A novel polysaccharide from the roots of Millettia Speciosa Champ: preparation, structural characterization and immunomodulatory activity

Journal Pre-proof A novel polysaccharide from the roots of Millettia Speciosa Champ: preparation, structural characterization and immunomodulatory activity

Zhi Huang, Ying-Jie Zeng, Xi Chen, Si-Yuan Luo, Lei Pu, FangZhou Li, Min-Hua Zong, Wen-Yong Lou PII:

S0141-8130(19)37907-3

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.166

Reference:

BIOMAC 14194

To appear in:

International Journal of Biological Macromolecules

Received date:

30 September 2019

Revised date:

17 December 2019

Accepted date:

19 December 2019

Please cite this article as: Z. Huang, Y.-J. Zeng, X. Chen, et al., A novel polysaccharide from the roots of Millettia Speciosa Champ: preparation, structural characterization and immunomodulatory activity, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.12.166

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© 2019 Published by Elsevier.

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A novel polysaccharide from the roots of Millettia Speciosa Champ: Preparation, structural characterization and immunomodulatory activity

Zhi Huanga, Ying-Jie Zenga, Xi Chena, Si-Yuan Luoa, Lei Pua, Fang-Zhou Lia,

Lab of Applied Biocatalysis, School of Food Science and Engineering, South China

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a

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Min-Hua Zonga,b, Wen-Yong Loua,b,*

Guangdong Province Key Laboratory for Green Processing of Natural Products and

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b

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University of Technology, No. 381 Wushan Road, Guangzhou 510640, China.

*

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Guangzhou 510640, China.

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Product Safety, South China University of Technology, No. 381 Wushan Road,

Corresponding author: Wen-Yong Lou, Professor

Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, No. 381 Wushan Road, Guangzhou 510640, China Tel.: +86-020-22236669 E-mail: [email protected]

1

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Abstract: A novel polysaccharide fraction (MSCP2) was extracted and isolated from the roots of Millettia Speciosa Champ. Structural characterization revealed that MSCP2 had an average molecular weight of 2.85 x 104 Da and was composed of fucose, arabinose, galactose, glucose and xylose with a ratio of 2.20 : 2.52 : 4.04 : 87.29 : 3.96. Methylation analysis and nuclear magnetic resonance (NMR) analysis showed that the main glycosidic linkage types of MSCP2 were proved to be

→3,4)-β-L-Fucp-(1→

and

→6)-β-D-Galp-(1→,

of

α-L-Araf-(1→,

→4)-α-D-Glcp-(1→, →4)-α-D-Xylp-(1→,

→4)-α-D-GalpA-(1→.

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α-D-Glcp-(1→,

The

-p

immunomodulatory assay suggested that MSCP2 could significantly improve the

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pinocytic capacity and increase the secretion of nitric oxide (NO) and cytokines by

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regulating the corresponding mRNA expression in RAW 264.7 cells. The data from

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the membrane receptor assay demonstrated that the potential mechanisms of MSCP2-induced macrophage activation were mainly through toll-like receptor 4

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(TLR4), scavenger receptor type A (SRA) and glucan receptor (GR)-mediated signaling pathways. These results suggested that MSCP2 can be developed as a promising immunomodulatory agent in functional foods. Keywords: Millettia Speciosa Champ polysaccharide; structural characterization; immunomodulatory activity

2

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1 Introduction Millettia Speciosa Champ. (M. speciosa) is a member of Leguminous family, which is mainly distributed in tropical and subtropical regions especially in southeast China.[1] The roots of M. speciosa are commonly applied as edible food and folk medicine to prevent and treat a variety of diseases, including hepatitis, arthritis,

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irregular menstruation and cough.[2, 3] These pharmacological properties were associated tightly with bioactive molecules in the roots of M. speciosa, such as

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flavonoids and polysaccharides. Previous researches primarily focused on the

-p

flavonoids, which exhibited good antioxidant activity.[4-6] Polysaccharides are also

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the major bioactive compositions in the roots of M. speciosa. In recent years, natural

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polysaccharides derived from plants, fungi and animals have attracted great attention

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due to their potent biological activities, for example, antibacterial,[7] anti-tumor,[8] anti-inflammatory[9] and immunological activities.[10] However, to the best of our

Jo ur

knowledge, there are limited reports available in the literature regarding the precise structure and biological activity of polysaccharides from the roots of M. speciose. The innate immune system is the first line of defense protecting the hosts against invading pathogens. Macrophage is an important kind of immune cell in the innate immune system and makes great contribution to defending against microbial infection, cancers and immunological diseases. In the immune response, the phagocytosis of macrophages was firstly activated to remove the pathogens and tumor. Moreover, macrophages can also kill these external intruders through secreting proinflammatory cytokines and cytotoxic components, such as NO, tumor necrosis factor-alpha (TNF-α) 3

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and interleukin-6 (IL-6). Thus, macrophages are generally applied as a classic cell model for evaluation of the immunological activity of the active ingredients. Studies showed that polysaccharides could be recognized by receptors on the macrophage surface, which triggered specific signaling transcription pathways and finally activated macrophage.[11, 12] Furthermore, such specific immunomodulatory

of

properties were found to be connected closely with certain characteristic structure of polysaccharides.[13]

ro

In the present study, the structural properties and immunomodulatory activity of

-p

a novel polysaccharide (MSCP2) from the roots of M. speciosa were elucidated. The

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detailed structure of MSCP2 was characterized by ion chromatography (IC), gas

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chromatography-mass spectrometer (GC-MS), Fourier transform infrared (FT-IR)

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and NMR. The immunomodulatory activity of MSCP2 was evaluated using murine macrophage RAW 264.7 cells. Additionally, the molecular mechanisms of

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MSCP2-induced macrophage activation were explored through membrane receptor analysis. The results from this study could provide critical information for understanding the relationship between structural properties and immunomodulatory activity of polysaccharides from the roots of M. speciosa. 2 Materials and methods 2.1 Materials and reagents The roots of M. speciosa was collected from Guangdong Province of China. AB-8 macroporous resin was obtained from Yuanye Biotechnology Co., Ltd (Shanghai, China). DEAE-52 and Sephadex G-100 were purchased from Dingguo 4

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Changsheng Biotechnology Co., Ltd (Beijing, China). Standard monosaccharides (including arabinose, fucose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid) were acquired from Boao Biotechnology Co., Ltd (Shanghai, China). RAW 264.7 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s

of

medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), streptomycin and penicillin were purchased from Gibco Life Technologies (Waltham,

ro

MA, USA). Mouse TNF-α, and IL-6 ELISA kits were obtained from Neobioscience

-p

Technology Co., Ltd (Shenzhen, China). NO detecting kit was purchased from

re

Biyotime Biotechnology Co., Ltd (Shanghai, China). Anti-toll-like receptor 4

antibody

(anti-SR),

anti-beta-glucan

receptor

antibody

(anti-GR),

na

receptor

lP

antibody (anti-TLR4), anti-toll-like receptor 2 antibody (anti-TLR2), anti-scavenger

anti-mannose receptor antibody (anti-MR) and anti-complement receptor 3 antibody

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(anti-CR3) were obtained from Bioswamp Biotechnology Co., Ltd (Wuhan, China). The above antibodies originated from rabbit are polyclonal. The endotoxin test kit was acquired from Xiamen Bioendo Technology Co., Ltd (Xiamen, China). All other chemical reagents used in this study were analytical grade. 2.2 Preparation of polysaccharides Fresh roots of M. speciosa were washed and dried at 60 °C by hot air drying and grinded into powder using a disintegrator. The powder was extracted with distilled water at a ratio of 1:20 (w/v) at 90 °C for 2 h with continuously stirring. After extraction twice, the combined extracts were centrifuged at 5000 g for 10 min to 5

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remove the residue. The supernatant was then collected and concentrated to one-fourth of the initial volume using a rotary evaporator at 60 °C. After that, the AB-8 macroporous resin was used to remove pigment and protein from concentrated solution. The protein in concentrated solution was also removed by the Sevag method for five times. Then, the resulting solution was precipitated by adding four-fold

lyophilized to obtain crude polysaccharides (MSCP).

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volume 100 % ethanol at 4 °C for 12 h. The precipitate was centrifuged, collected and

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MSCP was purified by DEAE-52 anion-exchange chromatography column (2 ×

-p

60 cm), which was eluted with distilled water and NaCl solution (0.2, 0.4, 0.6 M,

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respectively) at a flow rate of 2 mL/min. The eluent fractions were collected using an

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automatic collector and analyzed by the phenol-sulfuric acid method. Based on

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elution curve, two fractions named MSCP1 (eluted with distilled water) and MSCP2 (eluted with 0.2 M NaCl) were successfully obtained followed by concentration,

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dialysis (MW cut-off 3.5 kDa) and lyophilization. After that, the collected fractions were further purified by Sephadex G-100 chromatography column (1 × 60 cm), which was washed with distilled water at a flow rate of 0.1 mL/min. 2.3 Structural characterization of MSCP2 2.3.1 Determination of molecular weight The molecular weight of MSCP2 was determined using a Waters HPLC system according to our previous method.[14] The system includes two serially linked columns a TSK-GEL G-5000 PWXL column (300 mm × 7.8 mm inner diameter, 10 μm) and a TSK-GEL G-3000 PWXL column (300 mm × 7.8 mm inner diameter, 6 6

Journal Pre-proof μm), and a Waters 2410 differential refractive index detector. The column was eluted with 0.02 mol/L KH2PO4 at a flow rate of 0.6 mL/min. MSCP2 sample (3 mg) was dissolved in 1 mL of mobile phase and filtered through a 0.22 μm microporous filtering film. 2.3.2 Methylation analysis

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The methylation analysis of MSCP2 was carried out according to the method reported by Wang et al.[15] with some modifications. Briefly, MSCP2 (20 mg) was

ro

completely dissolved in 6 mL DMSO by ultrasound. Then 240 mg NaOH was added

-p

to the solution and sonicated for 1 h. The mixture was added to 3.6 mL methyl iodide

re

and stirred for 12 h in the dark. This methylation procedure was conducted for three

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times until the disappearance of OH band (3200-3700 cm-1) in the IR spectrum and

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stopped by adding 6 mL distilled water. The obtained solution was dialyzed against distilled water for 24 h and extracted with CHCl3 for three times. The resulting

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extracts were evaporated to dryness and the residue was hydrolyzed by 4 ml 2 M trifluoroacetic acid at 105 °C for 6 h. Afterwards, the hydrolysates were dissolved into 4 mL distilled water and the pH of solution was adjusted to 10-12 with 10% NaOH. Then, the hydrolysates was reduced by NaBH4 (100 mg) for 12 h and acetylated with 2 mL acetic anhydride and 2 mL pyridine at 90 °C for 1 h. The acetylated derivatives were extracted with 2 mL CH2Cl2 and analyzed by GC-MS (7890A-5975C, Agilent, USA) equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm). The initial temperature of column was 150 °C and held for 2 min, then increased to 180 °C at 10 °C min-1 and held for 2 min, and then increased to 260 °C at 15 °C min-1 and 7

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held for 5 min. The injection temperature and ion source temperature was 250 °C. 2.3.3 Monosaccharide composition analysis The monosaccharide composition of MSCP2 was measured according to the previous study.[16] Briefly, MSCP2 was hydrolyzed as described above. Then, trifluoroacetic acid was removed using a rotary evaporator. In order to completely

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remove residual trifluoroacetic acid, the residue was re-dissolved in methanol and evaporated to dryness for five times. Finally, the hydrolysate of MSCP2 was dissolved

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in distilled water, filtered through a 0.22 μm microporous filtering, and analyzed by a

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Dionex ion chromatography (ICS 3000, Sunnyvale, CA, USA). The monosaccharide

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contents of MSCP2 were calculated according to the calibration curves of their

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2.3.4 FT-IR analysis

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corresponding monosaccharide standards.

MSCP2 sample was mixed with spectroscopic grade potassium bromide powder

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and analyzed by Fourier transform infrared spectrophotometer (Bruker, Ettlingen, Germany) in the 400-4000 cm-1 vibration region. 2.3.5 NMR analysis

MSCP2 sample (30 mg) was dissolved in 0.5 mL of D2O. The 1D NMR (1H NMR, 13C NMR) and 2D NMR (COSY, HSQC and HMBC) spectra were recorded on a Bruker 600 MHz NMR apparatus (Bruker Crop, Fallanden, Switzerland). 2.3.6 Determination of triple-Helix structure The triple-helix structure of MSCP2 was measured according to the Congo red method.[17] Briefly, MSCP2 (2 mg) was dissolved in 1 mL of distilled water and then 8

Journal Pre-proof mixed with 1 mL Congo red solution (100 μmol/L). The transitions of maximum absorption wavelength of the complex were determined at different NaOH concentrations. 2.3.7 Ultrastructural analysis MSCP2 was coated with a thin gold layer and observed by a scanning election

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microscope (HT S-3400 N, Hitachi, Japan).

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2.4 Immunomodulatory activity of MSCP2 2.4.1 Cell culture

-p

RAW 264.7 cells were cultured in DMEM medium (containing 10% FBS, 100

CO2.

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2.4.2 Cell viability

lP

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μg/mL streptomycin, and 100 units/mL penicillin) at 37 °C in the incubator with 5%

The effect of MSCP2 on the viability of RAW 264.7 cells was performed by cell

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counting kit-8 (CCK-8) assay. The cells were incubated in 96-well plate at a density of 1×105 cells/well for 24 h. After removal of the old medium, 100 μL of MSCP2 with different concentrations (0, 6.25, 12.5, 25, 50, 100 μg/mL) and 5 μg/mL lipopolysaccharide (LPS) were seeded into 96-well plate and the cells were incubated for another 24 h. Then, 10 μL of CCK-8 solution was added into each well. After 0.5 h of incubation, the absorbance at 450 nm was determined by a microplate reader. 2.4.3 Determination of pinocytic capacity The effect of MSCP2 on pinocytic capacity of RAW 264.7 cells was determined by neutral red assay.[18] As described above, RAW 264.7 cells were treated with 9

Journal Pre-proof MSCP2 or LPS. 24 h later, the medium was replaced by 100 μL of 0.1% neutral red and the cells were cultured for 1 h. After each well was washed with PBS for three times, 100 μL of cell lysis solution (acetic acid : ethanol = 1:1, v/v) was injected into each well and the plate was kept overnight. Finally, the absorbance at 540 nm was measured using a microplate reader. 2.4.4 Measurement of NO, TNF-α and IL-6

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RAW 264.7 cells were seeded in 96-well plate at a density of 1×105 cells/well for

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24 h. The medium was discarded and the cells were incubated with MSCP2 or LPS

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for another 24 h. Next, the cell supernatants were collected and the levels of NO,

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TNF-α and IL-6 were measured using NO kit, TNF-α ELISA kit and IL-6 ELISA kit,

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2.4.5 QRT-PCR analysis

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respectively.

RAW 264.7 cells were loaded onto 6-well plate at a density of 1×106 cells/well

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followed by incubation of 24 h. Then, cells were incubated with MSCP2 or LPS for another 24 h. The cells were washed with cold PBS twice and the total RNA was extracted by 1 mL of Trizol reagent. The obtained RNA was reversed into cDNA using cDNA synthesis kit. Finally, the levels of cDNA encoding iNOS, TNF-α and IL-6 were amplified by a quantitative real-time polymerase chain reaction (QRT-PCR) assay. The sequences of specific primers were listed in Table S1. GAPDH was used for the internal reference, and the expression levels of mRNA were calculated through 2-△△Ct method. 2.4.6 Membrane receptor analysis 10

Journal Pre-proof RAW 264.7 cells were loaded onto 6-well plate at a density of 1×105 cells/well and incubated for 24 h. The old medium was removed, and then the cells were pre-treated with different antibodies (anti-TLR4, anti-TLR2, anti-SR, anti-GR, anti-MR, anti-CR3, 5 μg/mL ) for 2 h. The supernatant was discarded and the wells were washed by PBS for three times. Subsequently, the cells were stimulated with

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MSCP2 (50 μg/mL). The group treated with only MSCP2 was used as the control. The untreated cells and LPS-treated cells were used as negative and positive control,

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

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incubation.

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2.5 Statistical analysis

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All data were expressed as means ± standard deviation from three separate

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experiments. The significant differences between the groups was analyzed by

significant.

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one-way ANOVA (Origin 8.5). Differences at p < 0.05 were considered statistically

3 Results and discussions

3.1 Extraction, purification and chemical composition of MSCP2 The crude polysaccharides were isolated from the roots of M. speciosa with a yield of 2.86 % (w/w). After purification by DEAE-52 column (Fig. 1A), two fractions (MSCP1 and MSCP2) were obtained. In this study, we mainly focused on MSCP2, and MSCP1 will be investigated in the future work. The fraction MSCP2 was further purified by Sephadex G-100 chromatography column. As shown in Fig. 1B, a single and symmetrical peak was observed. After dialysis and lyophilization, the 11

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total carbohydrate, protein, and uronic acid content of MSCP2 were determined to be 91.86 ± 0.80 %, 5.73 ± 0.19 % and 0.76 ± 0.01 %, respectively. The endotoxin contents of MSCP2 (100 μg/mL) and the positive control (LPS, 0.01μg/mL) were 1.46 ± 0.02 EU/mL and 2.58 ± 0.13 EU/mL, respectively. It was found that the endotoxin content of MSCP2 was almost half that of LPS, however, the concentration

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of MSCP2 was 10,000 times that of LPS. The results indicated that the endotoxin content in MSCP2 was too small to affect the immune response in RAW 264.7 cells.

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The average molecular weight of MSCP2 was measured by HPGPC as

-p

demonstrated in Fig. 1C. A symmetrical single peak was obtained, indicating that

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MSCP2 was homogeneous polysaccharide. The average molecular weight of MSCP2

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was calculated as 2.85 x 104 Da, which indicated that MSCP2 had a relatively low

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molecular weight in comparation with other plant polysaccharides.[14, 19, 20] The molecular weight of polysaccharide had a great influence on its bioactivities.[21]

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Generally, as the polysaccharides with high molecular weight appeared high viscosity and poor water-solubility, it is difficult for them to bind to the receptors and exhibit their bioactivities.[22, 23] Therefore, low molecular weight might endow polysaccharides

some

properties

different

from

high

molecular

weight

polysaccharides. The monosaccharide composition of MSCP2 was determined by ion chromatography. The results were presented in Fig. 2A (monosaccharide standard) and Fig. 2B (MSCP2). It was found that MSCP2 was composed of fucose, arabinose, galactose, glucose and xylose at approximate percentages of 2.20, 2.52, 4.04, 87.29 12

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and 3.96%, respectively, which indicated that glucose might be the main backbone of MSCP2. 3.2 Structural characterization of MSCP2 3.2.1 FT-IR spectrum The major functional groups and chemical bonds of MSCP2 were analyzed by FT-IR spectrum. As showed in Fig. 2C, the absorption peaks observed at 3442 and

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2929 cm-1 corresponded to O-H and C-H stretching vibrations, respectively.[24] The

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peaks at 1640 and 1415 cm-1 were as assigned to the stretching vibration of C=O and

-p

the blending vibration of C-H, respectively.[25, 26] The peaks in region of 1000-1200

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cm-1 might be attributed to the characteristic absorbance of C-O-C and C-O-H bond,

lP

which indicated the presence of pyranose ring.[27, 28] Besides, the absorption at 860

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cm-1 was considered to the characteristic of α-glycosidic bond.[29] In consequence, FT-IR analysis indicated that MSCP2 possessed typical groups of polysaccharides.

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3.2.2 Glycosidic linkages

The glycosidic linkages of MSCP2 were measured according to the methylation method. Based on the mass spectrum patterns and the standard data in the CCRC Spectral Database, the results were listed in Table 1. It can be concluded that MSCP2 was

mainly

consisted

of

→4)-Xylp-(1→,

→6)-Galp-(1→,

Araf-(1→,

→3,4)-Fucp-(1→, Glcp-(1→ and →4)-Glcp-(1→ linkages. The results agreed well with monosaccharide composition analysis, which demonstrated that MSCP2 was a highly-branched heteropolysaccharide with several glycosidic linkages types. The main backbone of MSCP2 appeared to be →4)-Xylp-(1→, →6)-Galp-(1→, 13

Journal Pre-proof →3,4)-Fucp-(1→, and →4)-Glcp-(1→. The presence of →3,4)-Fucp-(1→ indicated that it constructed the linkage of backbone and branch. However, the signal of uronic acid was not detected in GC-MS analysis. The possible reason may be that uronic acid in MSCP2 would be translated into methyl esters and then degraded by β-elimination in highly alkaline condition during methylation treatment.[30] The degree of

of

branching (DB) value of MSCP2 was calculated as 29.16% following the equation: DB=(NB+NT)/(NB+NT+NL), where NB, NT and NL represented the number of the

-p

(1,4-Xylp, 1,6-Galp, 1,4-Glcp), respectively.

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branch residues (1,3,4-Fucp), terminal residues (T-Glcp, T-Araf), and line residues

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3.2.3 NMR spectrum

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The fine structure of MSCP2 was further elucidated by NMR spectrum. In the 1H

na

NMR spectrum (Fig. 3B), it was observed that seven anomeric proton signals appeared at δ 5.32, 4.97, 5.00, 4.57, 5.17, 4.56 and 4.57 ppm. The signal at δ 1.19

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ppm was assigned to methyl proton.[25] In the

13

C NMR spectrum (Fig. 3A), seven

kinds of anomeric carbon signals were found at δ 99.70, 100.56, 98.42, 102.44, 109.29, 103.48 and 100.75 ppm. Generally, the chemical shifts of pyranose residues in the region of δ 98-100 ppm were attributed to α-anomeric configuration, and the chemical shifts from δ 100 to 105 ppm were assigned to β-anomeric configuration.[31] The resonance at δ 16.58 ppm indicated the presence of Fucp.[15, 32] The weak signal at δ 175.42 ppm was assigned to carboxyl group of GalpA. Besides, in the HSQC spectrum (Fig. 3D), seven cross peaks were observed at δ 5.32/99.70, 4.97/100.56, 5.00/98.42, 4.57/102.44, 5.17/109.29, 4.56/103.48 and 4.57/100.75 ppm, 14

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which indicated the existence of seven residues in MSCP2 (named as residue A, B, C, D, E, F and G, respectively) and chemical shifts at δ 99.70, 100.56, 98.42, 102.44, 109.29, 103.48 and 100.75 ppm were assigned to C-1 of residues A-G, respectively. For residue A, the cross peaks at δ 5.32/4.56, 4.56/4.04, 4.04/3.69, 3.69/3.88 and 3.88/3.76 ppm were found in the COSY spectrum (Fig. 3C). The signal at δ 5.32

of

corresponded to H-1 of residue A. Meanwhile, resonances at δ 4.56, 4.04, 3.69, 3.88 and 3.76 ppm were attributed to H-2, H-3, H-4, H-5 and H-6 of residue A. Besides, in

ro

the HSQC, the cross peaks at δ 76.86/4.56, 73.33/4.04, 73.86/3.69, 71.17/3.88,

-p

60.46/3.76 ppm were observed, which implied that δ 76.86, 73.33, 73.86, 71.17, 60.46

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ppm corresponded to C-2, C-3, C-4, C-5 and C-6 of residue A, respectively. Based on

lP

NMR analysis, monosaccharide composition, methylation analysis and previous

Similarly,

residue

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literature[24], it could be inferred that residue A was (1→4)-linked-α-D-Glcp. B-F

be

identified

as

(1→6)-linked-β-D-Galp,

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(1→4)-linked-α-D-Xylp,

could

(1→)-linked-α-D-Glcp, (1→)-linked-α-L-Araf,

(1→3,4)-linked-β-L-Fucp and (1→4)-linked-α-D-GlcpA, respectively. The above results were in a good accordance with the reported studies.[25, 26, 32-34] The entire assignment of chemical shifts was summarized in Table 2. The sequence of glycosidic linkages can be assigned by the HMBC spectrum (Fig. 3E). The cross peaks at δ 3.69/99.70 and 3.67/98.42 ppm indicated the presence of →4)-α-D-Glcp-(1→4)-α-D-Xylp-(1→ and →4)-α-D-Xylp-(1→6)-β-D-Galp-(1→, respectively. Besides, the cross peaks at δ3.67/99.70, 3.55/103.48 and 3.67/100.75 ppm was determined as →4)-α-D-Glcp-(1→6)-β-D-Galp-(1→, →6)-β-D-Galp-(1→4) 15

Journal Pre-proof -β-L-Fucp-(3,1→ and →4)-α-D-GalpA-(1→6)-β-D-Galp-(1→, respectively. The cross peaks at δ 3.65/100.56 and 3.69/100.56 ppm suggested the existence of α-D-Glcp-(1→4)-α-D-Glcp-(1→ and α-D-Glcp-(1→4)-α-D-Xylp-(1→ in the branch chains, respectively. Based on above analysis, the backbone of MSCP2 may be composed of →4)-α-D-Glcp-(1→4)-α-D-Xylp-(1→, →4)-α-D-Glcp-(1→6)-β-D-Galp-(1→, →4)

of

→6)-β-D-Galp-(1→4)-β-L-Fucp-(3,1→

-α-D-Xylp-(1→6)-β-D-Galp-(1→,

and

residue.

And

the

branches

may

-p

Fucp

ro

→4)-α-D-GalpA-(1→6)-β-D-Galp-(1→. The branches were substituted at C-3 of consist

of

1→)-α-L-Araf,

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α-D-Glcp-(1→4)-α-D-Glcp-(1→ and α-D-Glcp-(1→4)-α-D-Xylp-(1→.

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3.2.4 Triple-Helix structure

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The Congo red method was applied to analyze the triple-helix structure of MSCP2. As displayed in Fig. 2D, a bathochromic shift of the maximum absorption

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wavelength from 497 nm to 502 nm was observed after mixing MSCP2 with Congo red solution, which indicated that MSCP2 had formed a complex with Congo red. Generally, the maximum absorption wavelength of the complex would appear a sharp decrease as NaOH concentration increased, indicating that the triple-helix structure of the complex was damaged. However, the result demonstrated that the maximum absorption wavelength decreased slightly with the increase in NaOH concentration, suggesting that MSCP2 did not present a triple helix structure. The results was similar to those of other polysaccharides, such as, MC-1[35], DG2[14] and CAVAP-1[17]. The reason why MSCP2 resulted in a bathochromic shift of the maximum absorption 16

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wavelength may be that MSCP2 owned another conformation which could form a stable complex with Congo red in NaOH solutions.[35] Therefore, the detailed conformation of MSCP2 was necessary to further investigate. 3.2.5 Ultrastructural analysis As presented in Fig. 2E, the SEM images (1000x and 2000x magnifications)

of

showed that the surface of MSCP2 was mainly in the form of sheets and rods, which

3.3 Immunomodulatory activity of MSCP2

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may be related to the branches and network structures of MSCP2.[36]

-p

3.3.1 Effect of MSCP2 on RAW 264.7 cells viability

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Prior to study the immunomodulatory activity of MSCP2, the cytotoxic effect of

lP

MSCP2 on RAW 264.7 cells was measured by CCK-8 assay. As shown in Fig. 4A,

na

after being stimulated with MSCP2 (100, 50, 25, 12.5, 6.25, μg/mL), the cell viabilities were 106.78 ± 4.95%, 105.35 ± 1.84%, 106.33 ± 6.49%, 104.75 ± 2.98%,

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102.45 ± 6.14%, respectively (p>0.05), indicating that MSCP2 (under 100 μg/mL) was nontoxic to RAW 264.7 cells. Therefore, the concentration of MSCP2 for the following experiments was below 100 μg/mL. 3.3.2 Effect of MSCP2 on pinocytic capacity of RAW 264.7 cells It is well known that the pinocytic activity is the prominent feature of macrophage activation.[37] The pinocytic capacity of macrophage could be evaluated by neutral red assay. As shown in Fig. 4B, in contrast to the control group, a dose-dependent increase of uptake rate was observed in MSCP2-treated group (12.5-100 μg/mL) (p<0.05). Specially, the uptake rate in the MSCP2-treated group 17

Journal Pre-proof (100 μg/mL) was 1.14 times more than that of the control group. The result indicated that MSCP2 could enhance the pinocytic capacity of RAW 264.7 cells. However, the pinocytic capacity of LPS-treated group was lower than that of the control group as a result of the low cell viability. A previous study stated that polysaccharides composed of fucose or mannose residues could attach to mannose receptors and trigger the

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pinocytosis.[38] Besides, another study reported that polysaccharides could improve the pinocytic capacity in immune responses by activating PI3k/Akt signaling pathway

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in the cells.[39]

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3.3.3 Effect of MSCP2 on the secretion of NO, TNF-α and IL-6

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In the immune responses, activated macrophage could secrete NO and a variety

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of cytokines (TNF-α, IL-6, etc.) to fight against cancer cells and pathogens.[12, 40]

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NO is synthesized by inducible nitric oxide synthase (iNOS), which is able to inhibit the proliferation and growth of pathogens.[41] TNF-α is one of the most powerful

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cytokines found to kill tumors so far, while IL-6 is a key cytokine that transmits signals between immune cells.[42] Therefore, in this study, the effects of MSCP2 on the secretion of NO, TNF-α and IL-6 were analyzed. As shown in Fig. 5A-C, in contrast with the control group, MSCP2 could remarkably promote the secretion of NO, TNF-α and IL-6 in a dose-dependent manner (p<0.01). Specifically, the levels of NO, TNF-α and IL-6 in the MSCP2-treated group (100 μg/mL) were 11.98, 4.15, 1.69 times more than those of the control group, respectively. The NO levels (e.g. 10.07 μmol/L of NO at 6.25 μg/mL of MSCP2 ) treated by MSCP2 much higher than those of the polysaccharide LPD2 (lower than 10 μmol/L of NO at 25 μg/mL of LPD2) 18

Journal Pre-proof from longan. [20]. The TNF-α levels (i.e. 2274.43 pg/mL of TNF-α at 6.25 μg/mL of MSCP2 ) treated by MSCP2 were higher than those of the polysaccharide FCPW80-2 (lower than 1500 pg/mL of TNF-α at 160 μg/mL of FCPW80-2) isolated from Ficus carica.[12] The IL-6 levels (e.g. 199.24 pg/mL of IL-6 at 6.25 μg/mL of MSCP2) treated by MSCP2 were higher than those of the polysaccharide MOP-2 (lower than

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40 pg/mL of IL-6 at 500 μg/mL of MOP-2) from Moringa oleifera.[16] The results suggested that MSCP2 exhibited immunomodulatory activity by promoting the

ro

secretion of NO, TNF-α and IL-6 in RAW 264.7 cells.

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3.3.4 Effect of MSCP2 on mRNA expression of iNOS, TNF-α and IL-6

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For the aim to study whether the increase of NO, TNF-α and IL-6 might be

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relevant to the gene expression, the gene transcription levels of iNOS, TNF-α and

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IL-6 were further examined using QRT-PCR. As illustrated in Fig. 5D, the mRNA expression levels of iNOS in the MSCP2-treated group were significantly improved

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compared with the control group (p<0.01). Similar trends were observed in the mRNA expression levels of TNF-α and IL-6 (Fig. 5E, F). The obtained results kept in line with the secretion levels of NO, TNF-α and IL-6. Generally, the mRNA expressions of NO and cytokines were regulated by the transcription factors (e.g. nuclear factor kappa B, NF-κB) in the nucleus, which were activated by specific signaling cascade.[43] The results indicated that MSCP2 facilitated the secretion of NO and cytokines through inducing intracellular signal transduction and up-regulating the related genes. 3.3.5 Membrane receptors for MSCP2 binding to macrophages 19

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It was considered that the first step for polysaccharides to activate macrophage was to bind to the pattern recognition receptors (PRRs) on the cell surface.[44] PRRs are non-clonal immune proteins that can recognize pathogen-associated molecular pattern, followed by activating the intracellular signal transduction pathways, leading to transcriptional regulation of inflammatory mediators that function in immune

of

responses.[45, 46] Therefore, in order to elucidate the molecular mechanisms of macrophage activation induced by MSCP2, it is of great significance to find out the

ro

PRRs that can recognize and combine with polysaccharides. In this study, RAW 264.7

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cells were treated with anti-TLR4, anti-TLR2, anti-SRA, anti-GR and anti-MR

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antibodies for 2 h before being stimulated with MSCP2, and the secretion levels of

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NO and cytokines were measured. As shown in Fig. 6A and B, after exposure to

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anti-TLR4, anti-SRA and anti-GR, it was observed that the levels of NO (30.07, 29.52 and 29.89 pg/mL in anti-TLR4, anti-SRA and anti-GR-treated group, respectively )

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were notably lower than that of group treated by only MSCP2 (32.96 pg/mL of NO). Similarly, after being treating by anti-TLR4, anti-SRA and anti-GR, the levels of TNF-α (4583.42, 4520.95 and 4478.08 pg/mL of TNF-α in the anti-TLR4, anti-SRA and anti-GR-treated group, respectively ) were also markedly lower than that of group treated by only MSCP2 (5265.71 pg/mL of TNF-α). The results demonstrated that TLR4, SRA and GR were PRRs for MSCP2 to induce the secretion of NO and TNF-α in macrophages. However, compared to the group stimulated by only MSCP2, no distinct decrease in the level of IL-6 was detected in all groups treated by antibodies (Fig. 6C). The result suggested that the PRRs for MSCP2 to induce the secretion of 20

Journal Pre-proof IL-6 were distinct from those for NO and TNF-α and other potential PPRs might exist. The different signaling pathways for the expression of NO, TNF-α and IL-6 might account for the above results. Similarly, a previous research reported that the polysaccharide (P1) purified from Prunella vulgaris stimulated NO level via the activation of toll-like receptor 2 (TLR2), while it stimulated IL-6 level via the

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activation of TLR4, TLR2 and complement receptor 3 (CR3). Besides, the receptors for P1 to induce TNF-α were different from those for NO and IL-6.[47]

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TLR4 is a pivotal Toll-like receptor and has been proved to be involved in the

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activation of myeloid differentiation protein 88 (MyD88)-dependent pathway, which

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can trigger the activation of mitogen-activated protein kinases (MAPKs) and

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NF-κB.[48] SRA belongs to a member of scavenging receptors family and can lead to

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the activation of macrophage phagocytosis, phosphoinositide-3- kinase (PI3K) and NF-κB.[49, 50] GR is a kind of nonclassical C-type lectin receptor and its signaling is

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mediated by the spleen tyrosine kinase (Syk)-caspase recruitment domain-containg protein 9 (CARD9) pathway, which results in the activation of NF-κB.[51] The activation of above signaling pathways can induce gene expression and the production of NO and cytokines. Taken together, we speculated that the potential mechanisms of MSCP2-induced macrophage immunomodulatory were mainly through TLR4, SRA and GR-mediated signaling pathways. The signaling pathways will be verified in the follow-up experiments. The biological activities of polysaccharides depend mainly on their structural characteristics, including monosaccharide composition, glycosidic linkages and 21

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conformation. Increasing evidences indicated that polysaccharides containing arabinose, galactose and glucose might have close relationship with the immunomodulatory activity,[46] and polysaccharides composed of glucose can be recognized by TLR4, SR, and GR.[52, 53] Therefore, several monosaccharide compositions (arabinose, galactose and glucose) in MSCP2 might contribute to the immunomodulatory activity. It was reported that the glycosidic linkages in

of

polysaccharides, for example, →4)-α-Glcp-(1→ linkage, were associated with the

ro

production of NO and cytokines,[54] which indicated the glucosidic linkage

-p

→4)-α-Glcp-(1→ in MSCP2 might be also conducive to its immunomodulatory

re

activity. It was noted that specific structure regions also have great influence on

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macrophage activation. A polysaccharide from Lemna minor L. did not exhibit

na

immunomodulatory activity after removal of the regions of a linear 1,4-αD-galactopyranosyluronan.[55] The rhamnogalacturonan Ⅱ-like region containing

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2-keto-3-deoxyoctulosonic acid, which is a component of LPS, might be the specific structure cross-linked with TLR4 and TLR2. Therefore, the specific active structure regions of MSCP2, which are relative to the immunomodulatory activity, need further in-depth research.[56] In addition, the triple-helix conformation of some polysaccharides is beneficial for immunomodulatory activity.[57] However, as mentioned above (3.2.4), MSCP2 exhibited a conformation distinct from triple-helix structure, thus, the effect of conformation in MSCP2 on the immunomodulatory activity needs to be further investigated. 4 Conclusions 22

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In summary, a new heteropolysaccharide MSCP2 with an average molecular weight of 2.85 x 104 Da was purified from the roots of M. speciosa. MSCP2 was composed of arabinose, fucose, xylose, glucose and galactose. The main linkage types of

MSCP2

were

proved

to

α-D-Glcp-(1→,

be

→4)-α-D-Glcp-(1→,

→4)-α-D-Xylp-(1→, →6)-β-D-Galp-(1→, α-L-Araf-(1→, →3,4)-β-L-Fucp-(1→ and

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→4)-α-D-GalpA-(1→. MSCP2 was confirmed to have significant immunomodulatory activity through improving the pinocytic capacity, and increasing the levels of NO,

ro

TNF-α, and IL-6. TLR4, SRA and GR were found to be the major PRRs for MSCP2

-p

to trigger signaling transcription and macrophage activation. These results indicated

re

that MSCP2 might be a potential candidate for application in medical and food

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Conflicts of interest

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industries.

Funding

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All authors declare no competing financial interest.

The authors are grateful for the financial support from the National Natural Science Foundation of China (21676104, 21878105, 21908070), National Key R&D Plan (2018YFC1603400, 2018YFC1602100), the Key Research and Development Program of Guangdong Province (2019B020213001), the Science and Technology Program of Guangzhou (201904010360), China Postdoctoral Science Foundation (BX20180102, 2019M652902) and the Fundamental Research Funds for the Central Universities (2019PY15, 2019MS100). References 23

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Figure Captions Fig.1

Chromatograms

of

MSCP2

purified

by

DEAE-52

anion-exchange

chromatography (A) and Sephadex G-100 (B); HPGPC profile of MSCP2 (C).

Fig.2 Ion-exchange chromatography profiles of the monosaccharide mixture (A) and

ro

analysis of MSCP2 (D); SEM images of MSCP2 (E).

of

MSCP2 sample (B); FT-IR spectrum of MSCP2 (C); Triple-helical conformation

-p

Fig.3 13C NMR (A) , 1H NMR (B) , COSY (C), HSQC (D) and HMBC (E) spectrum

lP

re

of MSCP2.

na

Fig.4 Effect of MSCP2 on the viability (A) and pinocytic capability (B) of RAW 264.7 cells. The group without MSCP2 was used as the negative control, and the

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group treated with LPS (5 μg/mL) was used as the positive control. Significant differences with the negative control group are designated as *, p<0.05, and **, p<0.01. The data are expressed as means ± SD.

Fig.5 Effect of MSCP2 on the secretion levels of NO (A), TNF-α (B), IL-6 (C) and mRNA expression levels of iNOS (D), TNF-α (E), IL-6 (F) in RAW264.7 cells. The group without MSCP2 was used as the negative control, and the group treated with LPS (5 μg/mL) was used as the positive control. Significant differences with the negative control group are designated as *, p<0.05, and **, p<0.01. The data are 32

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expressed as means ± SD.

Fig.6 Roles of TLR4, TLR2, GR, MR, SR, CR3 on the secretion of NO (A), TNF-α (B) and IL-6 (C) in RAW 264.7 cells. Significant differences between the group treated with antibodies and the negative control group (without MSCP2) are designated as *, p<0.05, and **, p<0.01; and significant differences between the

of

group treated with antibodies and the group treated with only MSCP2 (50 μg/mL) are

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na

lP

re

-p

ro

designated as #, p<0.05, and ##, p<0.01. The data are expressed as means ± SD.

33

Journal Pre-proof Author Statement:

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na

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re

-p

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Zhi Huang: Conceptualization, Data curation, Visualization, Writing- Original draft preparation, Writing- Reviewing and Editing Ying-Jie Zeng : Conceptualization, Methodology. Xi Chen: Investigation, Formal analysis. Si-Yuan Luo: Software, Validation. Lei Pu: Software, Investigation. Fang-Zhou Li: Software, Validation. Min-Hua Zong: Supervision, Project administration. Wen-Yong Lou: Conceptualization, Resources, Supervision.

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Journal Pre-proof Table 1 Glycosidic linkage composition of methylated MSCP2. Glycosidic linkage Glcp-(1→ →4)-Glcp-(1→ →4)-Xylp-(1→ →6)-Galp-(1→ Araf-(1→ →3,4)-Fucp-(1→

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Partial methylated alditol acetates 2,3,4,6-Me4-Glcp 2,3,6-Me3-Glcp 2,3-Me2-Xylp 2,3,6-Me3-Galp 2,3,5-Me3-Araf 2-Me-Fucp

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Peak area (%) 25.68 65.69 2.64 2.51 1.90 1.58

Journal Pre-proof Table 2 Assignment of 13C NMR and 1H NMR chemical shifts of MSCP2 (δ, ppm). C2/H2

C3/H3

C4/H4

C5/H5

C6/H6

→4)-α-D-Glcp-(1→ α-D-Glcp-(1→ →4)-α-D-Xylp-(1→ →6)-β-D-Galp-(1→

99.70/5.32 100.56/4.97 98.42/5.00 102.44/4.57

76.86/4.56 72.86/3.63 73.99/3.72 74.51/3.35

73.33/4.04 73.58/3.74 73.19/3.65 76.86/3.47

73.86/3.65 71.65/3.80 81.39/3.69 70.26/3.54

71.17/3.88 72.72/3.89 60.66/3.58 76.26/3.51

60.46/3.76 61.14/3.65 -/61.14/3.67

α-L-Araf-(1→ →3,4)-β-L-Fucp-(1→ →4)-α-D-GalpA-(1→

109.29/5.17 103.48/4.56 100.75/4.57

81.39/4.11 67.71/4.13 74.51/3.57

77.74/3.94 69.91/4.01 73.58/3.60

81.24/4.13 66.65/3.55 78.13/4.32

61.89/3.69 69.39/3.72 70.69/4.89

-/16.58/1.19 175.42/-

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C1/H1

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Journal Pre-proof

Highlights: 1. A novel polysaccharide (MSCP2) was isolated and purified from the roots of Millettia Speciosa Champ. 2. MSCP2 could significantly enhance the pinocytic capacity and promote the secretion of NO and cytokines in RAW 264.7 cells.

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3. The pattern recognition receptors for MSCP2 binding to macrophages were

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elucidated.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6