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Article

Structural Characterization and ImmunomodulatoryActivity of a Novel Polysaccharide from Lepidium meyenii

Mengmeng Zhang, Guang Wang, Furao Lai, and Hui WuJ. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05610 • Publication Date (Web): 17 Feb 2016

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1

Structural Characterization and Immunomodulatory Activity of 1

a Novel Polysaccharide from Lepidium meyenii 2

3

Mengmeng Zhang1, Guang Wang

1, Furao Lai

1, 2*, Hui Wu

1* 4

5

Affiliation 6

1 College of Light Industry and Food Sciences, South China University of Technology, 7

Guangzhou, Guangdong 510640, China 8

2 Guangdong Provincial Key Laboratory of Green Agricultural Products Processing，9

Guangzhou, Guangdong 510640, China 10

11

12

13

14

15

16

17

Co-corresponding authors: 18

Hui Wu, Department of Food Quality and Safety, South China University of 19

Technology, Wushan Road 381, Guangzhou, Guangdong, China. 20

Tel: (+86) 20-87112853; E-mail: [email protected] 21

Furao Lai, Department of Food Quality and Safety, South China University of 22

Technology, Wushan Road 381, Guangzhou, Guangdong, China. 23

Tel: (+86) 20-87112373; E-mail: [email protected] 24

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ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

2

ABSTRACT: A novel polysaccharide named as MC-1 was isolated from the roots 25

of Lepidium meyenii using a water extraction method. Structural characterization 26

revealed that MC-1 had an average molecular weight of 11.3 kDa and consisted of 27

arabinose (26.21%), mannose (11.81%), glucose (53.66%), and galactose (8.32%). 28

The main linkage types of MC-1 were proven to be (1→5)-α-L-Ara, (1→3)-α-L-Man, 29

(1→2,6)-α-L-Man, (1→)-α-D-Glc, (1→4)-α-D-Glc, (1→6)-α-D-Glc and 30

(1→6)-β-D-Gal by methylation analysis, periodate oxidation-Smith degradation and 31

NMR analysis. The immunostimulating assay indicated that MC-1 could significantly 32

enhance the pinocytic and phagocytic capacity and promote the NO, TNF-α and IL-6 33

secretion of RAW 264.7 cells, involving toll-like receptor 2, complement receptor 3 34

and mannose receptor mainly. These results suggested the potential utilization of 35

MC-1 as an attractive functional food supplement candidate for hypoimmunity 36

population. 37

KEYWORDS: polysaccharide, Lepidium meyenii, structural characterization, 38

immunomodulatory activity. 39

40

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42

43

44

45

46

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INTRODUCTION 47

Maca (Lepidium meyenii) is a food source in the Andes region and belongs to the 48

Brassicaceae. It grows in altitudes varying between 3700 and 4450 m.1 In the Andes, 49

people have used maca to enhance fertility for centuries.2 Recently, maca has attracted 50

interests as a dietary supplement due to its pharmacological activity, such as 51

enhancing fertility, anti-fatigue,

anti-depression, anti-osteoporosis, anti-oxidative, 52

anti-inflammation, and so on.3-12

And many effective compounds in maca were 53

identified, including macaenes, macamides, glucosinolates, alkaloid, and 54

flavonolignans.3, 13-16

However, little attention was devoted to the maca 55

polysaccharide and its activity. Especially, there was little research on the structural 56

information of maca polysaccharides. Polysaccharides obtained from natural sources 57

are known as kinds of biological activities, for example, immunomodulatory activity. 58

But only the antioxidant activity of maca polysaccharides was reported.1 More 59

activities need further exploration. 60

Macrophages play a unique role in the immune system. They can not only initiate 61

innate immune responses, but also contribute to fight against infection and 62

inflammation. Macrophages can kill pathogens directly by phagocytosis and indirectly 63

by releasing cytotoxic molecules such as NO and secreting cytokines including 64

TNF-α and IL-6.17-19

Thus, macrophages are usually used as an ideal cell model to 65

evaluate the immunomodulatory activity of bioactive compounds. 66

In the present study, a new polysaccharide, named as MC-1, was separated from 67

the roots of L. meyenii. The primary chemical structure of MC-1 was characterized. 68

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Also, the current experiments were designed to investigate the immunomodulatory 69

activity of MC-1 on the murine macrophage cell line, RAW 264.7 cells, by 70

determining the effect on the pinocytic and phagocytic capacity, production of NO, 71

TNF-α and IL-6, and explore the membrane receptors of MC-1 on RAW 264.7 cells. 72

The results from this study might supply useful information to further study on 73

polysaccharides in maca. 74

MATERIALS AND METHODS 75

Materials and Chemicals 76

The roots of L. meyenii were collected from Peru. Myoinositol and standard 77

monosaccharides (xylose, rhamnose, arabinose, fucose, mannose, glucose, galactose) 78

were purchased from Sigma Company (St. Louis, MO, USA). Diethylaminoethyl 79

(DEAE)-Sepharose Fast Flow were obtained from Shanghai Yuanye Bio-Technology 80

Company Limited (Shanghai, China). Sephadex G-100 was acquired from GE 81

Healthcare Life Science (Piscataway, NJ). RAW 264.7 cells were obtained from the 82

Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). 83

Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, 84

and streptomycin were purchased from Gibco Life Technologies (Grand Island, NY). 85

lipopolysacch aride (LPS), neutral red were purchased from Sigma Company 86

(St.Louis, MO, USA). Vybrant Phagocytosis Assay Kit was purchased from 87

Molecular Probes (Carlsbad, CA, USA). Griess reagent was purchased from 88

Sigma-Aldrich (NSW, Australia). Mouse TNF-α and IL-6 detecting ELISA kits were 89

from R＆D Systems. Trizol was purchased from Invitrogen, USA; Transcriptor First 90

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Strand cDNA Synthesis Kit, FastStart Universal SYBR Green Master (ROX) was 91

purchased from Roche. Anti-scavenger receptor I antibody (anti-SR), anti-mannose 92

receptor antibody (anti-MR), anti-beta glucan receptor antibody (anti-GR), 93

anti-toll-like 2 antibody (anti-TLR2), anti-complement receptor 3 antibody (anti-CR3), 94

and anti-toll-like 4 receptor antibody (anti-TLR4) were obtained from Abcam 95

(Cambridge, MA). All of the other chemical reagents used in this study were 96

analytical grade. 97

Extraction and Purification of Polysaccharides from the Roots of L. meyenii 98

The dried roots of L. meyenii were crushed into powder using a tissue triturator. 99

The powder was extracted with boiling water at a ratio of 1:30 (w/v) for 2 h, and the 100

obtained extract was centrifuged at 4000×g for 15 min. The supernatant was then 101

collected and concentrated at 60 ℃. After that, the protein in concentrated solution 102

was removed by the Sevag method. The deproteinated process was repeated 30 times. 103

The resulting solution was precipitated with four volumes of 100% ethanol at 4 ℃ for 104

overnight. The supernatant and precipitate were then separated by centrifugation at 105

4000×g for 15 min. Finally, the precipitates was centrifuged and redissolved in 106

distilled water before lyophilization to obtain crude polysaccharides. 107

A total of 50 mg of crude polysaccharides was dissolved in 10 mL of ultrapure 108

water and loaded onto a pre-equilibrated DEAE-Sepharose Fast Flow chromatography 109

column (1.6×35 cm) at a flow rate of 1 mL/min, then sequentially eluted with distilled 110

water and 0.05, 0.1, 0.2, 0.3, 0.5 M NaCl solution, respectively. The eluent fractions 111

were collected and analyzed by the phenol-sulfuric acid method. The fraction eluted 112

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by ultrapure water were concentrated at 60 ℃ by rotary vacuum evaporator, then 113

dialyzed (MW cut off 5.0 kDa) at 4 ℃ for 48 h and freeze-dried. Sephadex G-100 114

chromatography column (1.6×60 cm) was used to purify further the fraction. The 115

fraction (20 mg) was dissolved in distilled water (10 mL) at 25 ℃. The column was 116

washed with 0.3 L of distilled water at a flow rate of 1 mL/min for 300 min. The 117

eluent was detected with phenol-sulfuric acid method, then collected and concentrated 118

the polysaccharide solution. Three peaks were gotten. In this study, we mainly 119

focused on the research of MC-1. The resultant solution was dialyzed and 120

freeze-dried. 121

Molecular Weight Determination of MC-1 122

The weight-average molecular weight of MC-1 was conducted on 123

high-performance gel permeation chromatography (HPGPC) using a Waters HPLC 124

system including two serially linked columns a TSK-GEL G-5000 PWXL column 125

(300 mm×7.8 mm inner diameter, 10µm) and a TSK-GEL G-3000 PWXL column 126

(300 mm×7.8 mm inner diameter, 6µm), a Waters 2410 differential refractive index 127

detector, eluted with 0.02 mol/L KH2PO4 at a flow rate of 0.6 mL/min. MC-1 (2.5 mg) 128

was dissolved in 1 mL mobile phase. The polysaccharide solution was filtered through 129

a 0.22 µm microporous filtering film. 130

Infrared Spectrum Analysis 131

The MC-1 samples (2-3 mg) were ground to a fine powder and analyzed through 132

the potassium bromite pellet method with a Fourier transform infrared (FTIR) 133

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spectrophotometer (Bruker, Ettlingen,Germany) in the 400-4000 cm-1

vibrations 134

region.20

135

Determination of Triple-helix Structure 136

The conformational structure of MC-1 was determined following the Congo red 137

method.21

138

Monosaccharide Compositon 139

A total of 10 mg MC-1 sample was hydrolyzed in 4 mL of 2 mol/L trifluoroacetic 140

acid (TFA) for 8 h at 110 ℃. Polysaccharides hydrolysis transformed into alditol 141

acetates. The alditol acetates product of MC-1 was analysed by gas chromatography 142

(GC) (Agilent, US) fitted with a HP-5 capillary column (30 nm×0.32 mm×0.25 µm, 143

160~210 ℃ at 2 ℃/min, and then 210~250 ℃ at 10 ℃/min) equipped with a flame 144

ionization detector (FID). Glucose, galactose, fucose, rhamnose, mannose, xylose, and 145

arabinose were used as the monosaccharide standards. Myoinositol was used as the 146

interior reference. 147

Periodate Oxidation-Smith Degradation 148

25 mg of MC-1 sample was dissolved in 12.5 mL of ultrapure water and 149

incubated with 12.5 mL of NaIO4 (30 mmol/L) in the dark at room temperature. 150

During the incubation period, 0.1 mL of the reaction liquid was taken out from the 151

reaction system at different time intervals (0, 6, 12, 24, 36, 48 and 60 h) until the 152

absorbance value becoming stable under an ultraviolet visible spectrophotometer 153

(model UV-18000,Shimadzu, Japan) in the 233 nm. The periodate product (2 mL) was 154

used to quantity the production of formic acid by titration with 0.098 mol/L sodium 155

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hydroxide solution (screened phenolphthalein as indicater) after addition of glycol. 156

The rest solution was dialyzed in distilled water for 3 days at 4 ℃. The dialyzed was 157

concentrated and added with 70 mg sodium borohydride reacting for 12 h in the dark 158

to destroy the furfural. The solution was neutralized to pH 6.0-7.0 with 50% acetic 159

acid and dialyzed for another 3 days at 4 ℃. The dialyzed was concentrated and 160

freeze-dried. A total of 10 mg of the residues was hydrolyzed by 4 mL of 2 mol/L TFA 161

at 105 ℃ for 6 h. The lysate was acetylized with 1 mL of pyridine and 10 mg of 162

hydroxylamine hydrochloride at 90 ℃ for 30 min. Then, 1 mL of acetic anhydride 163

was added to the reaction system and continuously heating at 90 ℃ for another 30 164

min. The production of acetate derivative was analyzed by a GC (Aglient, USA) with 165

a DB-1701 capillary column (30m×0.25mm×0.25µm, J&W Scientific, Fulsom, CA) 166

and a flame ionization detector. The linearly heating program is from 80 to 220 ℃ at 167

a speed of 2 ℃/min, from 220 to 250 ℃ at a speed of 5 ℃/min, and kept at 250 ℃ 168

for 5 min. The temperature of the detector was set at 300 ℃. Phycite, glycol, glycerol, 169

rhamnose, xylose, arabinose, mannose, glucose, and galactose were used as standards. 170

Methylation Analysis 171

Methylation analysis of polysaccharide was performed according to the modified 172

method reported by Nie et al.22

The dried MC-1 (10 mg) was dissolved in 6 mL 173

anhydrous DMSO at 60 ℃ for 2 h and sonicated for 1 h to ensure a completed 174

solution. Sodium hydroxide (240 mg) was added to the solution at 60 ℃ throughout 175

the night. The mixture was added 3.6 mL methyliodide with stirring for 8 min. This 176

procedure was conducted at three times and stopped by the addition of 6 mL distilled 177

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water. The obtained solution was dialyzed against distilled water for 48 h at 4 ℃. The 178

methylated polysaccharide was extracted with dichloromethane three times. The 179

dichloromethane extract was then dried over sodium sulfate, and evaporated to 180

dryness. The dried methylated polysaccharide was hydrolyzed as describe above. The 181

hydrolysate was reduced by sodium borodeuteride (70 mg) and acetylated with acetic 182

anhydride (0.5 mL). Finally, the resultant was analysed by gas chromatography (GC) 183

coupled with mass spectrometry (MS) (Agilent, USA) using a TR-5MS capillary 184

column (30 m ×0.25 mm ×0.25 µm, 150~180 ℃ at 10 ℃/min, and then 180~260 ℃ 185

at 15 ℃/min). 186

NMR Spectroscopy 187

About 30 mg of MC-1 was dissolved with 0.55 mL of D2O in a NMR tube and 188

then the 13

C NMR and 1H NMR spectra were recorded on a Bruker 600 MHz NMR 189

apparatus (Bruker Corp, Fallanden, Switzerland) at 60 ℃. 190

Determination of Pinocytic and Phagocytic Capacity 191

Pinocytic Capacity RAW 264.7 cells were incubated at 37 ℃ in a humidified 192

atmosphere with 5% CO2. DMEM medium with 10% FBS, 100 µg/mL streptomycin, 193

and 100 units/mL, penicillin was used as the culture medium. Cells were adjusted to a 194

concentration of 1×106 cells/mL in the exponential phase, loaded onto the 96-well or 195

6-well plate, and continuously incubated for 24 h. Then, cells were treated with MC-1 196

at different concentrations (62.5, 125, 250, 500, 1000 µg/mL) or LPS (20 µg/mL) 197

were added into each well. After 24 h incubation, the medium was removed, and 100 198

µL of 0.1% neutral red dissolved in PBS was added to each well and incubated for 1 h. 199

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The cells were washed with PBS three times, and then 100 µL of 1% acetic acid 200

solution (v/v) in 50% ethanol (v/v) was added to each well. Cell culture plate was 201

statically placed overnight. The absorbance at 540 nm was measured using a 202

microplate reader (Biotek, USA). 203

Phagocytic Capacity RAW 264.7 cells were seeded on 6-well plates and 204

stimulated with MC-1 at different concentrations (62.5, 250, 1000 µg/mL) or LPS (20 205

µg/mL) for 6 h. A solution of FITC-labeled E. coli provided by Vybrant Phagocytosis 206

Assay Kit was added to the cells, and incubated for 2 h. The Bio Particle loading 207

suspension was removed and 100 µL of the prepared trypan blue suspension was 208

added. After 1 minute at room temperature, the excess trypan blue suspension was 209

removed immediately. Images of the cells were captured using a fluorescence 210

microscope (ECCIPSE 50, Nicon, Japan). The fluorescence intensity was measured 211

by flow cytometry using a FC500 flow cytometer (Beckman Coulter Ltd, USA). 212

Measurement of NO and cytokines 213

Cells were treated with the different concentrations of MC-1 (62.5, 125, 250, 500, 214

1000 µg/mL) or LPS (20 µg/mL) and incubated for 24 h. After that, the supernatants 215

of cells were collected and the levels of NO, TNF-α and IL-6 were measured using a 216

Griess reagent and ELISA kits, respectively. 217

QPCR Analysis 218

RAW 264.7 cells were seeded on 6-well plates at a concentration of 1×106 219

cells/mL. After 24 h, cells were treated with the different concentrations of MC-1 220

(62.5, 125, 250, 500, 1000 µg/mL) or LPS (20 µg/mL). After 12 h, cells were lysed to 221

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isolate total RNA. 222

Total RNA was isolated using TRIzol reagent according to the manufacturer’s 223

protocol, and the RNA was used for cDNA synthesis using reverse transcriptase. The 224

cDNA encoding iNOs, TNF-α, IL-6 genes was quantified by quantitative real-time 225

PCR assay (QPCR). GAPDH was used as the internal reference. The specific primers 226

were used (Table 1). Gene amplification was carried out with the ABI 7500 sequence 227

detection system (Applied Biosystems, Foster, USA). 228

Investigation of Membrane Receptors 229

The cells were pretreated with antibodies (5 µg/mL) of membrane receptors (SR, 230

MR, GR, CR3, TLR2 and TLR4) or the mixed antibodies of MR, CR3 and TLR2 for 231

2 h prior to stimulation with MC-1 (125 µg/mL). The group treated with only MC-1 232

(125 µg/mL) was used as a control. The untreated cells were used as the negative 233

control group. LPS was used as the positive control. The levels of NO, TNF-α, and 234

IL-6 were measured after 24 h .23

235

Statistical Analysis 236

Data are expressed as means± standard deviation (SD) for three replicates. 237

One-way ANOVA was used to analyze the significant differences between the groups 238

by SPSS 21.0. p <0.05 was considered statistically significant. 239

RESULTS AND DISCUSSION 240

Extraction and Purification of MC-1 Polysaccharide from L. meyenii 241

Crude polysaccharides were isolated from the roots of L. meyenii with a yield of 242

6.36%. The extracts were first purified by a DEAE-Sepharose Fast Flow 243

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chromatography (Figure 1A). Two independent fractions, eluted by ultrapuren water 244

and 0.05 mol/L NaCl, respectively, were obtained. The fraction eluted by ultrapuren 245

water which was at much higher content was further purified by a Sephadex G-100 246

column. As shown in Figure 1B, three peaks, MC-1, MC-2 and MC-3, were observed. 247

In the present study, we mainly focused on MC-1, and the property of MC-2 and 248

MC-3 will be studied in our future work. Finally, MC-1 was detected using the 249

phenol-sulfuric acid method for the determimation of 97.5% content. 250

Ultraviolet Rays (UV) Spectrum 251

UV absorption of MC-1 spectra did not show any absorption peaks in the 252

wavelength 260 and 280 nm (Figure 1C), indicating MC-1 didn’t contain any impurity 253

of nucleotides or protein.24

254

Characterization of MC-1 255

Molecular Weight of MC-1 256

The average molecular weight of MC-1 was measured by HPGPC as illustrated in 257

Figure 2A. A single peak was observed. It indicated that MC-1 was a homogeneous 258

polysaccharide. The average molecular weight of MC-1 was determined to be 11.3 259

kDa. Generally, the molecular weight of polysaccharides could reach to hundreds of 260

thousands of Da. So MC-1 was a low molecular weight polysaccharide. This might 261

give MC-1 some characters different from the high molecular weight polysaccharides. 262

FT-IR Spectrum 263

In the FT-IR spectrum of MC-1 polysaccharides (Figure 2B), most of the absorption 264

bands could be assigned according to previous literature.25, 26

The vibrations region 265

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3406 cm-1

corresponds to the O-H stretching vibration and that at 2927 cm-1

266

corresponds to the C-H stretching vibration. The band at 1637 cm-1

may be due to the 267

C=O stretching vibration. The absorption peaks at 1422 cm-1

represented the C=C 268

stretching vibration and the strong peak at 1043 cm-1

was ascribed to the stretching 269

vibrations of pyranose ring. A characteristic absorption at 897 cm-1

was also observed, 270

indicating the β-pyranoside linkage. Finally, an absorption peak at 573 cm-1

indicated 271

the existence of α-configurations. The spectra indicated that MC-1 had the typical 272

groups of sugars. 273

The Conformational Structure of MC-1 274

The Congo red method was applied at different concentrations of NaOH (0.05 to 275

0.5 N) solution to identify the triple helix conformation of MC-1. As shown in Figure 276

2C, an obvious shift of maximum absorption wavelength from 496 to 506 nm was 277

induced by the presence of the polysaccharides in Congo red solution, indicating that 278

polysaccharide-Congo red complexes had formed. Along with the increase of NaOH 279

concentrations, the maximum absorption wavelength decreased slightly. With the 280

increase of concentrations of NaOH, the triple-helix conformation will translate into 281

single coil conformation, and the maximum absorption wavelength will decrease 282

dramatically. So the result indicated that MC-1 didn’t exhibit a triple helical 283

conformation. But a bathochromic shift was observed after mixing MC-1 and Congo 284

red. The reason might be that MC-1 had another order conformation which could 285

form a complex with Congo red, and the conformation was stable in the NaOH. So the 286

precise conformation of MC-1 needs to study further. 287

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Monosaccharide Compositon 288

The monosaccharide composition of MC-1 was analyzed using gas 289

chromatography. The result was shown in Figure 3A (monosaccharide standard) and 290

Figure 3B (MC-1). The polysaccharide MC-1 was found to mainly consist of 291

arabinose, mannose, glucose and galactose in an approximate molar percentage of 292

26.21%, 11.81%, 53.66% and 8.32%, respectively. It indicated that MC-1 was a kind 293

of heteropolysaccharide. The monosaccharide composition of MC-1 was different 294

from the previous study.1 For example, the mannose in MC-1 wasn’t reported, and 295

there was not rhamnose in MC-1. The reason might be that the extraction methods 296

were different, or the rhamnose existed in MC-2 or MC-3. 297

Periodate Oxidation-Smith Degradation 298

The result of periodate oxidation analysis showed that 1 mol of hexose residue 299

consumed 1.13 mol of periodate and produced 0.467 mol of formic acid. The mole 300

number of periodate was more than two times of formic acid. It indicated that one or 301

more of (1→6), (1→2) or (1→4)-linked glycosidic bonds might exist. Then MC-1 302

was further analyzed by Smith degradation. Glycerol，phycite，arabinose，glucose 303

were observed after Smith degradation (Figure 3C). Glycerol corresponded to the 304

existence of (1→2) or (1→6)-linked glycosidic bonds. The appearance of erythritol 305

indicated that some of the linkages were (1→4) or (1→6)-linked glycosidic bonds. 306

The presence of monosaccharides revealed the appearance of (1→3)-linked glycosidic 307

bonds. More precise glycosidic bonds were confirmed in the methylation analysis. 308

Methylation Analysis 309

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Methylation analysis followed by GC-MS was employed to obtain more structural 310

information on MC-1. According to the result in Table 2, seven homogeneous peaks 311

were observed. According to the retention time, the peaks were identified as 312

2,3-Me2-Araf, 2,4,6-Me3-Manp, 3,4-Me2-Manp, 2,3,4,6-Me4-Glcp, 2,3,6-Me3-Glcp, 313

2,3,4-Me3-Glcp and 2,3,4-Me3-Galp with a molar ratio of 27.13:3.03: 314

1.83:8.75:43.37:14.01:1.88. The result suggested that MC-1 contained seven linkage 315

forms: (1→5) -linked arabinose, (1→3) -linked mannose, (1→2, 6) -linked mannose, 316

(1→)-linked glucose, (1→4)-linked glucose, (1→6)-linked glucose, and (1→6)-linked 317

galactose. This inference also agreed with the result from the periodate 318

oxidation-Smith degradation. The ratio between terminal units (T-Glcp) and the 319

branching points (1, 2, 6-Manp) was 4.78, indicating that number of terminal units 320

was much more than that of branching points. These results indicated that MC-1 321

contained the linear and branched polysaccharides. And, the linear polysaccharide was 322

approximately two times more than the branched polysaccharide. Additionally, the 323

degree of branching (DB) value of MC-1 was calculated as 10.58% following the 324

equation DB = (NT + NB)/ (NT + NB + NL), where NT, NB, and NL are the numbers 325

of the terminal residues (T-Glcp), branch residues (1,2,6-Manp), and linear residues 326

(1,5-Araf, 1,3-Manp, 1,4-Glcp, 1,6-Glcp, 1,6-Galp), respectively.27

327

NMR Analysis of MC-1 328

The spectra of 13

C NMR and 1H NMR of MC-1 was shown in Figure 4. Signals of 329

MC-1 in 13

C NMR and 1H NMR spectra were analyzed through referring to the 330

previous research.28-30

The resonances in the region of δ 95.0-107.0 in 13

C NMR were 331

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assigned to the anomeric carbon atoms of D-galactose, D-glucose, L-mannose, and 332

L-arabinose. The anomeric proton signals (δ 5.28, 5.12, 5.05, 4.96, 4.53, 5.03 and 333

4.51) and the anomeric carbon signals (δ 106.36, 95.72, 106.85, 99.48, 99.56, 99.69 334

and 103.62) corresponded with H-1 and C-1 of seven anomeric residues, including 335

(1→5)-α-L-Ara,(1→3)-α-L-Man,(1→2,6)-α-L-Man,(1→)-α-D-Glc,(1→4)-α-D-Glc,(1336

→6)-α-D-Glc,and(1→6)-β-D-Gal, respectively. The entire assignment of the 13

C and 337

1H chemical shifts was shown in Table 3. 338

Immunomodulatory Activity of MC-1 339

Effects of MC-1 on RAW 264.7 cells Viability 340

To investigate the toxic effect of MC-1 on the RAW 264.7 cells, after being treated 341

with MC-1 (62.5, 125, 250, 500, 1000 µg/mL) for 24 h, the viability of cells were 342

detected by MTT assay. As shown in Figure 5A, MC-1 didn’t affect the viability of 343

RAW 264.7 cells, which indicated that MC-1 under 1000 µg/mL was nontoxic to 344

RAW 264.7 cells. So the concentration of MC-1 used in the following study was 345

below 1000 µg/mL. 346

Effects of MC-1 on the Pinocytic and Phagocytic Capacity of RAW 264.7 cells 347

The pinocytic capacity was examined by the uptake of neutral red. As shown in 348

Figure 5B, compared to the control group, 62.5 µg/mL MC-1 significantly enhanced 349

the uptake of neutral red (p<0.05). With the increase of concentration, the 350

enhancement was stronger. Although the effect of 1000 µg/mL was lower than 500 351

µg/mL, there was no significant difference between two groups. The result indicated 352

that MC-1 could enhance the pinocytic capacity of RAW 264.7 cells. 353

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Meanwhile, we also determined the effect of MC-1 on phagocytic capacity to 354

E.coli (FITC-labeled) of RAW 264.7 cells. The photos of RAW 264.7 cells taken by 355

fluorescence microscope were shown in Figure 5C. The photo of the control group 356

showed that the untreated cells could phagocytose a little of E.coli. The fluorescence 357

intensity of cells treated with MC-1 or LPS was obviously stronger than the control 358

group. Then, the flow cytometer was used to further confirm the result (Figure 5D). 359

The cell percentage in gating area of the control group was 12.00%. The percentages 360

of groups treated with 62.5, 250, 1000 µg/mL MC-1 or 20µg/mL LPS were 38.00%, 361

54.98%, 62.82%, 65.46%, respectively. From the results, it was known that MC-1 362

could promote the phagocytic capacity of RAW 264.7 cells. 363

When the concentration of MC-1 was below 250 µg/mL, the enhancement on 364

pinocytic and phagocytic capacity increased rapidly. It increased slowly or 365

indistinctively as the concentration was higher than 250 µg/mL. This might suggest 366

that the enhancement effect of MC-1 was limited. In fact, previous studies showed 367

that macrophages over-activated would lead inflammatory damage.31, 32

Thus, it might 368

hint that MC-1 exhibited remarkable immunomodulatory activity by enhancing the 369

pinocytic and phagocytic capacity of RAW 264.7 cells moderately. 370

Effects of MC-1 on NO, TNF-α and IL-6 Production of RAW 264.7 cells 371

Macrophage activated can induce NO expression and enhance production of 372

cytokines(TNF-α, IL-6 etc).33

Thus, the effects of MC-1 on levels of NO, TNF-α, IL-6 373

in RAW 264.7 cells were analyzed. The results were shown in Figure 6A, Figure 6B 374

and Figure 6C. Cells of the control group secreted a basal level of TNF-α, IL-6 and 375

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NO. The addition of MC-1 resulted in remarked increase in TNF-α, IL-6 and NO 376

levels. The concentration of TNF-α, IL-6 and NO after treatment by 62.5 µg/mL 377

MC-1 improved 1017 pg/mL, 153 pg/mL, 9.31 µM more than the control group, 378

respectively. Like the result shown in Figure 5B-5D, the production cytokines or NO 379

increased rapidly at the low concentrations of MC-1. The increase was slow, or 380

inapparent at the high concentrations. To further confirm the result, we examined the 381

effect of MC-1 on mRNA expression of nitric oxide synthase (iNOs), TNF-α and IL-6 382

using QPCR. As shown in Figure 6D, Figure 6E and Figure 6F, compared to the 383

control group, the mRNA expression of TNF-α, IL-6 and iNOs were increased 384

significantly. The mRNA expression of TNF-α, IL-6 and NO after treatment by 62.5 385

µg/mL MC-1 were 2.21, 9.12, 12.9 times more than the control group, respectively. 386

But the mRNA levels of cytokines or iNOs still increased rapidly at the high 387

concentrations of MC-1. It might result from that the sampling time for RNA 388

extraction and collecting supernatants was different. These results suggested that 389

MC-1 could promote the expression and secretion of TNF-α, IL-6 and NO by 390

activating RAW 264.7 cells. 391

Receptors of MC-1 on RAW 264.7 cells 392

Macrophage activation is mediated primarily through the stimulation of pattern 393

recognition receptors (PRRs). The PRRs binding polysaccharides mainly include 394

scavenger receptor I (SR), mannose receptor (MR), beta glucan receptor (GR), 395

complement receptor 3 (CR3), toll-like receptors (TLR2 and TLR4).34

In the present 396

study, the roles of SR, MR, GR, CR3, TLR2 and TLR4 on immunemodulatory 397

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19

activity of MC-1 were investigated. As shown in Figure 7A, Figure 7B and Figure 7C, 398

after treatment of anti-CR3, anti-MR, anti-TLR2, the levels of NO, TNF-α and IL-6 399

were all visibly decreased in comparison to the group treated with MC-1 only. The 400

decrement was the most, after the treatment of anti-TLR2, especially. Indistinctive 401

decrease was found in the groups treated by anti-SR, anti-GR, and anti-TLR4. It 402

indicated that TLR2, CR3 and MR were receptors of MC-1. The levels of TNF-α and 403

NO in the group treated with the mixed antibodies of TLR2, CR3 and MR didn’t show 404

significant difference with the control group. Interestingly, there was a significant 405

difference between the two groups on the level of IL-6 (p<0.05). The result hinted that 406

TLR2, CR3 and MR were the major receptors of MC-1, but it could not be excluded 407

that the other receptors existed. The six receptors used in the study just were major 408

receptors of polysaccharides. There are more than the six receptors on the 409

cytomembrane of macrophages. So the receptors of MC-1 need further confirmed and 410

investigated in the future. 411

TLR2 ligation leads to the activation of IL-1R-associated kinase (IRAK) via an 412

adaptor myeloid differentiation protein 88 (MyD88), with subsequent activation of 413

TNF receptor-associated factor 6 (TRAF-6), Jun N-terminal kinase (JNK) and NF-κB. 414

35 CR3 has been proven to be involved in the activation of phosphoinositide -3-kinase 415

(PI3K), activation of the mitogen-activated protein kinase (MAPK), and NF-κB signal 416

transduction pathways.36

MR activation leads to activation of macrophage 417

phagocytosis, endocytosis and NF-κB. Activation of these transcription pathways 418

induces expression of cytokines and iNOs.37-39

Therefore, we speculated that the 419

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20

possible mechanism of immunomodulatory activity of MC-1 was mainly through the 420

activation of the pathways induced by the three receptor types cooperating with each 421

other (Figure 7D). These pathways will be characterized in our future study. 422

The immunomodulatory activity of polysaccharides has close contact with the 423

structure, such as monosaccharide composition and glycosidic bonds.40

Previous 424

studies have proven that polysaccharides containing mannose or glucose are most 425

likely recognized by TLR2, CR3 or MR.38, 41

Therefore, the mannose and glucose 426

composition of MC-1 may contribute to the immunomodulatory activity. The 427

glycosidic bonds in polysaccharides, such as the α-(1→3)-Man, α-(1→4)-Glc and 428

α-(1→6)-Glc, always contact with the immunomodulatory activity.40

Also, MC-1 429

possessed these glycosidic bonds. 430

In this study, a new polysaccharide, MC-1 of 11.3 kDa, was purified from the roots 431

of L. meyenii. MC-1 consisted of arabinose (26.21%), mannose (11.81%), and glucose 432

(53.66%), and galactose (8.32%). The main linkage type of MC-1 were proven to be 433

(1→5)-α-L-Ara, (1→3)-α-L-Man, (1→2,6)-α-L-Man, (1→)-α-D-Glc, (1→4)-α-D-Glc, 434

(1→6)-α-D-Glc and (1→6)-β-D-Gal. MC-1 possessed significant immunomodulatory 435

activity by enhancing the pinocytic and phagocytic capacity of RAW 264.7cells, and 436

increasing the levels of NO, TNF-α, and IL-6. TLR2, CR3 and MR were confirmed to 437

be the major membrane receptors of MC-1 on RAW 264.7. From the above results, 438

MC-1 could be explored as a functional food supplement candidate for hypoimmunity 439

population. 440

FUNDING 441

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This work was financially supported by the Open Project Program of Provincial 442

Key Laboratory of Green Processing Technology and Product Safety of Natural 443

Products (201304) and National Natural Science Foundation of China (Grant No. 444

31201330). 445

446

447

448

449

450

451

452

453

454

455

456

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33. Schepetkin, I. A.; Faulkner, C. L.; Nelson-Overton, L. K.; Wiley, J. A.; Quinn, M. T., Macrophage 535

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1996, 156, 1235-1246. 544

37. Schepetkin, I. A.; Quinn, M. T., Botanical polysaccharides: Macrophage immunomodulation and therapeutic 545

potential. Int Immunopharmacol 2006, 6, 317-333. 546

38. Liao, W.; Luo, Z.; Liu, D.; Ning, Z.; Yang, J.; Ren, J., Structure Characterization of a Novel Polysaccharide 547

from Dictyophora indusiata and Its Macrophage Immunomodulatory Activities. J Agr Food Chem 2015, 63, 548

535-544. 549

39. Gordon, S., Pattern recognition receptors: doubling up for the innate immune response. Cell 2002, 111, 550

927-930. 551

40. Ferreira, S. S.; Passos, C. P.; Madureira, P.; Vilanova, M.; Coimbra, M. A., Structure–function relationships 552

of immunostimulatory polysaccharides: A review. Carbohyd Polym 2015, 132, 378-396. 553

41. Figueiredo, R. T.; Bittencourt, V. C. B.; Lopes, L. C. L.; Sassaki, G.; Barreto-Bergter, E., Toll-like receptors 554

(TLR2 and TLR4) recognize polysaccharides of Pseudallescheria boydii cell wall. Carbohyd Res 2012, 356, 555

260-264. 556

557

558

559

560

561

562

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FIGURE CAPTIONS 573

Figure1. Chromatography of the polysaccharides from Lepidium meyenii by 574

DEAE-Sepharose Fast Flow chromatography (A) and Sephadex G-100 (B); UV 575

spectrum (C) of MC-1. 576

Figure2. HPGPC (A), FTIR spectrum (B), and triple helical conformation analysis (C) 577

of MC-1 578

Figure3. Ion-exchange chromatography of the monosaccharide mixture (A) and 579

MC-1 sample (B). GC of MC-1 after Smith degradation (C). The production of MC-1 580

after Smith degradation: 1.693 (glycol), 3.361 (glycerol), 8.260(phycite), 11.320 581

(arabinose), 17.620 (glucose), 19.555 (inositol). 582

Figure4. 13

C NMR spectrum (A) and 1H NMR spectrum (B) of MC-1 583

Figure5. (A) Effect of MC-1 on the viability of RAW 264.7 cells. (B) Effect of MC-1 584

on taking neutral red of RAW 264.7 cells. Fluorescence microscopic images (C) and 585

the fluorescence intensity (D) of RAW 264.7 cells phagocytosing FITC-labeled E.coli. 586

The group without MC-1 was used as the negative control, and LPS (20µg/mL) was 587

used as the positive control group. *, p <0.05 and **, p < 0.01, versus the negative 588

control group. The data shown are means ± SD. 589

Figure6. Effects of MC-1 on secretion levels of TNF-α(A), IL-6(B), NO(C) and 590

mRNA levels of TNF-α(D), IL-6(E), iNOs (F) in RAW 264.7 cells. The group without 591

MC-1 was used as the negative control, and LPS (20µg/mL) was used as the positive 592

control group. *, p <0.05 and **, p < 0.01, versus the negative control group. The data 593

shown are means ± SD. 594

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Figure7. Roles of SR, MR, GR, CR3, TLR2, TLR4 on MC-1 enhancing TNF-α (A), 595

IL-6 (B) and NO (C) secretion in RAW 264.7 cells. The cells were incubated with 596

antibodies of the receptors for 2h and then washed with PBS three times before 597

stimulating with MC-1 (125µg/mL). The anti-mix was the group combined treatment 598

of anti-TLR2, anti-CR3, and anti-MR. (D) Possible mechanism of MC-1 activating 599

RAW 264.7 cells. *, p <0.05 and **, p < 0.01, versus the negative control group; △, p 600

<0.05 and △△, p < 0.01, the groups treated with antibodies versus the group treated 601

with only MC-1 (125 µg/mL). The data shown are means ± SD. 602

603

604

605

606

607

608

609

610

611

612

613

614

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Table1. Primers used in QPCR.

genes primer sequences

iNOs

TNF-α

IL-6

GAPDH

Forward:5’-CGGCAAACATGACTTCAGGC-3’

Reverse :5’-GCACATCAAAGCGGCCATAG-3’

Forward:5’-GGGGATTATGGCTCAGGGTC-3’

Reverse:5’-CGAGGCTCCAGTGAATTCGG-3’

Forward:5’-TACTCGGCAAACCTAGTGCG-3’

Reverse:5’-GTGTCCCAACATTCATATTGTCAGT-3’

Forward:5’-TTTGTCAAGCTCATTTCCTGGTATG-3’

Reverse: 5’-TGGGATAGGGCCTCTCTTGC-3’

Table2. Glycosidic linkage composition of methylated MC-1

retention

time(min) methylated sugar linkage

molar

ratio

21.083 2,3-Me2-Araf 1,5- 27.13

23.064 2,4,6-Me3-Manp 1,3- 3.03

23.311 3,4-Me2- Manp 1,2,6- 1.83

22.232 2,3,4,6-Me4-Glcp T- 8.75

24.240 2,3,6-Me3-Glcp 1,4- 43.37

26.431 2,3,4-Me3-Glcp 1,6- 14.01

24.981 2,3,4 -Me3-Galp 1,6- 1.88

Table3. Chemical shifts of resonances in the 1H and

13C NMR spectra of MC-1

sugar residue chemical shift (ppm)

C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6

→5)-α-L-Ara(1→ 106.36/5.28 80.93/4.09 76.64/3.92 84.00/3.64 69.14/4.00 -/-

→3)-α-L-Man(1→ 95.72/5.12 76.64/4.18 69.89/3.83 69.14/3.61 71.12/3.65 60.42/3.83

→2,6)-α-L-Man(1→ 106.85/5.05 76.15/4.19 76.50/3.68 69.27/3.61 71.69/3.68 62.30/3.72

α-D-Glc(1→ 99.48/4.96 71.50/3.62 73.29/3.68 69.27/3.61 71.12/3.70 60.68/3.83

→4)-α-D-Glc(1→ 99.56/4.53 76.64/3.68 73.93/4.09 72.81/3.72 71.00/3.89 60.04/3.75

→6)-α-D-Glc(1→ 99.69/5.03 72.81/3.83 74.49/3.54 71.50/3.50 69.89/3.75 61.26/3.95

→6)-β-D-Gal(1→ 103.62/4.51 74.49/3.27 76.81/3.45 71.12/3.52 76.15/3.50 61.05/3.70

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Figure1

200 300 400 500 600 700 800

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Wavenumber(cm-1

)

Ab

sorb

an

ce

260nm

280nm

C

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A

mV

0.0 0.1 0.2 0.3 0.4 0.5485

490

495

500

505

NaOH(mol/L)

Congo Red

Congo Red+MC-1

（（（（））））

Con

go R

edn

m

C

Figure2

4000 3500 3000 2500 2000 1500 1000 500

20

30

40

50

60

70

80

90

100

89

7.0

0

57

3.8

5

104

3.7

8

142

2.0

0

16

37

.86

29

27.0

2

Tra

nsm

itta

nce

(%)

Wavenumber (cm-1)

34

06

.25

B

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Figure3

min 2 4 6 8 10 12 14 16 18

pA

20

40

60

80

100

120

140

1.693

3.361 8.260

11.320 17.620

19.555

C

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A

B

Figure4

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C

LPSMC-1(1000µg/mL)MC-1(250µg/mL)MC-1(250µg/mL)

MC-1(250µg/mL)MC-1(62.5µg/mL)

MC-1(62.5µg/mL)

0 62.5 125 250 500 1000 LPS

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

******

****

*

OD

54

0nm

MC-1(µg/mL)

B

62.5 125 250 500 1000

40

50

60

70

80

90

100

110

120

Cel

l su

rviv

al r

ate

%

MC-1(µg/mL)

A

103

102

10110

0

FITC

Control

LPS

ControlControl

Control

FITC10

310

210

110

0

Control

MC-1(62.5µg/mL)

FITC10

310

210

110

0

Control

MC-1(250µg/mL)

FITC10

310

210

110

0

Control

MC-1(1000µg/mL)

D

Figure5

Control

Magnification:×200

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Figure6

mmmMM

0 62 .5 1 25 250 500 1000 LPS

0

10

20

30

40

50

**

**

**

****

**

Rela

tive m

RN

A l

evel

s (i

NO

s)

MC-1 (µg/mL)

F

0 62 .5 1 25 250 500 1000 LPS

0

10

20

30

40

50

60

70

**

**

**

**

**

**Rela

tiv

e m

RN

A le

vel

s (I

L-6

)

MC-1 (µg/mL)

E

0 62 .5 1 25 250 500 1000 LPS

0

1

2

3

4

5

6

**

**

**

**

**

Re

lative

mR

NA

lev

els

(TN

F-a

)

MC-1(µg/mL)

**

D

0 62 .5 1 25 250 500 1000 LPS

0

10

20

30

40

50

60 **

****

****

**

Con

cen

trat

ion o

f N

O (µ

M)

MC-1 (µg/mL)

C

0 62 .5 1 25 250 500 1000 LPS

0

500

1000

1500

2000

2500

*

**

****

**

**

Con

centr

atio

n o

f IL

-6 (

pg/m

L)

MC-1 (µg/mL)

B

0 62 .5 1 25 250 500 1000 LPS

0

1000

2000

3000

4000

5000

**

******

**

Conce

ntr

atio

n o

f T

NF

-a (

pg

/mL

)

MC-1 (µg/mL)

**

A

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D

Figure7

cont

rol

anti-

SR

anti-

GR

anti-

TLR4

anti-

CR3

anti-

TLR2

anti-

MR

anti-

mix

MC-1

LPS

0

5

10

15

20

25

30

50

55

60

△

△△

△△△△

**

**

**

***

******

Con

centr

atio

n o

f N

O (

µM

)

C

cont

rol

anti-

SR

anti-

GR

anti-

TLR4

anti-

CR3

anti-

TLR2

anti-

MR

anti-

mix

MC-1

LPS

0

100

200

300

400

500

600

2000

2500

△△

△

△△

△△

**

**

*

**

**

**

****

**

Con

centr

atio

n o

f IL

-6 (

pg/m

L)

B

cont

rol

anti-

SR

anti-

GR

anti-

TLR4

anti-

CR3

anti-

TLR2

anti-

MR

anti-

mix

MC-1

LPS

1000

1500

2000

2500

3000

3500

4000

4500

△△

△△

△△

△△

**

**

**

*

**

******

Conce

ntr

atio

n o

f T

NF

-α (

pg

/mL

)

A

Page 34 of 35



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Page 35 of 35



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