Isolation, structural characterization, and immunostimulatory activity of a new water-soluble polysaccharide and its sulfated derivative from Citrus medica L. var. sarcodactylis

Accepted Manuscript Isolation, structural characterization, and immunostimulatory activity of a new water-soluble polysaccharide and its sulfated derivative from Citrus medica L. var. sarcodactylis

Peng Bao, Yuanyuan Luo, Xianjing Hu, Liyan Song, Jianing Yang, Jianhua Zhu, Yao Wen, Rongmin Yu PII: DOI: Reference:

S0141-8130(18)34155-2 https://doi.org/10.1016/j.ijbiomac.2018.11.113 BIOMAC 10990

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

9 August 2018 3 October 2018 12 November 2018

Please cite this article as: Peng Bao, Yuanyuan Luo, Xianjing Hu, Liyan Song, Jianing Yang, Jianhua Zhu, Yao Wen, Rongmin Yu , Isolation, structural characterization, and immunostimulatory activity of a new water-soluble polysaccharide and its sulfated derivative from Citrus medica L. var. sarcodactylis. Biomac (2018), https://doi.org/ 10.1016/j.ijbiomac.2018.11.113

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ACCEPTED MANUSCRIPT Isolation,

structural

characterization,

and

immunostimulatory activity of a new water-soluble polysaccharide and its sulfated derivative from Citrus

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medica L. var. sarcodactylis

Biotechnological Institute of Chinese Materia Medica, Jinan University, 601 Huangpu Avenue

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a

Rongmin Yu a*

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c,

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Peng Bao a, Yuanyuan Luo b, Xianjing Hu b, Liyan Song b, Jianing Yang b, Jianhua Zhu c*, Yao Wen

b

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West, Guangzhou 510632, China

Department of Pharmacology, College of Pharmacy, Jinan University, 601 Huangpu Avenue

Department of Natural Products Chemistry, College of Pharmacy, Jinan University, Guangzhou

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c

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West, Guangzhou 510632, China

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510632, China.

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* Corresponding authors. Tel: +86-20-85220386, Fax: +86-20-85224766, E-mail: [email protected] (R. M. Yu); Tel: +86-20-85222069, Fax: +86-20-85224766, E-mail: tzhujh@jnu. edu. cn (J. H. Zhu).

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ACCEPTED MANUSCRIPT

ABSTRACT The homogeneous heteropolysaccharide CMSPW90-1 was first purified from bergamot by DEAE Sepharose Fast Flow and Sephadex G-75 column with a molecular weight of 18.8 kDa. The structure of CMSPW90-1 was elucidated with high-performance gel permeation

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chromatography, gas chromatography-mass spectrometry, infrared spectrum, methylation,

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nuclear magnetic resonance (NMR), Congo red test, and circular dichroism. In comparison with

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CMSPW90-1, the sulfated derivative CMSPW90-M1 showed significant ultra-structural differences with a molecular weight of 75.4 kDa. The antioxidant and immunomodulatory

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activities of CMSPW90-M1 were examined to determine the relationships of structure-

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bioactivity. CMSPW90-M1 exhibited stronger scavenging activities for DPPH• and ABTS+• than those of CMSPW90-1. CMSPW90-M1 exhibited more immunomodulatory activity in

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vitro by promoting the proliferation of mouse splenocytes and the neutral red phagocytosis of

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RAW264.7 cells. The results demonstrated that CMSPW90-M1 could be developed as one of

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the potential free radical inhibitors and immunomodulators.

Keywords: Polysaccharide; Citrus medica L. var. sarcodactylis.; Structure determination;

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Antioxidant activity; Immunomodulatory activity

1. Introduction Citrus medica L. var. sarcodactylis Swingle (bergamot) belongs to Rutaceae family and is an irregularly branched shrub or small tree. It originated from India and is now widely distributed mainly throughout China, France, Italy, Germany and USA. The main cultivation areas of bergamot in China are Guangdong, Fujian, Sichuan, and Zhejiang. Bergamot contains 2

ACCEPTED MANUSCRIPT polysaccharides, alkaloids, saponins, flavonoids, limonene, phytosterols, coumarin, glycoside, hesperidin, nelipid, and other physiologically active substances. It is widely used as traditional medicine, an ornamental, and for preserved fruit in China. It is traditionally utilized for treatment of tracheitis, angiocardiopathy, hypertension, respiratory tract infections, and asthma

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[1]. Also, bergamot is considered beneficial to liver, pancreatic, and stomach function [2]. In

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recent years, bergamot has been gradually developed into a functional food due to its

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antioxidant, antitumor, antibacterial, and immunomodulatory activities.

Structural features of polysaccharides, including the molecular weight, monosaccharide

[3]. Natural plant

polysaccharides

generally display certain

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biological activities

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composition, types of glycosidic bonds, and types and degrees of substitution decide their

pharmacological activities, and their biological properties can be significantly enhanced after

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changes in conformation of polysaccharides through molecular modification and structural

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improvement, including by sulfation, selenization, and phosphorylation methods [4]. Sulfated polysaccharides exert a wider variety of stronger bioactivities in comparison with native

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polysaccharides, including antioxidant, immunostimulant, anticoagulant, antitumor, and

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hypoglycemic properties [5]. However, previous studies primarily focused on the structure and activities of bergamot essential oil [6]. The structural and biological properties of polysaccharides derived from bergamot have few reports on the sulfated modification and the biological activities of derivatives. At the present paper, we report for the first time that the structural characterization of a new water-soluble polysaccharide (CMSPW90-1) extracted, purified and elucidated from bergamot by DEAE Sepharose Fast Flow and Sephadex G-75 column chromatography as well

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ACCEPTED MANUSCRIPT as physicochemical methods combined with modern instrumental analysis techniques. Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and atomic force microscopy (AFM) were used to analyze and compare the morphological and structural characteristics of CMSPW90-1 and its sulfated derivative (CMSPW90-M1) by the

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chlorosulfonic acid-pyridine method. In addition, their antioxidant and immunomodulatory

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activities were also evaluated.

2. Materials and methods

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2.1. Plant materials and chemicals

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Bergamot was obtained from the branch of Beijing Tong Ren Tang Drugstore, Guangzhou, China. The material was identified by Professor R.M. Yu, College of Pharmacy, Jinan

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University, China. Hydrogen peroxide (H2O2), sulfuric acid (H2SO4), standard monosaccharide

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samples, T-series dextrans, trifluoroacetic acid (TFA), ascorbic acid (vitamin C, Vc), chlorosulfonic acid (CSA), and pyridine (Pyr) were obtained from Guangzhou Chemical

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Reagent Company, China. Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-y1)-2,5-

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diphenyltetrazolium bromide (MTT), trypan, concanavalin A (Con A), penicillin G, and streptomycin sulfate were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Gibico 1640 medium and fetal bovine serum (FBS) were purchased from Gibco Invitrogen Corp. (San Diego, CA, USA). DEAE Sepharose Fast Flow and Sephadex G-75 column were obtained from Whatman Ltd. Other chemicals and reagents were of analytical grade.

2.2. Preparation of crude polysaccharide

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ACCEPTED MANUSCRIPT Dried fruit pulps of bergamot were milled into powder. The dried powder (500 g) was pretreated with 95% ethanol (1:8, w/v) twice for 3 h to remove the fat, pigments, and other partially alcohol-soluble chemicals. Residue was dried at 55 °C for 6 h. The dried residues (260 g) were extracted with distilled water (1:15, w/v) at 90 °C three times (each for 3 h). The water

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extract was centrifuged at 8000 rpm for 15 min and concentrated at 60 °C by a reduced rotary

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evaporator. The supernatant was then precipitated at the final concentration of 90% (v/v) of

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ethanol for 24 h at 4 °C and centrifuged at 8000 rpm for 15 min, and the precipitate was collected and re-dissolved in distilled water. Sevag reagent (chloroform/n-butanol at a ratio of

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4:1, v/v) was added [7], shaken vigorously for 10 min and then centrifuged at 8000 rpm for 15

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min. The procedure was repeated several times to ensure no proteins remained in the polysaccharide. The solution was intensively dialyzed against tap water for 48 h and then

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distilled water for 24 h at 37 °C (MW cut off 3.5 kDa). Finally, the resulting portion in the

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dialysis bags was concentrated and lyophilized as the crude polysaccharide (CMSPW90; 1.2

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g).

2.3. Separation and purification of CMSPW90

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Anion-exchange and gel filtration column chromatography were employed for the purification of polysaccharides. CMSPW90 (10 g) was dissolved in distilled water (200 mL), centrifuged, and loaded on a DEAE Sepharose Fast Flow column (2.5 × 40.0 cm) and eluted in succession with distilled water and a linear gradient from 0 to 0.8 M NaCl at a flow rate of 1.6 mL/min. The elution (8 mL/tube) was detected at 490 nm by using the phenol−sulfuric acid method. A sharp peak (CMSPW90-S) was collected, dialyzed, lyophilized, and further purified by a Sephadex G-75 gel filtration column (1.5 × 100.0 cm) eluted with distilled water at a flow 5

ACCEPTED MANUSCRIPT rate of 0.3 mL/min. Consequently, a polysaccharide fraction was concentrated and lyophilized to obtain white fluffy pure polysaccharide namely as CMSPW90-1.

2.4. Analytical methods

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The phenol-sulfuric acid colorimetric method was used to determine the total sugar content [8,9]. Optical rotation was determined by a Jasco P-1020 polarimeter at 28 °C. Wang’s method

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[10] was used to determine the sulfur content with potassium sulfate as the standard to make a

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calibration curve. The degree of substitution (DS) was calculated according to the following

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equation:

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DS = (1.62×S%)/(32-1.02×S%)

where S% represents the content of sulfate groups.

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2.5. UV spectrum

The UV spectrum of CMSPW90-1 (100 μg/mL) was determined by a UV-2401

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spectrophotometer in the wavelength range of 200–350 nm.

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2.6. Determination of homogeneity and average molecular weight The homogeneity and molecular weights of CMSPW90-1 were determined by highperformance gel-permeation chromatography (HPGPC) performed on a Waters HPLC system outfitted with TSK G-5000PWxl column (7.8 × 300 mm) and TSK G-3000PWxl analytical columns (7.8 × 300 mm) in series. CMSPW90-1 (2 mg/mL) was detected by dissolving in KH2PO4 (0.02 M), filtrating through a 0.22 μM microporous filtering film, and loading to the

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ACCEPTED MANUSCRIPT columns, which were eluted with KH2PO4 (0.02 M) at a flow rate of 0.6 mL/min. The standard calibration curve was set with dextran [11].

2.7. FT-IR spectrum analysis

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The dried powder of CMSPW90-1 (2 mg) was mixed with KBr powder, pressed into pellets and analyzed with a Perkin Elmer spectrophotometer. The IR spectrum was recorded in

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the wavelength range of 4000-400 cm−1.

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2.8. Monosaccharide composition analysis

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CMSPW90-1 (5 mg) was hydrolyzed in 10 mL of 2 M trifluoroacetic acid (TFA) at 120 °C

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for 6 h. After the hydrolysis was complete, excess TFA was removed by co-distillation with methanol. The monosaccharide composition was analyzed by HPAEC-PAD. The hydrolysate

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was re-dissolved in 0.5 mL of distilled water and then filtered with a 0.45 μm filter. The

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resulting solution (20 μL) was injected on the Dionex ICS-2500 system, which was eluted with a solution of water and 200 mM NaOH in the volume ratio of 92:8 [12,13].

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2.9. Methylation analysis

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To analyze the glycosyl linkages, CMSPW90-1 was methylated several times according to the method [14] with some modifications. NaOH (500 mg) and CMSPW90-1 (10 mg) were fully dissolved in 10 mL DMSO. The solution was treated with an ultrasonic wave for 0.5 h. Methyl iodide (3 mL) was added for methylation for 1 h at room temperature. The reaction was kept in darkness for 8 h at 25 °C and terminated by distilled water (3 mL). The methylated products were extracted with 3 × 2 mL of chloroform and dried under low pressure on a rotary evaporator. Infrared (IR) was used to detect the degree of methylation. Disappearance of the O-

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ACCEPTED MANUSCRIPT H band (3200-3700 cm-1) in IR indicated successive methylation. The methylated products were hydrolyzed in 2 M of TFA for 6 h and then dissolved in distilled water (2 mL) after they were dried at low pressure. NaBH4 (25 mg) was added to reduce the hydrolysates. The reduction was kept at 40 °C for 0.5 h and then terminated by glacial acetic acid (100 μL). The reaction product

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was acetylated with acetic anhydride and pyridine at 100 °C for 1 h after drying by rotary

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vacuum evaporation. Distilled water (6 mL) was used to decompose the remaining acetic

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anhydride. The acetylated derivatives were extracted with 3 × 2 mL of chloroform followed by washing with 3 × 4 mL of distilled water. Finally, the methylated alditol acetates were analyzed

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via gas chromatography-mass spectrometry (GC-MS).

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2.10. Nuclear magnetic resonance spectroscopy

The dried CMSPW90-1 (50 mg) was dissolved in 0.6 mL of D2O and then filtered with a

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0.45 μm filter. One-dimensional (1D) and two-dimensional (2D) Nuclear magnetic resonance (NMR) spectra were recorded on an AVANCE 600 MHz NMR spectrometer at 25 °C.

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2.11. Congo red test

The Congo red test was used to determine the conformational transitions of CMSPW90-1

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according to the protocols with minor modifications [15]. CMSPW90-1 (0.5 mg) was dissolved in 1 mL of deionized water and mixed with 1 mL of Congo red solution (50 μmol/mL). The transition of maximum absorption wavelength of the complex was measured using a UV-2401 spectrophotometer in the region of 400–600 nm at different NaOH concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mol/L). 2.12. Circular dichroism analysis

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ACCEPTED MANUSCRIPT Circular dichroism (CD) spectra were used to determine the conformational transitions of the polysaccharide. The analysis was performed according to the reported method [16]. Data were collected from 190 to 260 nm at 1 nm intervals.

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2.13. Sulfated modification of CMSPW90-1 Sulfation of CMSPW90-1 was performed using the chlorosulfonic acid-pyridine (CSA-

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Pyr) method [17]. The esterification reagent was prepared in an ice bath by adding 6 mL of

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CSA and 12 mL of Pyr in a three-necked flask with continuous stirring for 40 min. CMSPW90-

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1 (100 mg) was dissolved in 4 mL of anhydrous formamide (FA) and then stirred for 30 min at room temperature. The mixture was allowed to react for 2 h at 80 °C after sulfated reagent was

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added drop by drop with stirring. After the reaction was complete, the pH of the solution was adjusted to neuter with 2.0 M NaOH solution at room temperature, and then precipitated with

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95% ethanol at 4 °C overnight. The precipitate was re-dissolved in distilled water and intensively dialyzed and lyophilizated to obtain the sulfated polysaccharides [18], coded as

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CMSPW90-M1.

2.14. FT-IR spectrum analysis

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The dried powders of CMSPW90-M1 (2 mg) were separately mixed with KBr powder, pressed into pellets and analyzed with a Perkin Elmer spectrophotometer. The IR spectrum was recorded in the wavelength range of 4000-400 cm−1. 2.15. Scanning electron microscopy and atomic force microscopy Scanning electron microscopy (SEM) images of CMSPW90-1 and CMSPW90-M1 were observed by scanning electron microscopy (Philips XL-30). The dried powder of each sample was directly placed on a metal stub and then sputtered with gold powder using a sputter coater 9

ACCEPTED MANUSCRIPT [19]. Finally, the samples were observed with 300- and 500-fold magnification at 5.0 kV under high vacuum conditions. The ultrastructures of CMSPW90-1 and CMSPW90-M1 were also observed using atomic force microscopy (AFM) (BioScope Catylyst Bruker, Bil-lerica, MA). Each sample was

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dissolved in distilled water (1 μg/mL) and stirred for 4 h with a magnetic stirrer apparatus at

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room temperature. 5 μL of the diluted solution was then dropped onto a freshly cleaved mica

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substrate and dried at room temperature. AFM was determined in the contact mode [20]. A tube-type piezoelectric scanner (5 × 5 μm) and a Si3N4 probe (Olympus, Japan) were employed,

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and images were obtained simultaneously with 256 × 256 pixels at a scanning rate of 1.0 Hz

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per line.

2.16. Measurement of DPPH• radical-scavenging activity

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DPPH• free radical scavenging activity was detected with established methods [21].

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Briefly, 20 μL of CMSPW90-1 and CMSPW90-M1 solutions (0-3.2 mg/mL) were mixed with 180 μL of DPPH of ethanol solution (0.2 mM). For the positive control, Vc was used as a

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standard. The solution was shaken immediately and maintained in the dark for 30 min at room

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temperature, and a value of A517 nm was measured. The DPPH• free radical scavenging capability was calculated according to the following equation: Scavenging ability (%) = (A0-A1)/A0 × 100%

where A0 is the absorbance in the absence of the test sample and A1 is the absorbance in the presence of the test sample. 2.17. Measurement of ABTS+• radical-scavenging activity ABTS+• free radical scavenging activity was measured according to published methods

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ACCEPTED MANUSCRIPT [22]. Briefly, to obtain the ABTS+• reagent, 0.2 mL of ABTS (7.4 mmol/L) was added into 0.2 mL of K2S2O8 (2.6 mmol/L) in a centrifuge tube. The reaction was kept in the dark for 12 h at room temperature. The mixture was then diluted 30 times with absolute ethanol until the absorbance reached 0.7 ± 0.02 at 734 nm. 40 μL of different concentrations (0-3.2 mg/mL) of

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CMSPW90-1 or CMSPW90-M1 solutions were added to 160 μL ABTS+• reagent. Vc was used

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as the positive control. The solutions were shaken and kept for 15 min at room temperature,

calculated according to the following equation:

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and a value of A517 nm was recorded. The ABTS+• free radical scavenging capability was

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Scavenging ability (%) = (A0-A)/A0×100%

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where A0 is the absorbance in the absence of the test sample and A is the absorbance in the presence of the test sample.

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2.18. Splenic lymphocyte proliferation assay The MTT assay [23] was used to analyze the splenic lymphocyte proliferation. Spleens

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were aseptically separated from killed Kunming mice and then crushed in phosphate-buffered saline (PBS) buffer. The homogeneous cell suspension was obtained by pressing through a steel

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mesh (200 mesh). The removal of red blood cells was performed with a Tris–HCl–NH4Cl solution (pH 7.2) after centrifugation at 1500 rpm for 5 min. Cell pellets were re-suspended in RPMI-1640 medium and diluted to 5 × 106 cells/mL. Splenocytes were further placed into a 96-well flat-bottom microplate (100 μL/well) with concentrations of CMSPW90-1 or CMSPW90-M1 at 0, 7.82, 15.63, 31.25, 62.5, 125, 250, and 500 μg/mL. 5 μg/mL of concanavalin A (Con A) served as the positive control. After incubation at 37℃ under 5% CO2 for 48 h, 20 μL of MTT (5 mg/mL) was added to each well with incubation for 4 h. After 11

ACCEPTED MANUSCRIPT removal of MTT by centrifugation at 3500 rpm for 10 min at 4℃, the formazan precipitate was solubilized in DMSO (200 μL/well). Splenic lymphocyte proliferation was detected by the optical density (OD) at 570 nm according to a multifunctional microplate reader.

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2.19. Phagocytosis assay The neutral red uptake assay was used to measure the phagocytic ability of the

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macrophages [24]. Confluent cultures of RAW264.7 cells in 96-well plates were exposed to

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medium alone, to CMPW90-1 or CMSPW90-M1 at concentrations of 0, 7.82, 15.63, 31.25,

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62.5, 125, 250, and 500 μg/mL, or to LPS solution and incubated for 48 h at 37°C. After removal of the supernatant, PBS was used to remove the non-adherent cells by washing twice and 100

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µL of neutral red solution was added to each well. After the plates were incubated for 1 h, the supernatant was removed. The cells were washed with PBS twice to remove excess neutral red

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solution. 100 µL of cell lysate (1.0 M acetic acid/ethanol=50:50, v/v) was then added to each well and incubated for 1 h at room temperature. The absorbance at 540 nm was measured by a

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multifunctional microplate reader. 2.20. Statistical analysis

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All tests were performed in triplicate and results are presented as mean value ± standard deviation (SD). Data were analyzed by an independent t-test using the statistical analysis software SPSS 11.5. Differences were considered statistically significant at P < 0.05 and P < 0.01.

3. Results and discussion

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ACCEPTED MANUSCRIPT 3.1. Isolation and Purification of CMSPW90-1 The crude polysaccharide CMSPW90 (yield 4.8% of dried material) was obtained from dried bergamot, through hot water extraction previously subjected to ethanol precipitation, the Sevag method, dialyzing with water, and lyophilization. CMSPW90 was separated and

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fractionated by anion-exchange chromatography on a DEAE Sepharose Fast Flow column

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stepwise with sequential elution with distilled water and a NaCl gradient (0-0.8 M) to obtain a

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single and symmetrical sharp peak (CMSPW90-S) with the phenol-sulfuric acid method (Fig. 1A). The fraction of CMSPW90-S was further purified by a Sephadex G-75 column (Fig. 1B).

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After lyophilization, the total sugar content of CMSPW90-1 was measured at 95.80% ± 1.2%

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(w/w), and the protein and nucleic acid content was below the detection limit as evidenced by the lack of the absorption peak at 260-280 nm in UV spectrum of CMSPW90-1 [25,26] (Fig.

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1C). The uronic acid content of CMSPW90-1 was below the detection limit, indicating that

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CMSPW90-1 was a neutral polysaccharide. The optical rotation of CMSPW90-1 was

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 (c 1.0, H2O) [ ]20 D  164.4

3.2. Homogeneity and determination of molecular weight of CMSPW90-1

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The molecular weight of CMSPW90-1 was determined by the HPGPC system (Fig. 1D). The profile of CMSPW90-1 exhibited a single and symmetrical narrow peak with elution time of 27.492 min, indicating that CMSPW90-1 was a homogeneous polysaccharide. The average molecular weight of CMSPW90-1 was calculated to be 18.8 kDa. CMSPW90-1 was considered to be a lower molecular weight polysaccharide because polysaccharides generally had high molecular weight with hundreds of thousands of Da [27]. 3.3. FT-IR spectral analysis of CMSPW90-1 13

ACCEPTED MANUSCRIPT The characteristic absorption peaks of CMSPW90-1 shown in the IR spectrum revealed the structure of the polysaccharides (Fig. 1E). In the FT-IR spectrum, the broad absorption at 3447, 2953, and 1644 cm−1 corresponded to O-H, C-H, and bound water stretching vibrations, respectively. The band at 1421 cm−1 was assigned to C-O bending. The region around 1043-

confirmed the existence of α-configuration. No absorption at 1740 cm−1 was observed,

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1

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1074 cm−1 indicated the presence of a pyranose-ring structure. The absorption peaks at 866 cm-

3.4. Monosaccharide composition of CMSPW90-1

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indicating that CMSPW90-1 did not contain uronic acid [28].

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The monosaccharide composition of CMSPW90-1 was determined by HPAEC-PAD (Fig.

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1F). The presence of arabinose, galactose, glucose, and mannose in CMSPW90-1 were at the molar ratio of 54.46:1.45:14.39:1.00, indicating that CMSPW90-1 was a type of

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heteropolysaccharide.

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3.5. Methylation and GC-MS analysis of CMSPW90-1 Methylation analysis by GC-MS was used to obtain more structural information for

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CMSPW90-1. According to the retention time and standard data in the CCRC Spectral

Gal,

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Database for PMAAs, the peaks of CMSPW90-1 were identified as 2,4-Me2-Man, 2,3,4,6-Me42,3,6-Me3-Glc,

2,5-Me2-Ara,

2,3-Me2-Ara

with

the

molar

ratio

of

1.00:1.05:14.16:21.47:32.89 (Table 1). These results suggested that CMSPW90-1 contained four linkage forms: (1 → 3,6)-linked Manp, (1 → )-linked Galp, (1 → 4)-linked Glcp, (1 → 3)linked Araf and (1 → 5)-linked Araf. These results indicated a strong correlation between terminal and branched residues. 3.6. NMR spectroscopy analysis

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ACCEPTED MANUSCRIPT The detailed structural features of CMSPW90-1 were further identified by 1D and 2D NMR spectroscopy. According to the characteristic signals, the 13C NMR and 1H NMR spectra of CMSPW90-1 were completely analyzed based on two-dimensional HSQC NMR (Fig. 2A) and HMBC NMR experiments (Fig. 2B), together with comparison to the references [29-34].

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The 13C NMR spectra showed four anomeric carbon signals from 95 to 110 ppm based on the

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HSQC spectrum, indicated the presence of five residues, with C and D being arabinose residues

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with different linkages in CMSPW90-1, and designated as A, B, C, D, and E. The peak at 95.49 ppm corresponded to C-1 of (1→4)-linked α-D-Glcp unit, 106.50 ppm corresponded to C-1 of

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(1→3)-linked α-L-Araf unit and (1→5)-linked α-L-Araf unit, 106.95 ppm corresponded to C-1

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of (1→3,6)-linked α-D-Manp unit, and 107.43 ppm corresponded to C-1 of (1→)-linked α-DGalp unit. The 1H NMR spectrum contained four signals at δ 5.18, 5.15, 5.10, and 5.02 for the

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anomeric protons, indicating that five residues, designated (1→3)-linked α-L-Araf unit and

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(1→5)-linked α-L-Araf unit, (1→4)-linked α-D-Glcp unit, (1→3,6)-linked α-D-Manp unit, (1→)-linked α-D-Galp unit, respectively, were present in CMSPW90-1 (Table 1). The chemical

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shifts of four anomeric protons were larger than 5.0 ppm, which suggested that these residues

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were α-linked. The detected α-linked residues were in agreement with the FT-IR results of CMSPW90-1 [35]. By means of 13C NMR, 1H NMR, and HSQC NMR spectra, all 13C and 1H chemical shifts of CMSPW90-1 were described (Table 2). Five residues were shown to be connected by the analysis of the heteronuclear multiplebond correlation (HMBC) spectrum of CMSPW90-1 (Fig. 2B). In the HMBC spectrum, the strong correlation peak at 5.18/83.81 ppm (C H1/C C3) suggested that C-3 of residue C was linked to O-1 of residue C. The strong signal at 5.18/80.85 ppm (D H1/D C5) showed that C-5

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ACCEPTED MANUSCRIPT of residue D was linked to O-1 of residue D. Meanwhile, the methylation and GC-MS results revealed that (1→3)-linked α-L-Araf and (1→5)-linked α-L-Araf

accounted for 30.42% and

46.61% of the total sugar residues, respectively. Thus, the presence of a repetitive unit of (1→3)-linked α-L-Araf and (1→5)-linked α-L-Araf were confirmed. Accordingly, the

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following correlations were observed: E H-1 (δ 5.15) and B C-6 (δ 66.48), D H-1 (δ 5.18) and

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E C-4 (δ 81.36), E H-1 (δ 5.15) and D C-5 (δ 80.85), C H-3 (δ 4.24) and D C-1 (δ 106.50), D

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H-5 (δ 4.20) and B C-1 (δ 106.95), B H-3 (δ 4.06) and C C-1 (δ 106.50), E H-4 (δ 3.90) and A C-1 (δ 107.43). Therefore, according to results from HPAEC-PAD, GC–MS, 1D and 2D NMR,

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the structure of CMSPW90-1 was established, with the main chain composed of (1→5)-α-L-

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Araf, (1→3)-α-L-Araf, (1→4)-α-D-Glcp and (1→ 3,6)-α-D-Manp, which branched at O-6. One side chain consisted of (1→4)-α-D-Glcp terminating with (1→)-α-D-Galp. The predicated

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3.7. Congo red test

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structure of the repeating unit of CMSPW90-1 was thus determined (Fig. 2C).

The spatial stereochemistry of a polysaccharide could be determined by changes in the

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wavelength of the complex [36]. The Congo red test is most commonly used to determine the

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triple-helical conformation of a polysaccharide. With the increasing concentration of NaOH, the complex maximum absorption wavelength (λ max) of CMSPW90-1 changed at alkali concentrations ranging from 0 to 0.6 M (Fig. 3A). Compared with Congo red, the maximum absorption wavelengths of Congo red + CMSPW90-1 did not show a sharp decline. Therefore, the triple-helical structure did not exist in the solution of CMSPW90-1. 3.8. CD spectroscopy The CD spectra of CMSPW90-1 + Congo red was compared with that of CMSPW90-1 +

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ACCEPTED MANUSCRIPT water at the range from 190 nm to 260 nm. After CMSPW90-1 was added to Congo red, it showed a red shift, indicating a change in the asymmetry between polysaccharide molecules (Fig. 3B). However, there was no alternating positive and negative Cotton effect, indicating that the triple-helical conformation did not exist in the solution of CMSPW90-1, which was

PT

consistent with the result from the Congo red test. Some researchers have shown that no triple-

RI

helical conformations in some polysaccharides. Researchers have reported that a

SC

polysaccharide from Fagopyrum tartaricum did not have triple-helical structure in solution [37].

chains in 0.05 to 0.15 M NaOH solution [38].

MA

3.9. Structure elucidation of CMSPW90-M1

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However, others reported that a Cordyceps militaris polysaccharide existed as triple-helical

The sulfated derivative (CMSPW90-M1) with increased water solubility from

D

CMSPW90-1 was prepared by the CSA−Pyr method. Based on the calibration curve, the sulfur

PT E

content and the DS value of CMSPW90-M1 were 15.72% (w/w) and 1.59, respectively. HPGPC analysis of CMSPW90-M1 showed a single and symmetric sharp peak with an average

CE

molecular weight of 75.4 kDa (Fig. 1D). In comparison with CMSPW90-1, two new absorption

AC

bands at 1275 cm-1 and 843 cm-1 appeared in the FT-IR spectrum of CMSPW90-M1 (Fig. 1E), representing an asymmetrical S=O stretching vibration and a symmetrical C–O–S deformation vibration associated with a C–O–SO3 group, respectively. These results indicated that the sulfation reaction of polysaccharide CMSPW90-1 occurred successfully. 3.10. Ultrastructures of CMSPW90-1 and CMSPW90-M1 The surface structure of polysaccharides could be clearly observed by SEM to effectively understand their common physical properties [39-42]. Sulfated modification can change the

17

ACCEPTED MANUSCRIPT structure of polysaccharides, affecting the structure-activity relationship [43]. SEM of CMSPW90-1 and CMSPW90-M1 revealed that CMSPW90-1 had a rough surface with a sheetlike and irregular appearance, which consisted of many small lumpish and flaky particles, while CMSPW90-M1 appeared to be a smooth surface composed of mainly randomly distributed

PT

individual spherical particles (Fig. 4A and 4B). The irregular shapes indicated that CMSPW90-

RI

1 was an amorphous solid, and the tight configuration of CMSPW90-M1 reflected strong

SC

interactions between the highly branched polysaccharide chains [44].

The shapes of polysaccharides are much more complex than those of nucleotides and

NU

proteins. AFM was used as a metrological tool to study surfaces, biopolymers, and the

MA

structures of polysaccharides on the nanometer scale of particles [45]. AFM images were used to describe CMSPW90-1 and CMSPW90-M1 (Fig. 4C and 4D). CMSPW90-1 aggregated to

D

form spherical lumps with diameters ranging from 300 to 700 nm and height ranging from 8.4

PT E

to 14.0 nm. The presence of many spherical and uneven lumps suggested that molecular aggregation had occurred and that the structures of CMSPW90-1 were branched and entangled

CE

[46]. This was consistent with the previously speculated structure of CMSPW90-1. However,

AC

CMSPW90-M1 particles aggregated to form a circular-like structure uniformly dispersed in aqueous solution, with diameters ranging from 60 to 90 nm in length and from 2.0 to 3.0 nm in height. Sulfated structural modification may cause differences in the physicochemical properties, resulting in different surface topographies of the polysaccharides. The decrease of the size of CMSPW90-M1 due to the substitution of the hydroxyl group of CMSPW90-1 by the sulfate group could cause twisting and converting of the sugar ring conformation. 3.11. Scavenging activities of CMSPW90-1 and CMSPW90-M1 against DPPH• Radicals

18

ACCEPTED MANUSCRIPT As a stable free-radical compound with maximum absorbance at 517 nm, DPPH• is widely used as a free radical to evaluate the antioxidant abilities of various anti-oxidative samples in vitro. Vc was used as a positive control group to evaluate the scavenging effects of CMSPW901 and CMSPW90-M1 (Fig. 5A). CMSPW90-1, CMSPW90-M1, and Vc were able to scavenge

PT

DPPH• radicals to different degrees in a dose-dependent manner when the concentration ranged

RI

from 0.05 to 3.2 mg/mL. The highest DPPH• radical scavenging rate of CMSPW90-M1 was

SC

71.64% (3.2 mg/mL).

3.12. ABTS+• radical-scavenging properties of CMSPW90-1 and CMSPW90-M1

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The ABTS+• free radical scavenging activity is a widely used model for determining the

MA

free radical scavenging capacity of various compounds [47]. The scavenging activities of CMSPW90-1 and CMSPW90-M1 and Vc against ABTS+• radicals were measured (Fig. 5B).

D

ABTS+• radical scavenging activity increased with the concentration of the samples. The

PT E

highest ABTS+• radical scavenging rate for CMSPW90-M1 was 74.99% (3.2 mg/mL). The sulfated derivative had a more noticeable effect on scavenging free radicals, indicating that

CE

CMSPW90-M1 could be used as a natural antioxidant to inhibit radicals during food processing

AC

and conserving.

3.13. Effects of CMSPW90-1 and CMSPW90-M1 on splenic lymphocyte proliferation One of the basic functions of polysaccharides is immunoregulation [48]. The spleen is the most important organ for antibacterial and antifungal immune reactivity because it combines the innate and adaptive immune system in a uniquely organized way. Therefore, the spleen is an ideal tissue for research on immunization [49]. To investigate the effect on spleen lymphocyte proliferation of CMSPW90-1 and CMSPW90-M1 on immune cells, the

19

ACCEPTED MANUSCRIPT immunoregulation activities were evaluated. Compared with CMSPW90-1 at the same concentrations, CMSPW90-M1 (7.82-500 µg/mL) could more effectively promote the proliferation of lymphocytes in a dose-dependent manner (Fig. 6A). A large number of T and B lymphocytes in the spleen could mediate humoral and cellular immunity, and CMSPW90-

PT

M1 could significantly activate T- and B-cells and enhance immunity. Therefore, CMSPW90-

RI

M1 might have immunoregulatory activity on lymphocytes.

SC

3.14. Effects of CMSPW90-1 and CMSPW90-M1 on the phagocytic activity of macrophages The phagocytosis of macrophages is the first step in an immune response, and increased

NU

phagocytic activity is characteristic of activated macrophages [50]. Thus, we used the neutral

MA

red uptake method to measure the phagocytic ability of RAW264.7 cells (with LPS as a positive group). CMSPW90-1 (125-500 μg/mL) could increase the phagocytosis of RAW 264.7 cells

D

over the control cells (Fig. 6B). Moreover, CMSPW90-M1 (15.63-500 μg/mL) was more

PT E

effective at increasing phagocytosis than CMSPW90-1. CMSPW90-1 and CMSPW90-M1 observably promoted phagocytosis activation of macrophage RAW264.7 cells to uptake neutral

CE

red within a certain concentration range, and thus resist foreign invasion and improve immune

AC

function of an organism. Phagocytic activities of macrophages treated by polysaccharides from C. medica L. var. Sarcodactylis Swingle can be markedly improved after sulfated modification. These results were consistent with those of Yu et al., who reported that the sulfated polysaccharide from Cyclocarya paliurus could enhance the immunomodulatory activity of RAW 264.7 macrophages [51].

4. Conclusion

20

ACCEPTED MANUSCRIPT In this study, a novel low molecular-weight polysaccharide CMSPW90-1 was purified from bergamot. The total sugar and sulfate contents of CMSPW90-1 were 95.80% and 15.72%, respectively. CMSPW90-1 had a molecular weight of 18.8 kDa without triple-helix conformation, while the molecular weight of CMSPW90-M1 was 75.4 kDa with different

PT

stereostructures described in the profiles of SEM and AFM. The main linkage types of

RI

CMSPW90-1 were (1→3)-linked Araf, (1→5)-linked Araf, (1→3,6)-linked Manp, (1→ 4)-

and NMR analysis, giving its primary structure.

SC

linked Glcp, and (1→)-linked Galp based on complete acid hydrolysis, methylation analysis,

NU

The biological activities of polysaccharides are closely related to their water solubility,

MA

molecular weight, monosaccharide composition, and chain conformation [52], and many studies have demonstrated that the sulfation of natural polysaccharides is an important method

D

for obtaining new antioxidant agents. For example, Wang reported that sulfated polysaccharide

PT E

derivatives from green algae Enteromorpha linza significantly enhance its antioxidant activities [53]. Cui reported that sulfated polysaccharides from seaweed Dictyopteris divaricata exhibited

CE

strong antioxidant activity in vitro [54]. The incorporation of OSO3H groups into

AC

polysaccharide molecules can lead to weaker dissociation energy of the hydrogen bond and activate the hydrogen atom of the anomeric carbon to provide strong hydrogen-donating capacity. The hydrogen atom can combine with the radical ions and form a stable radical to terminate the radical chain reaction, which improves the scavenging free radical abilities of sulfated derivatives [55,56]. Furthermore, the sulfate groups could significantly increase water solubility of the natural polysaccharides such that the scavenging activity of free radicals was enhanced. CMSPW90-M1 has better water solubility than CMSPW90-1, and thus sulfated

21

ACCEPTED MANUSCRIPT polysaccharide CMSPW90-M1 shows stronger free radical scavenging ability, which is consistent with the results reported previously. Polysaccharides containing arabinose, mannose, glucose, or galactose might be closely related to immunomodulatory activity [57-59]. The current study demonstrated that

PT

CMSPW90-1 and its sulfated derivative are composed of arabinose, galactose, glucose, and

RI

mannose at the ratio of 54.46:1.45:14.39:1.00, which might explain their immunomodulatory

SC

activity. Immunomodulatory activities were improved after the introduction of sulfate groups [60]. For example, the sulfated polysaccharides extracted from marine algae could regulate

NU

immune function because they could bind to receptors on macrophages and lymphocytes to

MA

activate cells through the recognition and transfer of a series of biological information [61]. The sulfate groups of CMSPW90-M1 may twist and convert sugar ring configuration and

D

orientation to induce exposure of hydroxyl groups, which improves the interaction between the

PT E

sulfated polysaccharide and the specific receptors, thus enhancing immunoregulatory activity. Thus, CMSPW90-M1 shows stronger immunomodulatory activity than CMSPW90-1. Our

CE

finding suggests that CMSPW90-M1 has potential applications as a potent inhibitor and

AC

immunomodulator in functional foods and pharmaceuticals.

Conflicts of interest There are no conflicts of interest to declare.

Acknowledgments This research work was financially supported by Major National Science and Technology 22

ACCEPTED MANUSCRIPT Projects / Significant New Drugs Creation (No. 2011ZX09102-001-33). The authors thank Dr. Dongbo Yu from The University of Chicago Medical Center, USA, for proof-reading our manuscript.

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Table 1

AC

CE

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MA

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SC

RI

PT

ACCEPTED MANUSCRIPT

Methylation analysis of CMSPW90-1.

Methylation sugar

Retention

Molar ratio

Mass fragments (m/z)

time (min)

Linkage type

2,3,4,6-Me4-Gal

15.21

1.05

61,71,87,101,117,129,186

T→

2,3,6-Me3-Glc

25.23

14.16

57,75,88,101,117,129,215

1→4

2,5-Me2-Ara

27.55

21.47

61,71,87,101,117,130,191

1→3

2,4-Me2-Man

28.59

1.00

51,71,87,117,129,189

1→3,6

30

ACCEPTED MANUSCRIPT 36.01

32.89

1→5

52,75,88,101,117,129,181

Table 2 13

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C and 1H NMR chemical shifts of CMSPW90-1 (ppm).

Chemical Shifts Sugar residue

α-D-Galp-(1→

C1/H1

C2/H2

C3/H3

C4/H4

C5/H5

C6/H6

107.43/5.02

82.26/3.90

84.49/4.24

79.85/3.95

76.57/3.90

60.99/3.66

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106.95/5.10

80.27/4.06

83.44/4.06

76.57/3.90

81.25/4.06

106.50/5.18

84.49/3.90

83.81/4.24

79.85/4.07

76.57/3.95

106.50/5.18

81.30/4.06

76.57/3.90

78.00/3.95

80.85/4.20

95.49/5.15

84.49/3.99

80.85/4.14

81.36/3.90

66.85/3.87

66.48/3.82

B →3)-α-L-Araf-(1→ C →5)-α-L-Araf-(1→ D →4)-α-D-Glcp-(1→

61.13/3.77

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Figure Legends

Fig. 1. Chromatography of polysaccharides from bergamot of CMSPW90 by DEAE-Sepharose Fast Flow chromatography (A); Chromatography of CMSPW90-1 by Sephadex G-75 (B); UV spectrum of CMSPW90-1 (C); HPGPC of CMSPW90-1 and CMSPW90-M1 (D); FTIR spectrum of CMSPW90-1

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and CMSPW90-M1 (E); HPAEC of standard monosaccharides and CMSPW90-1 peaks: (a) Fucose, (b) Rhamnose, (c) Arabinose, (d) Galactose, (e) Glucose, (f) Mannose, (g) Fructose (F). Fig. 2. HSQC spectra (A), HSBC spectra (B), and proposed primary structure of repeating unit (C) of CMSPW90-1, x + 13y = 33.

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various concentrations of NaOH (A); CD spectra of CMSPW90-1 (B).

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Fig. 3. Changes in absorption wavelength maximum of mixtures of Congo red and CMSPW90-1 at

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Fig. 4. SEM images of CMSPW90-1 (A) and CMSPW90-M1 (B), AFM images of CMSPW90-1 (C) and CMSPW90-M1 (D).

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Fig. 5. Antioxidant activities of CMSPW90-1 and CMSPW90-M1: DPPH• radical-scavenging activity

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(A); ABTS+• radical-scavenging activity (B). Values are the mean ± SD of three replicates. Fig. 6. Effect of CMSPW90-1 and CMSPW90-M1 on splenic lymphocyte proliferation. The OD 570 nm

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reflected the cell activity of spleen lymphocytes (A); Effect of CMSPW90-1 and CMSPW90-M1 on

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macrophage phagocytosis, as determined by the neutral red uptake assay (B). Values are the mean ± SD

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Graphical Abstract

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