Structural Characterization of a Novel Polysaccharide from Lepidium

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Structural Characterization of a Novel Polysaccharide from Lepidium meyenii (maca) and Analysis of Its Regulatory Function in Macrophage Polarization in vitro Mengmeng Zhang, Wenjia Wu, Yao Ren, Xiao-Feng Li, Yuqian Tang, Tian Min, Furao Lai, and Hui Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05218 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017

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Journal of Agricultural and Food Chemistry

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Structural Characterization of a Novel Polysaccharide from

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Lepidium meyenii (maca) and Analysis of Its Regulatory Function in

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Macrophage Polarization in vitro

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Mengmeng Zhang1, Wenjia Wu1, Yao Ren2, Xiaofeng Li1, Yuqian Tang1, Tian Min1, Furao Lai1*, and Hui Wu1*

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Affiliation

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1 College of Food Sciences and Engineering, South China University of Technology,

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Guangzhou, Guangdong 510640, China

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2 College of Light Industry, Textile and Food Engineering, Sichuan University,

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Chengdu, 610065, China

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Short title: Structural Characterization and Regulation in Macrophage Polarization of

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Maca Polysaccharide

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Co-corresponding authors:

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Hui Wu, Department of Food Quality and Safety, South China University of

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

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Tel: (+86) 20-87112853; E-mail: [email protected]; Fax: (+86)20-87112532

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Furao Lai, Department of Food Quality and Safety, South China University of

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Technology, Wushan Road 381, Guangzhou, Guangdong, China. Tel: (+86) 20-87112373; E-mail: [email protected]; Fax: (+86)20-87112532 1

ACS Paragon Plus Environment


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ABSTRACT: In our previous study, three novel polysaccharides, named MC-1,

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MC-2, and MC-3, were separated from the roots of maca (Lepidium meyenii), which

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is a food source from the Andes region. The structural information and

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immunomodulatory activity of MC-1 were then investigated. The structure and

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activity of MC-2 is still unknown. In this study, structural characterization revealed

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that MC-2 has an average molecular weight of 9.83 kDa and is composed of arabinose

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(20.9%), mannose (4.5%), glucose (71.9%), and galactose (2.7%). The main linkage

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types of MC-2 were proven to be (1→5)-α-L-Ara, (1→3)-α-L-Man, (1→)-α-D-Glc,

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(1→4)-α-D-Glc, (1→6)-α-D-Glc, and (1→6)-β-D-Gal by methylation and NMR

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analyses. Congo red assay showed that MC-2 possesses a triple helix conformation.

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Immunostimulating assays indicated that MC-2 could induce M1 polarization of

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original macrophages and convert M2 macrophages into M1 phenotype. Although

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MC-2 could not shift M1 macrophages into M2, it could still inhibit inflammatory

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reactions induced by LPS. Furthermore, toll-like receptor 2, toll-like receptor 4,

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complement receptor 3, and mannose receptor were confirmed as the membrane

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receptors for MC-2 on macrophages. These results indicate that MC-2 could

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potentially be used toward hypoimmunity and tumor therapies.

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KEYWORDS: polysaccharides, maca, structure, macrophage , polarization

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INTRODUCTION

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Maca (Lepidium meyenii) is a food source that has been used to enhance fertility for

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centuries in the Andes region.1, 2 Recently, maca has attracted interest as a dietary

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supplement owing to its various pharmacological properties, and as a result, many

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effective compounds in maca were identified, including macaenes, macamides,

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glucosinolates, alkaloid, and flavonolignans.3-7 However, little attention has been

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devoted to the maca polysaccharides. In our previous study, three new

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polysaccharides, named, MC-1, MC-2, and MC-3 were separated from the roots of

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maca. The primary chemical structure and immunomodulatory activity of MC-1 was

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then investigated.8 However, the structure of MC-2 is still unknown.

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Macrophages are an essential component of innate immunity and play a

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central role in inflammation and host defense. Macrophage functions are elicited

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in response to microenvironmental signals, which drive the acquisition of polarized

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programs, whose extremes are simplified in the M1 and M2 dichotomy.9 M1

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macrophages (classically activated) exert pro-inflammatory activity. They have been

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known to be induced by interferon (IFN) or lipopolysaccharide (LPS), and release

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pro-inflammatory cytokines, such as TNF-α and IL-6, and nitric oxide (NO). M1

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macrophages are considered to mediate host defense against microbial infections and

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tumors, but also cause autoimmune tissue damage.10 IL-4 and IL-13 were found to

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induce alternatively activated M2 macrophages, which are involved in inflammation

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resolution and tissue repair.11 IL-10 (anti-inflammation cytokine) and arginase-1

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(Arg-1) represent the signature molecules of M2 macrophages. However, M2 3



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macrophages are also involved in metazoan parasite containment, immune tolerance,

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and tumor progression.12 Macrophage M1 and M2 phenotype responses generate

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opposing activities in terms of killing and repairing processes. An imbalance in

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macrophage M1-M2 polarization is often associated with various diseases or

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inflammatory conditions.13

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Plasticity and flexibility are key features of mononuclear phagocytes and their

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activation states. The phenotype of polarized M1-M2 macrophages can, to some

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extent, be reversed both in vitro and in vivo.14 Polysaccharides obtained from

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natural sources are known to govern the immunomodulatory activity of macrophages.

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Many polysaccharides can induce macrophage-produced pro-inflammatory cytokines

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TNF-α, IL-1b, IL-6, and IL-12.15-17 Conversely, some polysaccharides have been

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shown to have anti-inflammatory activities.18-20 These observations indicate that the

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immunomodulatory activity of polysaccharides is relative to macrophage polarization.

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However, few reports about the immunomodulatory activity of polysaccharides have

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focused on the different macrophage phenotypes. Given the fact that MC-1 has

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stimulating effects on macrophages, we hypothesized that MC-2 displays a similar

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effect.

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In the present study, the primary chemical structure of MC-2 was characterized.

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The regulation of MC-2 on the polarization of RAW 264.7 cells, a murine

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macrophage cell line, was investigated by determining the markers of M1, IL-6 and

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inducible nitric oxide synthase (iNOs), and markers of M2, IL-10 and Arg-1. The

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potential membrane receptors of MC-2 on RAW 264.7 cells were also explored. The 4


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results obtained from this study might provide useful information toward enabling

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further in-depth studies on polysaccharides present in maca.

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MATERIALS AND METHODS

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Chemicals

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The roots of maca were collected from Peru. Myoinositol and standard

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

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galactose)

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Diethylaminoethyl (DEAE)-Sepharose Fast Flow was obtained from Shanghai

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Yuanye Bio-Technology Company Limited (Shanghai, China). Sephadex G-50 was

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acquired from GE Healthcare Life Science (Piscataway, NJ). RAW 264.7 cells were

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obtained from the Type Culture Collection of the Chinese Academy of Sciences

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(Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine

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serum (FBS), penicillin, and streptomycin were purchased from Gibco Life

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Technologies (Grand Island, NY). Griess reagent was purchased from Sigma-Aldrich

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(NSW, Australia). Mouse TNF-α, IL-10 and IL-6 detection ELISA kits were

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purchased from R＆ D Systems. TRIzol was purchased from Invitrogen, USA;

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Transcriptor First Strand cDNA Synthesis Kit and FastStart Universal SYBR Green

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Master (ROX) were purchased from Roche. Anti-scavenger receptor I antibody

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(anti-SR), anti-mannose receptor antibody (anti-MR), anti-beta glucan receptor

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antibody (anti-GR), anti-toll-like 2 antibody (anti-TLR2), anti-complement receptor 3

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antibody (anti-CR3), and anti-toll-like 4 receptor antibody (anti-TLR4) were obtained

were

purchased

from

Sigma-Aldrich

(St.

5


Louis,

MO,

USA).


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from Abcam (Cambridge, MA). All of the other chemical reagents used in this study

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were of analytical grade.

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Extraction and Purification of Polysaccharides from the Roots of Maca

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The extraction method was the same as in our previous study.21 Briefly, the powder

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of dried roots of maca was extracted with boiling water at a ratio of 1:30 (w/v) for 2 h,

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and the obtained extract was then centrifuged at 4000 × g for 15 min. After the

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supernatant was concentrated at 60 °C, the protein was removed according to the

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Sevag method. The resulting solution was precipitated with four volumes of 100%

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ethanol at 4 °C for overnight, and then centrifuged at 4000 × g for 15 min to collect

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the precipitates. Finally, the precipitate was redissolved in distilled water prior to

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lyophilization to obtain crude polysaccharides.

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The purification method is similar to that used in the previous study, with some

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modifications.22 A total of 50 mg of crude polysaccharides was dissolved in 10 mL of

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ultrapure water and loaded onto a pre-equilibrated DEAE-Sepharose Fast Flow

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chromatography column (1.6×35 cm) at a flow rate of 1 mL/min, followed by and

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elution step with distilled water. The eluent fractions were collected and concentrated

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at 60 °C by rotary vacuum evaporator, then dialyzed (MW cut off 5.0 kDa) at 4 °C for

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48 h and freeze-dried. In our previous study, a Sephadex G-100 column could not

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totally separate the fractions. Therefore, in this study, a Sephadex G-50

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chromatography column (1.6 × 60 cm) was used to yield a better purification process.

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The fraction (20 mg) was dissolved in distilled water (10 mL) at 25 °C. The column

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was washed with 0.3 L of distilled water at a flow rate of 1 mL/min for 300 min. The 6


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eluent was detected according to the phenol-sulfuric acid method and collected before

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concentrating the polysaccharide solution. As previously, when we studied MC-1,

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three peaks were obtained (Figure 1). In this study, we mainly focused on

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investigating of MC-2.

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MC-2 Molecular Weight Determination

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The weight-average molecular weight of MC-2 was determined through

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high-performance gel permeation chromatography (HPGPC) using a Waters HPLC

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system including two serially linked columns a TSK-GEL G-5000 PWXL column

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(300 mm×7.8 mm inner diameter, 10µm) and a TSK-GEL G-3000 PWXL column

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(300 mm×7.8 mm inner diameter, 6µm), a Waters 2410 differential refractive index

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detector, eluted with 0.02 mol/L KH2PO4 at a ﬂow rate of 0.6 mL/min. MC-2 (2.5 mg)

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was dissolved in 1 mL mobile phase.

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Infrared Spectrum Analysis

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The MC-2 samples (2-3 mg) were analyzed according to the potassium bromate

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pellet method with a Fourier transform infrared (FTIR) spectrophotometer (Bruker,

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Ettlingen, Germany) in the 400-4000 cm-1 vibrations region.23

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Monosaccharide Composition

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A total of 10 mg MC-2 sample was hydrolyzed in 4 mL of 2 mol/L trifluoroacetic

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acid (TFA) for 8 h at 110 °C. Polysaccharides were transformed into alditol acetates

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by hydrolysis. The MC-2 alditol acetates were analyzed by gas chromatography (GC)

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(Agilent, US) fitted with a HP-5 capillary column (30 nm×0.32 mm×0.25 µm,

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160~210 °C at 2 °C /min, and then 210~250 °C at 10 °C /min) equipped with a flame 7



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ionization detector (FID). Glucose, galactose, fucose, rhamnose, mannose, xylose, and

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arabinose were used as the monosaccharide standards. Myoinositol was used as the

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internal reference.

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Methylation Analysis

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The methylation analysis of polysaccharide was performed according to the 21

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method reported by Nie et al

,with some modifications. MC-2 (10 mg) was

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dissolved in 6 mL anhydrous DMSO at 60 °C for 2 h and sonicated for 1 h. NaOH

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(240 mg) was added to the solution, which was subsequently left to react at 60 °C

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overnight. The mixture was added to a 3.6 mL methyliodide solution and stirred for 8

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min. This procedure was repeated three times and stopped by adding 6 mL of distilled

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water. The solution obtained was dialyzed against distilled water for 48 h at 4 °C. The

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methylated polysaccharide was extracted with dichloromethane three times. The

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dichloromethane extract was then dried over sodium sulfate, and evaporated to

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dryness. The dried methylated polysaccharide was hydrolyzed as describe above. The

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hydrolysate was reduced by sodium borodeuteride (70 mg) and acetylated with acetic

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anhydride (0.5 mL). Finally, the resulting product was analyzed by gas

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chromatography (GC) coupled with mass spectrometry (MS) (Agilent, USA) using a

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TR-5MS capillary column (30 m ×0.25 mm ×0.25 µm, 150~180 °C at 10 °C /min, and

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then 180~260 °C at 15 °C /min).

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NMR Spectroscopy

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About 30 mg of MC-2 was dissolved in 0.55 mL of heavy water (D2O) in a NMR 13

C NMR and 1H NMR spectra were recorded on a Bruker 600

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tube and then, the

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MHz NMR apparatus (Bruker Corp, Fallanden, Switzerland) at 60 °C.

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Determination of Triple-helix Structure

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The conformational structure of MC-2 was determined following the Congo red

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method.22

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Measurement of cytokines

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RAW 264.7 cells were incubated at 37°C in a humidified atmosphere with 5%

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CO2. DMEM medium with 10% FBS, 100 µg/mL streptomycin, and 100 units/mL

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penicillin was used as the culture medium. Cells were adjusted to a concentration of 1

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× 106 cells/mL in the exponential phase, loaded onto 96-well or 6-well plates, and

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continuously incubated for 24 h.

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Cells were treated with different concentrations of MC-2 (62.5, 125, 250, 500,

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1000 µg/mL), MC-1 (1000 µg/mL), LPS (1 µg/mL) or IL-4 (20 ng/mL) and incubated

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for 24 h. After that, the cell supernatants were collected and the levels of IL-6 and

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IL-10 were measured using the ELISA kits.

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QPCR Analysis

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RAW 264.7 cells were seeded on 6-well plates at a concentration of 1 × 106

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cells/mL. After 24 h, cells were treated with different concentrations of MC-2 (62.5,

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125, 250, 500, 1000 µg/mL), MC-1 (1000 µg/mL), LPS (1 µg/mL) or IL-4 (20 ng/mL).

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After 12 h, the cells were lysed to isolate total RNA.

9



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Total RNA was isolated using TRIzol reagent according to the manufacturer’s

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protocol, and the RNA was used for cDNA synthesis using reverse transcriptase. The

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cDNA encoding iNOs and Arg-1 genes was quantified by quantitative real-time PCR

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assay (QPCR). GAPDH was used as the internal reference. The specific primers used

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are described in Table 1. Gene amplification was carried out with the ABI 7500

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sequence detection system (Applied Biosystems, Foster, USA).

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MC-2 Treatment After Polarization

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Cells within the control group were cultured in medium without any treatment.

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Cells in the other groups were polarized to M1 with LPS (1 µg/mL) for 12 h, and then

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treated with combinations of different concentrations of MC-2 (0, 31.25, 125, 500

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µg/mL) and LPS (1 µg/mL) for 24 h. M2 macrophages were polarized by IL-4 (20

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ng/mL) using the same procedure. Following polarization, cells were collected for the

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determination of iNOS and Arg-1 mRNA levels, and the supernatants were used to

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measure the levels of IL-6 and IL-10.

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Investigation of Membrane Receptors

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The cells were pretreated with antibodies (5 µg/mL) against membrane receptors

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(SR, MR, GR, CR3, TLR2 and TLR4) or a mixture of antibodies against MR, CR3,

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TLR4, and TLR2 for 2 h prior to stimulation with MC-2 (125 µg/mL). The group

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treated with only MC-2 (125 µg/mL) was used as the control. The untreated cells were

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used as the negative control group. LPS-treated cells represented the positive control.

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The levels of NO, TNF-α, and IL-6 were measured after 24 h .23 NO, TNF-α, and IL-6

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were measured using a Griess reagent and ELISA kits, respectively. 10


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Statistical Analysis

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Data are expressed as the mean± standard deviation (SD) of three replicates.

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Significant differences between the means of parameters were calculated by Duncan’s

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multiple-range test using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). p

Structural Characterization of a Novel Polysaccharide from Lepidium

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