Purification, characterization, and bioactivities of a polysaccharide from mycelial fermentation of Bjerkandera fumosa

Article abstract of DOI:10.1016/j.carbpol.2017.03.029

In this work, a novel polysaccharide (named DBFM3) was isolated from mycelia of Bjerkandera fumosa by DEAE-32 and Sepharose CL–6?B column chromatography. High-performance gel permeation chromatography showed that DBFM3 was homogeneous, with an average molecular weight of 1.8?×?10⁵?Da. Structural characteristics of the purified fraction were investigated by high-performance liquid chromatography (HPLC), FT-IR, and NMR. HPLC analysis indicated that DBFM3 was composed of mannose, galacturonic acid, and galactose at molar ratios of 1:18.16:0.702. Spectral analysis suggested that DBFM3 had (1?→?6), (1?→?3,6), (1?→?3) linkages and pyranose conformation. Antioxidant assay in vitro showed that DBFM3 exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical scavenging activity, and has protective effect against DNA damage and damage to SH-SY5Y cells induced by H₂O₂. Immunological tests indicated that DBFM3 significantly increased lymphocyte proliferation in vitro. Furthermore, DBFM3 increased the proliferation of lymphocytes in the presence of concanavalin A or lipopolysaccharide as mitogen.

Full text of DOI:10.1016/j.carbpol.2017.03.029

Accepted Manuscript
Title: Purification, characterization, and bioactivities of a
polysaccharide from mycelial fermentation of Bjerkandera
fumosa
Authors: Di Liu, Qianwen Sun, Jing Xu, Na Li, Jianan Lin,
Shan Chen, Fan Li
PII:
S0144-8617(17)30285-0
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2017.03.029
CARP 12118
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25-1-2017
8-3-2017
Please cite this article as: Liu, Di., Sun, Qianwen., Xu, Jing., Li, Na., Lin,
Jianan., Chen, Shan., & Li, Fan., Purification, characterization, and bioactivities of
a polysaccharide from mycelial fermentation of Bjerkandera fumosa.Carbohydrate
Polymers http://dx.doi.org/10.1016/j.carbpol.2017.03.029
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Purification, characterization, and bioactivities of a
polysaccharide from mycelial fermentation of Bjerkandera
fumosa
Di Liu a, Qianwen Sun a, Jing Xu a, Na Li b, Jianan Lin b, Shan Chena, *, Fan Li a, *
a
School of Life Sciences, Northeast Normal University, Changchun 130024. PR
China.
b
Changchun Research and Development Center, Changchun University of Chinese
Medicine, Changchun 130117. PR China.
*Corresponding author: Tel(Fax): +86-431-85098130; E-mail: Lif885@nenu.edu.cn.
1

Highlights
• A homogeneous polysaccharide was purified from B. fumosa.
• The chemical characterizations of the purified polysaccharide (designated as
DBFM3) were performed by HPLC, FT-IR, NMR.
• Antioxidant and immunoregulatory activities in vitro of purified polysaccharide
from B. fumosa were studied.
• DBFM3 presents a promising natural source of antioxidant and immunoregulatory
agents.
Abstract
In this work, a novel polysaccharide (named DBFM3) was isolated from mycelia
of Bjerkandera fumosa by DEAE-32 and Sepharose CL-6B column chromatography.
High-performance gel permeation chromatography showed that DBFM3 was
homogeneous, with an average molecular weight of 1.8×105 Da. Structural
characteristics of the purified fraction were investigated by high-performance liquid
chromatography (HPLC), FT-IR, and NMR. HPLC analysis indicated that DBFM3
was composed of mannose, galacturonic acid, and galactose at molar ratios of
1:18.16:0.702. Spectral analysis suggested that DBFM3 had (1→6), (1→3,6), (1→3)
linkages and pyranose conformation. Antioxidant assay in vitro showed that DBFM3
exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical scavenging
activity, and has protective effect against DNA damage and damage to SH-SY5Y cells
induced by H2O2. Immunological tests indicated that DBFM3 significantly increased
2

lymphocyte proliferation in vitro. Furthermore, DBFM3 increased the proliferation of
lymphocytes in the presence of concanavalin A or lipopolysaccharide as mitogen.
Keywords
Bjerkandera fumosa; Polysaccharide; Purification; Bioactivities
Chemical compounds studied in this article:
DPPH (PubChem CID: 2735032)
Ascorbic acid (PubChem CID: 54670067)
MTT (PubChem CID: 64965)
Hydrogen peroxide (PubChem CID: 784)
Rutin (PubChem CID: 5280805)
Galacturonic Acid (PubChem CID: 439215)
D-galactose (PubChem CID: 6036)
D-glucose (PubChem CID: 79025)
1. Introduction
A number of edible mushrooms have medicinal properties and have gained
attention as functional health promoters due to their biochemical composition (Zhang,
Cui, & Cheung, 2007). Polysaccharides are important bioactive constituents of
medicinal fungi and possess numerous bioactivities, such as immunomodulation,
anticancer, antioxidant, and hypoglycemic activities (Kardosova & Machova, 2006;
3

Liuet al., 2016; Schepetkin & Quinn, 2006). Polysaccharides are known for their roles
as structural elements of cell walls and have significant effects on multi-cytokine
inducers in cell-cell communication, for instance, in inducing gene expression of
various immunomodulatory cytokines (Ooi & Liu, 2000). Several studies have
demonstrated that the activities of polysaccharides are associated with the chemical
composition and structural characteristics (Methacanon, Madla, & Kirtikara, 2005).
Bjerkandera fumosa (Pers.) P. Karst, which is widely distributed in China
including Jilin, Hebei, Qinghai, and Liaoning provinces, is an annual wood-decaying
fungus used in traditional Chinese medicine. Water extraction of the fruiting body is
considered to be effective for treating uterine carcinoma and enhancing immunity.
However, the active components of B. fumosa have not been reported. Recently, many
natural polysaccharides and polysaccharide-protein complexes, such as lentinans from
Lentinus edodes (Xu, Yan, & Tang, 2014) and PSP from Coriolus versicolor (Cui &
Chisti, 2003), have been studied as immune modifiers and drugs for cancer therapies
(Giavasis, 2014; Stachowiak & Reguła, 2012; Yang & Zhang, 2009). Therefore, the
properties of polysaccharides from B. fumosa should be studied to better understand
the activity of the fungus.
In the present study, a polysaccharide (named DBFM3) was purified from B.
fumosa and characterized by chemical methods. The antioxidant effect of DBFM3
was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical
scavenging assay, as well as its protective effect on DNA damage and SH–SY5Y cells
damage induced by H2O2 was studied. In addition, the immunomodulatory effects on
4

splenocyte proliferation were investigated. This paper is the first to report the
identification and biological activities of polysaccharides from B. fumosa.
2. Materials and methods
2.1. Materials
B. fumosa was purchased from the China Forestry Culture Collection Center
(CFCC, Beijing, China). DEAE-32 cellulose and Sepharose CL-6B were purchased
from Amersham (Sweden). 3-(4,5-Dimethyl tiazol-2-yl)-2,5 diphenyl tetrazolium
bromide (MTT), bovine serum albumin (BSA), DPPH, lipopolysaccharide (LPS),
concanavalin A (ConA), and glucan standards were purchased from Sigma Co. (USA).
The solvents and other chemicals used were analytical grade.
2.2. Mycelia and polysaccharide prescription
B. fumosa was maintained on potato dextrose agar slants. A 10 mm2 portion of the
agar with mycelia was inoculated in the seed medium (0.1 g/L CaCl2, 1 g/L KH2PO4,
1.5 g/L MgSO4, 2 g/L yeast extract, 3 g/L peptone, and 20 g/L glucose) for 5 days at
28°C and in a rotary shaker at 150 rpm. Then, the seed medium was added to a 500
mL flask containing 200 mL of fermentation medium and inoculated at a rate of 10%
(v/v). The fermentation medium contained 0.5 g/L MgSO4, 0.05 g/L FeSO4, 1 g/L
KH2PO4, 0.02 g/L CuSO4, 0.4 g/L K2HPO4, 0.01 g/L ZnSO4, 0.01g/L CoCl2, 0.08 g/L
MnSO4, 3 g/L peptone, and 53 g/L corn powder. The medium was cultured in a rotary
shaker (150 rpm) at 28 °C for 8 days. Mycelia of B. fumosa were filtered with gauze
after cultivation.
5

The cultured mycelia were washed with distilled water two times, and extracted
with boiling water for 2 h. This process was repeated three times. All water extracts
were combined, concentrated, and precipitated by adding four volumes of 95%
ethanol under vigorous stirring and incubated at 4 °C overnight. The precipitate was
collected by centrifugation (4000 rpm for 15 min) and successively washed with
absolute ethanol and ether. Finally, the crude mycelium polysaccharide (BFM) was
lyophilized to obtain a taupe white powder.
BFM was dissolved in distilled water (5%), frozen-thawed, and centrifuged at
4 ℃ until no insoluble materials were present, and then deproteinized by a
combination of pronase and Sevag methods (Xu, Zhou, & Hao, 2015). The
deproteinized polysaccharide (DBFM) was passed through a DEAE-32 cellulose (2.6
cm×20 cm) column, and eluted first with distilled water, then with a linear gradient of
NaCl from 0 M to 1.0 M at a flow rate of 1 mL/min. The eluate was collected
automatically, and the polysaccharide content was monitored by the phenol-sulfuric
acid method. The eluted samples were collected, concentrated, and further purified by
gel filtration chromatography on Sepharose CL-6B column (2.5 cm×90 cm) using
0.9% NaCl as the eluant at a flow rate of 0.15 mL/min. The purified fractions were
collected, treated with four volumes of ethanol, centrifuged, and then lyophilized.
2.3. Molecular weight determination
The homogeneity and molecular weight of DBFM3 were determined by
high-performance gel permeation chromatography (HPGLC), which was performed
on a Shimadzu HPLC system with a TSK-G3000PWXL column (7.8 mm×30.0 cm)
6

and a Shimadzu RID-10A detector. The sample solution (2 mg/mL) was injected and
run with 0.2 mol/L NaCl as mobile phase at a flow rate of 0.6 mL/min.
2.4. Chemical components and monosaccharide composition
Polysaccharide
content
was
determined
by phenol-sulfuric
acid
spectrophotometric assay with D-glucose as a standard (Masuko et al., 2005). The
uronic acid content was evaluated aby the m-hydroxydiphenyl colorimetric method
with D-glucuronic acid as a standard, in which neutral sugars don’t interfere (Filisetti
& Carpita, 1991). The Bradford method was used to determine the protein content of
polysaccharides using BSA as a standard (Bradford, 1976).
Monosaccharide composition of DBFM3 was determined as follows. The
polysaccharide (2 mg) was hydrolyzed with 2 M trifluoroacetic acid at 120 °C for 2 h
in
a
sealed-tube. The released monosaccharides were derivatized by
1-phenyl-3-methyl-5-pyrazolone and analyzed on a DIKMA Inertsil ODS-3 column
(4.6 mm×150 mm) connected to a Shimadzu HPLC system (LC-10ATvp pump and
SPD-10AVD UV-vis detector).
2.5. UV, FT–IR, and NMR analyses
Ultraviolet and visible (UV-vis) absorption spectra were recorded using a
Shimadzu MPS-2000 spectrophotometer (200-700 nm).
Purified sample (2 mg) was mixed with KBr powder and pressed into a 1 mm
pellet for IR analysis. The FT-IR spectrum was recorded on SPECORDIR in the range
of 4000-500 cm−1.
13
1
The C-NMR and H-NMR spectroscopic analyses were conducted on a Bruker
7

-DRX Avance 600 MHz (Germany) spectrometer at 150 MHz and 500 MHz at 28°C,
respectively. The sample (20 mg) was dissolved in D2O (1 mL, 99.8%) with overnight
stirring at room temperature. Two-dimensional (2D) correlation spectroscopy (COSY)
techniques were used for general assignments of DBFM3.
2.6. Antioxidant activity test in vitro
2.6.1. DPPH scavenging assay
DPPH radical scavenging activity was estimated according to the literature with
minor modifications (Sharm & Bhat, 2009). The reaction mixture (OD1) contained 2
mL of DPPH (0.1 M in methanol) and 2 mL of different concentrations of the
polysaccharide solution (0.5, 1, 2, 3, 4, and 5 mg/mL). The reaction solution was
shaken vigorously and incubated in the dark at room temperature for 30 min, and the
absorbance of the mixture was measured at 517 nm. Ascorbic acid was used as a
positive control. OD0 was measured using distilled water (instead of sample solution)
as blank, and OD2 was measured using methanol (instead of DPPH solution) as
control. The DPPH antioxidant activity was calculated using the following formula:
OD0 - (OD1 - OD2 )
Scavenging rate(%) 
100%
OD0
2.6.2. Hydroxyl radical scavenging assay
The hydroxyl radical scavenging activity of purified polysaccharide was
determined as described previously in the literature (Li, Huang, & Lu, 2011). The
reaction mixture (Ax) contained 1 mL of samples at different concentrations (0.5, 1, 2,
3, 4, and 5 mg/mL), 1 mL of 9 mM salicylic acid-ethanol solution , 1 mL of 9 mM
FeSO4 solution, and 1 mL of 8.8 mM H2O2. After incubating at 37 °C for 1 h, the
8

mixture was centrifuged at 10000 rpm for 5 min. The absorbance was measured at
510 nm using ascorbic acid as positive control. A0 was measured using distilled water
(instead of sample solution) as blank, and Ax0 was measured using distilled water
(instead of H2O2) as control. The hydroxyl radical scavenging activity was expressed
as follows:
A0  (Ax  Ax0 )
Scavevging rate (%) 
100%
A0
2.6.3. Detection of DNA cleavage
Protection against DNA damage was evaluated as described previously (Akhilesh
& Sharmila, 2007; Russo, Acquaviva, & Campisi, 2000). Three microliters of pUC-19
plasmid DNA (10 nM), 4 µL of 2% H2O2, and 3 µL of 5 mg/mL sample were added to
a microfuge tube. The reaction was initiated by UV irradiation for 15 min on a clean
bench at room temperature. Reactionmixtures were subjected to 1% agarose gel
electrophoresis to determine the extent of DNA damage.
2.6.4. Protective effect of polysaccharides on H2O2-induced toxicity in SH-SY5Y
cells
Cytoprotective activity of polysaccharides on H2O2-induced cell injury was
assessed by the MTT assay (Nirmaladevi et al., 2014). The human neuroblastoma
SH-SY5Y cells (2×104/mL) were plated in sterile 96-well plates and incubated for 24
h to allow cells to adhere to the plate. Cells pretreated for 2 h with various
concentrations of polysaccharides (5, 10, 25, 50, 100 µg/mL) were treated with H2O2
(400 µmol/L) for 24 h at 37 °C in a humidified 5% CO2 incubator. Then, each well
was added with MTT (5 mg/mL) and incubated for another 4 h. After aspirating the
9

supernatant from the wells, 100 µL of DMSO was added to dissolve the formazan
crystals. The absorbance was measured at 570 nm using a microplate reader. Each
experiment was performed in triplicate.
2.7 Lymphocyte proliferation assay
Lymphocyte proliferation assays were assessed in vitro by an MTT-based
colorimetric assay. C57 mice (8-12 weeks old) were sacrificed by cervical dislocation.
Spleens were aseptically removed and placed in sterile RPMI-1640 medium, and
single cell suspension was prepared by disrupting spleen into small pieces under
aseptic conditions. The cells were filtered with gauze and red cells were lysed by
adding 0.75% NH4Cl. Cells were washed twice with RPMI-1640 and resuspended in
RPMI-1640 media. Aliquots of 100 µL of splenocytes (5×106/mL) were placed in a
96-well culture plate. Samples (concentrations of 10, 20, 40, 80, 200 µg/mL) were
added to each cell with or without ConA (5 µg/mL) or LPS (10 µg/mL) for 48 h at
37 °C in a humidified 5% CO2 incubator. Then, each well was added with 20 µL of
MTT (5 mg/mL) and the plate was incubated for another 4 h. After aspirating the
supernatant from the wells, 100 µL of DMSO was added to dissolve the formazan
crystals. Absorbance of each well was measured at 570 nm using a microplate reader.
Control experiments were performed without the polysaccharide.
2.8 Statistical analysis
Data were presented as mean ± SD (standard deviation). Statistical analyses were
performed by t-test and one way analysis of variance (SPSS, version 13.0).
Differences were considered statistically significant at p < 0.05.
10

3. Results and discussion
3.1. Isolation and purification of polysaccharide
DBFM was first separated through an anion-exchange DEAE-32 cellulose
column and was eluted consecutively with distilled water and a linear gradient of
NaCl from 0 M to 1.0 M. Three fractions corresponding to DBFN, DBFA1, and
DBFA2 were clearly separated in the elution profile of the anion-exchange
chromatogram (Fig. 1a). DBFN was a neutral polysaccharide, whereas both DBFA1
and DBFA2 were acidic polysaccharides. Each of the three fractions was further
purified by Sepharose CL-6B column chromatography using 0.9% NaCl as eluent. A
fraction from DBFA2 showing a single and symmetrically sharp peak was collected
and designated as DBFM3 (Fig. 1b).
3.2. Molecular weight and homogeneity of polysaccharides
The homogeneity and molecular weight of DBFM3 was determined by HPGLC.
The profile of DBFM3 showed a single and symmetrical narrow peak (Fig. 2), which
indicated that DBFM3 was a homogeneous and highly pure polysaccharide. HPGLC
analysis revealed that the molecular weight of DBFM3 was 1.8×105 Da, making it a
high molecular weight polysaccharide. A similar molecular weight has been reported
for an immunostimulatory polysaccharide (2.1×105 Da) from C. militaris mycelium
(Lee et al., 2010a, 2010b) and an acidic proteoglycan (1.5×105 Da) from P. Linteus
(Kim, Park, & Nam, 2003). Some studies have confirmed that polysaccharides with
11

higher molecular weights exhibit higher antitumor and immunological activity. A high
molecular weight is essential for enhancing the antitumor and immunological
activities of polysaccharides (Yan, Pei, & Ma, 2015).
3.3. Chemical components and monosaccharide composition
As a homogeneous polysaccharide, DBFM3 was chosen for further study.
Chemical analysis showed that the total sugar content, uronic acid, and protein
content of DBFM3 were 57.64%, 20.29%, and 1.75% respectively.
The monosaccharide composition of DBFM3 was measured by HPLC analysis.
Nine standard monosaccharides were separated rapidly within 30 min. The peaks of
standard monosaccharides were identified in the order of D-mannose, D-glucuronic
acid, L-rhamnose, galacturonic acid, D-glucose, D-galactose, D-xylose, L-arabinose,
and L-fucose (Fig. 3a). The sample was identified by matching retention time with
those of standard monosaccharides under the same analytical conditions. DBFM3 was
composed of mannose, galacturonic acid, and galactose at a molar ratio of
1:18.16:0.702. Galacturonic acid accounted for the largest proportion, indicating that
DBFM3 was a pectin-type polysaccharide just like the pectin polysaccharide PM II50
reported by Samuelsen et al. (1995)
3.4. Structural characterization of DBFM3
The UV absorption spectra of DBFM3 suggested that the polysaccharide had a
low absorption at the wavelength of 280 nm, indicating that DBFM3 contained trace
protein.
12

FT-IR spectroscopy is commonly used to determine organic the functional groups
(Wu, Zhu, & Zhang, 2012) in molecules. As shown in Figure 4, the spectrum of
DBFM3 displayed a broad and intense peak at 3366.64 cm−1 (3500-3100 cm−1), which
could be ascribed to the hydroxyl groups stretching vibration of polysaccharide and
water involved in hydrogen bonding. The weak absorption peak in the 2930.33cm−1
region (3000-2800 cm−1) was the characteristic of C-H stretching vibration of the
methyl group (Seedevi, Moovendhan, Viramani, & Shanmugam, 2017) and the strong
absorption at 1651.70 cm−1 was due to the C=O stretching vibration. The band at
1400.57 cm−1 represented the C-H bending vibration. Absorption peaks were detected
in the 1700-1300 cm−1 region, which indicated that the polysaccharide had a carboxyl
group (i.e., glycuronic acid in DBFM3). The absorbance at 1239.36 cm−1 was
attributed to the existence of sulfate radical. The stretching peak at 1086.72 cm−1
indicated the presence of pyranose ring (Park, 1971). The band at 819 cm−1 was
attributed to the D-galactopyranose unit (Barker, Bourne, & Stacey, 1954).
Signals corresponding to polysaccharide in the NMR spectra were identified
on the results of monosaccharide analysis and chemical shifts (Hu et al., 2011; Yang,
1
Lv, & Tian, 2010). The H-NMR spectrum of DBFM3 was complicated due to the
overlap of most signals. It was difficult to confirm the anomeric proton signals which
1
were merged with the signal from HOD (Fig. 5a). However, H-1H COSY revealed
three correlation peaks in the anomeric proton region (Fig. 5c), indicating the
existence of three anomeric protons at δ 4.329, 4.599 and 4.642, which were assigned
to H-1 of (1→6) -linked, (1→3,6) -linked and (1→3) -linked glucosyl residues,
13

respectively. The peaks of DBFM3 at around δ 4.7 were attributed to the chemical
shifts of D2O. Furthermore, δ 5.8-8.7 represented the aromatic protons, and weak
signals at δ 0.6-3.2 indicated the 1H chemical shift signal of β-H and γ-CH3, as well as
-CH2 and -CH from protein amino acid residues, which indicated that DBFM3 was a
glycopeptide.
13
The C-NMR spectrum of DBFM3 (Fig. 5b) suggested that the resonances of
98-109 ppm were anomeric carbon (Hu et al., 2011)，further indicating a β-anomeric
configuration for glucopyranosyl units (Hall & Johnson, 1969). The peak at 109.92
ppm was assigned to the C-1 signal of galactose. The signal at δ 61.009 should be
unsubstituted C-6 of (1→3)-linked glucosyl residues. In addition, the 13C-NMR
spectrum showed a signal at low field from δ 160-180, which indicated the presence
of uronic acid.
3.5. Antioxidant activity test analysis
3.5.1. DPPH scavenging assay
DPPH is a stable organic radical with peak absorption at 517 nm in methyl
alcohol, which can be extensively used to evaluate the free-radical scavenging ability.
As shown in Figure 6a, DBFM3 and DBFM demonstrated a dose-dependent DPPH
scavenging ability ranging from 0.5 mg/mL to 5 mg/mL. The DPPH scavenging
ability of DBFM3 was significantly stronger than that of unpurified DBFM with
approximately 14.25 % at 5 mg/mL, but lower than that of the positive control.
3.5.2. Hydroxyl radical scavenging assay
14

Hydroxyl radical is one of the most reactive oxygen radicals and can induce severe
damage to functioning biomolecules in living cells. Removing hydroxyl radicals is
important for protecting living systems. As shown in Figure 6b, with increasing
concentration, the scavenging capacity of DBFM3 and DBFM improved ranging from
0.5 mg/mL to 5 mg/mL. DBFM3 exhibited higher scavenging activity (with
approximately 13.5 % at 5 mg/mL) on hydroxyl radical than unpurified DBFM, but
lower than that of ascorbic acid.
3.5.3. Detection of DNA cleavage
The protective effect of DBFM3 on the damage caused by H2O2 and UV was
evaluated using pUC-19 plasmid DNA. DNA damage was detected by gel
electrophoresis. The results showed that DNA can be damaged by ·OH generated
from UV irradiation (Fig. 7a). UV irradiation of samples containing rutin, which has
strong UV absorption, revealed the presence of two forms of DNA, i.e., supercoiled
(SC)-DNA and open circular (OC)-DNA (Lane 2, Fig. 7a), indicating that rutin can
protect DNA. For this reason, we used rutin as positive control. Addition of DBFM3
revealed the existence of SC-DNA (Lane 3, Fig. 7a), indicating that DBFM3 had
some protective effect against DNA damage. SC-DNA was completely converted into
OC-DNA in the negative control to which PBS had been added instead of rutin (Lane
4, Fig. 7a), indicating that the selected conditions met the test requirements. Thus,
polysaccharide had protective effect on DNA damage, which could be attributed to
scavenging of hydroxyl radicals; this ability was associated with the antioxidant
property of polysaccharides.
15

3.5.4. Protective effect of polysaccharide on H2O2-induced toxicity in SH-SY5Y
cells
After exposure to H2O2 (400 µmol/L) for 24 h, cell viability decreased by 80 ±
2.08% compared to the normal group. However, pretreatment of cells with various
concentrations of DBFM and DBFM3 significantly restored cell viability in a
dose-dependent manner compared to the H2O2-model group (Fig. 7b). DBFM and
DBFM3 at 50 µg/mL almost neutralized H2O2-induced oxidative stress demonstrating
the effectiveness of polysaccharide in preventing oxidative stress to neuronal cells.
Some studies have indicated that the antioxidant activity of polysaccharides is
related to many factors, including monosaccharide composition (Gao et al., 2013),
uronic acid, and molecular weight. In addition, high GalA-containing polysaccharides
could have high antioxidant activity, owing to the functional group-COOH. It has
been shown that the functional groups (-COOH and CH3CO-) are good electron or
hydrogen donors and may thereby contribute to the antioxidant activity of
polysaccharides (Wang et al., 2015). In our study, DBFM3 was found to be mainly
composed of galacturonic acid, which contribute to the antioxidant activity of
polysaccharides.
3.6 Lymphocyte proliferation assay
DBFM3 and DBFM were subjected to immune tests to evaluate their effects on
lymphocyte proliferation. As shown in Figure 8a, splenic lymphocyte proliferation
was stimulated by polysaccharide alone, and DBFM3 had a greater impact than
16

DBFM. As shown in Figure 8b and Figure 8c, synergistic effect was observed
between polysaccharide and ConA or LPS. Lymphocyte proliferation was higher in
the DBFM3-treated groups compared to the DBFM-treated group (p<0.05). Many
polysaccharides have been demonstrated to possess immune modulation activity. For
example, purified polysaccharides from mycelial cultures of Phellinus linteus have
been found to stimulate the proliferation of T and B lymphocytes (Hanet al., 1999;
Kim et al., 1996), which polysaccharides from P. linteus act as immunopotentiators
partly by protecting T cells and enhancing mucosal IgA responses (Li et al., 2011).
Previous studies have demonstrated that structural features such as β-(1→3) linkages
and β-(1→6) linkages are important for increasing immune-competent cell activity
(Wasser, 2002). The result of our study demonstrated that glycosidic linkage may be
had related to the immune modulation activity of DBFM3.
(a) Polysaccharides, values represent mean ± SD; *P < 0.05, **P < 0.01 vs. negative
control; (b) Polysaccharides + ConA, values represent mean ± SD; *P < 0.05, **P <
0.01 vs. ConA;
(c) Polysaccharides + LPS, values represent mean ± SD; *P < 0.05, **P < 0.01 vs.
LPS.
4. Conclusion
A new polysaccharide, named DBFM3 in this paper, was isolated from B. fumosa
mycelia. The water-soluble polysaccharide was purified by DEAE-32 cellulose and
Sepharose CL-6B column chromatography. HPGLC showed that DBFM3 is
17

homogenous with a molecular weight of 1.8×105 Da. DBFM3 was found to be
composed of mannose, galacturonic acid, and galactose at molar ratios of
1:18.16:0.702. Spectral analysis suggested that DBFM3 has (1→6), (1→3,6) and
(1→3) linkages and pyranose conformation. Moreover, DBFM3 showed positive
antioxidant potential on DPPH and hydroxyl radical invitro, as well as protective
effect against DNA damage and SH-SY5Y cell damage induced by H2O2.
Proliferation study on immunological activities indicated that DBFM3 has the
potential to promote splenocyte proliferation, and increased the proliferation of
lymphocytes in the presence of ConA or LPS as mitogen. These findings indicate
that DBFM3 might be a candidate for the development of antioxidative or
immunopotentiating agents in food and pharmaceutical industries.
Acknowledgements
This work was supported by National Natural Science Foundation of China
(Grant No. J1210045).
18

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Fig. 1. Elution profile of DBFM on DEAE-32 cellulose column (a) and elution curve
of polysaccharide fraction DBFA2 on Sepharose CL-6B column (b).
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Fig. 2. HPGLC Profile of DBFM3
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Fig. 3. HPLC spectra of derivatives of (a) standard monosaccharides and (b) DBFM3.
Peaks: (1) D-mannose, (2) D-glucuronic acid, (3) L-rhamnose, (4) galacturonic acid,
(5) D-glucose, (6) D-galactose, (7) D-xylose, (8) L-arabinose, and (9) L-fucose.
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Fig. 4. FT–IR spectrum of DBFM3 from B. fumosa
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1
Fig. 5. NMR spectra of DBFM3 from B. fumosa: (a) H-NMR, (b) 13C-NMR,
(c)1H-1H COSY spectrum.
29