International Immunopharmacology 21 (2014) 269–278

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Polysaccharides from Inonotus obliquus sclerotia and cultured mycelia stimulate cytokine production of human peripheral blood mononuclear cells in vitro and their chemical characterization Xiangqun Xu ⁎, Juan Li, Yan Hu Department of Chemistry, Zhejiang Sci-Tech University, Hangzhou 310018, China

a r t i c l e

i n f o

Article history: Received 5 January 2014 Received in revised form 29 April 2014 Accepted 11 May 2014 Available online 24 May 2014 Keywords: Inonotus obliquus Immunomodulator Cytokine Polysaccharides Sclerotia Submerged fermentation

a b s t r a c t Inonotus obliquus is an edible and medicinal mushroom to treat many diseases. In the present study, polysaccharides and fractions were isolated and purified by DEAE-52 and Sephadex G-200 chromatography from I. obliquus wild sclerotia, culture broth and cultured mycelia under submerged fermentation. The extracts and fractions could significantly induce the secretion of TNF-α, IFN-γ, IL-1β, and IL-2 in human peripheral blood mononuclear cells (PBMCs) and showed no toxicity to PBMCs. The stimulation effect of the six extracts and eight fractions on the four-cytokine production was dose-dependent. Sclerotial polysaccharides were more effective in the fourcytokine production at 150 μg/ml while exopolysaccharides and endopolysacchrides showed a much better effect on IL-1β production at 30 μg/ml. Purified fractions from exopolysaccharides and endopolysaccharides were more effective than the fraction from sclerotia in most cytokine production. These heteropolysaccharide-protein conjugates mainly contained glucose, galactose, and mannose. Protein content, molecular weight, monosaccharide molar ratio, and anomeric carbon configuration differed from each other and had effects on the cytokine induction activity of the polysaccharides to some extent. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Edible and medicinal mushrooms including Ganoderma lucidum, Phellinus linteus, Agaricus campestris, Lentinus edodes, Agaricus blazei, Antrodia cinnamomea, and Inonotus obliquus (I. obliquus) have been popularly used in Asia and the Slav regions for a long time [1]. In recent years, they have been widely studied due to their health-promoting properties and relatively low toxicity [1]. I. obliquus, a rare medicinal macrofungus in folk medicine, has been documented to contain polysaccharides, polyphenols, triterpenes, melanin and steroid, showing various biological activities [2–4]. Studies have repeatedly demonstrated that many mushroomextracted polysaccharides are major biologically active substances, which have significant effects on the innate and adaptive immune response, and therefore have the potential as immunostimulators with wide clinical and medicinal applications [5–7]. The polysaccharides extracted from fruiting body of I. obliquus are proven to be effective in promoting tumor necrosis factor-α (TNF-α) secretion, phagocytic uptake in macrophages, cell proliferation, and comitogenic effect [8–10]. The endopolysaccharides from submerged culture of I. obliquus strongly stimulate humoral immunity related to B lymphocytes and macrophages [11].

⁎ Corresponding author. Tel.: +86 571 86843228; fax: +86 571 87055836. E-mail address: [email protected] (X. Xu).

http://dx.doi.org/10.1016/j.intimp.2014.05.015 1567-5769/© 2014 Elsevier B.V. All rights reserved.

Submerged fermentation of mushrooms is a promising alternative for efficient production of polysaccharides instead of that from wild resource owning to its high productivity and low cost. For fermented polysaccharides, it is necessary to prove that they possess nutritional and medicinal values comparable to those of mushroom fruiting bodies. Some researches have already shown that the biomass of different medicinal mushrooms possesses pharmacologic properties as compared with those of mushroom fruiting bodies [2]. Our previous studies have reported that the exopolysaccharides (EPS) and endopolysaccharides (IPS) from the submerged culture of I. obliquus perform stronger antioxidant activity than the wild sclerotial polysaccharides (FSPS) [12]. The antioxidant activity of the EPS can be further enhanced by lignocellulose degradation under submerged fermentation [13,14]. However, there is very little research on the immunomodulatory activity of I. obliquus FSPS, EPS and IPS by submerged fermentation. The polysaccharide extracts and fractions from the wild sclerotia and culture broth of I. obliquus under submerged fermentation showed strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and hydroxyl radical-scavenging activities in our previous work [12,14], suggesting that the polysaccharide preparations could be potentially used as a natural antioxidant. In the present study, we investigated the immunomodulatory activity of three crude extracts (FSPS, EPS, IPS), three deproteinated PS extracts (DFSPS, DEPS, DIPS), four fractions from culture broth, three from cultured mycelia, and one from wild sclerotia by determining their ability to enhance human peripheral blood

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mononuclear cells (PBMCs) proliferation and induce cytokine production by human PBMCs. The PS fractions were obtained by DEAEcellulose and SephadexG-200 chromatography. Monosaccharide composition and molecular weight of the fractions were analyzed by gas chromatography and size exclusion chromatography. 2. Materials and methods 2.1. Cultured mycelia of I. obliquus Cultured mycelia of I. obliquus were obtained by submerged fermentation using a strain of CBS314.39. The seed culture was prepared by adding mycelia on a malt extract agar slant into the seed medium and incubating for 4–5 days in a rotary shaker (150 rpm) at 28 °C. The harvested seed culture was added into 250-ml Erlenmeyer flasks containing 100-ml liquid fermentation medium composed of (g/kg) corn flour 53, peptone 3, KH2PO4 1, ZnSO4·2H2O 0.01, K2HPO4·0.4, FeSO4·7H2O 0.05, MgSO4·7H2O 0.5, CuSO4·5H2O 0.02, CoCl2 0.01, MnSO4·H2O 0.08 at pH 6.0. The inoculate rate was 10% (v/v). The medium used was optimized by the response surface methodology (RSM) based on the basal medium in our previous paper [15]. The culture was incubated for 9 days under the conditions of 150 rpm at 28 °C and mycelia with the culture broth were harvested on the 9th day.

concentrated, dialyzed against distilled water for 72 h and then lyophilized. The fractions were named as DEPS1, DEPS2, DEPS3, DEPS4, DIPS1, DIPS2, DIPS3, and DFSPS1, respectively. The eight fractions were further applied to a gel permeation column over a Sephadex G-200 (50 × 2.4 cm) column and eluted at 0.3 ml/min with 0.1 mol/l of NaCl. A single fraction (purified fraction) of each sample was gathered and coded as PDEPS1, PDEPS2, PDEPS3, PDEPS4, PDIPS1, PDIPS2, PDIPS3 and PDFSPS1 for molecular weight measurement, infrared spectrum analysis, and nuclear magnetic resonance spectroscopy (NMR) analysis. 2.4. Chemical content analysis The total carbohydrate and protein content was determined by phenol–sulfuric acid method [16] and the Bradford's method with bovine serum albumin as a standard [17]. 2.5. Monosaccharide composition analysis Monosaccharide composition analysis was performed by gas chromatography (Varian Inc., Palo Alto, CA, US) equipped with a Varian CP-Sil 5 CB capillary chromatography column (25 m × 0.53 mm, 0.25 μm film thickness) and a flame-ionization detector [13,14].

2.2. Extraction and isolation

2.6. Homogeneity and molecular weight determination

The harvested culture broth was isolated from mycelia by using a filter paper and concentrated to one tenth of its original volume with a rotary evaporator under reduced pressure at 60 °C. The concentrated liquid was mixed with 4 volumes of chilled 95% (v/v) ethanol, stirred vigorously and left at 4 °C overnight. The precipitate was collected, then centrifuged at 6500 g for 10 min and lyophilized. The lyophilized sample was exopolysaccharide extract termed as EPS. The EPS aqueous solution reacted with 3% (w/w) neutrase for 3 h at 42 °C in neutral environment, and then was further mixed with the Sevage reagent i.e. chloroform: butanol = 5:1 (v/v) at the proportion 5:1 (v/v) and oscillated intensively. The supernatant was concentrated, precipitated, and lyophilized. The deproteinated EPS was named as DEPS and used for fractionation [13,14]. The mycelial masses were washed three times with distilled water and dried to constant weight under reduced pressure at 60 °C. The mycelial masses suspended in distilled water were first heated for 1.5 h and then heated for 3 h in an autoclave at 121 °C to extract heat-stable mycelial product, followed by filtration [12]. The filtrate was put through ethanol precipitation, dialysis, and lyophilization. The lyophilized sample was endopolysaccharide extract termed as IPS. The deproteinated IPS (denoted as DIPS) was obtained for fractionation [12]. The wild sclerotia (first-rate production) were purchased from a local Chinese traditional medicine store in Jilin, China and dried to constant weight at 60 °C for several hours before grinding into fine powder. The powder (50 g) was extracted with 3 × 500 ml of petroleum ether at 70 °C for 3 h to remove fatty and fat-soluble substances. The residue was then extracted with 3 × 500 ml of distilled water at 95 °C for 2 h. The filtrate was combined, concentrated, precipitated with four volumes of ethanol, and lyophilized. The lyophilized sample was named as FSPS (4.9 g). The deproteinated FSPS (denoted as DFSPS) was obtained for fractionation [12].

Molecular weight (MW) and molecular size (MN) of the purified fractions were determined by size exclusion chromatography with laser light scattering (SEC-LLS). An eight-angle laser light scattering instrument equipped with a He–Ne laser (λ = 658 nm, HELEOS8, Wyatt Technology Co., US) was utilized to state molecular mass parameters. The value of 0.125 ml/g was used as the specific refractive index increment of the fractions in 0.2 mol/l NaCl [14]. Data acquisition and processing was performed with the ASTRA software (Wyatt Technologies).

2.3. Fractionation and purification DEPS, DIPS, and DFSPS in sodium phosphate buffer were subjected to a DEAE-cellulose anion-exchange column (50 × 2.4 cm, i.d.) that had been equilibrated with 0.01 mol/l sodium phosphate buffer, pH 7.8 before. Fractions were eluted with a linear gradient of 0 to 0.5 mol/l of NaCl in sodium phosphate buffer at a flow rate of 72 ml/h. The four fractions of DEPS, three of DIPS, and the first fraction of DFSPS were collected,

2.7. Infrared spectrum analysis The infrared spectra of the purified fractions were determined using a Fourier transform infrared spectrophotometer (Thermo Nicolet Avatar 370 FT-IR). The freeze-dried samples were ground with KBr powder and then pressed into pellets for transformation infrared spectra measurement in a frequency range of 4000–500 cm−1 [7,19]. 2.8. 1HNMR analysis The purified and freeze-dried samples were kept over P2O5 in vacuum for several days and dissolved in DMSO. The 1HNMR spectra were recorded with an AVANCE AV 400 MHz Digital FT-NMR spectrometer at room temperature. All the chemical shifts were in relative to Me4Si [7,19]. 2.9. Cytotoxicity test The cytotoxicity test was determined in vitro using the colorimetric MTT assay [18]. Human peripheral blood mononuclear cells (PBMCs) were collected from six healthy volunteers according to the direction of HISTO-PAQUE-1077 (Sigma-Aldrich, USA). Ninety microliters of 2 × 106/ml mononuclear cell suspension in RPMI 1640 medium and 10% fetal serum (Hangzhou Four Seasons Clear Biological Engineering Co., Ltd, China) was added in each well of a replicate 96-well plate. Then, 10 μl of EPS, IPS, FSPS at 0.14, 0.28, 0.42, 0.56, and 1.68 mg/ml or phytohem-agglutinin (PHA) (Sigma-Aldrich, USA) at 200 μg/ml or PBS was added to the wells. The PHA was used as a positive control and the PBS was used as a negative control. The mixture was cultured at 37 °C in a 5% CO2 cell incubator for 48 h, and for another 3 h after being added with 10 μl of CCK-8 as a dye used in the MTT assay (Nanjing

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KeyGen Biotechnology Co., Ltd, China). The absorbance of each well was read at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. All determinations were done in triplicate samples. 2.10. Cytokine production measurement The PBMCs were isolated and pooled. Human PBMCs were plated in 96-well plates at a density 2 × 105 cells in 100 μl per well and incubated for 24 h in the culture medium supplemented with 3% (v/v) endotoxinfree fetal bovine serum, with the EP extracts (15, 30, 150 μg/ml) or fractions (15, 30, 150 μg/ml) or Escherichia coli (E. coli) LPS (15 μg/ml) (Sigma-Aldrich, USA) as a positive control or PBS as a blank control. A human cytokine multi-analyte ELISA kit (Shanghai Senxiong Biotechnology Co., Ltd, China) at the wavelength of 450 nm on an automatic ELISA plate was utilized to evaluate various cytokines of interleukin (IL)-1β and IL-2, interferon-γ (IFN-γ), and TNF-α in supernatants of PBMCs. The triplicate experiments were done for each sample. 2.11. Statistical analysis The results were expressed as means ± standard deviation (SD) of three independent experiments performed. The data were analyzed by one-way ANOVA and a level of P b 0.05 was regarded as statistically significant with Duncan's new multiple ranges. Origin 7.5 software (OriginLab Corp., Northampton, MA) was used for statistical analysis. 3. Results 3.1. Chemical characterization of extracts and fractions Table 1 is a summary of the carbohydrate and protein contents and monosaccharide components of the extracts and fractions from the wild sclerotia, culture broth and mycelia. The carbohydrate content of the EPS and IPS from the cultured I. obliquus was significantly higher than that from the wild sclerotia (FSPS) but with an inverse result for the protein content. The IPS had a higher content of carbohydrate than the EPS but with a similar protein level. Deproteinization of EPS, IPS, and FSPS (yielding DEPS, DIPS, and DFSPS) increased the carbohydrate content by 29.8%, 26.4%, and 47.7%, and decreased the protein content by 41.6% and 37.0% for DIPS and DFSPS, respectively. The remained proteins in DEPS, DIPS, and DFSPS were in the polysaccharide–protein complex. The deproteinated extract DEPS was found to have higher protein content as compared to its untreated sample EPS. It is consistent with our previous work [14]. The result may be explained by the fact that the untreated sample contained other compounds such as small molecular sugar and non-saccharide components except exopolysaccharide and

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protein. The deproteinated process and the following dialysis process removed not only free protein but also small molecular impurities. Thus, the remaining protein complex (glycoprotein) in EPS was purified, resulting in higher protein content in the deproteinated extract DEPS. Fig. 1 shows the elution curves of DFSPS (Fig. 1a), DEPS (Fig. 1b), and DIPS (Fig. 1c) subjected to DEAE-52, respectively. DEPS1, DEPS2, DEPS3, and DEPS4 from DEPS, DIPS1, DIPS2, and DIPS3 from DIPS, and DFSPS1 and DFSPS2 from DFSPS were eluted. The yield of the four fractions from DEPS were 28.3%, 10.7%, 13.8%, and 9.0%, the three fractions from DIPS were 31.5%, 13.3%, and 7.0%, and the first fraction DFSPS1 was 23.3% (w/w, fraction/deproteinated extract), respectively. DFSPS2 was not further studied due to the small amount. It should be noted that, from Fig. 1, the peak of DEPS2 is higher than that of DEPS3, but the yield of DEPS2 (10.7%) is less than that of DEPS3 (13.8%). The reason was that the elution curves were plotted on the basis of the sugar content which was detected by phonel–sulfate method but the yield was calculated based on the weight. The protein content could cause the consistency. The carbohydrate content in the fractions from DEPS and DIPS decreased as the elution time increased. Only the first fraction in DEPS, DIPS, and DFSPS had a higher carbohydrate content than DEPS, DIPS, and DFSPS itself, correspondingly. DIPS1 contained the highest carbohydrate content (86.1%) and the lowest protein content (11.4%) among all the fractions. DEPS, DIPS, and DFSPS were composed of rhamnose (Rha), arabinose (Ara), xylose (Xyl), mannose (Man), glucose (Glu), and galactose (Gal) with various molar ratios (Table 1). DEPS had much less amount of Rha, Ara, and Xyl than DIPS and DFSPS. In addition, every fraction from DIPS and DFSPS had six monosaccharides but the fractions from DEPS only contained three, four or five of the six (Table 1). Gal was the dominant component in DEPS and greater than 55% in DEPS1, DEPS2, and DEPS4 except DEPS3. In DEPS3, Man at 43.7% was the dominant component. Glu was the least among the three major components in four fractions in DEPS. However, Glu was the dominant component in DIPS and DFSPS. DIPS1 contained more Man than Gal but inverse results were found in DIPS2 and DIPS3 (Table 1). Table 2 displayed the average MW of the purified fractions. The three fractions of PDIPS1, PDIPS2, and PDIPS3 from DIPS had much larger MW of 108–120 kDa than the four fractions of PDEPS1, PDEPS2, PDEPS3, and PDEPS4 from DEPS with MW of 36–58 kDa. The MW of PDFSPS1 (32 kDa) from DFSPS was the smallest among all the fractions. The MN of three fractions from DIPS was between 89 and 96 kDa, much larger than that of four fractions from DEPS (28–55 kDa) and the fraction from DFSPS (21 kDa). The MW/MN value of PDFSPS1 was much greater than that of the fractions from DEPS and DIPS. From PDEPS1, PDEPS2 to

Table 1 Chemical contents and monosaccharide compositions of the extracts and fractions from the wild sclerotia (FSPS), culture broth (EPS), and cultured mycelia (IPS) of I. obliquus.

EPS DEPS DEPS1 DEPS2 DEPS3 DEPS4 IPS DIPS DIPS1 DIPS2 DIPS3 FSPS DFSPS DFSPS1 ND: not detected.

Sugar content (wt.%)

Protein content (wt.%)

53.4 69.3 74.1 59.9 50.6 46.7 64.0 80.9 86.1 51.4 30.6 40.5 59.8 72.3

21.3 25.5 17.0 33.0 16.8 17.1 22.1 12.9 11.4 33.4 22.0 51.3 32.3 24.7

Sugar component (mol%) Rhamnose

Arabinose

Xylose

Mannose

Glucose

Galactose

0.95 ND 3.06 ND ND

3.53 ND 1.14 ND ND

1.52 ND ND ND 3.00

25.4 23.3 20.2 43.7 17.9

13.1 21.6 17.5 24.3 11.3

55.5 55.1 58.1 32.0 67.8

7.09 3.78 5.11 6.30

10.3 1.36 7.63 15.8

8.81 5.16 7.26 9.6

21.3 35.1 24.8 19.7

37.8 30.3 23.5 20.3

14.7 24.3 31.7 28.3

3.4 1.2

5.2 2.1

14.4 15.8

20.3 20.6

40.5 43.3

16.2 17.0

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X. Xu et al. / International Immunopharmacology 21 (2014) 269–278 Table 2 Molecular weight (MW) and molecular size (MN) of the fractions from the wild sclerotia, culture broth, and cultured mycelia by gel permeation chromatography.

PDEPS1 PDEPS2 PDEPS3 PDEPS4 PDIPS1 PDIPS2 PDIPS3 PDFSPS1

MN (Da)

MW (Da)

MW/MN

40,000 28,000 47,000 55,000 90,000 96,000 89,000 21,000

48,000 36,000 50,000 58,000 120,000 119,000 108,000 32,000

1.20 1.29 1.06 1.05 1.33 1.24 1.21 1.52

3.2. Structure characterization of fractions The FT-IR spectra of the eight fractions shown in Fig. 2 were similar. The different absorption bands were assigned according to the literature [7,19]. The strong and broad band at 3200–3600 cm−1 was attributed to the hydroxyl stretching vibration of the polysaccharide, and that at 2930 cm−1 was due to the C\H stretching vibration absorption of CH2. The weak band near 1725 cm−1 indicated that the fractions might contain carboxylic acid groups. Strong overlapped IR bands in the region of 1200–1000 cm−1 were ascribed to C\O stretching vibrations of pyranose ring. The characteristic bands at 930–960 cm−1 suggested the presence of pyranose form of the glucosyl residue (Fig. 2a–h). NMR spectroscopy has become the most powerful technique for the structure analysis of carbohydrates. The anomeric proton signals of polysaccharides are mostly in the range of δ 4.0 to 5.5, including proton signals of C1 in δ 4.8–5.5, and C2–C6 in δ 4.0–4.8. The proton signals of C1 of pyranose with α-configuration usually are more than δ 5.0, those with β-configuration are less than δ 5.0, which can be used to distinguish the two configurations. The 1H NMR spectra shown in Fig. 3 were notably different among the eight fractions. Several signals appeared in the anomeric region of the 1H spectra, suggesting the presence of different linkage patterns for most fractions. For the EPS fractions, PDEPS1 and PDEPS2 had no signals in the range of δ 4.8–5.0, suggesting that their C1 was of α-configuration. The anomeric proton signals of PDEPS1 at δ 5.42 and 5.38 corresponded at H − 1 of (1 → 4)-α- and (1 → 4,6)-α-residues, respectively (Fig. 3a) [19]. PDEPS2 only had one signal at δ 5.14, suggesting one linkage (Fig. 3b). The C1 of PDEPS3 was totally of β-configuration with only one signal at δ 4.88 (Fig. 3c). The characteristic absorption of β-mannopyranose near 870 cm−1 was observed in FT-IR analysis (Fig. 2c), in good agreement with the high mannose content of 43.7% in DEPS3 by GC monosaccharide composition measurement (Table 1). The C1 of PDEPS4 was of α- and β-configuration but α-configuration was dominant with three different linkage patterns (Fig. 3d). For the IPS fractions, PDIPS1 had four different linkage patterns at δ 4.63, 4.84, 5.05, and 5.22, suggesting the C1 was of α- and β-configuration but more β-configuration (Fig. 3e). The C1 of PDIPS2 was totally of β-configuration with the signal at δ 4.88 (Fig. 3f). The C1 of PDIPS3 was of α- and β-configuration with the signals at δ 4.92, 5.13, and 5.29 (Fig. 3g). For the fraction form the wild sclerotia, PDFSPS1 had six different linkage patterns at δ 4.28, 4.49, 4.67, 4.83, 4.93, and 5.40, suggesting that the C1 was of α- and β-configuration but much more of β-configuration (Fig. 3h).

Fig. 1. Elution curves of DFSPS (a), DEPS (b), and DIPS (c) over DEAE-cellulose column.

PDEPS3, PDEPS4, and from PDIPS1 to PDIPS3, the MW/MN value decreased notably. The values of PDEPS3 and PDEPS4 were closer to 1 than the others, indicating more homogeneous.

3.3. Effects of extracts on the cell proliferation Fig. 4 shows that all the extracts (EPS, IPS, and FSPS) of I. obliquus from the wild sclerotia and cultured mycelia at the concentrations of 0.14–1.68 mg/ml were effective in propagating human PBMCs compared to the PHA group. PHA was reported to have certain activity in cell proliferation at the concentration of 0.02 mg/ml. FSPS

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Fig. 2. IR spectra of purified fractions PDEPS1 (a), PDEPS2 (b), PDEPS3 (c), and PDEPS4 (d) from the culture broth, PDIPS1 (e), PDIPS2 (f), and PDIPS3 (g) from the cultured mycelia, and PDFSPS1 (h) from the wild sclerotia.

exhibited stronger effect than EPS and IPS in promoting the growth of PBMCs at concentrations of 0.56 to 1.68 mg/ml. Generally, the results revealed that the PS extracts from the cultured I. obliquus as

well as the wild sclerotia had the activity on cell proliferation. In other words, both the PS extracts had no cytotoxicity on human PBMCs.

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Fig. 3. 1H NMR spectra of purified fractions PDEPS1 (a), PDEPS2 (b), PDEPS3 (c), and PDEPS4 (d) from the culture broth, PDIPS1 (e), PDIPS2 (f), and PDIPS3 (g) from the cultured mycelia, and PDFSPS1 (h) from the wild sclerotia.

3.4. Effects of extracts and fractions on cytokine secretion

140

PHA EPS IPS FSPS

Proliferarion activity (%)

120 100

e d bb

b

b

bb

c

c bc a

b bc

ba

0.42

0.56

1.68

80 60 40 20 0

0.02

0.14

0.28

Concentration of polysaccharides (mg/ml) Fig. 4. Effect of the extracts from the wild sclerotia and cultured mycelia under submerged fermentation on human PBMC proliferation at the concentrations of 0.14–1.68 mg/ml. PHA was as the positive control. The value of PHA (0.02 mg/ml) was set to 100% as a positive control and the level of the extracts were relative values compared with PHA. The different letters on the bars indicated that there was significant difference between groups (P b 0.05).

Fig. 5 shows the level of TNF-α (Fig. 5a), IFN-γ (Fig. 5b), IL-1β (Fig. 5c), and IL-2 (Fig. 5d) secreted by human PBMCs treated with EPS, DEPS, IPS, DIPS, FSPS, and DFSPS at concentrations of 15, 30, and 150 μg/ml. The level of four cytokines in the six polysaccharidestreated groups was much higher than that in the control and similar to that in the LPS-treated group at 15 μg/ml. The differences in TNF-α, IFN-γ, IL-1β and IL-2 content of six tested extracts vs. the control were significant (P b 0.05). The results suggested that the extracts from the cultured I. obliquus as well as the wild sclerotia had the ability to enhance production of TNF-α, IFN-γ, IL-1β, and IL-2 by human PBMCs. It is noted that all the cytokine production was increased in a dosedependent manner for the six extracts. The effects of six extracts on the production of TNF-α, IFN-γ, IL-2, and IL-1β were similar when the concentrations were 15 and 30 μg/ml except DFSPS at 15 μg/ml had the highest effect on the production of TNF-α (Fig. 5a) and IFN-γ among the six extracts (Fig. 5b) (P b 0.01), and EPS and DEPS delivered a much bigger increase of IL-1β at 30 μg/ml than the others (Fig. 5c). There were great differences in the effects among the six extracts as the concentration increased to 150 μg/ml. DEPS and FSPS showed a stronger effect on the secretion of TNF-α and IFN-γ than the others. IPS, FSPS and DIPS, FSPS, DFSPS were the best in IL-1β and IL-2 production, respectively. Fig. 6 shows the stimulatory effects of the eight fractions on the production of TNF-α (Fig. 6a), IFN-γ (Fig. 6b), IL-1β (Fig. 6c), and IL-2 (Fig. 6d) by human PBMCs. Similar to the extracts, there was no big

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(a)

(b)

(c)

(d)

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Fig. 5. TNF-α (a), IFN-γ (b), IL-1β (c), and IL-2 (d) production of human PBMCs incubated with FSPS and DFSPS from the wild sclerotia, EPS and DEPS from the culture broth, and IPS and DIPS from the cultured mycelia of I. obliquus. Suspension of human PBMCs in the medium was used as control under the same conditions. Four cytokine levels were measured by ELISA. Each value is presented as mean ± SD (n = 3). The different letters on the bars indicated that there was significant difference between groups (P b 0.05).

difference in effect among the eight fractions at low concentrations of 15 and 30 μg/ml, but significant difference among them at 150 μg/ml (P b 0.01). DIPS1 and DIPS3 were the most potent in TNF-α induction (Fig. 6a). DEPS3 was the most effective in IFN-γ and IL-2 secretion. DEPS4 was the best for IL-1β production. 4. Discussion Cytokines play important roles in the development, function and control of the cells of the immune system as well as many other systems. They are potent molecules that can cause changes in differentiation, migration and cell proliferation. The ability to enhance production of interleukins by macrophages or other leucocytes has been widely used as an experimental model for studying immunomodulatory activity of polysaccharide preparations [6,20]. TNF-α and IL-1β are cytokines that are predominantly secreted by innate immune cells. IL-2 is a general cytokine necessary for growth, proliferation and differentiation of T-cells. IFN-γ is specifically secreted by Th1 cells. Many reports confirmed that

mushroom polysaccharides exerted anti-tumor and immunomodulating activities because of the augmentation of cytokine production [21–23]. The β-glucan lentinan induced a marked increase in the mRNA levels of IL-1α, IL-1β, TNF-α, and IFN-γ [24]. Treatment of human PBMC with Lentinula edodes water-soluble extract increased levels of TNF-α, IL-1β, IL-10, and IL-12 [25]. Water extract from G. lucidum, with β-D-glucan as the major component, has been shown to be effective in stimulating T cells to release cytokines like IL-1β, IFN-γ, TNF-α, IL-2, and IL-6 which are proven to be effective in combating tumor cells [20]. In the present work, we observed that the PS extracts and fractions from both the sclerotia and cultured mycelia enhanced the human PMBC proliferation and promoted all the cytokines tested in this study. The PS extracts and fractions from the three sources were heteropolysaccharides composed of three to six kinds of monosaccharides. The DFSPS (DFSPS1) and DIPS (DIPS1, DIPS2, and DIPS3) had much higher Glu content and lower Gal content than DEPS (DEPS1, DEPS2, DEPS3, and DEPS4). DIPS and the fractions had higher Rha and Ara content than DFSPS and DEPS (Table 1). The activity of the PS extracts and

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(a)

(b)

250

(c)

(d)

Fig. 6. TNF-α (a), IFN-γ (b), IL-1β (c), and IL-2 (d) production of human PBMCs incubated with one fraction from the sclerotia, four fractions from the culture broth, and three fractions from the cultured mycelia of I. obliquus. Suspension of human PBMCs in the medium was used as control under the same conditions. Four cytokine levels were measured by ELISA. Each value is presented as mean ± SD (n = 3). The different letters on the bars indicated that there was significant difference between groups (P b 0.05).

fractions from the three resources differed in the various kinds of cytokines. Therefore, it is complicated to correlate carbohydrate composition and biological activity. Kim et al. reported that endopolysaccharide fractions from cultivated mycelia of I. obliquus with Man as the major component had an anti-cancer effect [26]. Mannan was the major component of some active hetero-polysaccharides, e.g., α-(1 → 3)-mannans from Dictyophora indusiata, glucuronoxylomannan from Tremella fuciformis, glucomannan from A. blazei, and galactoglucomannan from L. edodes [27]. As shown in Table 1, all the six PS extracts before and after Sevage reagent treatment and the eight fractions were polysaccharide–protein conjugates (Table 1). The order of protein contents of the three crude PS and deproteinated PS extracts was FSPS N IPS N EPS, and DFSPS N DEPS N DIPS. The protein content was markedly decreased by deproteinated treatment for FSPS and IPS. However, the deproteinated extract (DEPS) was found to have higher protein content as compared to their untreated samples (EPS). The results may be explained by the fact that the EPS from the culture broth contained other compounds such as small molecular sugar and non-saccharide components except

exopolysaccharide and protein. The deproteinated process and the following dialysis process removed not only free protein but also small molecular impurities. Thus, the remaining protein complex (glycoprotein) in DEPS was purified, resulting in higher protein content in deproteinated extract (DEPS). The presence of protein has been reported to be important for biological activities and especially important for the biological activities of the heteropolysaccharides [28]. Our previous results demonstrated that with increase of the protein content, the antioxidant activities of the three polysaccharide–protein conjugates from the wild sclerotia and I. obliquus culture increased [12,14]. In the present work, FSPS was better than DFSPS in argumentation of IL-1β, TNF-α, and IFN-γ production at 150 μg/ml (Fig. 5a, b, c), but DFSPS was better than FSPS for TNF-α and IFN-γ secretion at 15 μg/ml. IPS was more effective than DIPS for IL-1β production (Fig. 5c), but inverse result for IL-2 production at 150 μg/ml (Fig. 5d). Except for these results, protein content did not cause significant influence between FSPS and DFSPS, EPS and DEPS, and IPS and DIPS (Fig. 5). However, DEPS1 and DEPS3 (DIPS1 and DIPS3) with lower protein contents of 17.0% and 16.8% (11.4% and 22.0%) were more effective

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for IFN-γ and IL-2 (TNF-α) (Fig. 6a, b, d) production than the other fractions at 150 μg/ml, respectively. DEPS2 with higher protein content of 33.0% was also more effective for IL-2 production (Fig. 6d). This inconsistency on IL-2 production indicated that protein content was not the sole factor. DEPS4 with lower protein content (17.1%) and the highest Gal content (67.8%) had the strongest activity in IL-1β (Fig. 6c). Considering the molecular weight difference, we found that DIPS1 of 120 kDa and DIPS3 of 108 kDa had a higher activity in TNF-α production than the fractions from the culture broth with 36–58 kDa and DFSPS1 of 32 kDa at 150 μg/ml (P b 0.05) (Table 2, Fig. 6). DFSPS1 had a relatively lower effect on the four cytokine secretion. The results seemed likely to demonstrate that the higher MW, the higher activity in TNF-α production at the high concentration except for DIPS2. The molecular weight of DIPS2 was 119 kDa and comparable with that of DIPS1 and DIPS3. However, the TNF-α production activity of DIPS2 was lower than that of DIPS1 and DIPS3, indicating that there were other factors (P b 0.01). At lower concentrations, differences in molecular weight had no marked effect on the activity in cytokine production. The β-(1 → 3)-glucans with medicinal properties are strongly dependent on high molecular weight, ranging from 500 to 2000 kDa [29]. However, α-(1 → 3)glucuronoxylomannans, which are characteristic of Jelly mushrooms, are not strongly dependent on molecular weight. The NMR results demonstrated that the EPS fractions with more Gal except DEPS3 had C1 of more α-configuration, but the IPS and FSPS fractions with more Glu had C1 of more β-configuration (Fig. 3). The effects of monosaccharide composition, molecular weight, and C1 configuration on each cytokine production differed. In Figs. 5 and 6, all figure data show that the secretions of four cytokines in PBMCs by high dosage of these polysaccharides were higher than LPS. It tells something about the potency but has some degree of complexity to be explained. The complicated effects of monosaccharide composition, anomeric carbon configuration, molecular weight, and protein contents of the PS extracts and fractions on the enhancement of the secretion of the different cytokines suggested a differential role of them in the signaling pathways for the different cytokines. Except for the above factors, immunomodulating actions of polysaccharides are closely related with its glycosidic bond of the main chain, degree of substitution and branching, conformation of the main chains, etc. [30,31]. A separation of the cells present in peripheral blood would have been useful to make this issue clear as different cells have a different function in signaling. In addition, the production of some cytokines was low. Therefore, additional experiments by using human monocytes/macrophages, THP-1 cell line, and murine macrophages as well as RAW 264.7 were needed to further demonstrate the immunomodulator activity of I. obliquus polysaccharides. Biologically active polysaccharides are widespread among bacteria and mushrooms. Significant progress has been made over the past years in the understanding of the mechanism on inducing pro-inflammatory cytokine production, such as, Toll-like receptor (TLR) function. TLR signaling consists of at least two distinct pathways: a MyD88-dependent pathway that leads to the production of inflammatory cytokines, and a MyD88-independent pathway associated with the stimulation of IFN-β and the maturation of dendritic cells [32]. TLRs are essential receptors in host defense against pathogens by activating the innate immune system, a prerequisite to the induction of adaptive immune responses [33]. The PS extracts and fractions had similar (at same concentrations) or stronger (at higher concentrations) effects to or than E. coli LPS used as a positive control in this study, which is a TLR4 ligand. Whether the PS extracts and fractions affect the TLR signaling pathway is unknown. Some experiments using specific inhibitors to identify the signaling pathways are worthy of being further performed. In conclusion, the PS extracts and fractions from both the wild sclerotia and cultured mycelia of I. obliquus enhance the cell proliferation and stimulate the secretion of four cytokines of human PBMCs, suggesting immunomodulatory activity. In addition, the PS extracts from

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