Immunostimulatory activity and structure of polysaccharide from Streptococcus equi subsp. zooepidemicus

International Journal of Biological Macromolecules 57 (2013) 218–225

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Immunostimulatory activity and structure of polysaccharide from Streptococcus equi subsp. zooepidemicus Chunlin Ke a,b , Deliang Qiao a , Jianguang Luo a , Zuomei Li a,b , Yi Sun a , Hong Ye a,∗ , Xiaoxiong Zeng a,∗∗ a b

College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China Department of Biotechnology and Food Engineering, Bengbu College, Bengbu 233030, PR China

a r t i c l e

i n f o

Article history: Received 30 December 2012 Received in revised form 21 January 2013 Accepted 9 March 2013 Available online 16 March 2013 Keywords: Capsule polysaccharide Streptococcus zooepidemicus Immunostimulatory activity Structural characterization

a b s t r a c t In the present study, crude capsule polysaccharides from Streptococcus equi subsp. zooepidemicus C55129 (CP) were purified by DEAE cellulose-52 column and Sephadex G-100 column chromatography to afford purified fractions of CP-1, CP-2 and CP-3. The relative molecular weights of CP-1, CP-2 and CP-3 were 29.0, 891.0 and 1338.0 kDa, respectively. CP-1 was composed of mannose, glucose, arabinose and galactose in a percentage ratio of 66.15:28.97:2.43:2.45. CP-2 was composed of mannose, glucose, galactose and glucuronic acid in a percentage ratio of 40.94:27.71:5.96:25.39. In CP-1, there were pyranose rings, ␣configuration glycosidic bond residues, one backbone chain (1→6 glycosidic bonds) and two branch chains (1→3 and 1→4 glycosidic bonds) in one repeating unit. In vitro immunostimulatory assay, it was found that CP could promote the splenocyte proliferation and increase the activity of acid phosphatase in peritoneal macrophages. The immunostimulatory activity of CP-1 might be related to its monosaccharide composition, molecular weight and ␣-configuration glycosidic bond. © 2013 Elsevier B.V. All rights reserved.

1. Introduction There are increasing evidences indicating that many kinds of polysaccharides, which are widely distributed in animals, plants, and microorganisms, possess potential immunomodulatory activity by stimulating immune system and strengthening the specific and non-specific immune responses in mice [1–3]. And the immunostimulatory activities of polysaccharides isolated from different natural sources depend on their monosaccharide compositions, molecular weights, chain conformation and so on. For example, it has been reported that the ratios of arabinose, mannose and galactose in monosaccharide compositions of polysaccharides are related to their macrophage stimulatory activities [4–6]. Streptococcus equi subsp. zooepidemicus, a commensal of the mucous membranes and skin of animals (notably equine), is associated with various infections in animals and humans. The capsule polysaccharides produced by Streptococcus equi subsp. zooepidemicus may be effective to protect the animal against phagocytes during infection. It has been reported that the main component of capsule polysaccharides from Streptococcus equi is hyaluronic acid [7]. Furthermore, hyaluronic acid has been reported to have immunostimulatory activity both in vitro and in vivo [8]. The Streptococcus equi subsp. zooepidemicus is mainly isolated from pig in

China, and it has pathogenicity only to pig but not to people. Therefore, it is quite different from the Streptococcus zooepidemicus strains that mainly isolated from horse in other worldwide areas. Recently, we have reported that the main component of capsule polysaccharides from Streptococcus equi subsp. zooepidemicus C55129 (CP) was hyaluronic acid and the crude CP and its purified fraction had direct and potent antioxidant activities [9]. However, the other components in the capsule polysaccharides and their immunostimulatory activities are still unknown. Therefore, evaluation of the immunostimulatory activity and structural analysis of the capsule polysaccharides would be important for the elucidation of function and utilization of the polymers. In this paper, we report in detail the immunostimulatory activities of the purified factions (CP-1, CP-2 and CP-3) from crude CP by using in vitro cell models. Furthermore, we also present the structural characterization of CP-1 by Fourier-transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, periodic acid oxidation, smith degradation, methylation analysis combined with high performance liquid chromatography (HPLC), gas chromatography (GC) and GC–mass spectrometry (GC–MS). 2. Materials and methods 2.1. Reagents and materials

∗ Corresponding author. Fax: +86 25 84396791. ∗∗ Corresponding author. Tel.: +86 25 84396791; fax: +86 25 84396791. E-mail addresses: [email protected] (H. Ye), [email protected] (X. Zeng). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.03.033

Strain of Streptococcus equi subsp. zooepidemicus C55129 was obtained from China Institute of Veterinary and Drug Control

C. Ke et al. / International Journal of Biological Macromolecules 57 (2013) 218–225

(Beijing, China). Concanavalin A (ConA), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and monosaccharide references (N-acetylglucosamine, arabinose, fucose, galactose, glucose, glucuronic acid, mannose, rhamnose and xylose) were purchased from Sigma Chemical Co. (St. Louis, USA). Fetal bovine serum (FBS) and RPMI-1640 medium were obtained from Shanghai Sangon Biological Engineering Technology & Services Co. (Shanghai, China). The male Kunming mice were purchased from the Experiment Animal Center of Academy of Military Medical Sciences (Beijing, China). All other chemicals used were ultra pure or analytical grade. 2.2. Preparation of capsule polysaccharides The purified fractions of CP (CP-1, CP-2 and CP-3) were prepared from crude CP according to our reported method [9]. Briefly, Streptococcus equi subsp. zooepidemicus C55129 was cultivated at 37 ◦ C with a shaking at 200 rpm for 24 h in medium containing (g/L): glucose, 40; yeast extract, 20; peptone, 10; K2 HPO4 , 2.5; NaCl, 2 and MgSO4 ·7H2 O, 1.0. The fermentation broth was centrifuged at 10,500 × g for 10 min, and the supernatant was precipitated by addition 2.5 volume of ethanol. The resulting precipitate was collected and dried to afford the crude CP. Then, crude CP (50 mg) dissolved in de-ionized water was applied to a DEAE cellulose-52 column (2.6 cm × 20 cm), and the column was stepwise eluted with 0.0, 0.3 and 0.5 M sodium chloride (NaCl) solutions at a flow rate of 1 mL/min. The fractions (5 mL/tube) were collected automatically and the carbohydrates were determined by the phenol–sulfuric acid method [10], carbazole assay [11] and Elson Morgan’s assay [12], respectively. As results, 3 purified fractions of CP were obtained. They were concentrated, dialyzed against distilled water and purified by a column of Sephadex G100 (2.6 cm × 60 cm), respectively, affording CP-1, CP-2 and CP-3. The three purified fractions were collected, concentrated, dialyzed and lyophilized for further study, respectively.

219

2.3.2. Assay of acid phosphatase activity in peritoneal macrophages The activity of acid phosphatase in peritoneal macrophages was determined according to the reported method with minor modifications [14]. In brief, sterile 3% thioglycollate medium (50 mL/kg body weight) was injected intraperitoneally into male Kunming mice as a stimulant to elicit peritoneal macrophages. Three days later, peritoneal exudates cells were harvested by a lavage of the peritoneal cavity with 5 mL of ice-cold RPMI-1640 medium. The resulting cell suspension was centrifuged at 1700 rpm for 5 min, washed twice with RPMI-1640 medium and adjusted to a density of 2 × 106 cell/mL in the RPMI-1640 medium supplemented with FBS (10%), penicillin (100 unit/mL) and streptomycin (100 ␮g/mL). The cell suspension was added into a 96-well flat-bottom plate (100 ␮L/well) and the cells were allowed to adhere to the bottom of the plate at 37 ◦ C in a humidified 5% CO2 incubator for 3 h. The non-adherent cells were removed by washing three times with RPMI-1640 medium. Then, fresh medium (50 ␮L/well, control group) or test sample (50 ␮L/well, purified faction of CP at a final concentration 25, 50, 100 and 200 ␮g/mL as lower dose, low dose, medium dose and high dose, respectively) was added to each well and the plate was incubated with macrophages at 37 ◦ C for 24 h. The culture medium was removed by rapid inversion and flicking of the plate, and the macrophage monolayer in each well was solubilized by addition of 1% Triton X-100 (25 ␮L). Thereafter, 150 ␮L freshly prepared p-nitrophenyl phosphate (1 mg/mL) in 0.1 M citrate buffer (pH 5.0) was added as a substrate for acid phosphatase, and the plate was incubated at 37 ◦ C for 1 h. The reaction was stopped by addition of 50 ␮L of 3.0 M NaOH solution, and the Abs of the culture well was measured at 405 nm using an ELISA plate reader. The stimulating index of acid phosphatase activity was calculated by the following equation: Acid phosphatase activity =

Abssample − Abscontrol Abscontrol

2.3. Determination of immunostimulatory activity of CP in vitro

2.4. Structural characterization of CP-1

2.3.1. Assay of splenocyte proliferation The assay of splenocyte proliferation was done according to the MTT-based colorimetric method with slight modifications [13]. Briefly, male Kunming mice were killed by cervical dislocation and the spleens were removed aseptically. A single spleen cells suspension was prepared by homogenization in 5.0 mL RPMI-1640 medium. The suspension was centrifuged to afford cell pellet. The erythrocytes in cell pellet were lysed with Tris–NH4 Cl lysing buffer (0.15 M NH4 Cl and 20 mM Tris) for 3 min. The lysed solution was then centrifuged, washed twice with RPMI-1640 medium and adjusted to a density of 1 × 107 cells/mL in the RPMI-1640 medium supplemented with FBS (10%), penicillin (100 unit/mL) and streptomycin (100 ␮g/mL). The spleen cell suspension was pipetted into 96-well flat-bottom plate (50 ␮L/well). Then, group I (normal control group) was treated with RPMI-1640 medium (50 ␮L/well). Group II (lower dose), group III (low dose), group VI (medium dose) and group V (high dose) were treated with purified fraction of CP (50 ␮L/well, final concentration was 25, 50, 100 and 200 ␮g/mL, respectively). Group VI (positive control group) was treated with ConA (50 ␮L/well, final concentration 2.5 ␮g/mL). After incubation at 37 ◦ C in a humidified 5% CO2 incubator for 72 h, MTT solution (10 ␮L/well, 5.0 mg/mL) was added and the plate was further incubated for 4 h. Then, 100 ␮L of 10% SDS in 0.01N HCl solution was added to each well and the plate was kept overnight for the dissolution of formazan crystals. The absorbance (Abs) of each well at 570 nm was measured by an ELISA plate reader (TECAN Infinite F200, Switzerland).

2.4.1. Determination of relative molecular weight and monosaccharide composition The molecular weight of CP was determined on an Agilent 1100 HPLC system equipped with a refractive index detector (RID) and a TSK-GEL G3000SWxl column (7.8 mm × 300 mm, Tosoh Corp., Tokyo). The column was eluted with 0.1 M Na2 SO4 solution (dissolved in 0.01 M phosphate buffer, pH 6.8) at a flow rate of 0.8 mL/min. Temperature of column and detector was set at 25 ◦ C. Pullulan P-800, P-400, P-200, P-100, P-20, P-10 and P-5 (Shodex standard P-82, Showa denko, Japan) were used as standards for molecular weight measurement. Analysis of monosaccharide composition was performed according to the reported method [15] with minor changes. Polysaccharide sample (5 mg) was hydrolyzed in 2 M trifluoroacetic acid (4 mL) to monosaccharides at 120 ◦ C for 2 h. The hydrolyzed product was evaporated to dryness and derivatized by using the following method. Hydroxylammonium chloride (10 mg), inositol (5 mg, used as the internal reference) and pyridine (0.6 mL) were added to the hydrolyzed sample. The mixture was placed in water bath of 90 ◦ C to react for 30 min. After cooling to room temperature, acetic anhydride (1.0 mL) was added, and then the reaction system was placed in a water bath of 90 ◦ C for 30 min again. The reaction products were analyzed by GC (GC-6890N, Agilent) with a flame ionization detector and a HP-5 capillary column (30 m × 0.32 mm × 0.25 ␮m). The operation conditions of GC were as follows: flow rate of N2 , H2 and air was 25, 30 and 400 mL/min, respectively; the temperature of detector and inlet was 280 ◦ C and

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250 ◦ C, respectively; the oven temperature program was set from 120 ◦ C (standing for 3 min) up to 210 ◦ C (standing for 4 min) at a rate of 3 ◦ C/min. Standard monosaccharides (rhamnose, arabinose, fucose, xylose, mannose, glucose and galactose) were also derivatized and analyzed by GC in the same way. 2.4.2. FT-IR spectrometric analysis FT-IR analysis was carried out by the potassium bromide pellet method on FT-IR spectrometer (model MB154S, Bomen, Canada) in the wave-number range of 500–4000 cm−1 .

(A) 1.6 A490 nm

Absorbance

220

1.2

2.4.5. NMR spectral analysis CP-1 was dried in vacuum for several days by using P2 O5 , and then exchanged with deuterium by lyophilizing with D2 O four times. The 1 H and 13 C NMR spectra were recorded at 27 ◦ C on a Bruker Advance DPX-500 spectrometer.

F -3

F -2

0.4

0

10

20

30

40

50

60

70

80

Number of tube

Absorbance (490 nm)

(B) 2.0 1.6 1.2 0.8 0.4 0.0

0

10

20

30

40

50

60

70

80

90

100

70

80

90

100

70

80

90

100

Number of tube

(C) 2.0

Absorbance (490 nm)

2.4.4. Methylation analysis Polysaccharide sample (20 mg) was methylated three times according to the reported method [17], and complete methylation was confirmed by the disappearance of the OH band (3200–3700 cm−1 ) in the FT-IR spectrum. After completed methylation, the permethylated polysaccharide was hydrolyzed, reduced and acetylated by using the reported procedure [18]. The derivatized product was analyzed by GC–MS (GC: model CP 3800, MS: model Satum 2200, Varian) with quartz capillary column (model DB-5MS, 30 m × 0.25 mm × 0.25 ␮m). Temperature program was set rising from 80 ◦ C (standing for 1 min) up to 210 ◦ C (standing for 1 min) at a rate of 8 ◦ C/min and then up to 260 ◦ C (standing for 1 min) at a rate of 20 ◦ C/min. Range of mass charge ratio (m/z) was 30–450.

F -1

0.8

0.0

2.4.3. Periodic acid oxidation and Smith degradation The periodate oxidation and Smith degradation analysis was performed based on the reported method [16]. The reaction solution was dialyzed, dried, hydrolyzed, derivatized and analyzed by GC using the same way as described in Section 2.4.1. Standard monosaccharides (rhamnose, arabinose, fucose, xylose, mannose, glucose and galactose), glycerol and erythritol were used as references.

A530 nm

1.6 1.2 0.8 0.4 0.0 0

10

20

30

40

50

60

Number of tube

(D) 2.0

2.5. Statistical analysis

3. Results and discussion 3.1. Immunostimulatory activity of CP in vitro 3.1.1. Effect of CP on splenocyte proliferation The lymphocyte-mediated immunity plays an important role in the cellular and humoral immune responses. The capacity to elicit an effective T- and B-lymphocyte immunity can be shown by the stimulation of lymphocyte proliferation response [13]. Therefore, we investigated the effect of CP on splenocyte proliferation. Firstly, crude CP was purified through DEAE cellulose-52 chromatography (Fig. 1A) and Sephadex G-100 chromatography in the present study, affording three purified fractions of CP-1 (Fig. 1B), CP-2 (Fig. 1C) and CP-3 (Fig. 1D). As shown in Fig. 2A, the stimulating indexes of CP-1, CP-2 and CP-3 were all exceed 1.0 at a concentration of 100 ␮g/mL, which indicating that CP could promote the

Absorbance (530nm)

1.6

Data was statistically analyzed using SPSS 16.0 software package by one-way analysis of variance (ANOVA). Significant differences between two means were observed by Student–Newman–Keuls test. Difference was considered to be statistically significant if P < 0.05.

1.2 0.8 0.4 0.0

0

10

20

30

40

50

60

Number of tube

Fig. 1. Elution curve of CP by DEAE cellulose-52 anion-exchange chromatography (A) and by Sephadex G-100 column chromatography (B, C and D).

proliferation of splenocyte. In addition, the promoting effects significantly increased (P < 0.05) with the increase of CP concentration. Spleen is one of the major immune organs. There is Tlymphocyte (40%) and B-lymphocyte (60%) in spleen and splenocyte proliferation is related to immunity improvement of T-lymphocyte or B-lymphocyte, especially the T-lymphocyte. The present results indicated that CP had directly mitogen effect on mouse splenocytes and could stimulate proliferation of spleen lymphocyte, strengthen immunological response and improve

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221

(A)

Proliferation index

1.60

CP-1

CP-2

50

100

CP-3

1.20 0.80 0.40 0.00

25

200 ConA

Concentration (ug/mL) (B)

CP-1

Growth rate (%)

60

CP-2

CP-3

50 40 30 20 10 0

25

50

100

200

Concentration (ug/mL) Fig. 2. Effects of CP on splenocyte proliferation (A) and acid phosphatase activity in peritoneal macrophages in vitro (B). Group II: lower dose of CP (25 ␮g/mL), group III: low dose of CP (50 ␮g/mL), group IV: medium dose of CP (100 ␮g/mL), group V: high dose of CP (200 ␮g/mL), and group VI: positive control group (Con A).

immunity. And CP tended to stimulate the immune activity of B cell mediated immune response. 3.1.2. Effect of CP on acid phosphatase activity in peritoneal macrophages Acid phosphatase, a marker enzyme of lysosome, is a signal enzyme of macrophage activation. The activity of acid phosphatase in macrophages increases with the activation of macrophages and decreases with the inhibition of macrophages. Therefore, the immune function of macrophages can be represented by the activity of acid phosphatase [19]. In the present study, we found that the activities of acid phosphatase in macrophages increased significantly by CP-1, CP-2 and CP-3 in a dose-dependent manner (25–100 ␮g/mL, Fig. 2B). However, CP exhibited weaker stimulating activity in high dose (200 ␮g/mL), which indicated that CP possessed the ability of stimulating or immunosuppressive acid phosphatase in peritoneal macrophages in a dose-dependent manner. Macrophages, one of the major immunocyte, possess many immune functions, such as phagocytizing extraneous material, processing antigen, secreting cytokine and so on. Macrophages are

Fig. 3. GC spectra of derivatives from standard monosaccharides (A), monosaccharide compositions of CP-1 (B) and CP-2 (C). 1, rhamnose; 2, arabinose; 3, fucose; 4, xylose; 5, mannose; 6, glucose; 7, galactose; 8, inositol.

known to be the first defense line in preventing foreign invasion [3]. Usually, macrophage is in a state of dormancy and its phagocytosis is weaker. But macrophages may be activated by many immunoenhancers. After activation, macrophages can inhibit the growth and transferring of tumor cells and may have stronger immune functions of endocellular sterilization, exocellular oncolysis, phagocytosis and pinocytosis. The present results suggested that CP could have the function of activating macrophages due

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Fig. 4. FT-IR spectrum of CP-1 (A), FT-IR spectrum (B) and GC–MS chromatogram (C) of CP-1 after methylation reaction.

to its activation on acid phosphatase in peritoneal macrophages. Therefore, CP might practice improving the immunity by activating macrophages and strengthening acid phosphatase activity. The results suggested that CP might practice improving immunity by strengthening macrophages phagocytosis, and it also might be involved in activating both the adaptive and innate immunities to different extent [1,3]. 3.2. Structural characterization of CP-1 3.2.1. Relative molecular weight and monosaccharide composition To determine the relative molecular weights of purified fractions of CP, size-exclusive HPLC was employed and standard glucans were used as references. As results, the relative molecular weights of CP-1, CP-2 and CP-3 were 29.0, 891.0 and 1338.0 kDa, respectively. The GC chromatograms of derivatives from standard monosaccharides and purified fractions of CP are shown in Fig. 3, and Table 1 presents the monosaccharide compositions of CP-1, CP-2 and CP-3. For CP-1, mannose and glucose were the main monosaccharide compositions (Fig. 3B), while other monosaccharides (arabinose and galactose) occurred in less percentage. CP-2 was mainly composed of mannose, glucose and glucuronic acid (Fig. 3C). For CP-3, it was composed by glucuronic acid and N-acetylglucosamine as reported [9]. Because CP-3 is hyaluronic acid, and the

Fig. 5. GC spectra of derivatives from references (A), CP-1 (B) after periodic acid oxidation and Smith degradation. 1, glycerol; 2, erythritol; 3, rhamnose; 4, arabinose; 5, fucose; 6, xylose; 7, mannose; 8, glucose; 9, galactose; 10, inositol.

monosaccharide composition of CP-2 was similar to that of CP-1 except glucuronic acid, the structure of CP-1 was further characterized in the present study. 3.2.2. FT-IR spectrum In the FT-IR spectrum of CP-1 (Fig. 4A), two characteristic absorptions of polysaccharide at about 3100–3700 cm−1 and 2800–3000 cm−1 , were observed. Three strong absorption peaks existing in the range of 1010–1100 cm−1 suggested that the monosaccharide in CP-1 had a pyranose-ring. There was an absorption peak at about 815 cm−1 , but no absorption band at about 891 cm−1 was observed. These results implied that sugar ␣-linked residues in CP-1 were existed [20]. 3.2.3. Results of periodic acid oxidation and Smith degradation Glycosidic linkage location of polysaccharides may be preliminarily determined by consumption of NaIO4 and production of HCOOH in periodic acid oxidation. When CP-1 was oxidized, 1 mol of sugar residue consumed 1.084 mol of NaIO4 and produced 0.262 mol of HCOOH. The results indicated that the non-reducing terminal residue or 1→6 linked glycosidic bond was existed in CP-1. The consumption amount of NaIO4 was more than double amount of HCOOH produced, which

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223

(A) 50

200

40 30 20 10

0

150

-10

5.50 ppm (t1)

5.00

150

100

100 50 0

ppm (t1)

4.00

3.50

50

3.00

0

10.0 ppm (t1)

5.0

0.0

(B)

5000 15000 0

ppm (t1)

105.0

100.0

95.0

90.0

10000

10000

5000

5000

0

80.0 ppm (t1)

ppm (t1)

75.0

70.0

200

65.0

60.0

0

150

Fig. 6.

1

100

50

0

H NMR (A) and 13 C NMR (B) spectra of CP-1.

Table 1 Monosaccharide compositions of purified fractions of CP. Item

Arabinose (%)

CP-1 CP-2 CP-3

2.43 0 0

Mannose (%) 66.15 40.94 0

Glucose (%) 28.97 27.71 0

indicating that there were 1→3, 1→2 or 1→4 linked glycosidic bonds existed in CP-1. In order to obtain more information about glycosidic linkage location, the product of periodic acid oxidation was degraded (Smith degradation) and analyzed by GC [20].

Galactose (%)

Glucuronic acid (%)

N-Acetylglucosamine (%)

2.45 5.96 0

0 25.39 43.75

0 0 40.31

The GC chromatograms of derivatives from references and CP-1 after periodic acid oxidation and Smith degradation are presented in Fig. 5. Notably, the content of glycerol was more than that of erythritol or monosaccharide in CP-1 (Fig. 5B). The results indicated

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Table 2 GC/MS results of methylated sugar residues of CP-1. Relative retention time (min)

Methylated sugar

Model of glycosidic linkage

10.779 13.633 14.629 15.561 15.958 16.725 16.940 17.185 18.448 18.880

2,4,6-Me3 -Man 2,3,6-Me3 -Glc 2,3,4,6-Me4 -Man 2,3,4,6-Me4 -Glc 2,4-Me2 -Man 2,3,4,6-Me4 -Man 2,3,4-Me3 -Gal 2,3,4-Me3 -Glc 2,3,4-Me3 -Man 2,4-Me2 -Man

1→3 1→4 1→ 1→ 1→3,6 1→ 1→6 1→6 1→6 1→3,6

that glycosyl residues of CP-1 were mainly linked by 1→6 or 1→2 glycosidic bond. The amount of mannose decreased in the smith degradation of CP-1, which meant that some sugar residues were oxidated partly in periodic acid oxidation and 1→3, 1→6 or 1→2 linked glycosidic bonds were existed in CP-1. There was a very small amount of glucose in the smith degradation of CP-1, indicating that 1→6 or 1→2 linked glycosidic bond was existed in CP-1. Other monosaccharides were observed in GC chromatogram of Smith degradation. The results suggested that other glycosyl residues were connected mainly by 1→2 or 1→3, or 1→4 or 1→6 glycosidic bond. 3.2.4. Results of methylation analysis The FT-IR spectrum of methylated CP-1 is showed in Fig. 4B. The absorption band at about 3400 cm−1 for O H stretching vibration existed in CP could not be observed, while absorption peak at about 3000–2800 cm−1 for C H stretching vibration was stronger than that of CP. The results suggested that CP-1 had been completely methylated [20]. Fig. 4C presents the GC–MS chromatogram of CP-1 after methylation, and Table 2 shows the analyzable methylated sugar residues of CP-1 and their retention times, molar ratios and linkage modes. Backbone chains of CP-1 were composed of glycose linked by 1→6 glycosidic bond. Branch chains with non-reducing terminal were attached to backbone chain by 1→3 and 1→4 glycosidic bonds. Other monosaccharide residues could not be detected by GC–MS that might be due to their minor contents. In CP-1, three glycosyl residues were connected to form one polysaccharide repeating unit, and one polysaccharide repeating unit was constituted by ten glycosyl residues. There were one backbone chain (one linked by 1→6 glycosidic bond) and two branch chains (one linked by 1→3 glycosidic bond and another connected by 1→6 glycosidic bond) in one repeating unit of CP-1. 3.2.5. NMR spectra 1 H NMR spectrum of CP-1 is shown in Fig. 6A. Chemical shifts at 5.288, 5.026 and 4.979 ppm indicated that sugar rings of CP-1 were pyranose rings and saccharide residues of CP-1 were linked by ␣configuration glycosidic bonds. The results were in good agreement with the results of FT-IR. A group of signals at 3.2–4.2 ppm were produced by C2 C6 protons [21]. Fig. 6B presents the 13 C NMR spectrum of CP-1. Signals at 102.137, 100.508 and 98.165 ppm suggested that the sugar residues of CP-1 were linked by ␣-configuration glycosidic bonds. The results were also in good agreement with the results of FT-IR and 1 H NMR analysis. Signals in the range of 77.839–78.707 ppm were produced by substituted C3 and C4, signals at 73.255 ppm were assigned to un-substituted C2, and signals at 69.599–71.124 ppm were assigned to un-substituted C3 and C4. Signals at 66.617–66.827 ppm were created by substituted C6. Signals at 61.046 ppm were created by un-substituted C6. There was no signal near ı 20.0 ppm, indicating that CP-1 had no methyl

carbon resonance peak. And there was no uronic acid carboxyl resonance signal in the range of ı 160–180 ppm. These results of NMR spectra were consistent with those of FT-IR [22]. It has been reported that polysaccharide from Dioscorea opposita Thunb roots can stimulate ConA-induced T lymphocyte proliferation in vitro and its branches (␣-d-mannopyranosyl and ␤-d-galactopyranosyl residues) are extremely important for the expression of the enhancement of the immunological activity [6]. Lo also reported that the ratios of arabinose, mannose and galactose as the monosaccharide compositions of polysaccharide from different strains of Lentinula edodes could be related to their macrophage stimulatory activities [5]. Therefore, it was possible that the stimulating activity of CP-1 was related with the two branch chains (1→3 glycosidic bond and 1→4 glycosidic bond) and the compositions of arabinose, mannose and galactose [4]. 4. Conclusions In the present study, it was demonstrated that the purified fractions (CP-1, CP-2 and CP-3) of CP could promote the splenocyte proliferation and increase the activity of acid phosphatase in peritoneal macrophages in vitro. The relative molecular weights of CP-1, CP-2 and CP-3 were 29.0, 891.0 and 1338.0 kDa, respectively. For CP-1, mannose and glucose were the main monosaccharide compositions. Structure study demonstrated that the sugar rings of CP-1 were pyranose rings, and glycosyl residues were linked mainly by ␣-configuration glycosidic bonds. Furthermore, there was one backbone chain (1→6 glycosidic bonds) and two branch chains (one linked by 1→3 glycosidic bond and another connected by 1→4 glycosidic bond) in one repeating unit for CP-1. The immunostimulatory activity of CP-1 might be related to its monosaccharide composition, molecular weight and ␣-configuration glycosidic bond. Acknowledgements This work was partly supported by the Fundamental Research Funds for the Central Universities (KYZ201218) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. References [1] T. Liu, L.G. Ye, X.Q. Guan, X.S. Liang, C. Li, Q. Sun, Y. Liu, S.R. Chen, B.G. Liu, D. Pang, International Journal of Biological Macromolecules 54 (2013) 225–229. [2] D.L. Qiao, J.G. Luo, C.L. Ke, Y. Sun, H. Ye, X. Zeng, International Journal of Biological Macromolecules 47 (2010) 676–680. [3] K.H. Wong, C.K.M. Lai, P.C.K. Cheung, Food Hydrocolloids 25 (2011) 150–158. [4] S. Karnjanapratum, M. Tabarsa, M.L. Cho, S.G. You, International Journal of Biological Macromolecules 51 (2012) 720–729. [5] T.C.T. Lo, Y.H. Jiang, A.L. Chao, C.A. Chang, Analytica Chimica Acta 584 (2007) 50–56. [6] G.H. Zhao, J.Q. Kan, Z.X. Li, Z.D. Chen, Carbohydrate Polymers 61 (2005) 125–131. [7] J.B. Woolcock, Journal of General Microbiology 8 (1974) 372–375.

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