), number average molecular weight (Mn), and PDI (Mw/Mn), radius of gyration z1/2 of CMP-Ⅲ in 0.02 M KH2PO4 aqueous

solution

were

determined

using

high-performance

size-exclusion

chromatography combined with multi-angle laser light scattering (SEC-MALLS, Wyatt Technology, USA). The system consisted of an Agilent 1260 HPLC system (Agilent, USA), an Optilab TREX refractive index detector (RID, Wyatt Technology Co., USA), a Shodex OHpak SB-806M HQ column (8 mm×300 mm, Showa Denko

Journal Pre-proof K.K., Tokyo, Japan), and a multi-angle laser light scattering instrument (λ = 658, Dawn Heleos, Wyatt Technology Co., USA).

The mobile phase was 0.02 M K2HPO4 aqueous solution at a flow rate of 0.6 mL/min. Moreover, CMP-Ⅲ was dissolved in the same solvent at 1.0 mg/mL and filtered through 0.22 μm membrane (Millipore, USA) prior to injection. The injection

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volume was 100 μL. The specific refractive index increment (dn/dc) value used to calculate the molecular weight was 0.138 mL/g. The online ASTRA 6.1 software

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package (Wyatt Technology Co., USA) was used for data collection and analysis.

2.3.4 FT-IR analysis

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CMP-Ⅲ (1 mg) was mixed with KBr powder (100 mg) and then pressed into

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pellets for infrared spectral analysis within a range of 4,000−400 cm−1. The FT-IR

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spectrum of the polysaccharide was measured by a spectrophotometer (VERTEX 70, Bruker, Germany).

2.3.5 Methylation analysis

The glycosyl linkage analysis was carried out by methylation of CMP-Ⅲ followed by hydrolysis, reduction and acetylation as described in the previous study [21].

The

partially

methylated

alditol

acetates

were

analyzed

by

gas

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chromatography-mass spectrometry (GC-MS) using a GCMS-QP2010 Ultra instrument equipped with a DB-1701 capillary column (30 m × 0.32 mm × 0.25 µm) and a flame-ionization detector (FID). The injection port temperature was 250ºC. The initial column temperature was set at 150ºC and held for 2 min, then increased to 180ºC at a heating rate of 10ºC /min and held for 2 min before further increased to

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µL. The mobile phase flow rate was 1 mL/min.

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260ºC at a heating rate of 15ºC /min and held for 5 min. The injection volume was 1

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2.3.6 NMR spectroscopy analysis

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The freeze-dried CMP-Ⅲ (100 mg) was dissolved in 2 mL of 99.9% D2O and 13

C

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lyophilized three times for replacing exchangeable protons. The 1H NMR and

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NMR spectra were recorded using a Bruker-600 MHz NMR spectrometer (Bruker,

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Rheinsteten, Germany) at 25ºC. Data was obtained and analyzed using the standard Bruker NMR software.

2.4 Immunomodulatory activity assay

2.4.1. Cell culture

The RAW 264.7 cell line was purchased from Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) FBS, 100 unit/mL penicillin

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and 100 µg/mL streptomycin at 37ºC in a humidified 5% CO2 incubator (CCL-1708-8, ESCO, SG). The cells were cultured for 36−48 h to reach the logarithmic phase and then used for experiments.

2.4.2. Cytotoxicity assay

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RAW 264.7 cells were cultured in 96-well microplates at a density of 5 × 103

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cells/well (100 µL/well) overnight, and then cells were treated with CMP-Ⅲ (0, 25,

e-

50, 100 and 200 µg/mL) and LPS (2 µg/mL, positive control) for 24 h, respectively.

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Subsequently, 10 μL of MTT (5 mg/mL) were added to each well and the cells were incubated for another 4 h at 37ºC. The supernatant was discarded and the formazan

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crystals presented in cells were dissolved by 150 µL of DMSO. The absorbance at 570

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nm was measured using a microplate reader (Biotek, USA).

2.4.3. Phagocytosis assay

RAW 264.7 cells were cultured in 96-well microplates at a density of 5 × 105 cells/well (100 µL/well) overnight, and then cells were treated with CMP-Ⅲ (0, 25, 50, 100 and 200 µg/mL) and LPS (2 µg/mL, positive control) for 24 h, respectively. Then the supernatant was removed, and 100 µL of 0.1% neutral red solution (dissolved in phosphate buffer solution) were added to each well and incubated for another 1 h at 37ºC. The cells were washed with PBS for three times, then 100 µL of

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the cell lysis buffer (ethanol: glacial acetic acid = 1:1) were added to each well. Cell culture plate was statically placed overnight at 4ºC. The absorbance at 570 nm was measured using the microplate reader.

2.4.4. Nitric oxide and cytokines assay

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RAW 264.7 cells were cultured in 96-well microplates at a density of 5 × 105

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cells/well (100 µL/well) overnight, and then cells were treated with CMP-Ⅲ (0, 25,

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50, 100 and 200 µg/mL) and LPS (2 µg/mL, positive control) for 24 h, respectively.

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The culture medium containing NO and cytokines secreted from active RAW 264.7 macrophages was collected for further analyses. The levels of NO and cytokines were

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measured using the nitric oxide assay kit, TNF-α and IL-6 ELISA kits, respectively.

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tests.

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All pre-experiments were done to confirm that the dilution rate was appropriate for

2.4.5. RT-qPCR analysis

RAW 264.7 cells were cultured in 6-well plates at a density of 106 cells/well (2 mL/well) overnight, then cells were treated with CMP-Ⅲ (0, 25, 50, 100 and 200 µg/mL) and LPS (2 µg/mL, positive control) for 24 h, respectively. The cells were washed by cold PBS and the total RNA was extracted by E.Z.N.A. Total RNA Kit Ⅰ (OMEGA, USA) based on the manufactures' procedures. The isolated RNA was used

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for cDNA synthesis with reverse transcriptase. Finally, the levels of cDNA encoding induced NO synthase (iNOS), TNF-α and IL-6 were measured using a real-time quantitative PCR instrument (Bio-Rad, CA, USA) with GAPDH as the internal reference. The sequences of the specific primers were displayed in Table 1. The mRNA expression levels were expressed as the ratio of optimal density to GAPDH by

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calculating ΔΔCt, and then analyzed using 2 -ΔΔCt method.

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2.4.6. Western blot analysis

RAW 264.7 cells were cultured in 6-well plates at a density of 106 cells/well (2

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mL/well) overnight, then cells were treated with CMP-Ⅲ (0, 25, 50, 100 and 200

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µg/mL) and LPS (2 µg/mL, positive control) for 30 min, respectively. The cells were

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rinsed twice with cold PBS, then collected into 150 μL of RIPA lysis buffer containing

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1% protease inhibitor (Meilun Biotech, Dalian, China) to extract total proteins. Nuclear proteins were extracted using the nuclear protein isolation kits (Meilun Biotech, Dalian, China). The protein content was measured with the BCA protein assay kit (Takara Biomedical Tech, Beijing, China). The proteins (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore, USA). The membrane was immediately sealed with 5% non-fat dry milk in TBST for 1 h at room temperature, and hybridized with primary antibodies in TBST containing 5% BSA overnight at 4ºC. After rinsing three times with TBST for 5 min, the membrane was

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incubated with 1:1000 goat anti-rabbit IgG-HRP for 1 h at room temperature. The bands were finally detected using the ECL Detection Kit and photographed using the Amersham Imager 600 (GE Healthcare, USA). GAPDH was used to normalize the relative band intensity. The optical densities of bands were detected and quantified

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using the Image J soft-ware (National Institute of Health, USA).

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2.5. Statistical analysis

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The data obtained from at least three separate experiments, was presented as means ± standard deviation. The significance of difference between CMP-Ⅲ and LPS

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was analyzed using Student’s t-test, and multi-group comparisons were analyzed by

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one-way analysis of variance (ANOVA) using the statistical analysis software SPSS

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11.5. The result showing p < 0.05 was considered significantly different, and p < 0.01

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was accepted as highly significant difference.

3. Results and discussion

3.1. Extraction, purification and characterization

The CMP was isolated from the dried fruiting bodies of C. militaris with a yield of 4.83 ± 0.18% (w/w). After the preliminary separation by the DEAE-52 column, three fractions with the recovery rates of 23.18%, 9.54% and 12.27% were obtained (Fig. 1A). These fractions were further purified through a Sephadex column to collect

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the more homogeneous polysaccharide. The HPGPC analysis indicated that the molecular weights of CMP-Ⅰ and CMP-Ⅱ were similar to the C. militaris polysaccharide reported by Zhu et al., [22] and Yu et al., [23]. Therefore, we mainly focused on the CMP-Ⅲ in this study. Fig. 1B shows that CMP-Ⅲ appeared as a single and symmetrical peak with a yield of 0.19% after being further purified by the

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Sephadex G-200 column chromatography.

The total sugar, protein, uronic acid and total polyphenol contents of CMP-Ⅲ

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were 89.47%, 0.895%, 2.628% and 0.815%, respectively. As shown in Fig. 1C, there

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was no absorption at 260 or 280 nm in the UV spectrum of CMP-Ⅲ, indicating the

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absence of nucleic acids, proteins, peptides and other impurities. The lyophilized CMP-Ⅲ was white solid like cotton fiber (Fig. 1D) and the iodine-potassium iodide

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reaction showed that CMP-Ⅲ is a non-starch polysaccharide (results not shown).

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These results suggested that the CMP-Ⅲ obtained had a high purity and was suitable

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for structure analysis and immunomodulatory activity study.

3.2. Structural characterization

3.2.1. Monosaccharide composition, molecular weight determination and chain conformation analysis Fig. 2A shows the GC results that CMP-Ⅲ was composed of mannose, glucose and galactose in the molar ratio of 1.00:8.09:0.25.

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As shown in Fig. 2B, a single symmetrical peak with the PDI value of 1.60 revealed that CMP-Ⅲ was homogeneous and the molecular weight distribution was narrow. The Mw value of CMP-Ⅲ in 0.02 M KH2PO4 aqueous solution was calculated to be 4.796 × 104 kDa (Table 2). The conformation of CMP-Ⅲ can be analyzed based on the theory of dilute

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polymer solutions, which suggests that the α values of 0.3 for sphere-like, 0.5−0.6 for

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flexible random coil, and 0.6−1.0 for rigid rod conformation, respectively [24].

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According to the MALLS data (Fig. 2B-3 and Table 2), the slope of the log (RMS

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radius)/log (Mw) curve was 0.15 in 0.02 M KH2PO4 aqueous solution, indicating that

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CMP-Ⅲ existed both in a compact coil conformation in the aqueous solution as a

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result of its relatively high branching.

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3.2.2. FT-IR spectroscopy

The IR spectrum of CMP-Ⅲ is shown in Fig. 2C. The strong and broad absorption peak centered at 3392.8 cm−1 arose from O-H stretching vibrations of hydroxyl groups because of intramolecular or intermolecular. The weak band at 2930.6 cm-1 was due to C–H stretching vibrations of -CH3 or -CH2 groups [25]. The band in the region of 1645.9 cm−1 corresponded to the associated water. The absorption band at 1415.6 cm−1 was the characteristic absorption peak caused by the stretching and bending vibrations of C-H or O-H bonds. There were typical absorption peaks of polysaccharides. The bands at 1155.0 cm−1, 1080.6 cm−1 and

Journal Pre-proof 1023.7 cm−1 were the characteristic absorptions of the pyranose ring [26], and the absorption bands at about 1080.6 cm−1 and 1023.7 cm−1 were mainly associated with C-O-C stretching vibrations and C-O-H bending vibrations. The characteristic absorptions at 933.7 cm−1, 857.2 cm−1 and 762.9 cm−1 suggested that CMP-Ⅲ is a D-glucopyranose derivative [27]. Therefore, it can be concluded that CMP-Ⅲ belongs

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to the α-type glucans with a pyran group.

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3.2.3. Methylation analysis

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To measure the sugar linkage type, CMP-Ⅲ was completely methylated, hydrolyzed and converted to alditol acetates before GC-MS analysis. Based on the

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retention time and standard data in the CCRC Spectral Database for PMAA’s, the

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results of methylation analysis were summarized in Table 3. The peaks were

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confirmed as T-Manp, 1,4-Glcp, 1,4,6-Manp and 1,2,6-Galp, with molar ratio of 10.79: 70.08: 9.59: 3.93.

The results in Table 3 demonstrated that the amount of 1,4-Glcp residues was 70.08%, indicating that they might form the backbone of CMP-III, which agreed with the monosaccharide composition analysis (Fig. 2A). According to the contents of branching, terminal and linear residues, the degree of branching (DB) of CMP-III was calculated as 0.258. Based on the proportion of branch and the possibility of connection, it is speculated that multiple types of linkage exist in the backbone of CMP-III. For example, the branch point may appear at the O-6 position of 1,4,6-Manp,

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or the O-6 position of 1,2,6-Galp. Furthermore, the non-reducing terminal residue was T-Manp, accounting for 10.79%.

3.2.4. NMR spectroscopy analysis The 1H and 13C NMR spectrum of CMP-III are shown in Fig. 3 and the signals in

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the spectra were analyzed according to the previous studies [28–31]. The anomeric

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proton signals (δ 5.32, 5.12, 5.16 and 5.09) and the anomeric carbon signals (δ 106.

residues

→4)-α-D-Glcp-(1→,

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13, 100.64, 100.63 and 99.82) corresponded with H-1 and C-1 of four anomeric →4,6)-α-D-Manp-(1→,

α-D-Manp-(→1 1

13

C

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NMR chemical shifts of CMP-III.

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→2,6)-α-D-Galp-(1→. Table 4 gives the information about assignment of the H and

and

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3.3. Immunostimulatory activities

3.3.1. Macrophage activation The cytotoxic effect of CMP-Ⅲ on RAW 264.7 cells was analyzed by the MTT assay. CMP-Ⅲ did not show any cytotoxicity up to 200 μg/mL in vitro (Fig. 4A). Thus, concentrations of 25, 50, 100 and 200 μg/mL were set as testing concentrations for the following analyses.

Macrophage activation was determined by phagocytosis activity, NO releasing and cytokines (IL-6 and TNF-α) secretion. As shown in the supporting information

Journal Pre-proof (Fig. 4B), compared to the control group, CMP-Ⅲ significantly (p < 0.01) enhanced the phagocytic uptake of the cells in the concentration range of 25−200 μg/mL, and the phagocytic capacity of CMP-Ⅲ was slightly higher than that of LPS (2 μg/mL). At the concentration of 100 μg/mL, the phagocytic uptake of the cells reached the maximum.

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As shown in Figs. 5A, B and C, the releases of IL-6 and TNF-α were

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significantly stimulated by CMP-Ⅲ of 25−200 μg/mL in a clearly dose-dependent

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manner (p < 0.01). CMP-Ⅲ stimulated the production of NO only at high

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concentrations (100 and 200 μg/mL), which was comparable to the LPS group.

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To confirm the activation of macrophages by CMP-Ⅲ, quantitative RT-PCR

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analysis was further employed to determine the mRNA levels of iNOS, IL-6 and

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TNF-α in RAW 264.7 cells. It has been reported that the iNOS is a critical controller responsible for a major amount of NO synthesized in macrophages [32]. As shown in

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Figs. 5D, E and F, the cells treated with CMP-Ⅲ showed a significant increase in the mRNA levels of iNOS, IL-6 and TNF-α compared to the control group. These findings were consistent with the results of cytokine secretion in the gene expression level, and further supported the conclusion that macrophages can be activated by CMP-Ⅲ in the tested concentration range.

3.3.3. MAPKs and NF-κB signaling pathways in RAW 264.7 cells

The defensive mechanisms of macrophages can be activated when various

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stimulus molecules bind to the pattern recognition receptors (PRRs) on the surface of macrophages and trigger several different signaling pathways including MAPKs and NF-κB. Western blot analysis was conducted to detect the expression of target proteins.

MAPKs play critical roles in the regulation of cell proliferation, differentiation,

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growth, migration and apoptosis as well as in cellular responses to cytokines and

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stresses. As illustrated in Fig. 6A, CMP-Ⅲ increased the expression of p-ERK1/2,

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p-JNK1/2 and p-p38 proteins of RAW 264.7 cells in a dose-dependent manner.

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Moreover, according to the values of “p-ERK1/2/total ERK 1/2”, “p-JNK1/2/total

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JNK1/2” and “p-p38/total p38”, the degrees of phosphorylation of ERK1/2, JNK1/2

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and p38 proteins significantly increased (p < 0.05) than the control group.

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NF-κB is formed by hetero- or homodimerization of proteins in the Rel family, including p65 and p50. Inactive NF-κB forms a complex with the inhibitor of κB (IκB)

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and exists in a latent form in the cytosol [33]. Upon activation by upstream signals, IκB-α phosphorylation at specific amino-terminal serine residues and degraded, thereby initiating NF-κB phosphorylation, leading to NF-κB translocation into the nucleus and then regulate the expression of a variety of genes [34]. Therefore, Western blotting was performed to examined the effect of CMP-Ⅲ on the phosphorylation of NF-κB p65, IκB-α and degradation of IκB-α. As shown in Fig. 6B, the level of total IκB-α remained nearly degraded upon CMP-Ⅲ treatment, whereas the phosphorylation of IκB-α (p-IκB-α) was significantly enhanced compared to that of the untreated cells (p < 0.01). We also found that the levels of NF-κB p65

Journal Pre-proof phosphorylation markedly increased for the CMP-Ⅲ treated groups (p < 0.05), however the total of IκBα did not decrease. Furthermore, the nuclear level of NF-κB p65 protein increased after the cells were treated with CMP-Ⅲ. Accordingly, the cytoplasmic level of p65 protein decreased with the CMP-Ⅲ concentration, suggesting the fact that CMP-Ⅲ stimulated the translocation of NF-κB p65 from cytoplasm to nucleus in a dose-dependent manner. Thus, the findings indicated that

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the NF-κB signaling pathway was indeed involved in the CMP-Ⅲ mediated activation

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of macrophages.

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Based on the results of increased secretion of NO, IL-6 and TNF-α in RAW

Pr

264.7 cells via the activation of MAPKs and NF-κB signaling pathways (Fig. 6C), we

rn

al

identified the potential of CMP-Ⅲ as an immunomodulatory agent.

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3.4. Structure-activity relationship

Many factors are involved in the biological activity of polysaccharides, including molecular weight, glycosidic bonds and solution property [35]. In this study, the novel high-molecular-weight polysaccharide (4.796 × 107 Da) was isolated from C. militaris. The high-molecular-weight polysaccharide with Mw higher than 106 Da have exhibited great bioactivity. For example, a homogeneous polysaccharide RPS-1 (1.617 × 107 Da) was extracted from liquid-cultured mycelia of Rhizopus nigricans by Wei et al. [36], which showed potent immunomodulatory activity. In addition, He et al. [37] reported that a novel arabinogalactan (MOP-1)

Journal Pre-proof isolated from leaves of Moringa oleifera with a molecular weight of 7.65 × 107 Da had significant antioxidant activity in vitro. These studies also demonstrated that polysaccharides with high molecular weights may have brilliant bioactivity, however, opposite findings have also been reported. Kakutani et al. [38] found that the glycogens with a molecular weight of more than 107 Da hardly activated macrophage

f

RAW 264.7 cells and suggested that the macrophage-stimulating activity of glycogen

oo

is strictly related to its molecular weight rather than structural properties.

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Furthermore, glycosidic bond is an important factor for the biological activities

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of polysaccharide. In this study, CMP-Ⅲ was proved with an α-D-(1→4)-Glcp

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backbone structure, and it had a similar chain linkage to several polysaccharides from parasitic Cordyceps fungi, such as Cordyceps gunnii [13] and Cordyceps sinensis [14].

al

The main chain of these polysaccharides is primarily composed of α-(1→4) glucose,

rn

and they exhibit strong immuno-stimulatory or anti-tumor activity. Therefore, we

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speculate that the stronger immune-modulatory activity of these polysaccharides may be related to the presence of α-1,4 glucosidic bond in their structures, which agree with the previous studies [36,39].

Solution properties, such as solubility, viscosity and chain conformation of polysaccharide in solvent, also have an important effect on polysaccharide bioactivities [40]. For example, solutions with high viscosity can impede the diffusion and absorption of polysaccharide samples [41]. In this study, CMP-Ⅲ had relatively high solubility and low viscosity in water, which were beneficial for its immune-modulatory activity.

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4. Conclusions

In summary, the present study is the first systematic characterization of the structure and bioactivities of a homogeneous high-molecular-weight C. militaris polysaccharide. This bioactive water-soluble polysaccharide, named CMP-Ⅲ, was

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f

composed of mannose, glucose and galactose in the molar ratio of 1.00: 8.09: 0.25. The main linkage types of CMP-Ⅲ consisted of 1→4)-α-D-Glc, 1→4,6)-α-D-Man,

pr

1→)-α-D-Man and 1→2,6)-α-D-Gal based on methylation and NMR analysis. In addition,

e-

the analyses of immunomodulatory functions revealed that CMP-Ⅲ was able to

Pr

increase macrophage phagocytosis and secretion of NO, TNF-α and IL-6. Further

al

study suggested that CMP-Ⅲ may function through activating the MAPKs and NF-κB

rn

signaling pathways. These findings implied that CMP-Ⅲ could be used as a natural

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agent in immunoregulation functional food.

Acknowledgments

This research was supported by the State key research and development plan “modern food processing and food storage and transportation technology and equipment”, (Grant No. 2017YFD0400204-3), the National Natural Science Foundation of China (Grant No. 31901693, Grant No. 21462026 and Grant No. 31772373), and the Guangzhou Planned Program in Science and Technology (No. 201903010094).

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Conflict of interest

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The authors declare no conflict of interest.

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Figure captions

Fig.1.

Chromatographic

profiles

and

physicochemical

analysis.

Crude

polysaccharides on DEAE-52 (A), fraction Ⅲ on Sephadex G-200 column (B), UV spectrum (C) and the lyophilized sample of CMP-Ⅲ (D).

Fig.2. Gas chromatograms of monosaccharide composition of the standard substance

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mixture (A-1) and CMP-Ⅲ (A-2), SEC-MALLS profile of CMP-Ⅲ for determination of homogeneity (B-1), molecular mass (B-2) and chain conformation (B-3), FT-IR

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spectrum of CMP-Ⅲ (C).

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Fig. 3. NMR spectra of CMP-Ⅲ. 1H spectrum (A), 13C spectrum (B).

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RAW264.7 cells.

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Fig. 4. Effect of CMP-Ⅲ on the viability (A) and phagocytic activity (B) of

Fig. 5. Effects of CMP-Ⅲ on production levels of NO (A), IL-6 (B), TNF-α (C) and

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mRNA levels of iNOS (D), IL-6 (E), TNF-α (F) in RAW 264.7 cells.

Fig. 6. Effect of CMP-Ⅲ on MAPKs (A) and NF-κB (B) signaling pathway, and the proposed signal transduction pathways involved in macrophage activation by CMP-Ⅲ (C).

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Author contributions: Bao-Lin He and Qian-Wang Zheng were responsible for the concept and design of the study; Bao-Lin He, Shi-Shi Huang and Qian-Wang Zheng performed experiments; Jen-Yi Huang and Fan Yun provided technical assistance and contributed to results analysis; Bao-Lin He, Qian-Wang Zheng, Li-Qiong Guo and Jun-Fang Lin drafted, edited and finalized the manuscript. All authors reviewed the

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results and approved the final version of the manuscript.

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Table 1. Primers used in RT-PCR

Genes

Primer sequences

GAPDH

Forward: 5’-TGTTGCCATCAATGACCCCTT-3’ Reverse: 5’-CTCCACGACTGACTCAGCG-3’ Forward: 5’-TGTTCTTTGCTTCTGTGCTAATGC-3’

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iNOS

Forward: 5’-TTCCAGCCAGTTGCCTTCTTG-3’

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IL-6

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Reverse: 5’-AGTTGTTCCTCTTCCAAGGTGTTT-3’

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Forward: 5’- CCACCACGCTCTTCTGTCTACTG -3’

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Reverse: 5’- GGGCTACGGGCTTGTCACTC -3’

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TNF-α

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Reverse: 5’-GGTCTGTTGTGGGTGGTATCCTC-3’

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Table 2. Molecular parameters of CMP-Ⅲ in 0.02 M KH2PO4 aqueous solution

Parameter

Detection results

Molar mass moments (g/mol)

Mn

3.004×107 (±0.19%)

Mw

4.796×107(±0.22%)

Mz

5.365×107(±0.49%)

PDI

1.60 (±0.29%)

Polydispersity

f

Molecular characteristic

α

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Rn Rw

e-

Rz

24.9 ± 2.5

pr

R.M.S. radius (nm)

0.15

25.6 ± 2.1 26.8 ± 2.6

Pr

Mn, Mw and Mz refer to number-, weight-, z-average molecular weights, respectively.

al

PDI refer to Mw/Mn, indicates the polydispersity ratio.

rn

α refer to the slope of the log (RMS radius)/log (Mw) curve.

respectively.

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Rn, Rw and Rz refer to number-, weight-, z-average square mean radii of gyration,

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Table 3. Methylation analysis and linkage mode of CMP-Ⅲ Retention time (min)

Methylated sugars

Type of linkage

Molar ratios(%)

Mass fragments (m/z)

12.956

2,3,4,6-Me4-Manp

T-Manp

10.79

43, 71, 87, 102, 118, 129, 145, 162, 205

16.597

2,3,6-Me3-Glcp

1,4-Glcp

70.08

43, 87, 102, 113, 118, 129, 131, 143, 162, 173, 233

19.428

2,3-Me2-Manp

1,4,6-Manp

9.59

19.541

3,4-Me2-Galp

1,2,6-Galp

3.93

l a

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f o

o r p

43, 85, 99, 102, 118, 162, 261

r P

e

43, 87, 100, 130, 190

Journal Pre-proof Table 4. Chemical shifts of the signals in the 1H and 13C NMR spectra of CMP-Ⅲ Chemical shifts (ppm) Sugar Residue H1/C1

H2/C2

H3/C3

H4/C4

H5/C5

H6/C6

5.32

3.57

3.77

3.88

4.01

3.63

99.82

73.32

72.88

76.89

71.20

60.54

5.16

3.57

3.35

4.10

3.77

3.77

100.63

70.37

71.20

76.23

72.70

66.90

5.12

4.12

3.63

3.88

3.15

3.57

100.64

71.20

72.88

70.37

69.33

62.28

5.09

4.12

4.16

3.94

3.62

3.56

106.13

86.91

74.52

81.62

70.36

69.34

→4)- α-D-Glcp→1

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rn

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Pr

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α-D-Manp-(1→

→2,6)-α-D-Galp-(1→

f

→4,6)-α-D-Manp-(1→

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Highlights  A novel high-molecular-weight polysaccharide (CMP-Ⅲ) was isolated from Cordyceps militaris.  The structure of CMP-Ⅲ was established based on methylation and NMR analysis.

f

 CMP-Ⅲ can promote macrophage phagocytosis and cytokines secretion.

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 CMP-Ⅲ induced macrophage activation mainly via MAPK and NF-κB signaling

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Pr

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pathways.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6