Water-soluble polysaccharide from Erythronium sibiricum bulb: Structural characterisation and immunomodulating activity

Accepted Manuscript Title: Water-soluble polysaccharide from Erythronium sibiricum bulb: Structural characterisation and immunomodulating activity Authors: Rena Kasimu, Chunli Chen, Xiangyun Xie, Xue Li PII: DOI: Reference:

S0141-8130(17)30262-3 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.07.060 BIOMAC 7868

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

21-1-2017 3-6-2017 10-7-2017

Please cite this article as: Rena Kasimu, Chunli Chen, Xiangyun Xie, Xue Li, Water-soluble polysaccharide from Erythronium sibiricum bulb: Structural characterisation and immunomodulating activity, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.07.060 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Water-soluble polysaccharide from Erythronium sibiricum bulb: Structural characterisation and immunomodulating activity

Rena Kasimua,*, Chunli Chena, Xiangyun Xiea and Xue Lib

a

Pharmacy College of Xinjiang Medical University, Urumqi 830011, China

b

Supervision and Testing Center for Quality and Safety of Agri-products of Xinjiang Uygur

Autonomous Region, Urumqi 830049, China *

Correspondence: Tel.:+86 991 4362473; fax: +86 991 4362473

E-mail address: [email protected] (Renakasimu)

Highlights 

a water-soluble polysaccharide named ESBP2-1 was isolated and purified from the crude polysaccharides of E. sibiricum bulb.



The structure of ESBP2-1 was characterised through a combination of chemical and spectroscopic methods.



Immunomodulatory assays showed ESBP2-1 from Erythronium sibiricum bulb had a positive immunomodulatory activity.

ABSTRACT: A water-soluble polysaccharide named ESBP2-1 was isolated from Erythronium sibiricum bulb through anion-exchange and size-exclusion chromatography. ESBP2-1 is a neutral

polysaccharide fraction with an average molecular weight of 9.4×105 Da and is composed of glucose, galactose and arabinose in a ratio of 24.3:1.1:1. Methylation analysis, partial hydrolysis and NMR studies revealed that the backbone of ESBP2-1 primarily consists of repeating →1)-α-D-Glcp-(4→ units where the disaccharide side chains of Arap-(1→6)-α-D-Galp-(1→ residue are attached to the O-6 position of Glc. An in vitro assay showed that ESBP2-1 significantly promoted the proliferation and neutral red phagocytosis of RAW 264.7 macrophage cells. In addition, ESBP2-1 stimulated the production of secretory molecules (nitric oxide, TNF-α and IL-1β) of RAW264.7 cells in a dose-dependent manner. These results suggest that ESBP2-1 is a potential immunostimulator. Keywords: Erythronium sibiricum; polysaccharide structure; immunomodulating activity

1. Introduction Erythronium sibiricum (Fisch. et Mey.) Kryl belongs to the Liliaceae family, and its bulb has been extensively applied as traditional herbal medicine and health food for centuries by the Kazak ethnic population in Xinjiang. Kazak ethnic medical text listed the traditional usage of E. sibiricum bulb as “body improvement” [1]. The efficacy of the herb for physical strength is consistent with the immunological activity of polysaccharides. In our previous study, we first reported the extraction and bioactivities of crude polysaccharides from E. sibiricum bulb. In the current study, a water-soluble polysaccharide named ESBP2-1 was isolated and purified from the crude polysaccharides of E. sibiricum bulb. The structure of ESBP2-1 was characterised through a combination of chemical and spectroscopic methods. Moreover, the immunomodulatory effects of ESBP2-1 on RAW264.7 were studied. The results of this study could be used in the selection of active polysaccharides from E. sibiricum bulb. 2. Materials and methods 2.1. Materials and chemicals E. sibiricum bulbs were collected in Altai or Fuhai regions in Xinjiang Province (2013, 2014). Monosaccharide standards (mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose), lipopolysaccharide (LPS), m-hydroxydiphenyl, DMSO and HPLC-grade acetonitrile were purchased from Sigma–Aldrich Co., USA. Murine macrophage RAW264.7 cell lines were obtained from Shanghai Institutes for Biological Sciences, CAS (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) supplemented with high glucose, foetal bovine serum (FBS), penicillin–streptomycin and phosphate-buffered saline (PBS, pH 7.4) was supplied

by Hyclone (GE Healthcare Life Sciences, USA). Cell counting kit-8 (CCK-8) was supplied by DOJINDO (Japan). The Griess reagent system was obtained from Promega (G2930, USA). Mouse IL-1β and TNF-α ELISA kits were purchased from Cusabio Biotech Co. (Wuhan, China). Neutral red was obtained from Solarbio Life Sciences (Beijing, China). All other chemicals and solvents used were of analytical grade and produced in China unless otherwise specified. 2.2. Isolation and purification Crude polysaccharides (ESBP) were separated from E. sibiricum bulbs with hot water as previously described [2]. In brief, 2 g of ESBP was dissolved in distilled water and then centrifuged. The supernatant was loaded on a DEAE Sepharose CL-6B column (Ф55 cm × 3.5 cm) connected with a fraction collector (CBS-B, Shanghai Huxi Analysis Instrument Factory Co., Ltd., China) and a peristaltic pump (BT100-1L, Longer Precision Pump Co., Ltd., China) by eluting with 0–0.8 M NaCl at a flow rate of 50 mL/h [3, 4]. The elution was monitored using the phenol–sulfuric acid method at 490 nm. The fraction eluted with 0.05 M NaCl was collected, dialysed, lyophilised and further purified on a Sephadex G-100 column (Ф75 cm × 2.5 cm), which was eluted with distilled water at a flow rate of 1 mL/min. Each fraction with 5 mL of eluate was collected [5, 6]. The fractions were combined based on the chromatography profile obtained using high-performance gel-permeation chromatography (HPGPC) (the same with 2.3). A purified polysaccharide (ESBP2-1) was obtained, dialysed and lyophilised for further study. 2.3. Determination of purity and molecular weight The homogeneity and molecular weight of ESBP2-1 were identified by HPGPC [7, 8]. The HPGPC instrument was equipped with a Waters e2695 HPLC system (Waters, USA) coupled with a TSKgel G5000PWXL column (Ф7.8 mm × 30 cm, TOSOH, Japan) and a Waters 2414 refractive

index detector (Waters, USA). The column was eluted with water at a flow rate of 0.8 mL/min and was kept at 30 °C during the experiment. A 10 µL aliquot was injected for each run. A standard linear calibration curve was formed with the Dextran T-series standard of different molecular weights (Dextran T2000, T500, T70, T40 and T10) 2.4. Physicochemical characterisation UV spectra in the range of 200–700 cm−1 were recorded on a UV spectrometer (UV-2550, Shimadzu, Japan). Total sugar content was determined using the phenol–sulphuric acid method with glucose as the standard [9, 10]. Uronic acid content was estimated using the m-hydroxydiphenyl method with galacturonic acid as the standard [11, 12]. Iodine-potassium iodide reagent was added to the polysaccharide solution to detect the presence of starch. 2.5. Monosaccharide composition analysis The monosaccharide composition assay was performed as previously described [2]. ESBP2-1 was hydrolysed with trifluoroacetic acid (TFA), derivatised with PMP and NaOH, and then analysed by HPLC using a Waters2690 instrument a Dual λ absorbance Detector (2487, Waters, USA). A Hypersil-BDS C18 column (250 mm × 4.6 mm, 5 μm, Dalian Elite, China) was used in the analysis. Elution was conducted at a flow rate of 1.0 mL/min at 25 °C. The mobile phase was a mixture of 0.02 M ammonium acetate buffer (pH 5.5) and acetonitrile (78:22). The injection volume was 10 μL. Mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose were used as the monosaccharide standards. 2.6. Methylation analysis The polysaccharides were methylated four times in accordance with the method described by Needs and Selvendran [13–15] with slight modifications. A 10 mg polysaccharide sample was

dissolved in 3 mL of DMSO, and 50 mg of dried NaOH power was added under the protection of N2. After NaOH was dissolved, 1.0 mL of methyl iodide (CH3I) was added in triplicate to the reaction system under N2 atmosphere. Ultrasonic treatment was conducted for 30 min after each addition of CH3I. After allowing to stand for an hour in the dark, the reaction was stopped by adding 2.0 mL of distilled water, and the methylated polysaccharides were extracted with 3 × 4 mL of CH2Cl2. The chloroform layer was washed with 3 × 4 mL of distilled water. CH2Cl2 was dried at reduced pressure. The complete methylation was examined by the disappearance of the OH band (3200–3700 cm−1) in the IR spectrum. The methylated product was dissolved with formic acid (90%, 1 mL) at 100 °C for 6 h and then hydrolysed with 2 mol/L TFA (1 mL) at 120 °C for 3 h, followed by the reduction of the hydrolysates with NaBH4 (50 mg). After reduction, the product was acetylated with acetic anhydride (1 mL) and pyridine (1 mL) for 1 h at 100 °C. The methylated alditol acetate was analysed by GC (7890A, Agilent, USA)–MS (Quattro micro GC, Waters, USA) fitted with a HP-5 column (Agilent, USA, 30 m×0.25 mm×0.2 μm). The temperature program was set as follows. The initial column temperature of 80 °C was increased to 210 °C at the rate of 5 °C/min. The rate was changed to 10 °C/min from 210 °C to 280 °C. Afterwards, the temperature was maintained for 5 min at 280 °C. The injection temperature and ion source of the mass spectrometer were set at 250 °C and 280 °C, respectively. 2.7. Partial acid hydrolysis In brief, 50 mg of ESBP2-1 was dissolved in 5 mL of 0.1 M TFA and then hydrolysed at 100 °C for 1 h in a sealed tube. The solution was evaporated to dryness and dialysed for 24 h [16]. The retentate, which was named ESBP2-1-P1, was collected and then freeze-dried. The

methylation of ESBP2-1-P1 was conducted as described in Section 2.6. 2.8. FT-IR analysis The FT-IR spectrum of the polysaccharide sample was recorded with an IR Prestige-21 spectrometer (Shimadzu, Japan) using the potassium bromide disk method. The spectrum was obtained between 400 and 4000 cm−1 with a resolution of 2 cm−1. 2.9. NMR spectroscopy analysis All 1D and 2D NMR spectra were generated with a Varian Inova-600 NMR spectrometer at room temperature. About 50 mg of the polysaccharide was exchanged with deuterium by lyophilising with D2O (99.98%) for three times. The final sample was dissolved in 0.5 mL of D2O. Chemical shifts were expressed in ppm using acetone as the internal standard at 2.225 ppm for 1H and 31.07 ppm for13C [17, 18]. 2.10. Measurement of immunomodulatory activities 2.10.1. Cell culture RAW 264.7 cells were cultured in DMEM medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat-inactivated FBS. The cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C [19]. 2.10.2. Macrophage proliferation assay The effects of ESBP2-1 on RAW 264.7 cell proliferation were assessed using a CCK-8 assay [20, 21]. The cell suspension (1.2 × 105 cells/mL, 100 μL) was planted in a 96-well plate for 24 h and then cultured with different concentrations of ESBP2-1 (0, 12.5, 25, 50, 100, 200 and 400 μg/mL) for 24, 48 or 72 h; LPS (1 μg/mL) was used as a positive control. After incubation, CCK-8 solution (10 μL) was added to each well. Keeping away from light, the plates were further

incubated for 1 h, and the absorbance at 450 nm was measured on a microplate ELISA reader (Multiskan Go 1510, Thermo Fisher Scientific Inc., USA). The stimulated cell viability was calculated as follows: Cell viability (%) = (Adrug-Ablank)/ (Acontrol-Ablank) × 100 where Adrug is the absorbance value of the cell groups treated with ESBP2-1 or LPS, Acontrol is the absorbance of the cell groups treated with the complete medium and Ablank is the absorbance of the complete medium without cells. 2.10.3. Phagocytosis of macrophage test The phagocytic ability of macrophages was evaluated as previously described [22, 23]. After the cells were cultured with ESBP2-1 at different concentrations (0, 25, 50, 100, 200 and 400 μg/mL) or with LPS (1 μg/mL) for 48 h, neutral red solution (0.075%, 100 μL) was added and then incubated for an additional hour. The supernatant was discarded, and the 96-well plates were washed three times with PBS to remove any neutral red that was not phagocytised by the RAW264.7 cells. Subsequently, 100 μL of cell lysates (Ethanol: water: acetic acid 50:49:1) were added and stored for 4 h at room temperature. Finally, the absorbance at 540 nm was measured on a microplate ELISA reader. 2.10.4. Nitric oxide (NO) determination NO secretion from macrophages and nitrite concentration were determined by Griess reaction [24, 25]. RAW 264.7 cells were seeded into a 96-well plate (1.2×105 cells/mL, 100 μL) and then incubated for 24 h at 37 °C. The cultured cells were treated with 100 μL of ESBP2-1 at different concentrations (0, 25, 50, 100, 200 and 400 μg/mL) or with LPS (1 μg/mL). After incubating for 48 h at 37 °C, 50 μL of the cultured cell supernatant was added to the wells of another 96-well

plate, and then 1% sulfanilamide solution in 5% phosphoric acid was dispensed to all experimental samples. The mixture that was protected from light was incubated for 5–10 min at room temperature. Then, 50 μL of 0.1% N-1-napthylethylenediamine dihydrochloride solution was dispensed to all wells. Keeping away from light, the mixture was again incubated at room temperature for 5–10 min. Finally, the absorbance at 520 nm was measured on a microplate reader within 30 min. The NO secretion from RAW 264.7 cells was calculated with reference to a standard curve obtained with NaNO2. 2.10.5. Cytokine production assay The effects of ESBP2-1 on cytokine secretion in RAW264.7 macrophages were evaluated by measuring the TNF- α and IL-β levels in culture supernatants of RAW264.7 treated with ESBP2-1 through ELISA [26]. Cells were seeded into a 24-well plate (2.0×105 cells/mL, 1 mL) and incubated for 24 h at 37 °C. The cultured cells were then treated with 1 mL of ESBP2-1 at different concentrations (0, 25, 50, 100 and 200 μg/mL) or with LPS (1 μg/mL). The supernatants were collected after incubation for 48 h. The levels of TNF-α and IL-1β were measured at 450 nm by using commercial ELISA kits in accordance with the manufacturers’ instructions. Cytokine quantities in the samples were calculated from the standard curves of recombinant cytokines using a linear regression method. 3. Results and discussion 3.1. Purification and preliminary characterisation of ESBP2-1 The fraction of ESBP2-1 was obtained through further purification of ESBP by using an ion-exchange chromatography of DEAE Sepharose CL-6B and gel-filtration chromatography of Sephadex G-100. The molecular weight of ESBP2-1 was determined by HPGPC on a

TSKgelG5000PWXL column. As shown in Fig. 1, ESBP2-1 exhibited a single and symmetric peak in the HPGPC chromatogram, indicating that ESBP2-1 is a homogeneous polysaccharide. The retention time of ESBP2-1 was 8.23 min. According to the equation of the standard curve (Log Mw = −0.5228t+10.274 R=0.9759, where Mw represents the molecular weight and t represents the retention time), the average molecular weight of ESBP2-1 was calculated as 9.4×105 Da. ESBP2-1 is a light yellow and water-soluble power. Its total sugar and uronic acid contents were 99.3% and 0.02%, respectively, which indicated that ESBP2-1 is a neutral polysaccharide. On the basis of the UV scanning spectrum of ESBP2-1 solution (Fig. 2), the absence of absorption at 260 and 280 nm confirmed that ESBP2-1 does not contain protein and nucleic acid. The iodine-potassium iodide reaction showed that ESBP2-1 is a non-starch polysaccharide. The monosaccharide composition of ESBP2-1 was analysed through HPLC. The monosaccharide component of ESBP2-1 was identified by comparing the retention time with the standards (Fig. 3). The result showed that ESBP2-1 is composed of glucose, galactose and arabinose in a ratio of 24.3:1.1:1, which indicated that the polysaccharide is a glucan that contains a small amount of non-glucose residues.

2.5

2.0

AU

1.5

1.0

0.5

0.0 0

2

4

6

8

10

12

14

16

18

20

time(min)

Fig. 1. HPGPC chromatogram of ESBP2-1 on a TSKgel G5000PWXL Column

2.0

Absorbance

1.5

1.0

0.5

0.0 200

300

400

500

wavelength(nm)

Fig. 2. UV scanning spectrum of ESBP2-1

600

700

0.5

0.4

5

AU

0.3

A B

1

0.2

2

3

PMP

0.1

7

4 6

0.0 0

2

4

6

8

10 12 14 16 18 20 22 24 26 28 30 32 34

time(min)

Fig. 3. HPLC analysis of monosaccharide composition in ESBP2-1. The standards were separated under the conditions as described in (A), and the derivatives of hydrolysate were separated as described in (B). (1 mannose, 2 rhamnose, 3 glucuronic acid, 4 galacturonic acid, 5 glucose, 6 galactose and 7 arabinose) 3.2. Infrared spectra of ESBP2-1 The FT-IR spectrum illustrated in Fig. 4 exhibited the typical signals of the polysaccharide in the range of 4000–400 cm−1. A strong and wide stretching peak at 3500–3000 cm−1 and a weak stretching peak at 2923 cm−1 were attributed to O–H and C–H stretching vibrations, respectively. The band towards 1640 cm−1 occurred due to the absorbance of COO− deprotonated carboxylic group or bound water [27]. The peak at 1410 cm−1 corresponded to C-H deformation vibration, and the region of 1200–950 cm−1 was dominated by ring vibrations overlapping with the stretching vibrations of the C-OH side groups and the (C–O–C) glycosidic band vibration [28]. The absorptions at 1149, 1080 and 1026 cm−1 showed that ESBP2-1 is a pyranose form of carbohydrates [29]. The characteristic peak at 856 cm−1 in the IR spectrum indicated the existence of α-glucopyranosyl residues. No apparent peak was observed at 1750–1700 cm−1, indicating that

the uronic acid content of ESBP2-1 is negligible [30].

Transmittance(%)

100

80

60

40

4000

3500

3000

2500

2000

1500

1000

500

-1

wavenumber(cm )

Fig. 4. FTIR spectrum of ESBP2-1 3.3. Methylation analysis of ESBP2-1 and ESBP2-1-P1 Methylation analysis provides important information about the linkage pattern of sugar residues. The preparation of partially O-methylated alditol acetates (PMAAs) involves successive methylation, hydrolysis, reduction and acetylation identified with GC–MS using EI-MS fingerprints. On the basis of this analysis of PMAA, the methylated sugar residues, molar ratios and

linkage

modes

of

ESBP2-1

are

shown

in

Table

1.

Large

amounts

of

2,3,6-tri-O-methyl-triacetate-D-glucitol were detected in ESBP2-1, indicating the presence of the →1)-α-D-Glcp-(4→

residue

(A).

2,3,4-tri-O-methyl-triacetate-D-Galactitol, 3-di-O-methyl-tetraacetate-D-glucitol →1)-α-D-Galp-(6→

residues

(B),

In

addition,

small

amounts

2,3,4-tri-O-methyl-β-L-arabinopyranoside

were terminal

detected, unit

indicating

Arap-(1→(C)

the and

of

and

presence branching

2, of

point

→1)-α-D-Glcp-(4,6→ residues (D). The molar ratio for A, B, C and D was 22.8:1:1.1:1.2 in ESBP2-1. The ratio between the terminal unit and branching point was 0.92, which is consistent

with the fact that the number of branching points is approximately equal to the number of terminal units. Compared with ESBP2-1, ESBP2-1-P1 was mainly composed of A. Other residues were not detected, which indicated that →1)-α-D-Glcp-(4→ residues were in the main chain of ESBP2-1. The presence of C and D revealed that they are located at the branch of ESBP2-1 and are easily removed through hydrolysis. As the branching point, the ratio of C and D almost approached 1:1, suggesting their linkage with one another. The data from the methylation analysis revealed that ESBP2-1 is a branched glucan that is mainly composed of →1)-α-D-Glcp-(4→ residues. The branches were at the O-6 position of the main chain and were composed of one molecule Arap-(1→residue connecting one molecule →1)-α-D-Galp-(6→residue.

Table 1 GC-MS analysis of the methylated products Molar ratio PMAA

Reside ESBP2-1

ESBP2-1-P1 1

2,3,6-Me3-Glcp (A)

→1)-α-D-Glcp-(4→

22.8

2,3,4-Me3- Galp (B)

→1)-α-D-Galp-(6→

1

2,3,4,6-Me4- Arap (C)

Arap-(1→

1.1

2,3-Me2- Glcp (D)

→1)-α-D-Glcp-(4,6→

1.2

3.4. 1D and 2D NMR spectra of ESBP2-1 The structural features of ESBP2-1 were further elucidated by NMR spectral analysis. The 1H and13C NMR spectra (Figs. 5–6) of ESBP2-1 were crowded in a narrow region within 3.0–5.3 ppm ( 1H NMR) and 60–101 ppm ( 13C NMR), which are typical for polysaccharides. The

anomeric proton signals at δ5.23, 5.22 and 4.63 ppm, as well as the anomeric carbon signals at δ100.51, 100.10 and 97.70 ppm, revealed that ESBP2-1 contains three types of monosaccharide residues. Combined with COSY, HMBC, HSQC spectra (Figs. 7–9) and documented data, the anomeric proton and carbon signals at δ5.22/100.10, 5.23/100.51 and 4.63/97.70 ppm were assigned to the H-1/C-1 of →1)-α-D-Glcp-(4→, →1)-α-D-Galp-(6→ and Arap-(1→ residues, respectively. The signals at 72.54, 74.33, 77.71, 72.16 and 61.36 ppm corresponded to C-2 to C-6 of →1)-α-D-Glcp-(4→ residues, respectively. The signals at 73.68, 74.93, 76.56, 76.76 and 70.39 ppm were attributed to the C-2 to C-6 of →1)-α-D-Galp-(6→ residues, respectively. The signals at 71.15, 72.34, 73.52 and 61.51 ppm were attributed to the C-2 to C-5 of Arap-(1→ residues, respectively.

Fig. 5. 1H NMR spectrum of ESBP2-1

Fig. 6. 13C NMR spectrum of ESBP2-1

Fig. 7. gHMBC spectrum of ESBP2-1

Fig. 8. gCOSY spectrum of ESBP2-1

Fig. 9. gHSQC spectrum of ESBP2-1

Table 2 Chemical shift (δ) assignments of 1H NMR and 13C NMR spectra of ESBP2-1 based on 2D NMR Residue

C1

C2

C3

C4

C5

C6

→1)-α-D-Glcp-(4→

100.10

72.54

74.33

77.71

72.16

61.36

→1)-α-D-Galp-(6→

100.51

73.68

74.93

76.56

76.76

70.39

Arap-(1→

97.70

71.15

72.34

73.52

61.51

→1)-α-D-Glcp-(4,6→

100.10

72.54

74.33

77.71

72.16

70.11

After the methylation analysis, partial hydrolysis experiment and NMR studies, the suggested ESBP2-1 repeat unit was concluded as the following: OH O HO

HO

O OH O

OH

HO OH O

O O

HO

O OH

O

OH

HO

x

O

OH O

HO OH O

y

x+y≈23 3.5. Immunomodulatory activities 3.5.1. Effect of ESBP2-1 on macrophage proliferation Macrophage cell plays a key role in host defence and innate immune response. Activation of macrophages is a self-defence mechanism that protects the host against pathogen infection [31]. In the present study, RAW 264.7 cells were activated by ESBP2-1 at 12.5–400 μg/mL for 24–72 h. The CCK-8 method was used to detect the proliferative effect of ESBP2-1. The results are shown in Fig. 10. Experimental data revealed that the cells activated by ESBP2-1 for 48 and 72 h have greater viability than the cells activated for 24 h. When the activation time was 48 h, all

concentrations of polysaccharides significantly stimulated the proliferation of RAW 264.7 cells (p<0.001).The results of 72 h were similar to 48 h, except for the result of 400 μg/mL. When the concentration was 400 μg/mL, the cell viability was similar to that of the control group (p>0.05). Therefore, 48 h was applied in the following tests.

240 220

24h 48h 72h

***

200

***

cell viability(%)

180 160

*** *** *** ***

140

***

*** *

**

*

***

***

*** ***

**

120 100 80 60 40 20 0 control

LPS

12.5

25

50

100

200

400

concentration(μg/ml)

Fig. 10. Effects of ESBP2-1 on the proliferation of RAW264.7 cells. The data were shown as mean±SD (n=5). *p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control. 3.5.2. Effect of ESBP2-1 on macrophage phagocytosis

Phagocyte phagocytosis is the first and pivotal step in the immune response [32]. In this work, the phagocytic activity of RAW264.7 macrophages treated with ESBP2-1 was evaluated by the neutral red. As shown in Fig. 11, a dose-dependent enhancement of phagocytic activity was observed in the macrophages treated with ESBP2-1 at 25–400 μg/mL for 48 h. Compared with the negative control group, 400 μg/mL ESBP2-1 significantly stimulated the phagocytic activity of RAW 264.7 cells (p<0.05), whereas the phagocytosis index of ESBP2-1 reached a maximum of 1.27±0.10.

2.0

control LPS ESBP2-1

phagocytosis index

1.5

*

*

1.0

0.5

0.0

control

LPS

25

200 100 50 concentration(μg/ml)

400

Fig. 11. Effect of ESBP2-1 on macrophage phagocytosis. The data were shown as mean±SD (n=3). *

p<0.05 vs. control.

3.5.3. Effect of ESBP2-1 on NO production in macrophages Nitric oxide is a chemical messenger and an important effector molecule for macrophages. NO produced by activated macrophages is cytotoxic or cytostatic in fighting against invading pathogens and tumour cells [33]. This study measured the changes in NO production in the macrophages culture with ESBP2-1. As shown in Fig. 12, NO production increased with increasing polysaccharide concentration. The productions of NO were 6.73, 7.41 and 10.75 μM when the ESBP2-1 concentrations were 100, 200 and 400 μg/mL, respectively. Compared with the negative control group, these three concentrations of ESBP2-1 significantly enhanced NO production in RAW 264.7 cells (p<0.05, p<0.01 and p<0.001, respectively).

22 20 18

control LPS ESBP2-1 ***

NO production(μM)

16 14 12

***

10 8

*

**

6 4 2 0

control

LPS

25

200 100 50 concentration(μg/ml)

400

Fig. 12. Effect of ESBP2-1 on NO production in RAW264.7 cells. The data were shown as mean±SD (n=3). *p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control. 3.5.4. Effect of ESBP2-1 on cytokine production in macrophages Cytokines play important roles in immunomodulatory responses and inflammatory reactions. The induction of cytokine synthesis evaluates the augmentation activity of the innate immunity. In this study, the immunomodulatory activity of ESBP2-1 was measured by analysing the cytokine secretions of TNF-α and IL-1β from RAW264.7 cells. As shown in Fig. 13, ESBP2-1 increased the secretion of IL-β in a dose-dependent manner, and the secretion levels significantly increased after treatment with 50, 100 and 200 μg/mL ESBP2-1 (p < 0.01, p < 0.001 and p<0.001, respectively). A dose-dependent enhancement of TNF-α was also observed in the macrophages treated with ESBP2-1 at 25–50 μg/mL, whereas the uptake slightly decreased when the concentration of the polysaccharide was further increased (100–200 μg/mL). However, all test concentrations of ESBP2-1 significantly up-regulated the macrophage production of TNF-α compared with the control group (p<0.001). In summary, the effect of ESBP2-1 on cytokine production further revealed that ESBP2-1

effectively activates macrophages and exerts significant immunomodulatory activities.

100

control LPS ESBP2-1

A

***

IL-1β production(pg/ml)

80

***

60

***

40

** 20

0

control

control LPS ESBP2-1

B 2000

TNF-α production(pg/ml)

LPS

25

50 100 concentration(μg/ml)

200

***

1500

*** ***

***

***

1000

500

0

control

LPS

25

50 100 concentration(μg/ml)

200

Fig. 13. Effect of ESBP2-1 on TNF-α and IL-1β production in macrophages. The data were shown as mean±SD (n=3). **p<0.01 vs. control; ***p<0.001 vs. control. 4. Conclusion A homogeneous and water-soluble polysaccharide (ESBP2-1) with a molecular weight of 9.4×105 Da was extracted from E. sibiricum bulb, which was purified through an ion-exchange chromatography of DEAE Sepharose CL-6B and gel-filtration chromatography of Sephadex

G-100. Physicochemical characterisation and monosaccharide composition analysis revealed that ESBP2-1 is a glucan that is composed of a large number of glucose and a small amount of galactose and arabinose in a ratio of 24.3:1.1:1. The repeating unit of ESBP2-1 is a →1)-α-D-Glcp-(4→ backbone where the branching occurs at the O–6 positions of the residues. The side chains were composed of terminal Arap-(1→residue linking →1)-α-D-Galp-(6→residue. Immunomodulatory assays showed that ESBP2-1 significantly promotes the proliferation and neutral red internalisation in RAW 264.7 macrophage cells in vitro. Furthermore, ESBP2-1 promotes innate immunity by up-regulating NO production and cytokine (TNF-α and IL-1β) secretion. These results suggest that ESBP2-1 from Erythronium sibiricum bulb has a positive immunomodulatory activity; however, its precise mechanisms and structure–activity relationship need to be further explored in future studies. Acknowledgement This work was supported by the Natural Science Foundation of Xinjiang Uygur Autonomous Region of China (No. 2016D01C181). References [1] X. Xu, B.G. Huangerhan, Kazakh drug chi, Nationalities Press, Beijing, 2009:397-399(in Chinese) [2] C.L. Chen, R. Kasimu, X.Y. Xie, Y.L. Zheng, W.H. Ding, Optimized extraction of Erythronium sibiricum bulb polysaccharides and evaluation of their bioactivities, International Journal of Biological Macromolecules. 82(2016):898-904 [3] L. Tang, Y.C. Chen, Z.B. Jiang, S.P. Zhong, W.Z. Chen, F.C. Zheng, G.G. Shi, Purification, partial characterization and bioactivity of sulfated polysaccharides from Grateloupia livida,

International Journal of Biological Macromolecules. 94 (2017) 642–652 [4] W.R. Cai, H.L. Xu, L.L. Xie, J. Sun, T.T. Sun, X.Y. Wu, Q.B. Fu, Purification, characterization and in vitro anticoagulant activity of polysaccharides from Gentiana scabra Bunge roots, Carbohydrate Polymers. 140 (2016) 308–313 [5] Y.X. Mei, H. Zhu, Q.M. Hu, Y.Y. Liu, S.M. Zhao, N. Peng, Y.X. Liang, A novel polysaccharide from mycelia of cultured Phellinus linteus displays antitumor activity through apoptosis, Carbohydrate Polymers. 124 (2015) 90–97 [6] Y. Zhang, C.J. Ren, G.B. Lu, W.Z. Cui, Z.M. Mu, H.J. Gao, Y.W. Wang, Purification, characterization and anti-diabetic activity of a polysaccharide from mulberry leaf, Regulatory Toxicology and Pharmacology. 70 (2014) 687–695 [7] P. Yi, N.S. Li, J.B. Wan, D.Z. Zhang, M.Y. Li, C.Y. Yan, Structural characterization and antioxidant activity of a heteropolysaccharide from Ganoderma capense, Carbohydrate Polymers. 121 (2015) 183–189 [8] J. Dou, Y.H. Meng, L. Liu, J. Li, D.Y. Ren, Y.R. Guo, Purification, characterization and antioxidant activities of polysaccharides from thinned-young apple, International Journal of Biological Macromolecules. 72 (2015) 31–40 [9] C.K. Balavigneswaran, T. Sujin Jeba Kumar, R. Moses Packiaraj, A. Veeraraj, S. Prakasha, Anti-oxidant activity of polysaccharides extracted from Isocrysis galbana using RSM optimized conditions, International Journal of Biological Macromolecules.60 (2013) 100–108 [10] M. DuBois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Analytical Biochemistry. 28(1956) 350–356. [11] N. Blumenkranta, G. Asboe-Hansen, New method for quantitative determination of uronic

acids, Analytical Biochemistry. 54 (1973) 484–489 [12]Nastaran Khodaei , Salwa Karboune, Valérie Orsat, Microwave-assisted alkaline extraction of galactan-rich rhamnogalacturonan I from potato cell wall by-product, Food Chemistry. 190 (2016) 495–505 [13]P.W.

Needs,

R.R.

Selvendran,

Avoiding

oxidative

degradation

during

sodium

hydroxide/methyl iodide-mediated carbohydrate methylation in dimethyl sulfoxide. Carbohydrate Research. 245 (1993) l-10 [14] C.P. Zheng, Q. Dong, Z.Y. Du, P.P. Wang, K. Ding, Structural elucidation of a polysaccharide from Chrysanthemum morifolium flowers with anti-angiogenic activity, International Journal of Biological Macromolecules. 79 (2015) 674–680 [15]L.J. You, Q. Gao, M.Y. Feng, B. Yang, J.Y. Ren, L.J. Gu, C. Cui, M.M. Zhao, Structural characterisation of polysaccharides from Tricholoma matsutake and their antioxidant and antitumour activities, Food Chemistry. 138 (2013) 2242–2249 [16]C.P. Zheng, Q. Dong, H.J. Chen, Q.F. Cong, K. Ding, Structural characterization of a polysaccharide from Chrysanthemum morifolium flowers and its antioxidant activity, Carbohydrate Polymers. 130 (2015) 113–121 [17] H.D. Huang, M.M. Wu, H.P. Yang, X.Y. Li, M.N. Ren, G.Q. Li, T. Ma, Structural and physical properties of sanxan polysaccharide from Sphingomonas sanxanigenens, Carbohydrate Polymers. 144 (2016) 410–418 [18] N. Li, W.J. Mao, M.X. Yan, X. Liu, Z. Xia, S.Y. Wang, B. Xiao, C.L. Chen, L.F. Zhang, S.J. Cao, Structural characterization and anticoagulant activity of a sulfated polysaccharide from the green alga Codium divaricatum, Carbohydrate Polymers. 121 (2015) 175–182

[19] C. Li, Q. Huang, X. Fu, X.J. Yue, R.H. Liu, L.J You, Characterization, antioxidant and immunomodulatory activities of polysaccharides from Prunella vulgaris Linn, International Journal of Biological Macromolecules. 75 (2015) 298–305 [20] S.Y. Dong, Y.J. Guo, Y. Feng, X.X. Cui, S.H. Kuo, T. Liu, Y.C. Wu, The epigenetic regulation of HIF-1αby SIRT1 in MPP+ treated SH-SY5Y cells, Biochemical and Biophysical Research Communications. 470 (2016) 453-459 [21] S.S. Lin , X.D. Lyu, J. Yu, L. Sun , D.Y. Du, Y.Q. Lai , H.Y. Li , Y. Wang , L.Y. Zhang , H.P. Yin, S.T. Yuan, MHP-1 inhibits cancer metastasis and restores topotecan sensitivity via regulating epithelial-mesenchymal transition and TGF-βsignaling in human breast cancer cells, Phytomedicine. 23 (2016) 1053–1063 [22] Z.L. Sun, Y. Peng, W.W. Zhao, L.L. Xiao, P.M. Yang, Purification, characterization and immunomodulatory activity of a polysaccharide from Celosia cristata, Carbohydrate Polymers. 133 (2015) 337–344 [23] 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 [24] H. Zhao, Q.H. Wang, Y.P. Sun, B.Y. Yang, Z.B. Wang, G.F. Chai, Y.Z. Guan, W.G. Zhu, Z.P. Shu, X. Lei, H.X. Kuang, Purification, characterization and immunomodulatory effects of Plantago depressa polysaccharides, Carbohydrate Polymers. 112 (2014) 63–72 [25] W. Liu, H. Wang, J.P. Yu, Y.M. Liu, W.S. Lu, Y. Chai, C. Liu, C. Pan, W.B. Yao, X.D. Gao, Structure, chain conformation, and immunomodulatory activity of the polysaccharide purified from Bacillus Calmette Guerin formulation, Carbohydrate Polymers. 150 (2016) 149–158

[26] M.M. Liu, P. Zeng, X.T. Li, L.G. Shi, Antitumor and immunomodulation activities of polysaccharide from Phellinus baumii, International Journal of Biological Macromolecules. 91 (2016) 1199–1205 [27] W. Wang, Y. Zou, Q. Li, R.W. Mao, X.J. Shao, D. Jin, D.H. Zheng, T. Zhao, H.F. Zhu, L. Zhang, L.Q. Yang, X.Y. Wu, Immunomodulatory effects of a polysaccharide purified from Lepidium meyenii Walp. on macrophages, Process Biochemistry. 51 (2016) 542–553 [28] L.J. Wang, Y. Yao, W. Sang, X.S. Yang, G.X. Ren, Structural features and immunostimulating effects of three acidic polysaccharides isolated from Panax quinquefolius, International Journal of Biological Macromolecules. 80 (2015) 77–86 [29] G.H. Zhao, J.Q. Kan, Z.X. Li, Z.D. Chen, Characterization and immunostimulatory activity of an (1→6)-α-D-glucan from the root of Ipomoea batatas, International Immunopharmacology. 5 (2005) 1436– 1445 [30] C. Li, Q. Huang, X. Fu, X.J. Yue, R.H. Liu, L.J. You, Characterization, antioxidant and immunomodulatory activities of polysaccharides from Prunella vulgaris Linn, International Journal of Biological Macromolecules. 75 (2015) 298–305 [31]M.L. Wang, Y.Y. Hou, Y.S. Chiu, Y.H. Chen, Immunomodulatory activities of Gelidium amansii gel extracts on murine RAW 264.7 macrophages, Journal of food and drug analysis. 21(2013) 397-403 [32] Y.G. Bai, P.Y. Zhang, G.C. Chen, J.F. Cao, T.T. Huang, K.S. Chen, Macrophage immunomodulatory activity of extracellular polysaccharide (PEP) of Antarctic bacterium Pseudoaltermonas sp.S-5, International Immunopharmacology. 12 (2012) 611-617 [33] Foo Y. Liew, Regulation of lymphocyte functions by nitric oxide, Current opinion in

immunology, 7(1995) 396-399