Characterization and immunological activity of polysaccharides from Ixeris polycephala

Characterization and immunological activity of polysaccharides from Ixeris polycephala

Accepted Manuscript Characterization and immunological activity of polysaccharides from Ixeris polycephala Bi Luo, Li-Mei Dong, Qiao-Lin Xu, Qiang Zh...

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Accepted Manuscript Characterization and immunological activity of polysaccharides from Ixeris polycephala

Bi Luo, Li-Mei Dong, Qiao-Lin Xu, Qiang Zhang, Wen-Bin Liu, Xiao-Yi Wei, Xu Zhang, Jian-Wen Tan PII: DOI: Reference:

S0141-8130(17)33426-8 doi:10.1016/j.ijbiomac.2018.02.165 BIOMAC 9222

To appear in: Received date: Revised date: Accepted date:

6 September 2017 26 February 2018 28 February 2018

Please cite this article as: Bi Luo, Li-Mei Dong, Qiao-Lin Xu, Qiang Zhang, Wen-Bin Liu, Xiao-Yi Wei, Xu Zhang, Jian-Wen Tan , Characterization and immunological activity of polysaccharides from Ixeris polycephala. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), doi:10.1016/j.ijbiomac.2018.02.165

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ACCEPTED MANUSCRIPT Characterization and Immunological Activity of Polysaccharides from Ixeris polycephala

Bi Luo 1,2,4, Li-Mei Dong 1,2, Qiao-Lin Xu 3,*, Qiang Zhang 1,4, Wen-Bin Liu 1,4,

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Xiao-Yi Wei 1, Xu Zhang 2, and Jian-Wen Tan 2,**

Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical

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1

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Affiliation

Garden, Chinese Academy of Sciences, Guangzhou 510650, China; State Key Laboratory for Conservation and Utilization of Subtropical

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2

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Agro-bioresources/ Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, College of Forestry and Landscape

Guangdong Provincial Key Laboratory of Bio-Control for Forest Diseases and Pests,

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3

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Architecture, South China Agricultural University, Guangzhou 510642, China;

Guangdong Academy of Forestry, Guangzhou 510520, China; University of Chinese Academy of Sciences, Beijing 100049, China

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* Corresponding author: E-mail: [email protected]; Tel.: +86-20-87029780. ** Corresponding author: E-mail: [email protected]; Tel/Fax: +86-20-85280256.

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ACCEPTED MANUSCRIPT ABSTRACT A water-soluble polysaccharide, named KMCP, was isolated and purified from edible plant Ixeris polycephala by using DEAE-52 cellulose chromatography. Its structure was determined by chemical analysis, methylation analysis, and NMR

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analysis, coupled with characterization by scanning electron spectroscopy (SEM). The

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resulting data indicated that KMCP was an arabinogalactan, with an average

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molecular weight of 1.95×106 Da, which was mainly composed of arabinose and galactose in a relative molar ratio of 28.1% and 70.3%, respectively. The structure of

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KMPC was characterized as 72.5% of (1→4)-β-Galp residues interspersed with 27.5%

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of (1→4,6)-β-Galp residues in the main chain, and the branches were composed of (1→5)-α-Araf moieties or α-Araf (1→5) α-Araf (1→ disaccharide moieties attached at

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O-6 of the (1→4,6)-β-Galp residues. KMCP was revealed to be capable of exhibiting

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macrophage-mediated innate immune responses via enhancing phagocytosis of macrophages and increasing production of NO, activating NF-κB signaling pathway

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and promoting the mice spleen cells proliferation in a dose-dependent manner within

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the test concentrations (10.0-200.0 μg/mL). These results suggested that KMCP could potentially be an effective and safe immunomodulator valuable to be utilized in pharmacological fields or in the development of functional foods. Keywords: Ixeris polycephala, polysaccharide, KMCP, structural characterization, immunological activity

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ACCEPTED MANUSCRIPT INTRODUCTION The genus Ixeris, comprising fifty plant species, is belonging to the Cichorieae tribe of the Asteraceae family[1], plants of which were believed to be rich in bioactive substances. At present, the chemical constituents reported from this genus plants

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include sesquiterpene lactones, triterpenes and steroids, phenylpropanoids, phenols,

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amino acids, and fatty alcohol/acids[2], among which sesquiterpene lactones were

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addressed as a major class of natural bitter compounds that occurring in vegetables and culinary herbs, as well as in medicinal plants[3]. Up to date, pharmacological have

revealed such

some

as

identified

chemicals

cardiovascular-protective,

exhibited

diverse

anti-inflammatory,

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bioactivities[4-9],

that

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studies

neuroprotective, hepatoprotective and antimicrobial activities that can enhance the

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human immune system, as well as antioxidant, antidiabetic[10] and antitumor

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activities[11, 12], etc. So far, as mentioned above, though extensive phytochemical investigations on plants of genus Ixeris have been carried out, their potential bioactive

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macromolecular substances such as polysaccharides have yet not been reported.

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Polysaccharides are a primary and essential class of biomacromolecules, which are widely used in different areas such as food and feed, medicine and pharmaceutics, as well as in papermaking[13]. It was reported that polysaccharides are closely related to significant physiological processes and vital phenomena, such as cell-cell communication, cell adhesion and molecular recognition, structural support, information transfer and immune defense, among which the potential modulatory effects of plant polysaccharides on the immune system were often emphasized[14]. 3

ACCEPTED MANUSCRIPT The immune system highly depends on accurate cell-cell communication for optimal function, and any damage to the signaling systems involved will cause an impaired immune responsiveness[15]. Therefore, effective and safe immunomodulatory agents would significantly important for hypoimmunity population. As compared with those

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immunomodulatory bacterial polysaccharides and synthetic ones, most plant derived

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polysaccharides are relatively nontoxic and do not cause significant side effects[16,

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17]. Due to these reasons, searching for safe and effective plant polysaccharides with immunomodulatory potential has gradually become a hot research field which has

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attracted more and more attention during the last decades.[18, 19]

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Ixeris polycephala (Cass.) is an annual and edible herb belonging to the genus Ixeris, which is widely cultivated and distributed in the east, southeast and south part

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of Asia. The fresh leaf of cultivated I. polycephala, commonly called ‘Ku-Mai-Cai’ in

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China, is consumed by the local people as a green vegetable, part of which is also processed into beverages, canned or dehydrated vegetables and feed additive to

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increase its usage and commercial value[20]. In Chinese folklore, the whole plants of

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I. polycephala has also been used for treatment of appendicitis, measles, contusion, analgesic and hemostasis[21], and its immunomodulatory potential was also suggested, indicating that I. polycephala might be a promising source for bioactive substances. While, so far, few such bioactive substances have been reported from this plant species. With the aim to clarify whether bioactive polysaccharides with significant immunomodulatory potential would exist in this edible and medicinal plant, we carried out this study, by which a water-soluble polysaccharides, named as KMCP, 4

ACCEPTED MANUSCRIPT was obtained and identified. Furthermore, to investigate the potential KMCP-induced immunomodulatory responses, the effects of KMCP on phagocytosis of macrophages and increasing production of NO, activating NF-κB signaling pathway and promoting the mice spleen cells proliferation were evaluated in vitro.

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MATERIALS AND METHODS

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Material and Animals

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The aerial part material of I. polycephala, collected at Hunan province in China, was identified and authenticated by Dr. Qi-Fei Yi at the South China Botanical

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Garden, Chinese Academy of Sciences (CAS). A voucher specimen (No.151215) was

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deposited at the Laboratory of Bioorganic Chemistry at the South China Botanical Garden, CAS. Trifluoroacetic acid (TFA), D-glucose, and galacturonic acid were

tiazol-2-yl)-2,5

Aladdin diphenyl

Reagent

Int.

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from

tetrazolium

(Shanghai,

bromide

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purchased

China).

(MTT),

3-(4,5-dimethyl

T-series

Dextran,

lipopolysaccharide (LPS), Medium RPMI-1640 and DEAE-cellulose were purchased

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from Sigma-Aldrich., and Concanavalin A (ConA) was from Sigma Chemical Co.. All

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other chemicals were of analytical grade as available. The RAW264.7 cell line was from our laboratory. Male Kunming mice (weighing 18.0-22.0 g) were purchased from Laboratory Animal Center of South China Agricultural University, China. All mice were allowed to acclimatize for 7 days in the animal room and were kept on standard pellet and tap water ad libitum at a temperature of 25± 2 °C. All experiments were performed in accordance with the Regulations of Experimental Animal Administration issued by 5

ACCEPTED MANUSCRIPT the State Committee of Science and Technology of the People’s Republic of China. General Experimental Procedures The 1H (500 MHz) and

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C NMR (125 MHz) spectra were obtained with an

Avance-600 NMR spectrometer using standard pulse sequences with 5 mm BBO

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H-1H correlation spectroscopy (COSY),

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dimensional NMR spectra including

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probe at 25 °C. All the chemical shifts are reported relative to tetramethylsilane. Two

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hetero-nuclear singular quantum correlation (HSQC), hetero-nuclear multiple bond correlation(HMBC), distortion less enhancement by polarization transfer (DEPT) and

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nuclear Overhauser effect spectroscopy (NOESY) were recorded on the same

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apparatus when required. ESI-MS spectra were measured on a MDS SCIEX API 2000 LC/MS/MS apparatus (Applied Biosystems Inc., Forster, CA, USA).

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Extraction and Purification of Crude Polysaccharide

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The dried plant material (3.0 Kg), crushed into powder by a disintegrator, was soaked in 95% ethanol to remove lipophilic constituents. Subsequently, the dried

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ethanol-extracted residue was extracted with boiling water for 2h (three times), at the

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ratio of 1:10 (w/v). The extracts were combined, filtered and concentrated to appropriate volume (1.0-1.5 L), and then mixed with four volumes of 95% ethanol, stood still overnight and centrifuged at 3500 rpm for 30 min, the precipitate was re-dissolved in distilled water, and dialyzed against distilled water (molecular weight cut off 1000 Da) for 3d. Finally, the dialysis solution was collected and lyophilized, the maroon crude polysaccharides (396.0 g) of I. polycephala was obtained and detected by high-performance gel-permeation chromatography (HPGPC). Further 6

ACCEPTED MANUSCRIPT purification of the crude polysaccharides was conducted as the following. In brief, the crude polysaccharides was dissolved in distilled water, centrifuged at 3000 rpm for 10 min. The supernatant was loaded on a DEAE-52 cellulose column (5 × 50 cm, OH-1 form) for further purification. Distilled water and different

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concentrations of gradient NaCl solution (0.1 M, 0.2 M, 0.3 M NaCl) were used to

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fractionate and elute the column with constant flow rate. Each fraction was gathered

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and phenol-sulfuric acid method was used to assay the content of carbohydrate. The water eluent was further purified with DEAE-52 cellulose column, and the main

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polysaccharides fraction (KMCP) was collected, dialyzed and lyophilized. The KMCP

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solution was filtered through 0.22 μm membrane and detected by HPGPC. Molecular weight Analysis of KMCP

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The molecular weight of KMCP was determined by HPGPC[22, 23] with three

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columns (Waters Ultra hydrogel 250, 1000 and 2000; 30 cm ×7.8 mm; 6 μm particles) in series. The calibration curve was made of T-series Dextran standards (molecular

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weights of 5.2, 48.6, 668, 2000 kDa) under the conditions described above for

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estimating the molecular weight of KMCP. Sodium acetate (0.003 M) was used as eluent at a flow rate of 0.5 mL/min and 37°C. The elution was monitored by a refractive index detector (Waters 2414, USA). A 100 μL aliquot was injected for each run. The calibration curve of log (Mw) vs. elution time (T) is: log (Mw) = −0.1297T + 11.158. Chemical Composition Analysis The total carbohydrate content was measured according to the phenol-sulphuric 7

ACCEPTED MANUSCRIPT acid method and D-glucose was used as standard[24]. Uronic acid content was determined by m-hydroxydiphenyl-sulphuric acid method[25], with galacturonic acid as the standard. Protein content was assessed by Bradford’s with bovine serum albumin (BSA) as standard[26]. Neutral monosaccharide compositions were analyzed

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by pre-column derivatization high performance liquid chromatographic (HPLC)

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method [27-29]. Polysaccharide (10.0 mg) was hydrolyzed with 4 M TFA at 110 °C

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for 4 h. After the solution was concentrated under vacuum, the residue was washed by methanol and lyophilized, and this procedure was repeated three times in order to

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remove TFA completely. Then the residue was re-dissolved in distilled water.

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Subsequently, 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution and NaOH solution were added to the hydrolysate. The mixture was kept for 2 h at 70 °C and was

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neutralized with HCl. Then, chloroform was added and extracted in triplicate, organic

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phase was discarded. Finally, the solution was passed through a 0.45 µm syringe filter for HPLC analysis. The monosaccharide standards (mannose, rhamnose, glucose,

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fucose, galactose, ribose, xylose, galacturonic acid and arabinose) were dissolved in

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deionized water and analyzed as mentioned above. The monosaccharides of KMCP would be identified by comparison of their retention times with those of monosaccharide standards and quantitatively determined by their peak areas. Scanning Electron Microscope (SEM) Analysis The SEM images of KMCP were obtained by field emission scanning electron microscope (FESEM, S-4800, Hitachi, Japan). KMCP was fixed with a thin layer of gold under reduced pressure. The morphological feature was observed at 10 kV 8

ACCEPTED MANUSCRIPT acceleration voltages under a high vacuum condition with image magnification of 400×, 2K×, 5K×, 35K×. Methylation Analysis The methylation analysis of KMCP was carried out according to the method as

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described by Hakomori and Fang with slight modifications [30-32]. Briefly, KMCP

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(10.0 mg) was dried by P2O5, followed by dissolving in anhydrous DMSO (3 mL),

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and then stirred at room temperature with dropwise addition of sodium methylsulfinylmethylide (SMSM) (1.5 mL) under an atmosphere of nitrogen.

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Subsequently, iodomethane (1.5 mL) was added dropwise under ice bath by

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ultrasounding. Finally, the methylated polysaccharides were recovered by dialysis against distilled water and freeze-drying. The methylation procedure was repeated till

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completeness of methylation which was confirmed by the disappearance of the OH

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band (3000-3500 cm−1) in the IR spectrum. The methylated polysaccharide was hydrolyzed with 98% formic acid (100 °C, 6 h), and then 2 M TFA (110 °C, 2 h),

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reduced with NaBH4 and acetylated with acetic anhydride. The methylated alditol

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acetate was recovered after extracting with chloroform-water system and analyzed by GC-MS.

NMR Analysis

A dried KMCP (30.0 mg) was exchanged 3 times in 99.9% D2O to replace H with D completely, with intermediate freeze-drying, and finally dissolved in D2O overnight before NMR analysis. The NMR analyses were then conducted with an Avance-600 NMR spectrometer by the procedures as describe in the aforementioned 9

ACCEPTED MANUSCRIPT “General Experimental Procedures” section. Assay of Immunological Activities in vitro Assay for NO Production RAW264.7 cells were seeded in 96-well plate at a density of 1×106/mL with 1

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μg/mL LPS (for the positive group)[33, 34], PBS (as the control group) or different

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concentration of KMCP (10.0, 100.0, 200.0 μg/mL) at 37 °C for 24 h (six replicates

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per concentration). Nitrite content (NO) secreted by macrophage RAW264.7 was

Assay of Macrophages Phagocytosis

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determined by Griess reaction and NaNO2 was used as a standard.

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Neutral red uptake was used to evaluate phagocytic ability of macrophages[35]. Cells (1×106 cells/well) were pipetted into 96-well plate and incubated at 37 °C in a

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humidified 5% CO2 incubator for 2 h, followed by treatment with various

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concentration of KMCP (10.0, 100.0, 200.0 μg/mL) at 37 °C for 12 h. Then, 0.075% neutral red solution was added into each well and incubated for 2 h. The supernatant

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was discarded and the cells in plate were washed with PBS twice to remove excess

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neutral red. The cell lysate (ethanol and 1% glacial acid at the ratio of 1:1, 100 μL/well) was added. After the plates were kept overnight at room temperature, the Abs at 540 nm of each well was measured by a microplate reader. Complete RPMI-1640 medium and LPS (final concentration, 2.0 µg/mL) [36] alone were used as positive and blank control, respectively. Phagocytosis index was calculated by the following equation: Phagocytosis index =

𝐴𝑏𝑠𝑠𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠𝑏𝑙𝑎𝑛𝑘 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 10

(1)

ACCEPTED MANUSCRIPT Effects of KMCP on the nuclear factor-kappa B (NF-κB) activation The cells were immunofluorescence-labeled in reference to the manufacturer’s instruction using a Cellular NF-κB Translocation Kit (Beyotime Institute of Biotechnology, Shanghai)[37, 38]. In brief, cells were incubated with a blocking

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buffer for 1 h to suppress non-specific binding after washing and fixing. Next, cells

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were incubated with the primary NF-κB p65 antibody for 1 h, followed by incubation

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with a Cy3-conjugated secondary antibody for 1 h, then with DAPI for 5 min before observation. The p65 protein and nuclei fluoresce red and blue, respectively, can be

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simultaneously viewed by laser confocal microscope at an excitation wavelength of

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350 nm for DAPI and 540 nm for Cy3. To create a two-color image, the red and blue images were overlaid, producing purple fluorescence in areas of co-localization. The

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figures and labels of imported resultant images were added by using Adobe

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Photoshop.

Assay of Splenic Lymphocyte Proliferation in vitro

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The spleen is an important immune organ of the body, most of lymphocytes are

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proliferated in the Spleen. The Spleen lymphocyte proliferation was assessed by using 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay[39, 40]. In brief, the mice spleen were removed, washed with PBS and the single cell suspension was obtained after grinding, filtering through screen. The cells were centrifuged at 1000 rpm for 5 min and the supernatant was discarded. 0.75% NH4Cl was added to lyse red cells. After red blood broken completely, cells were washed twice with PBS and resuspended to a final density of 1 × 107/mL in 11

ACCEPTED MANUSCRIPT RPMI-1640 media supplemented with 10% fetal calf serum. Then, the splenocyte solution was transferred into a 96-well plate with wells containing different concentrations of KMCP (10.0, 100.0, 200.0 μg/mL) in the presence of ConA (final concentration 5.0 μg/ml) or LPS (final concentration 10.0 μg/mL)[41, 42]. Wells

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containing ConA (final concentration 5 μg/ml) or LPS (final concentration 10 μg/ml)

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alone served as positive control. After the plates were cultured at 37 °C in 5% CO2

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atmosphere for 48 h, 10 μL MTT (5 mg/mL) was added to each well, evenly mixed and incubated for another 4 h. The absorbance of each well at 570 nm was obtained

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on an ELISA reader. The percentage of proliferation was calculated using the

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following equation: Proliferation(%) = (

ODcontrol

) × 100

(2)

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Statistical Analysis

ODsample −ODcontrol

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All data were presented as the mean ± SD. The results obtained were analyzed using t-test with the Prism software for Windows (version 5.00; Graph Pad Software).

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p values of 0.05 or less were considered statistically significant.

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RESULTS AND DISCUSSION Characterization of KMCP Molecular Weight and Monosaccharide Composition of KMCP In this study, the extraction yield of crude polysaccharides from I. polycephala was 13.2%. As shown in Figure 1, KMCP exhibited as the main portion of a single, symmetrical, narrow peak on HPGPC, which indicated that KMCP was a homogeneous polysaccharide[43]. After collection and lyophilization, the dried and 12

ACCEPTED MANUSCRIPT pure polysaccharide, named as KMCP, was obtained in the yield of 3.6%. The average molecular weight of KMCP was measured by HPGPC. As shown in Figure 1, the average molecular weight of KMCP was calculated to be 1.95×106 Da (T = 37.54 min) according to the calibration curve with Dextran T-series standards of

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known molecular weight. The pharmacological effects of polysaccharides usually

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vary with their molecular weight, which is generally classified as low (<10 kDa),

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medium (10 - 10,000 kDa), or high (>10,000 kDa) [44], and KMCP could be classed as a typical medium molecular weight polysaccharide. As demonstrated in Table 1,

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KMCP was further identified as an arabinogalactan which composed of arabinose and

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galactose with a relative molar ratio of 28.1% and 70.3%. SEM Analysis

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KMCP was a water-soluble, white and fluffy polysaccharide. The SEM images

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recorded for KMCP at different magnifications were displayed in Figure 2. Among them, Figure 2a displayed that KMCP had a flat surface in 400×. Figure 2b-c showed

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that KMCP was sponge-like and has significant pore and protrusions. Figure 2d

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further showed morphology changes and surface cracks of KMCP in 35000×. Methylation and NMR Analysis Methylation analysis is an indispensable experimental means which can provide the information of glycosidic linkage of sugar chains. In this study, methylation analysis on KMCP was carried out, by which the resulting data correlated to glycosidic linkage of KMCP was provided and summarized in Table 2. As shown in the table, the reduced KMCP was mainly composed of five derivatives, i.e. 13

ACCEPTED MANUSCRIPT 2,3,6-Me3-Galp, 2,3-Me2-Galp, 2,3,4,6-Me4-Galp, 2,3,5-Me3-Araf, and 2,3-Me3-Araf, with their molar ratios consistent with the overall monosaccharide composition (Table 1), which supported that the arabinose residues are 1- and 1,5-linked, and the galactose residues are 1-, 1,4- and 1,4,6-linked.

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In order to facilitate the description of the further structural elucidation, the five

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C NMR spectrum of KMCP, the

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residues A-E, respectively (see Table 2). In the

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sugar residues characterized by the methylation analysis were briefly described as

signals appeared in the sugar anomeric carbon region at 108.5 ppm and 106.4 ppm

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were designated as C-1 of residues E and D, respectively, and the intensity of the

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signal at 104.7 ppm can be ascribed to the contribution of the carbon C-1 of both residues A and B. Take the proportion of the five major linkage styles that revealed by

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the methylation analysis into consideration (Table 2), these signals of 67.5 and 68.7

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ppm that demonstrated the substitution at C-5 of the arabinosyl residues and C-6 of the galactosyl residues were ascribed to C-5 of residue E and C-6 of residue B,

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respectively. Besides, there have three signals appeared in the 75-85 ppm region at

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77.2, 80.0, 81.3 ppm, of which the stronger signal at 77.2 ppm was attributed to C-4 of the residue A and the other two were assigned to C-4 and C-5 of arabinosyl residues. Generally, in the 1H NMR spectrum, the signals derived from α-anomeric protons usually appeared in the 5.0-6.0 ppm region, while most of the β-anomeric protons usually appeared in the 4.0-5.0 ppm region. In the HSQC 1

(Figure 3A) and

H-1H COSY (Figure 3B) spectra, KMCP correspondingly exhibited signals at 5.1 and

4.6 ppm, assignable to H-1 of all the arabinosyl and the galactosyl residues, 14

ACCEPTED MANUSCRIPT respectively, which indicated that the arabinosyl residues were α-configuration and the galactosyl residues were β-form. Specifically, in the HMBC spectrum (Figure 3C), significant cross-peaks of (AC4, AH1) and (AC1, AH4) were exhibited, which supported the backbone linkages of some residues A with other residues A or B at O-4

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position. The appearance of cross-peaks (DC6, DH1), (BC1, BH6) and (EC6, EH1),

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(BC1, BH6) indicated that residue D, E were linked to O-6 of residue B [45].

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Furthermore, comparison of integrated intensities of anomeric signals for the different sugar residues provided important data on the relative proportions of basic units in the

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arabinogalactan. These data indicated that the molar ratio of galactose residues

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branched at O-6 to free units was 5:2 and the ratio of the arabinose units linked through O-5 to terminal arabinose was 1:2, respectively[46].

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Based on these results described above, we can propose for the structure of

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KMCP as the following repeating unit (Figure 4), but other variations are possible. The structure of this arabinogalactan possesses a backbone of the (1→4)-β-Galp

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residues with 27.5% of those units branched at O-6. The side-groups consisted either

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of single (1→) α-Araf moieties or (1→) α-Araf (1→5)-linked disaccharide moieties[47].

Immunological activities of KMCP KMCP Induced NO Secretion from RAW264.7 cells Macrophages are immune cells that remove cell debris and pathogens by phagocytosis and digestion. As secretory cells, macrophages produce a wide array of chemical substance, including nitric oxide, enzymes, complement proteins, and 15

ACCEPTED MANUSCRIPT regulatory factors[48]. NO produced by macrophages act as an antimicrobial molecule and also a signaling molecule in response to danger signals. The measurement of NO may be used to assess the modulatory effects of polysaccharides [49]. As demonstrated in Figure 5A, KMCP was reveal to stimulate the NO release in

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a dose-dependent manner at concentrations ranging from 10.0 to 200.0 µg/mL. When

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KMCP was tested at 100.0 µg/mL, the secretion of NO increased significantly (p <

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0.05) as compared to the control, but it was lower than that of the positive control (LPS). When KMCP increased to 200.0 µg/mL, the NO content further significantly

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increased to 7.1 times the amount of the control (p < 0.001) , and this is already reach

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to 1.2 times the amount of the positive control. The results demonstrated that KMCP effectively induced NO secretion and activated RAW264.7 cells within the tested

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concentrations.

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Effect of KMCP on the Phagocytosis Activity of RAW264.7 cells The human immune system, the body’s first line of defence, combats a range of

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invasive microorganisms and cancers through macrophages[50]. Activation of

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macrophages is a key event for effective innate and adaptive immunity. Macrophages are acted as a scavenger to engulf any apoptotic or necrosis cells. They are important components of the innate immune system and playing a crucial role in host defence against infection through immuno-inflammatory responses, recognition of pathogens and phagocytosis[51]. The phagocytic activity of RAW264.7 cells when treated with KMCP was examined by neutral red uptake test. As shown in Figure 5B, the phagocytosis indexes 16

ACCEPTED MANUSCRIPT of the RAW264.7 cells showed an upward trend along with the increasing of KMCP concentrations (from 10.0 to 200.0 µg/mL). When KMCP was treated at 100.0 µg/mL, the phagocytosis index increased to a significant extend (p < 0.05) as compared to control, but it was inferior to that of the positive control (LPS). When KMCP was

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tested at 200.0 µg/mL, the phagocytosis index reached to 2.1 times as compared to

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that of the control (p< 0.01). The result indicated that KMCP could activate, trigger

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and improve the phagocytosis of RAW264.7 cells, which was similar to polysaccharides from Collybia radicata mushroom[52].

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Effect of KMCP on Splenocyte Proliferation

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The proliferation of spleen cells is one of the most important steps in the activation pathway of cell-mediated or humoral immunity[53]. The immune response,

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characterized by T cells and B cells respectively, plays an important role in the body’s

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immune system. ConA was used as mitogen for T lymphocytes, and LPS was used as mitogen for B lymphocytes. As observed in Figure 6, the facilitation induced by

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KMCP on LPS-activated or ConA-activated splenocyte proliferation had a

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prominent-related increment at the concentration ranged 10-200 µg/mL, as compared to the group that treated with LPS or ConA alone. When KMCP was tested at 100.0 µg/mL in the presence of LPS or ConA, significant increase of the proliferation rates were displayed (p < 0.001), as compared to that triggered by LPS or ConA alone. When KMCP reached to 200.0 µg/mL, the proliferation rates increased to an even higher extent than tested at 100.0 µg/mL (p < 0.001). Overall, KMCP stimulated both the LPS-activated and ConA-activated splenocyte proliferations in a dose-dependent 17

ACCEPTED MANUSCRIPT relationship. At the same time, the results also suggested that KMCP was low or nontoxic to the spleen cells within tested concentrations. Effect of KMCP on NF-κB Activation NF-κB is a major transcription factor that regulate the transcription of many of

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the immunomodulatory mediators[54]. It is a converging point of various immune and

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inflammatory responses[55]. Upon activation of the immune cell receptor, NF-κB

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becomes activated and enters the nucleus to upregulate genes involved in T-cell development, maturation, and proliferation. Firstly, RAW264.7 cells were treated with

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1.0 µg/mL LPS for 3 hours. Then, using Cellular NF-κB Translocation Kit, the

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process of p65 protein transferring to the nucleus in RAW264.7 cells after stimulation with LPS can be visualized to determine whether NF-κB is activated. As shown in

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Figure 7a, NF-κB p65 protein (red) translocated into nuclei (blue) were observed by

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confocal laser microscopy. The results showed that, the relative density of p65 protein observed in RAW264.7 cells after treatment with KMCP at 100.0 µg/mL is much

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higher than that of the control (PBS), but the intensity is somewhat lower than that of

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the positive control (LPS). The Order of fluorescence intensity of p65 protein with different treatments is: PBS < KMCP < LPS (Figure 7b). These data suggested that KMCP could activate NF-κB nuclear translocation of p65 protein from cytosol to nucleus. In summary, the obtained data supported that KMCP could enhance the immunomodulatory activity through activating NF-κB signaling pathway, and this is in whole consistence with literature reports that the immunoregulatory effects of polysaccharides are related to the NF-κB signaling pathway[56-58]. 18

ACCEPTED MANUSCRIPT In the present paper, a water-soluble polysaccharide named KMCP, with an average molecular weight of 1.95 × 106 Da, was isolated and purified from I. polycephala, a widely cultivated edible and medicinal herb in China. It was characterized as an arabinogalactan, mainly composed of arabinose and galactose in a

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relative molar ratio of 28.1% and 70.3%, respectively. Based on methylation analysis

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and NMR analysis, the structure of KMCP was identified as 72.5% of (1→4)-β-Galp

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residues interspersed with 27.5% of (1→4,6)-β-Galp residues in the main chain, and the branches were composed of (1→5) α-Araf moieties or α-Araf (1→5)α-Araf (1→

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disaccharide moieties attached at O-6 of the (1→4,6)-β-Galp residues. KMCP was

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further revealed to be capable of exhibiting potent immunomodulatory effects via stimulating NO production in RAW264.7 cells, enhancing the phagocytic capacity of

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RAW264.7 cells, and inducing the mice spleen cells proliferation in a dose-dependent

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manner within the test concentrations (10.0-200.0 μg/mL). In addition, the obtained data supported that KMCP enhanced the immunomodulatory activity via activating

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NF-κB signaling pathway. These results supported that KMCP could potentially be an

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effective immunopotentiating agent valuable to be explored for utilization in pharmacological fields or in the development of functional foods. Funding Sources

This work was financially supported by the National Natural Science Foundation of China (31470422 and 30970453), and the Natural Science Foundation of Guangdong Province (2014A030313742), China.

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ACCEPTED MANUSCRIPT References [1] Z. Shi, Flora of China, Beijing: Science Press, 1997. [2] G. Cui, W. Zhang, A. Zhang, H. Mu, H. Bai, J. Duan, C. Wu, Variation in antioxidant activities of polysaccharides from Fructus Jujubae in South Xinjiang area, International journal of biological macromolecules 57 (2013) 278-284. [3] A. Brockhoff, M. Behrens, A. Massarotti, G. Appendino, W. Meyerhof, Broad tuning of the human bitter taste receptor hTAS2R46 to various sesquiterpene lactones, clerodane and labdane diterpenoids, strychnine, and denatonium, J. Agric. Food Chem 55 (2007) 6236-6243.

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[4] S. Yuuya, H. Hagiwara, T. Suzuki, M. Ando, A. Yamada, K. Suda, Takao Kataoka, K. Nagai, Guaianolides as immunomodulators. Synthesis and biological activities of dehydrocostus lactone,

RI

mokko lactone, eremanthin, and their derivatives, Journal of Natural Products 62 (1999) 22-30. [5] J.Y. Yeon, J.T. Hong, S.B. Han, P.Y. Hyun, S.D. Ju, Effect of Ixeris dentata Nakai extract on nitric

SC

oxide production and prostaglandin E2 generation in LPS-stimulated RAW264.7 Cells, Immune network 15 (2015) 325-330.

[6] S.B. Kim, J.E. Kim, O.H. Kang, S.H. Mun, Y.S. Seo, D.H. Kang, D.W. Yang, S.Y. Ryu, Y.M. Lee,

NU

D.Y. Kwon, Protective effect of ixerisoside A against UVB-induced pro-inflammatory cytokine production in human keratinocytes, International journal of molecular medicine 35 (2015) 1411. [7] S. Karki, H.J. Park, A. Nugroho, E.J. Kim, H.A. Jung, J.S. Choi, Quantification of major

MA

compounds from Ixeris dentata, Ixeris dentata Var. albiflora, and Ixeris sonchifolia and their comparative anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 cells, Journal of medicinal food 18 (2015) 83-94. [8]

H.

Kang,

Anti-neuroinflammatory

effects

of

Ixeris

dentata

Nakai

against

D

lipopolysaccharide-induced Bv-2 microglial cells via suppressing NF-κB signaling, Tropical Journal of Pharmaceutical Research 13 (2014) 1629-1635.

PT E

[9] S.B. Kim, O.H. Kang, D.K. Joung, S.H. Mun, Y.S. Seo, M.R. Cha, S.Y. Ryu, D.W. Shin, D.Y. Kwon, Anti-inflammatory effects of tectroside on UVB-induced HaCaT cells, International journal of molecular medicine 31 (2013) 1471-1476. [10] D. Chaturvedi, P.K. Dwivedi, Chapter 6 – Recent Developments on the Antidiabetic Sesquiterpene Natural Products

CE

Lactones and Their Semisynthetic Analogues, Discovery & Development of Antidiabetic Agents from (2017) 185-207.

[11] Y.C. Zhang, L. Zhou, K.Y. Ng, Sesquiterpene lactones from Ixeris sonchifolia Hance and their

AC

cytotoxicities on A549 human non-small cell lung cancer cells, Journal of Asian natural products research 11 (2009) 294-298. [12] S. Zhang, M. Zhao, L. Bai, T. Hasegawa, J. Wang, L. Wang, H. Xue, Q. Deng, F. Xing, Yuhua Bai, J.-i. Sakai, J. Bai, R. Koyanagi, Y. Tsukumo, Takao Kataoka, K. Nagai, K. Hirose, M. Ando, Bioactive guaianolides from siyekucai (Ixeris chinensis), Journal of Natural Products 69 (2006) 1425-1428. [13] G. Franz, Poysaccharides in pharmacy: current applications and future concepts, Planta Medica 55 (1989) 493-497. [14] Y.Q. Du, Y. Liu, J.H. Wang, Polysaccharides from Umbilicaria esculenta cultivated in Huangshan Mountain and immunomodulatory activity, International journal of biological macromolecules 72 (2015) 1272-1276. [15] X. Shang, Y. Chao, Y. Zhang, C. Lu, C. Xu, W. Niu, Immunomodulatory and Antioxidant Effects of Polysaccharides from Gynostemma pentaphyllum Makino in Immunosuppressed Mice, Molecules 20

ACCEPTED MANUSCRIPT 21 (2016) 1085. [16] C. Zhang, K. Huang, Characteristic immunostimulation by MAP, a polysaccharide isolated from the mucus of the loach, Misgurnus anguillicaudatus, Carbohydrate polymers 59(1) (2005) 75-82. [17] J.E. Ramberg, E.D. Nelson, R.A. Sinnott, Immunomodulatory dietary polysaccharides : a systematic review of the literature, Nutrition Journal 9(1) (2010) 54--75. [18] L. Lei, Polysaccharide structure: a hint from gut bacteria, Nature Plants 3 (2017) 17062. [19] D. Belhaj, D. Frikha, K. Athmouni, B. Jerbi, M.B. Ahmed, Z. Bouallagui, M. Kallel, S. Maalej, J. Zhou, H. Ayadi, Box-Behnken design for extraction optimization of crude polysaccharides from Tunisian Phormidium versicolor cyanobacteria (NCC 466): Partial characterization, in vitro

PT

antioxidant and antimicrobial activities, International journal of biological macromolecules 105(Pt 2) (2017) 1501-1510.

RI

[20] Z. Xiaomei, The use of Ixeris, Ningxia Journal of Agriculture and Forestry Science and Technology (6) (2002) 51.

SC

[21] Y.F. Han, K. Gao, z.J. Jia, Two new norsesquiterpenes from Ixeris polycephala, Chinese Chemical Letters 17 (2006) 913-915.

[22] J. Duan, X. Wang, Q. Dong, J.-n. Fang, X. Li, Structural features of a pectic arabinogalactan with

NU

immunological activity from the leaves of Diospyros kaki, Carbohydrate Research 338(12) (2003) 1291-1297.

[23] D. Zheng, Y. Zou, S.J. Cobbina, W. Wang, Q. Li, Y. Chen, W. Feng, Y. Zou, T. Zhao, M. Zhang, L.

MA

Yang, X. Wu, Purification, characterization and immunoregulatory activity of a polysaccharide isolated from Hibiscus sabdariffa L, Journal of the science of food and agriculture 97 (2017) 1599-1606. [24] T. Masuko, A. Minami, N. Iwasaki, T. Majima, S. Nishimura, Y.C. Lee, Carbohydrate analysis by a phenol-sulfuric acid method in microplate format, Analytical Biochemistry 339 (2005) 69-72.

D

[25] N. Blumenkranz, G. Asboe-Hansen, New method for quantitative determination of uronic acids, Analytical Biochemistry 54 (1973) 484-489.

PT E

[26] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analytical Biochemistry 72 (1976) 248-254. [27] S. Honda, E. Akao, S. Suzuki, M. Okuda, K. Kakehi, J. Nakamura, High-performance liquid chromatography of reducing carbohydrates as strongly ultraviolet-absorbing and electrochemically

CE

sensitive 1-phenyl-3-methyl5-pyrazolone derivatives, ANALYTICAL BIOCHEMISTRY 180 (1989) 351-357

[28] Y.J. Liu, X.L. Mo, X.Z. Tang, J.H. Li, M.B. Hu, D. Yan, W. Peng, C.J. Wu, Extraction

AC

optimization, characterization, and bioactivities of polysaccharides from Pinelliae Rhizoma Praeparatum Cum Alumine employing ultrasound-assisted extraction, Molecules 22 (2017) 1-19. [29] X. Sun, H. Wang, X. Han, S. Chen, S. Zhu, J. Dai, Fingerprint analysis of polysaccharides from different Ganoderma by HPLC combined with chemometrics methods, Carbohydrate polymers 114 (2014) 432-9. [30] J.-N. Fang, Methods for methylation analysis of polysaccharides, Journal of International Pharmaceutical Research (04) (1986) 222-226. [31] S. Hakomori, A rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsnlfinyl carbanion in dimethyl sulfoxide, Journal of Biochemiitry 55 (1964) 205-208. [32] Z. Zhao, M. Liu, P. Tu, Characterization of water soluble polysaccharides from organs of chinese Jujube (Ziziphus jujuba Mill. cv. Dongzao), European Food Research and Technology 226 (2007) 985-989. 21

ACCEPTED MANUSCRIPT [33] X.Q. Han, B.C. Chan, H. Yu, Y.H. Yang, S.Q. Hu, C.H. Ko, C.X. Dong, C.K. Wong, P.C. Shaw, K.P. Fung, P.C. Leung, W.L. Hsiao, P.F. Tu, Q.B. Han, Structural characterization and immuno-modulating activities of a polysaccharide from Ganoderma sinense, International journal of biological macromolecules 51(4) (2012) 597-603. [34] R. Kasimu, C. Chen, X. Xie, X. Li, Water-soluble polysaccharide from Erythronium sibiricum bulb: Structural characterisation and immunomodulating activity, International journal of biological macromolecules 105(Pt 1) (2017) 452-462. [35] X. Li, L. Zhao, Q. Zhang, Q. Xiong, C. Jiang, Purification, characterization and bioactivity of polysaccharides from Glossaulax didyma, Carbohydrate polymers 102 (2014) 912-919.

PT

[36] W. Zhang, D. Song, D. Xu, T. Wang, L. Chen, J. Duan, Characterization of polysaccharides with antioxidant and immunological activities from Rhizoma Acori Tatarinowii, Carbohydrate polymers 133

RI

(2015) 154-62.

[37] Z. Xu, S. Lin, W. Wu, H. Tan, Z. Wang, C. Cheng, L. Lu, X. Zhang, Ghrelin prevents protective mechanisms, Toxicology 247 (2008) 133-138.

SC

doxorubicin-induced cardiotoxicity through TNF-alpha/NF-kappaB pathways and mitochondrial [38] G. Cui, W. Zhang, Q. Wang, A. Zhang, H. Mu, H. Bai, J. Duan, Extraction optimization,

NU

characterization and immunity activity of polysaccharides from Fructus Jujubae, Carbohydrate polymers 111 (2014) 245-55.

[39] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to

MA

proliferation and cytotoxicity assays, Journal of lmmunological Methods 65 (1983) 55-63. [40] A. Manosroi, A. Saraphanchotiwitthaya, J. Manosroi, Immunomodulatory activities of Clausena excavata Burm. f. wood extracts, Journal of ethnopharmacology 89 (2003) 155-160. [41] Y. Wu, L. Yi, E. Li, Y. Li, Y. Lu, P. Wang, H. Zhou, J. Liu, Y. Hu, D. Wang, Optimization of

D

Glycyrrhiza polysaccharide liposome by response surface methodology and its immune activities, International journal of biological macromolecules 102 (2017) 68-75.

PT E

[42] Z. Ren, C. He, Y. Fan, H. Si, Y. Wang, Z. Shi, X. Zhao, Y. Zheng, Q. Liu, H. Zhang, Immune-enhancing activity of polysaccharides from Cyrtomium macrophyllum, International journal of biological macromolecules 70 (2014) 590-5. [43] J. Li, L. Ai, Q. Yang, Y. Liu, L. Shan, Isolation and structural characterization of a polysaccharide 83-87.

CE

from fruits of Zizyphus jujuba cv. Junzao, International journal of biological macromolecules 55 (2013) [44] W. Jin, W. Zhang, G. Liu, J. Yao, T. Shan, C. Sun, Q. Zhang, The structure-activity relationship

AC

between polysaccharides from Sargassum thunbergii and anti-tumor activity, International journal of biological macromolecules 105(Pt 1) (2017) 686-692. [45] S. Bagchi, K.J. Kumar, Studies on water soluble polysaccharides from Pithecellobium dulce (Roxb.) Benth. seeds, Carbohydrate polymers 138 (2016) 215-21. [46] Y. Habibi, M. Mahrouz, M.F. Marais, M.R. Vignon, An arabinogalactan from the skin of Opuntia ficus-indica prickly pear fruits, Carbohydrate Research 339 (2004) 1201-1205. [47] X. Zhao, J. Li, Y. Liu, D. Wu, P. Cai, Y. Pan, Structural characterization and immunomodulatory activity of a water soluble polysaccharide isolated from Botrychium ternatum, Carbohydrate polymers 171 (2017) 136-142. [48] D.A. Ovchinnikov, Macrophages in the embryo and beyond: much more than just giant phagocytes, Genesis 46 (2008) 447-62. [49] V.C. Castro-Alves, D. Gomes, N. Menolli, Jr., M.L. Sforca, J.R. Nascimento, Characterization and 22

ACCEPTED MANUSCRIPT immunomodulatory effects of glucans from Pleurotus albidus, a promising species of mushroom for farming and biomass production, International journal of biological macromolecules 95 (2017) 215-223. [50] N. Abdullah, R. Abdulghani, S.M. Ismail, M.H.Z. Abidin, Immune-stimulatory potential of hot water extracts of selected edible mushrooms, Food and Agricultural Immunology 28 (2017) 374-387. [51] W. Fang, D. Bi, R. Zheng, N. Cai, H. Xu, R. Zhou, J. Lu, M. Wan, X. Xu, Identification and activation of TLR4-mediated signalling pathways by alginate-derived guluronate oligosaccharide in RAW264.7 macrophages, Scientific reports 7 (2017) 1-13. [52] Y. Wang, Y. Tian, J. Shao, X. Shu, J. Jia, X. Ren, Y. Guan, Macrophage immunomodulatory

PT

activity of the polysaccharide isolated from Collybia radicata mushroom, International journal of biological macromolecules 108 (2017) 300-306.

RI

[53] P. Galanaud, Generalities on immune response. Humoral immunity, cellular immunity, cell interactions, complement. Study in clinical practice, La Revue Du Praticien 41 (1991) 747.

SC

[54] M.C. Kuo, C.Y. Weng, C.L. Ha, M.J. Wu, Ganoderma lucidum mycelia enhance innate immunity by activating NF-kappaB, Journal of ethnopharmacology 103 (2006) 217-22. [55] A.M. Goldminz, S.C. Au, N. Kim, A.B. Gottlieb, P.F. Lizzul, NF-κB: an essential transcription

NU

factor in psoriasis, Journal of Dermatological Science 69 (2013) 89.

[56] P.A. Baeuerle, T. Henkel, Function and activation of NF-kappa B in the immune system, Annual Review of Immunology 12 (1994) 141.

MA

[57] T. Nakamura, H. Suzuki, Y. Wada, T. Kodama, T. Doi, Fucoidan induces nitric oxide production via p38 mitogen-activated protein kinase and NF-kappaB-dependent signaling pathways through macrophage scavenger receptors, Biochemical & Biophysical Research Communications 343 (2006) 286-294.

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[58] M. Karin, F.R. Greten, NF-kappaB: linking inflammation and immunity to cancer development

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CE

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and progression, Nature Reviews Immunology 5 (2005) 749.

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ACCEPTED MANUSCRIPT FIGURE CAPTIONS Figure 1. Chromatogram of KMCP on HPGPC Figure 2. SEM images of KMCP showing its morphological characteristics (shape and surface) at different magnifications. (a) KMCP (400×), (b) KMCP (2200×), (c)

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KMCP (5000×), (d) KMCP (35000×).

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Figure 3. NMR spectra of KMCP in D2O. (A) HSQC spectra, (B) 1H/1H COSY

Figure 4. Repeating unit structure of KMCP

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spectra, (C) HMBC spectra.

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Figure 5. Effects of KMCP on the secretion of NO and on phagocytosis capacity of

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RAW264.7 cells. Cells were incubated with KMCP at different concentrations (10.0-200.0 µg/mL) for 24 h. (A) Effect of KMCP on the secretion of NO. (B) Effect

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of KMCP on phagocytosis capacity of RAW264.7 cells. These results were expressed

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as mean ± SD. Significant difference from the control group was designated as * p < 0.05, ** p < 0.01 and *** p <0.001

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Figure 6. Effect of KMCP on splenocyte proliferation. Splenocytes were incubated

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with KMCP at different concentrations (10.0-200.0 µg/mL) in the presence of mitogens LPS (10.0 µg/mL) (A) or ConA (5.0 µg/mL) (B) for 48 h. The Spleen lymphocyte proliferation was assessed by using MTT assay. These results were expressed as mean ± SD. Significant difference from the control group was designated as * p < 0.05, ** p < 0.01 and *** p <0.001 Figure 7. Effects of KMCP on activation of nuclear factor-κB (NF-κB). (a) Cultured RAW264.7 cells were treated with PBS, 1 μg/mL LPS, 100.0 μg/mL KMCP. After 3 h 24

ACCEPTED MANUSCRIPT of treatment, the images (400×) were scanned by confocal laser microscopy, and NF-κB p65 protein (red) translocated into nuclei (blue) was observed. (b) The fluorescence intensity of p65 protein and DAPI in RAW264.7 cells by treating with

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PBS, 1.0 μg/mL LPS, 100.0 μg/mL KMCP.

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ACCEPTED MANUSCRIPT Table 1. The composition of KMCP Neutral monosaccharide Carbohydrate

Protein

Uronic acid composition

content (%)

2.32 ± 0.62

4.14 ± 0.29

arabinose

galactose

28.1%

70.3%

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95.37 ± 0.12

content (%)

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KMCP

content (%)

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Molar ratio

Residue

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2,3,6-Me3-Galp

5.0

→4-Galp-(1→

B

2,3-Me2-Galp

1.9

→4,6-Galp-(1→

C

2,3,4,6- Me4-Galp

trace

Galp-(1→

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2,3,5-Me3-Araf

1.8

Araf-(1→

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2,3-Me3-Araf

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