The fruiting bodies of D. indusiata were purchased from Xinguan Ecological Agriculture Development Co., Ltd (Hubei, China). Dimethyl sulfoxide (DMSO), LiCl, EDTA, trypsin, MTT, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium and fetal bovine serum (FBS) were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). Standard monosaccharides, including glucose, xylose, rhamnose, arabinose, mannose and galactose were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Sepharose CL-6B was purchased from GE Healthcare (Little Chalfont, UK). MCF-7 human breast cancer cells and B16 mouse melanoma cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were authenticated by morphological characteristic checks under an inverted phase contrast microscope (Optec BDS200; Chongqing Optec Instrument Co., Ltd, Chongqing, China). Chlorosulfonic acid (CAS), pyridine (Pyr), BaCO₃, hydroxylamine hydrochloride, acetic anhydride, ferrous sulfate (FeSO₄), hydrogen peroxide (H₂O₂), salicylic acid, sodium phosphate, potassium ferricyanide, potassium bromide (KBr) and other chemicals and solvents were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

DIP was isolated from the fruiting bodies of D. indusiata, as previously described (18). In brief, the dried fruiting bodies were crushed into powder and then defatted with ethyl acetate and ethanol for 6 h using a Soxhlet extraction apparatus (Sigma-Aldrich). Subsequently, the degreased powder was extracted using distilled water at 100°C until the water-soluble polysaccharide was completely removed. The residues were then dried and extracted using 5% NaOH containing 0.05% NaBH₄ at 25°C for 2 h and the mixture was then centrifuged at 14,000 × g for 30 min. The supernatant was decanted and then neutralized with glacial acetic acid and the product was lyophilized on a vacuum freeze dryer (Free Zone 2.5 l, Labconco Co., Ltd, Kansas, MO, USA) to derive the DIPs. Sepharose CL-6B (1 cmx100 cm) was used to identify the homogenicity of the DIP. The column was eluted with 0.5 M NaOH at a flow rate of 0.5 ml/min. Aliquots of the fractions (3.0 ml) were then collected using a fraction collector. The total sugar content of all the fractions was detected using the phenol-sulfuric acid method (19).

Sulfation of DIP was performed using the chlorosulfonic acid (CAS)-pyridine (Pyr) method, as described previously (20). The degrees of substitution were controlled via a molar ratio of chlorosulfonic acid to monosaccharide residues, with ratios of 3:1 (S1-DIP), 4:1 (S2-DIP) and 5:1 (S3-DIP), respectively.

The water solubilities of the sulfated derivatives were measured at room temperature according to the Pharmacopoeia of the People’s Republic of China (21).

The total sugar content was determined using the phenol-sulfuric acid method (19) with glucose as a standard, the uronic acid content was estimated using the sulfuric acid-carbazole method with glucuronic acid as a standard (22) and the sulfur content was determined using the barium chloride-gelatin method (23). The degree of substitution (DS) was calculated from the sulfur content (S) using the following formulas:

The monosaccharide composition of DIP was determined using gas chromotography-mass spectrometry (GC-MS) (QP2010s; Shimadzu, Kyoto, Japan). Briefly, 20 mg monosaccharide was hydrolyzed with 2 ml 1 mol/l sulfuric acid at 100°C for 4 h. The residual sulfuric acid was removed by neutralization with excess BaCO₃ following hydrolysis. The hydrolyzate was evaporated under reduced pressure, dissolved in 0.5 ml pyridine and reacted with 10 mg hydroxylamine hydrochloride at 90°C for 30 min. Subsequently, 0.5 ml acetic anhydride was added and incubated for 30 min at 90°C. Following cooling down, the solution obtained was filtered with a 0.22-μm organic filter and loaded onto a Rxi-1 ms capillary GC column (30 m × 0.25 mm × 0.25 μm; Restek Corp., Bellefonte, PA, USA) at 120–250°C at a rate of 5°C/min. Rhamnose, arabinose, xylose, mannose, glucose and galactose were used as monosaccharide standards.

The molecular weight (Mw) of the sample was determined by gel permeation chromatography (GPC) using the methods described in a previous study by our group (8). GPC measurements of the samples were performed on an Agilent 1200 LC (Agilent Technologies Inc., Santa Clara, CA, USA) with a pump G1310A equipped with PL aquagel OH column (7.5 mm × 300 mm) and differential refractive index detector (G1362A) at 25°C. The eluent was NaNO₃ aqueous solution (0.2 M), and the flow rate was set at 0.8 ml/min. All the solutions were filtered with 0.45 μm sand filter.

FTIR spectroscopy was performed on a Nicolet Nesux 470 spectrophotometer (Thermo Scientific Corporation, Pittsburgh, PA, USA) at a wavenumber range between 4,000 and 400 cm⁻¹ using the potassium bromide disc method (24). Briefly, samples mixed with potassium bromide were ground in an agate mortar for 10 min, while the mass ratio of potassium bromide to added sample was ~100–200. Subsequently, the obtained powder was placed in casting equipment and compressed to form a disc, which was fixed to the sample holder and tested.

¹³C NMR analysis was performed at 400 MHz using a Bruker Advance spectrometer (Aduance III 400MHz; Bruker Coporation, Madison, WI, USA). The DIP was dissolved in DMSO and the sulfated derivatives were dissolved in D₂O. The temperature was set at 70°C.

Fenton’s reaction was performed to determine the hydroxyl radical scavenging activity of the sulfated derivatives at concentrations of 25–1,000 μg/ml according to the previously described method (25). The reaction volume consisted of 0.2 ml DIP and sulfated derivatives, 2.0 ml EDTA-FeSO₄ (0.15 mM), 2.0 ml H₂O₂ (6.0 mM), 0.8 ml salicylic acid (2.0 mM) and 0.8 ml distilled water. The hydroxyl radical was determined by monitoring the absorbance at 510 nm using a UV VIS spectrophotometer (UV-2550; Shimadzu, Kyoto, Japan) following incubation for 60 min at 37°C. The hydroxyl radical scavenging activity was expressed according to the following formula:

where A_control510 was the absorbance obtained using water and sodium phosphate to replace the sample and H₂O₂, respectively; A_blank510 was the absorbance obtained using water to replace the sample and H₂O₂.

The DPPH radical scavenging activity of the sulfated derivatives was measured using the previous description with minor modifications (26). Initially, the samples (0.5 ml; 25–1,000 μg/ml) were added to a 0.004% ethanol solution of DPPH (2 ml). Following incubation at room temperature for 15 min in the dark, the absorbance at 517 nm was determined and the DPPH scavenging activity was calculated as follows:

where A_blank517 was the absorbance obtained using water to replace the sample solution; and A_sample517 was the absorbance of polysaccharide solution.

The reduction ability of the sulfated derivatives was determined according to the method reported by Yen and Chen (27) with slight modifications. Briefly, 1 ml DIP and the sulfated derivatives (S1-DIP, S2-DIP and S3-DIP) in 2.5 ml sodium phosphate buffer (2 M; pH 6.6) were mixed with 2.5 ml aqueous potassium ferricyanide (1% w/v) and incubated at 50°C for 20 min. Subsequently, 2.5 ml trichloroacetic acid (10%, w/v) was added to terminate the reaction. The solution was then centrifuged at 3,000 xg for 10 min, following which 2.5 ml supernatant was mixed with 3.0 ml distilled water and 0.2 ml 0.1% ferric chloride (w/v). Absorbance at 700nm was measured using a UV VIS spectrophotometer (UV-2550; Shimadzu).

The MCF-7 cells were cultured in RPMI-l640 complete medium supplemented with 10%FBS, 200 mmol/l glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 5 μl/ml insulin. The B16 cells were cultured in RPMI-l640 complete medium supplemented with 10% FBS, 200 mmol/l glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. All the cells were then incubated at 37°C in a humidified atmosphere with 5% CO₂.

To compare the antitumor effects of the DIP and the sulfated derivatives, DMSO/LiCl, a polar aprotic solvent that dissolves polar and nonpolar compounds, was added to dissolve the DIP. DMSO has been routinely used in biopreservation, particularly in cell cryopreservation; however, it is able to induce cell differentiation in the culturing process. Thus, the effects of different concentrations of DMSO/LiCl on cell growth were investigated in the present study, which demonstrated that DMSO/LiCl had no effects on cell growth at concentrations of 0.01–0.1% (data not shown). Therefore, the concentration of DMSO/LiCl in the solvent used for DIPs in the in vitro experiments to determine tumor inhibitory effects was maintained at <0.1%.

The inhibitory effects of DIP on the cellular proliferation of MCF-7 and B16 cells were determined using an MTT assay. In brief, the MCF-7 or B16 cells were seeded into 96-well cell culture plates at a density of 3×10⁴ cells/well. The DIP and its sulfated derivatives were dissolved in 0.05% (v/v) DMSO/LiCl and water, respectively. Following incubation for 24 h, the cells were cultured with either DIP or the sulfated derivatives at concentrations of 25, 50, 100, 200, 500 and 750 μg/ml. Cell lines treated with medium only were used as a control. Subsequently, 20 μl MTT (5 mg/ml) was added to each well and plates were further incubated at 37°C for 4 h. Following removal of the supernatant, 100 μl DMSO was added to each well. The mixture was agitated in a horizontal direction for 10 min to dissolve the produced formazan crystals. The optical densities were measured at 570 nm using an ELISA microplate reader and each experiment was performed in triplicate. The cell viability was calculated as follows:

where Abs_sample was the absorbance of the cells treated with the samples, while Abs_control was the absorbance of the control cells.

Values are expressed as the mean ± standard deviation. SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) was used for data analysis. One-way analysis of variance followed by the least significant difference test was performed for the inter-group comparison. P<0.05 was considered to indicate a statistically significant difference.

The final yield of DIP was ~3.5% of the dried fruiting body powder. The tracer elution curve (Fig. 1) was single and symmetrical, which indicated purity of the DIP. The total sugar content and uronic acid content of DIP were 99.07 and 0.62%, respectively (Table I), indicating that DIP was the neutral polysaccharide. In addition, the DIP was hydrolyzed to a monosaccharide by trifluoroacetic acid and further acetylated for GC-MS analysis. The results indicated that the DIP was a homopolysaccharide, which consisted only of glucose (Fig. 2).

The CAS-Pyr method was adapted to determine the synthesis of sulfated polysaccharide derivatives (S-DIP) in the present study (28). A total of three sulfated derivatives, including S1-DIP, S2-DIP and S3-DIP, with different DS were obtained from the DIP by controlling the molar ratio of chlorosulfonic acid to monosaccharide residues (Table II). The DS of S1-DIP, S2-DIP and S3-DIP ranged between 0.58 and 1.55. The Mw of S1-DIP, S2-DIP and S3-DIP was 69, 74 and 85 kDa, respectively. A marked increase was noted in the DS and Mw of the sulfated derivatives as the molar ratio of the chlorosulfonic acid to monosaccharide residues increased. Additionally, S1-DIP, S2-DIP and S3-DIP exhibited high levels of water solubility (Table II), indicating that sulfation improved the water solubility of the original polysaccharide.

The absorption of DIP ranged between 1,060 and 1,000 cm⁻¹, which demonstrated that the monosaccharide of DIP was in a pyranose form. In addition, a characteristic absorption band at 842 cm⁻¹ was observed, which was indicative of an α-configuration of the glycosidic bond. Compared with DIP, two new absorption bands were observed in the spectra of sulfated derivatives at 1,240 cm⁻¹ and 810 cm⁻¹, which were characteristic of an asymmetric S=O stretching vibration and a symmetric C-O-S vibration, respectively (29). Additionally, the absorption bands at 2,930 cm⁻¹ and 1,350 cm⁻¹, which stemmed from the C-H stretching vibration, were weaker as the DS increased (Fig. 3). These changes in the IR peaks confirmed the sulfation of DIP.

The results of the ¹³C-NMR analysis were consistent with the IR substitution measurements. Due to its wide range of chemical shifts and limited overlapping peaks, ¹³C-NMR has been frequently used to examine polysaccharide structure. The NMR spectra of DIP and S-DIP are shown in Fig. 4. The substitution of a sugar ring on the polysaccharide caused changes in chemical shifts.

The ¹³C-NMR spectrum of DIP (Fig. 4A) contained six peaks at C-1 (100.2 ppm), C-2 (71.5 ppm), C-3 (83.1 ppm), C-4 (70.1 ppm), C-5 (72.6 ppm) and C-6 (60.8 ppm), which correlated with the carbons in [α-Glc (1→3)-]_n. In addition, the peak at 100.2 ppm was due to due to an anomeric carbon chemical shift, which indicated that the polysaccharides were formed by glycosidic bonds. With the monosaccharide analysis and infrared spectrum in consideration, these results confirmed that DIP was an α-(1→3)-D-glucan.

As shown in Fig. 4B and Table II, the -OH groups at the C-6 position were substituted according to the peak areas of C-6 and C-6s, at 61.2 and 66.5 ppm, respectively. The peak at 61.2 ppm was significantly weaker compared with that of the DIP signal and new signals appeared at 63.704–67.374 ppm, indicating that partial sulfation of C-6 had occurred. Additionally, as shown in Table II, substitution of S-DIP also occurred in the C-4 and C-2 positions.

As shown in Fig. 5A, the hydroxyl radical scavenging activity of S1-DIP, S2-DIP and S3-DIP was markedly higher compared with that of DIP at concentrations of 100, 300, 500 and 1,000 μg/ml, respectively. The scavenging activity of DIP was weak at <15%, including that at 1,000 μg/ml. The hydroxyl radical scavenging activity of S1-DIP, S2-DIP and S3-DIP was concentration-dependent. Furthermore, S2-DIP with a DS value of 0.989 exhibited more marked scavenging of hydroxyl radicals compared with S1-DIP and S3-DIP, which had a DS of 0.584 and 1.549, respectively. This suggested that the changes in molecular structure induced by sulfation may be associated with the scavenging activity of hydroxyl radicals in addition to DS. These results indicated that the sulfate group was important in the scavenging capacity of hydroxyl radicals.

The DPPH assay was used as a substrate to determine the antioxidative activity of compounds. The DPPH scavenging activity of DIP and its derivatives is shown in Fig. 5B. It was noted that the scavenging capability of DPPH was enhanced as the concentrations of S1-DIP, S2-DIP and S3-DIP increased. However, no increase was observed in the scavenging activity of DPPH by DIP. Significant differences were observed in the DPPH scavenging activities of the S1-DIP, S2-DIP and S3-DIP groups compared with those of the DIP group at concentrations >100 μg/ml (P<0.05). The maximal scavenging activity was observed in S2-DIP (63.6%), followed by S3-DIP (60.58%) and S1-DIP (58.1%) at 1,000 μg/ml. All sulfated derivatives exhibited a significantly higher scavenging effect compared with that of DIP (P<0.05). Overall, the results demonstrated that sulfation improved the antioxidant activity of DIP.

The reduction ability of DIP and its sulfated derivatives is shown in Fig. 5C, which revealed that the absorbance value was positively associated with the reduction ability. The reduction ability increased as the concentrations of S1-DIP, S2-DIP and S3-DIP increased. However, no significant increase was observed in the reduction ability with increasing concentration of DIP. The reduction ability of S1-DIP, S2-DIP and S3-DIP was 0.645, 0.705, 0.675 at a concentration of 1,000 μg/ml, respectively, which was ~6.0-fold higher compared with that of DIP. In addition, compared with the absorbance rate of DIP, higher absorbance values were obtained for S1-DIP, S2-DIP and S3-DIP at the same concentration. Furthermore, significant differences were noted in the reduction potential among the different DS of the sulfated polysaccharides at 300–500 μg/ml (P<0.05). The maximum reduction ability was observed for S2-DIP. These results indicated that sulfation enhanced the reduction potential of the original polysaccharide.

The inhibitory effects of DIP and S-DIPs on MCF-7 cells are shown in Fig. 6A. The S-DIPs exhibited greater inhibitory effects compared with that of DIP (P<0.05), while the unmodified DIP had no effect on the growth of the MCF-7 cells. At concentrations of 200–750 μg/ml, statistically significant differences were noted for the inhibitory effects on the MCF-7 cells between S1-DIP, S2-DIP and S3-DIP (P<0.05). Additionally, these inhibitory effects were enhanced with increasing DS.

In the B16 cells (Fig. 6B), no significant difference was identified in cell growth following treatment with DIP at concentrations of 25–750 μg/ml. However, a significant inhibition was noted in the cell growth of the cells treated with the sulfated derivatives (P<0.05). Additionally, the inhibitory effects were more marked as the DS and concentration increased. S3-DIP exerted its maximum inhibitory effect at a concentration of 750 μg/ml.