Physicochemical properties and antioxidant activities of polysaccharide from floral mushroom cultivated in Huangshan Mountain

Article abstract of DOI:10.1016/j.carbpol.2015.05.052

Abstract In this paper, a polysaccharide fraction (FMPS) was purified from the floral mushroom cultivated in Huangshan Mountain for the first time. Physicochemical properties and antioxidant activities of FMPS were investigated. FMPS had an average molecular weight of 7.2 × 10⁵ Da and was composed of glucose. On the basis of FT-IR, NMR and methylation analysis, the repeating unit of FMPS was established as (1 → 3)-linked β-d-glucopyranosyl backbone with 1-linked β-d-glucopyranosyl branches substituted at O-6 position of (1 → 3)-linked β-d-glucopyranosyl residues. The advanced structure studies indicated that FMPS was a triple-helical polysaccharide. The main hydrodynamic radius (R_h) of FMPS was 23.4 nm and it could form a stable system with water in 1.2 × 10^-2 g/mL solutions. In addition, FMPS exhibited high DPPH radical scavenging activities (79.46% at 5 mg/mL) and hydroxyl radical scavenging activities (74.18% at 5 mg/mL), as well as Fe²⁺chelating activities and ABTS radical scavenging activities to some extent.

Full text of DOI:10.1016/j.carbpol.2015.05.052

Accepted Manuscript
Title: Physicochemical properties and antioxidant activities of
polysaccharide from floral mushroom cultivated in Huangshan
Mountain
Author: Jun-Hui Wang Jin-Long Xu Jing-Cheng Zhang Yong
Liu Han-Ju Sun Xueqiang Zha
PII:
S0144-8617(15)00465-8
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2015.05.052
CARP 9956
To appear in:
Received date:
Revised date:
Accepted date:
31-3-2015
15-5-2015
25-5-2015
Please cite this article as: Wang, J.-H., Xu, J.-L., Zhang, J.-C., Liu, Y., Sun, H.-J., and
Zha, X.,Physicochemical properties and antioxidant activities of polysaccharide from
floral mushroom cultivated in Huangshan Mountain, Carbohydrate Polymers (2015),
http://dx.doi.org/10.1016/j.carbpol.2015.05.052
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

1
2
3
4
5
6
Highlights
 A polysaccharide fraction (FMPS) was extracted from Huangshan floral mushroom for
the first time.
The primary structure and advanced structure of FMPS was studied.
Investigated the antioxidant activity of FMPS by different manners.
1
Page 1 of 31

6
7
8
Physicochemical properties and antioxidant activities of polysaccharide
from floral mushroom cultivated in Huangshan Mountain
Jun-Hui Wang1*, Jin-Long Xu1, Jing-Cheng Zhang2, Yong Liu1, Han-Ju Sun1，Xueqiang Zha1
9
10
11
12
(1 School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei
230009, People’s Republic of China)
(2 Center of Analysis and Measurement, Hefei University of Technology, Hefei 230009,
People’s Republic of China)
13
14
15
16
17
----------------------------------------------------------------------------------------------------------------
* Correspondence Author: Jun-Hui Wang
Fax: 86-551-62901516
E-mail: junhuiwang@hfut.edu.cn
2
Page 2 of 31

18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Abstract:
In this paper, a polysaccharide fraction (FMPS) was purified from the floral mushroom
cultivated in Huangshan Mountain for the first time. Physicochemical properties and
antioxidant activities of FMPS were investigated. FMPS had an average molecular weight of
7.2 × 105 Da and was composed of glucose. On the basis of FT-IR, NMR and methylation
analysis, the repeating unit of FMPS was established as (1→3)-linked β-D-glucopyranosyl
backbone with 1-linked β-D-glucopyranosyl branches substituted at O-6 position of
(1→3)-linked β-D-glucopyranosyl residues. The advanced structure studies indicated that
FMPS was a triple-helical polysaccharide. The main hydrodynamic radius (Rh) of FMPS was
23.4 nm and it could form a stable system with water in 1.2 × 10-2 g/mL solutions. In addition,
FMPS exhibited high DPPH radical scavenging activities (79.46% at 5 mg/mL) and hydroxyl
radical scavenging activities (74.18% at 5 mg/mL), as well as Fe2+chelating activities and
ABTS radical scavenging activities to some extent.
Keywords: Floral mushroom; Polysaccharides; Physicochemical properties; Antioxidant
activities.
3
Page 3 of 31

33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
1. Introduction
Mushrooms have been appreciated as important edible and medical resources (Villares,
Mateo-Vivaracho, & Guillamón, 2012; Wani, Bodha, & Wani, 2010; Wasser, & Weis, 1999).
A lot of mushrooms are known for their good flavor and texture, such as lentinus edodes,
flammulina velutipes and pleurotus ostreatus (Xiong, 2009). In recent decades, the medicinal
values of mushrooms attract people’s attention greatly. Mushrooms are rich in many
biologically active compounds of high medicinal values, such as lectins, terpenoids,
polyphenolics, polysaccharides, ergosterols, and volatile organic compounds (Kalač, 2013).
Especially, polysaccharide is a very important active ingredient of mushrooms. People have
made much research of polysaccharides from edible mushrooms and find they exhibit
antitumor (Zhang, Cui, Cheung, & Wang, 2007; Zhang, et al., 2013), antioxidant (Chen, Xie,
Nie, Li, & Wang, 2008; Popov, et al., 2014; Xie, et al., 2010), and immunoregulation (Luo,
Sun, Wu, & Yang, 2012; Yin, et al., 2012) activities. Thus, the deep study on polysaccharides
from edible mushrooms together with its application is very meaningful.
A lot of polysaccharides have been extracted from edible mushrooms and many of them
are β-linked glucans (Bohn, & BeMiller, 1995; Dai, et al., 2012; Villares, 2013; Wang, et al.,
2009). Structures and functions of polysaccharides extracted from different edible
mushrooms are different. It was reported that Zhuling polysaccharide, a water-soluble
polysaccharide with (1→6, 1→4)-linked β-D-glucopyranosyl backbone, was a potent
activator of B cells, macrophages and dendritic cells (Dai, et al., 2012). The polysaccharide
isolated from the fruiting bodies of Dictyophora indusiata, consisting mainly of
(1→3)-β-D-glucan with (1→6)-β-glucosyl side branches, had antitumor activities (Deng, et
4
Page 4 of 31

55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
al., 2013). The chain conformations of polysaccharides are closely related to their functions.
For instance, the antitumor activity of lentinan with triple-helix chains is much higher than
that in single flexible chains (Zhang, Li, Xu, & Zeng, 2005). It is necessary to study the
structure of a novel polysaccharide and its conformations in aqueous solution.
Huangshan floral mushroom, hereinafter refer to as floral mushroom, grows in
Huangshan Mountain, Anhui province, China (Fig. 1A). It is a rare species of edible
mushrooms and looks like a flower with some grains in its top surface. Huangshan floral
mushroom tastes delicious and has a high nutritional value. Though, polysaccharide is a kind
of important active ingredients in floral mushroom. There are few reports on the
polysaccharides from Huangshan floral mushroom. This limits the application of Huangshan
floral mushroom. In this study, for the first time, a polysaccharide fraction was isolated from
the fruiting body of floral mushroom (FMPS). The physicochemical properties and structural
features of FMPS were elucidated by infrared spectroscopy, gas chromatography, gas
chromatography-mass spectrometry, NMR and chemical methods. For further understanding
of FMPS, the antioxidant activity of FMPS, including DPPH radical, hydroxyl radical, ABTS
radical scavenging activities and Fe2+chelating activities were tested. This study would
provide meaningful data for the development of Huangshan floral mushroom.
2. Materials and methods
2.1 Materials and reagents
Dried fruiting bodies of floral mushroom were obtained from Huangshan city, Anhui
province, China. Standard monosaccharides (D-glucose, D-mannose, D-xylose, L-galactose,
L-rhamnose, and L-arabinose) and DMSO-d6 were purchased from Sigma–Aldrich Co., Ltd.
5
Page 5 of 31

77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
(St. Louis, MO, USA). All other reagents were of analytical grade.
2.2 Isolation and purification of polysaccharides
The dried fruiting bodies of floral mushroom (Fig. 1A) were pulverized by a pulverizer
and went through a 60-mesh screen before experiments. The powders（150 g） were defatted
with petroleum ether and acetone successively by soxhlet extraction methord. The residues
were dried at 50 oC and extracted twice with hot distilled water for 2 h at 100 oC. The rest of
materials were further extracted with 5 L 0.05% NaBH4/1.25 M NaOH solution twice at 25
oC for 2 h. The extracted solution was filtered with gauze to get the supernatants. The
supernatants were neutralized by acetic acid and centrifuged to remove precipitation. 4 times
volume of 95% ethanol was slowly added into the supernatants. The resulting mixture was
centrifuged to get the precipitation after placed 24 h. The precipitation was dissolved in water
again and removed proteins by Sevage methods. The resulting solution was decolorized with
30% H2O2 at 45 oC for 4h, dialyzed with tap water for 4 days and distilled water for 3 days.
Dialysate was lyophilized to get the polysaccharide fraction FMPS (Fig. 1B). The yield was
4.7%.
93
94
Fig. 1. Pictures of Huangshan floral mushroom (A) and the FMPS (B).
6
Page 6 of 31

95
2.3 Homogeneity and molecular weight
96
97
The homogeneity and average molecular weight of FMPS were identified by
high-performance gel-permeation chromatography (HPGPC) on a Waters E2695 HPLC
system (Waters Corporation, USA) equipped with a Waters 2424 evaporative light scattering
detector (ELSD). The UltrahydrogelTM 2000 column (7.8 mm×300 mm) and UltrahydrogelTM
500 column (7.8 mm×300 mm) were used as the separation medium, which were connected
in series. The test conditions were as follows: sample concentration: 1mg/mL; mobile phase:
ultrapure water; flow rate: 0.5 mL/min; injection volume: 20 μL. The calibration curve was
created using a set of dextran standards.
98
99
100
101
102
103
104
2.4 Fourier-transform infrared spectra (FT-IR) analysis
105
106
107
FT-IR spectrum of FMPS was recorded on FT-IR spectrometer (Nicolet 5700, Thermo
Nicolet, USA) in the wave-number range 4000–400 cm−1. The testing method was potassium
bromide (KBr) disc method (Xie et al., 2013).
108
2.5 Monosaccharide composition analysis
109
110
111
112
113
114
115
The monosaccharide composition of FMPS was analyzed by gas chromatography (GC).
Sample (5 mg) was hydrolyzed by 4 mL 2 mol/L trifluoroacetic acid (TFA) at 120 oC for 4 h
in a sealed glass tube. The residual TFA was removed using a rotary vacuum evaporator and
the monosaccharides were reduced with 30 mg NaBH4 for 3 h at room temperature after
dissolved in distilled water. The reaction solution was neutralized with 25% CH3COOH until
there were no air bubbles. After removed the water phase, the residue was reacted with 3 mL
acetic anhydride and 3 mL pyridine for 1 h. The reaction products were alditol acetate
7
Page 7 of 31

116
117
derivatives and analyzed by GC. The operation method was reported in the previous study
(Wang, Luo, & Zha, 2010).
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
2.6 Methylation analysis
The polysaccharide, FMPS (10 mg), was methylated using DMSO, sodium hydride and
methyl iodide according to the Hakomori method (Needs & Selvendran, 1993). Complete
methylation was confirmed by the disappearance of the OH band (3200–3700 cm−1) in the IR
spectrum. The methylated polysaccharide was depolymerized with 88% HCO2H (3 mL) for 6
h at 100 oC in a sealed glass tube. After removing the residual formic acid, the polysaccharide
o
was hydrolyzed by 4 mL 2 mol/L trifluoroacetic acid (TFA) at 120 C for 4 h. The following
treatment methods were the same to section 2.5. The resulting products were methylated
alditol acetates and analyzed by GC–MS.
2.7 NMR spectroscopy
Sample (25 mg) was dried in P2O5 for 24 h, and then its 13C NMR spectrum was
recorded in DMSO-d6 with NMR spectrometer (VNMRS600, Agilent) (Liu, Du, Wang, Zha,
& Zhang, 2014).
2.8 Advanced structure studies
2.8.1 Congo red analysis
2 mL sample solution (2 mg/mL) was added to 2 mL Congo red solution (80 μmol/L) in
a concentration gradient of NaOH solutions (0.00, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35,
0.40, 0.45 and 0.50 mol/L). The absorbance was measured by UV-Vis spectrometer in the
range of 200~800 nm. The distilled water without adding FMPS was served as the control.
This method was reported in the previous literature (Liu, et al., 2014).
8
Page 8 of 31

138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
2.8.2 Viscosity of FMPS in NaOH solutions
A certain volume of distilled water and the NaOH solution were added to 2 mL sample
solution (2 mg/mL). The final solution volume was 4 mL with a concentration gradient of
NaOH (0.00, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 and 0.50 mol/L). The
viscosity was tested by the rotational rheometer (DHR-3, TA, USA) with a 40 mm parallel
plate.
2.8.3 Dynamic light scattering
Dried polysaccharide was dissolved in distilled water with the concentration of 1mg/mL.
The solution was centrifuged to remove the impurities before testing. The test was performed
on a dynamic light scattering instrument (MS800, Dynapro).
2.8.4 Dynamic strain sweep
The dependences of storage modulus (G') and loss modulus (G") on the strain were
investigated by the rotational rheometer (DHR-3, TA, USA) with a 60 mm parallel plate. The
dynamic strain sweep measurements were repeated three times for 1.2×10-2 g/mL FMPS
aqueous solution at 15 oC.
2.9 Antioxidant activity test
2.9.1 DPPH radical scavenging activity
The DPPH radical scavenging activities of FMPS were determined using the reported
method (Chaiklahan, et al., 2013) with some modifications. The FMPS solutions were
prepared with the concentrations of 1.0, 2.0, 3.0, 4.0 and 5.0 mg/mL, respectively. 2.0 mL
sample solution was mixed with 0.5 mL 100 μmol/L DPPH in methanol. Then the mixtures
were shaken and allowed to equilibrate at room temperature for 30 min. The sample
9
Page 9 of 31

160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
absorbances were measured at 517 nm. In this study, the distilled water was used as the blank
control and ascorbic acid (Vc) was served as positive control. The ability to scavenge the
DPPH radical was calculated according the following equation:
DPPH radical scavenging activity (%) = (A0－A1＋A2 ) /A0×100
Where, A0, A1 and A2 were the absorbances of the control solution (water instead of FMPS),
sample in reactive system and final absorbance of sample (methanol instead of DPPH),
respectively.
2.9.2 Hydroxyl radical scavenging activity
The activity of FMPS to scavenge hydroxyl radical was determined according the
reported method (Gao, Wang, Wang, & Wang, 2013). The FMPS solutions were prepared
with the concentrations of 1.0, 2.0, 3.0, 4.0 and 5.0 mg/mL, respectively. 1.0 mL sample
solution was mixed with 1.0 mL FeSO4 of 9 mmol/L, 1.0 mL salicylic acid of 9 mmol/L in
o
ethanol and 0.05 mL H2O2 of 9 mmol/L. Then the mixtures were incubated at 37 C for 30
min, the sample absorbances were measured at 510 nm. The distilled water was used as the
blank control and ascorbic acid (Vc) was served as positive control. The scavenging activity
of hydroxyl radical was calculated using the equation as follows:
Hydroxyl radical scavenging activity (%) = (A0－A1＋A2 ) /A0×100
Where, A0, A1 and A2 were the absorbances of the control solution (water instead of FMPS),
sample in reactive system and final sample, respectively.
2.9.3 ABTS radical scavenging activity
The ABTS radical scavenging activity of FMPS was measured according to the reported
method (Cheng, et al., 2013). The FMPS solutions were prepared with the concentrations of
10
Page 10 of 31

182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
1.0, 2.0, 3.0, 4.0 and 5.0 mg/mL, respectively. 0.2 mL sample solution was mixed with 4.0
mL 7 mmol/L ABTS. Then the mixtures were incubated at room temperature for 30 min, the
sample absorbances were measured at 734 nm. The distilled water was used as the blank
control and ascorbic acid (Vc) was served as positive control. The scavenging activity of
ABTS radical was calculated using the equation as follows:
ABTS radical scavenging activity (% )= (A0－A1＋A2 ) /A0×100
Where, A0, A1 and A2 were the absorbances of the control solution (water instead of FMPS),
sample in reactive system and final sample, respectively.
2.9.4 Fe2+chelating activity
The Fe2+chelating activity of FMPS was tested according to the previous method
(Manivasagan, et al., 2013). 1 mL sample containing different concentrations of FMPS (1~5
mg) was mixed with deionized water (2.75 mL), FeCl2 (0.05 mL 2 mM) and ferrozine (0.2
mL, 5 mM). The mixture was incubated for 10 min at room temperature and the absorbance
was determined at 562 nm. The distilled water was used as the blank control and ethylene
diamine tetraacetic acid disodium salt (EDTA-2Na) was served as positive control. The
Fe2+chelating activity was calculated according the following equation:
Fe2+chelating activity (%)=(A0－A1＋A2 ) /A0×100
Where, A0, A1 and A2 were the absorbance of the control solution (water instead of FMPS),
sample in reactive system and sample, respectively.
3. Results and discussions
3.1 Isolation, purification and composition analysis of FMPS
A water-soluble polysaccharide FMPS was isolated and purified from the fruiting body
11
Page 11 of 31

204
205
206
207
208
209
of floral mushroom cultivated in Huangshan Mountain. The homogeneity and average
molecular mass of FMPS were identified by HPGPC and it showed a single and symmetrical
peak (Fig. 2A) with an estimated molecular weight 7.2 × 105 Da based on dextran standards.
The monosaccharide composition of FMPS was analyzed by GC (Fig. 2B). As shown in Fig.
2B, FMPS was composed of glucose only based on the GC analysis of standard
monosaccharide.
12
Page 12 of 31

210
211 Fig. 2. (A) HPGPC chromatogram of FMPS; (B) GC chromatogram of monosaccharides
212 composition of FMPS; (C) FT-IR spectrum of FMPS.
213
214
3.2 FT-IR spectrum analysis
13
Page 13 of 31

215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
The FT-IR spectrum of FMPS was showed in Fig. 2C. A typical broad peak at 3342
cm-1 was due to the stretching vibration of O-H in the sugar ring. The peaks at 1160 cm-1 and
1051cm-1 suggested the presence of C-O bonds in FMPS (Du, Liu, & Wang, 2015). The
absorption band at 2919 cm-1 was due to the C-H stretching vibration (Suvakanta, Narsimha,
Pulak, Joshabir, & Biswajit, 2014). A little absorption peak around 1413 cm-1 was attributed
to the deformation vibration of C-H. The absorption peak at 889 cm-1 indicated a β-D-glucan
existing in the polysaccharide (Wang, et al., 2009). No absorption peaks at 1730 cm−1
suggested that there were no uronic acids in FMPS (Zhang, Xiao, Deng, He, & Sun, 2012).
3.3 Linkage types analysis
The FMPS was methylated twice according to the Hakomori method (Needs &
Selvendran, 1993). FT-IR spectrum of methylated polysaccharide was recorded on a FT-IR
spectrometer and the result was shown in Fig. 3A. FT-IR spectrum indicated that the
methylation was complete indicative of the absences of absorption peaks at 3342 cm-1. The
absorption peak of C-H stretching vibration at 2919 cm-1 had obvious enhancement due to
the addition of methyl.
14
Page 14 of 31

230
231 Fig. 3. (A) FT-IR spectrum of methylated FMPS; (B) Total ion chromatogram of FMPS by
232 GC–MS; (C) 13C NMR spectrum of FMPS in DMSO-d6.
233
234
235
236
237
238
239
The methylated polysaccharides were converted into partially methylated alditol
acetates and analyzed by GC-MS (Fig. 3B). The methylation analysis revealed mainly three
partially methylated alditol acetates 1, 5-di-acetyl-2, 3, 4, 6-tetra-O-methyl glucitol, 1, 3,
5-tri-acetyl-2, 4, 6-tri-O-methyl glucitol, and 1, 3, 5, 6-tetra-acetyl-2, 4-di-O-methyl glucitol
with a molar ratio of 1.50: 3.44: 1.45. And their corresponding peak times were 24.28 min,
26.10 min and 29.36 min, respectively (Table 1). The other absorption peaks in Fig. 3B were
15
Page 15 of 31

240
241
242
243
244
245
246
247
248
attributed to solvent and other chemical reagents added during methylation. Accordingly, the
following three glucosidic linkages existed in FMPS: non-reducing terminal glucosyl,
(1→3)-linked glucosyl and (1→3, 6)-linked glucosyl. It showed that (1→3)-linked-glucosyl
was the largest amount residue in FMPS and (1→3, 6)-linked-glucosyl was the branched
residue, revealing that (1→3)-linked-glucosyl should be possible to form the backbone
structure (Wang, et al., 2013). The relative molar ratio of (1→3)-linked-glucosyl and (1→3,
6)-linked-glucosyl was 2.29 indicating that average one branching point for each three or
four residues of backbone. The residues of branch structure were 1-linked glucosyl residues.
249 Table 1. GC-MS data for methylation analysis of FMPS.
Retention
time
Methylated
sugars
Linkage
types
Molar
ratios
Mass fragments (m/z)
24.28
26.10
29.36
2,3,4,6-Me4-Glcp
2,4,6-Me3-Glcp
2,4-Me2-Glcp
Terminal
1.50 45, 71,87,101,117,129,145,161,205
3.44 45,58,71,87,97,101,117,129,161,173,233
1.45 58,87,99,101,117,129,159,173,189,233
1,3-Linked-
Glcp
1,3,6-Linked-
Glcp
250
251
252
253
254
255
256
257
3.4 NMR spectroscopy analysis
The 13C NMR spectrum of FMPS in DMSO-d6 was shown in Fig. 3C. Compared with
the data on similarity substituted sugar residues in previous literatures (Wang, et al., 2009;
Corsaro, et al., 2005; Zhang, Zhang, Dong, Guo, & Song, 2001), the chemical shifts at 106.1
(C1), 75.9 (C2), 89.4 (C3), 71.6 (C4), 79.3 (C5) and 64.0 (C6) ppm were assigned to the
carbon atoms of the (1→3)-β-D-glucan backbone. The peaks at C3′ (90.1, 89.1, 79.9), C5′
(77.6), C2′ (76.8), and C6′ (73.1) were resulted from branching effect. The structure features
16
Page 16 of 31

258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
of FMPS were similar to those reported polysaccharides such as JQPs (Liu, et al., 2014) and
PD3 (Wang, et al., 2009), which had a (1→3)-β-D-glucan backbone with (1→6)-β-glucosyl
side chains.
Combining the 13C NMR spectrum and methylation analysis of FMPS, it was concluded
that the FMPS had (1→3)-β-D-glucopyranose backbone, and O-6 position of (1→3)-linked
β-D-glucopyranose was substituted by 1-linked-β-D-glucopyranosyl branches. There was
average one branching point for each three or four residues of backbone.
3.5 Advanced structure of FMPS
Congo red could form special complexes with polysaccharides possessed an ordered
three-dimensional structure, generally triple-helical conformation in solution, and the
maximum absorption wavelength (λmax) in spectral scan would shift (Nitschke, et al., 2011).
The complex was stabilized by hydrophobic interactions or strong hydrogen bonds between
the polysaccharide and the dye molecule (Palacios, García-Lafuente, Guillamón, & Villares,
2012). The interactions of FMPS with Congo red were shown in Fig. 4A. The λmax increased
to a maximum with the CNaOH increased initially and decreased sharply when the CNaOH
researched 0.2mol/L, indicating that FMPS displayed a triple-helical structure and which was
destroyed in high concentrate NaOH solution (Du, Liu, & Wang, 2015).
Moreover, the viscosities of FMPS in a series of NaOH solutions were tested at the
o
shear rate of 100 s-1 by the rotational rheometer on the temperature 25 C. The result was
shown in Fig. 4B. In order to eliminate the influence of solvent, the viscosities of NaOH
solutions were also tested. The viscosities of NaOH solutions had no obvious change in the
selected concentration range. The viscosities of FMPS increased when the concentration of
17
Page 17 of 31

280
281
282
283
284
285
286
287
288
289
290
291
292
NaOH reached 0.05 mg/mL. When the concentration of NaOH more than 0.05 mg/mL, the
viscosities of FMPS reduced gradually with the increasing of NaOH concentration and
reached a steady value. The reason that the viscosities of FMPS increased in low
concentration of NaOH solution was due to that part of hydrogen bonds in the triple-helical
chains was destroyed and the extension of the molecular chain was intensified. With the
further increasing of concentration of NaOH solutions, the conformation of FMPS had
transformed from triple-helical chain into flexible chain. This conformation transformation
made the molecular chains fully collapse and they were less contact with each other, which
resulted in the decreasing of viscosities of FMPS. Similar phenomenon was observed in
Schizophyllan (Zhou, Guo, Cai, & Li, 2005). It had been also reported that the
(1→3)-β-D-glucan from Poriacocos sclerotium could form flexible chain conformation in
the alkaline aqueous solution (Chen, Xu, Zhang, & Kennedy, 2009).
18
Page 18 of 31

293
294
Fig. 4. (A) The maximum absorption wavelength of Congo red with FMPS and Congo red in
various concentrations of sodium hydroxide solution; (B) The viscosities of FMPS in
various concentrations of sodium hydroxide solution and the viscosities of sodium
hydroxide solutions; (C) Dynamic light scattering spectrum of FMPS; (D) Strain
dependence of shear storage modulus G' (solid symbols) and loss modulus G" (open
symbols) determined by dynamic strain sweep measurement for FMPS solutions with
concentration of 1.2×10-2 g/mL at 15 oC.
295
296
297
298
299
300
301
302
303
304
For further understanding of the polysaccharide chain morphology in aqueous solution,
1 mg/mL FMPS solution was tested by dynamic light scattering (Fig. 4C). The results
indicated that the hydrodynamic radius (Rh) of FMPS was composed of two parts, 23.4 nm
19
Page 19 of 31

305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
and 96.5 nm. The percentage of them was 95.7% and 4.3%, respectively. This suggested that
FMPS mainly presented as single triple-helical chains in dilute solution. Rh of 96.5 nm maybe
resulted from a little aggregation (Zhang, Li, & Zhang, 2010). The results of dynamic light
scattering showed that the MW of FMPS was 6.18 ×105 Da. It was very close to the results of
HPGPC.
Lentinan, which presenting triple-helical conformation in aqueous solution, could form
a stable system with water in appropriate condition. This system was stable in a certain strain
range and had good reproducibility even it was destroyed (Zhang, Xu, & Zhang, 2008). Since
FMPS had a triple-helical structure, dynamic strain sweep was repeated three times for 1.2 ×
o
10-2 g/mL FMPS aqueous solution at 15 C. The second and third sweep began immediately
after the end of the former sweep. Results of the above experiments were shown in Fig. 4D.
As shown in Fig. 4D, G′ and G″ were relatively stable before the strain research 100%. When
strain was more than 100%, the values of G′ and G″ reduced sharply, indicating that the
structure of FMPS in solution had been destroyed. Moreover, three curves coincided very
well, suggesting it was able to immediately return to original state even the structure was
widespread destruction in previous sweep. The results revealed that FMPS could form a
stable system with water under appropriate condition and the FMPS/water system had good
reproducibility. The property of FMPS was meaningful for its application in food industry.
3.6 Antioxidant activity analysis
In this study, the antioxidant activities of FMPS were measured (Fig. 5). The DPPH free
radical is a stable free radical, which has been widely used to determine the free radical
scavenging abilities of antioxidants (Xie, et al., 2012). As shown in Fig. 5A, the DPPH
20
Page 20 of 31

327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
radical scavenging activities increased with the increase of concentration from 1 to 5 mg/mL.
When the concentration increased to 5 mg/mL, the scavenging ratio of FMPS and Vc were
79.46% and 95.75%, respectively. The IC50 value of FMPS was 1.97 mg/mL within the
concentration of test range. The results showed that FMPS had a strong DPPH radical
scavenging ability. However, it was lower than Vc at the same concentration.
Hydroxyl radical is considered as one of the most reactive oxygen radicals, which can
easily cross cell membranes, readily react with most biomolecules and lead to tissue damage
or cell death (Gao, et al., 2013). Fig. 5B showed that the hydroxyl radical scavenging ability
of FMPS had concentration dependence. It increased to 74.18% when the concentration
increased to 5mg/mL. Within the test concentration range, the IC50 value of FMPS was 2.58
mg/mL. It showed that FMPS had a strong hydroxyl radical scavenging ability.
Previous study suggested that there were two type of mechanism on hydroxyl radical.
One suppressed the generation of the hydroxyl radical and the other scavenged the hydroxyl
radicals generated (Qi, et al., 2005). The former referred to that the polysaccharide ligated to
the metal ions which reacted with H2O2 to give a hydroxyl radical (Qi, et al., 2005). It
indicated that the polysaccharide may have Fe2+chelating activity. Thus, the Fe2+chelating
activity of FMPS was tested.
21
Page 21 of 31

345
346 Fig. 5. Antioxidant activity tests of FMPS. (A) DPPH radical scavenging activity; (B)
347 Hydroxyl radical scavenging activity; (C) Fe2+chelating activity; (D) ABTS radical scavenging
348 activity.
349
350
351
352
353
354
355
356
Iron was an essential microelement for organisms, but overloaded iron would catalyze
the oxidation of protein, lipid and other components (Liu, et al., 2014).The Fe2+chelating
activity of FMPS was shown in Fig. 5C. The results showed that the chelating activity had
concentration dependence. The value was only 3.59% at the concentration of 1mg/mL, but it
had a sharp increase with the increase of concentration. The chelating activity increased to
42.68% when the concentration was 5 mg/mL. These results indicated that the FMPS had a
moderate Fe2+chelating activity at the concentration of 1-5 mg/mL.
22
Page 22 of 31

357
358
359
360
361
362
363
364
365
366
367
The test of ABTS radical scavenging activity is also a widely used method to measure
the antioxidant activity of chemical components (Re, et al., 1999; Shao, Chen, & Sun, 2013).
In this study, the ABTS radical scavenging activity of FMPS was tested and the results were
shown in Fig. 5D. The scavenging effect of FMPS on ABTS radical increased as the
concentration increasing. The scavenging ratio of FMPS was 22.51% at the concentration of
5 mg/mL. The result showed that FMPS exhibited some ABTS radical scavenging activity.
In this study, the purified polysaccharide (FMPS) showed different antioxidant activities
in various reactive systems, which may be due to its specific structures. Future research
should focus on the relationship between the antioxidant mechanism and the structure of the
polysaccharide. Furthermore, the antioxidant activity of the FMPS in vivo should be also
investigated.
23
Page 23 of 31

368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
4. Conclusion
In this study, a polysaccharide fraction FMPS was isolated and purified from the fruiting
body of floral mushroom cultivated in Huangshan Mountain, a precious edible mushroom.
FMPS had an average molecular weight of 7.2 × 105 Da and was composed of glucose.
Structural analysis revealed that FMPS had (1→3)-β-D-glucopyranose backbone and O-6
position
of
(1→3)-linked-β-D-glucopyranosyl
residues
was
substituted
by
1-linked-β-D-glucopyranosyl branches. There was average one branching point for three or
four residues of backbone. Congo red analysis showed that the FMPS was a triple-helical
polysaccharide. Its viscosity increased slightly and then reduced with the increase of NaOH
concentration. Dynamic light scattering test indicated that the hydrodynamic radius (Rh) of
FMPS was mainly 23.4 nm and it presented as single triple-helical chains in 1 mg/mL
solutions. Dynamic strain sweep showed that FMPS could form a stable system with water
in 1.2×10-2 g/mL solutions. The antioxidant activity tests in vitro indicated that FMPS had
high DPPH radical scavenging activities (79.46% at 5 mg/mL), hydroxyl radical scavenging
activities (74.18% at 5 mg/mL), some Fe2+chelating activity and ABTS radical scavenging
activity. These results indicated that FMPS could be used as a natural antioxidant. Future
research should focus on the relationship between the antioxidant mechanism and the
structure of the polysaccharide FMPS.
24
Page 24 of 31

386
387
388
389
390
Acknowledgements
This research was financially supported by the National Natural Science Foundation of
China (No. 31370371, No. 31271814, and No. 31171787), the Natural Science Foundation
of Anhui Province (No. 1408085MC45) and the Anhui International Science and
Technology Cooperation Project (No. 12030603020).
25
Page 25 of 31

391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
References
Bohn, J. A., & BeMiller, J. N. (1995). (1→3)-β-D-Glucans as biological response modifiers:
a review of structure-functional activity relationships. Carbohydrate Polymers, 28(1),
3-14.
Chaiklahan, R., Chirasuwan, N., Triratana, P., Loha, V., Tia, S., & Bunnag, B. (2013).
Polysaccharide extraction from Spirulina sp. and its antioxidant capacity. International
Journal of Biological Macromolecules, 58, 73-78.
Cheng, H., Feng, S., Jia, X., Li, Q., Zhou, Y., & Ding, C. (2013). Structural characterization
and antioxidant activities of polysaccharides extracted from Epimedium acuminatum.
Carbohydrate Polymers, 92(1), 63-68.
Chen, X., Xu, X., Zhang, L., & Kennedy, J. F. (2009). Flexible chain conformation of (1→
3)-β-D-glucan from Poriacocos sclerotium in NaOH/urea aqueous solution.
Carbohydrate Polymers, 75(4), 586-591.
Chen, Y., Xie, M. Y., Nie, S. P., Li, C., & Wang, Y. X. (2008). Purification, composition
analysis and antioxidant activity of a polysaccharide from the fruiting bodies of
Ganoderma atrum. Food Chemistry, 107(1), 231-241
Corsaro, M. M., Castro, C. D., Naldi, T., Parrilli, M., Tomás, J. M., & Regué, M. (2005). 1H
and 13C NMR characterization and secondary structure of the K2 polysaccharide of
Klebsiella pneumoniae strain 52145. Carbohydrate Research, 340(13), 2212–2217.
Dai, H., Han, X. Q., Gong, F. Y., Dong, H., Tu, P. F., & Gao, X. M. (2012). Structure
elucidation and immunological function analysis of a novel β-glucan from the fruit
bodies of Polyporus umbellatus (Pers.) Fries. Glycobiology, 22(12), 1673-1683.
26
Page 26 of 31

413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
Deng, C., Fu, H., Teng, L., Hu, Z., Xu, X., Chen, J., & Ren, T. (2013). Anti-tumor activity of
the regenerated triple-helical polysaccharide from Dictyophora indusiata. International
Journal of Biological Macromolecules, 61, 453-458.
Du, Y. Q., Liu, Y., & Wang, J. H. (2015). Polysaccharides from Umbilicaria esculenta
cultivated in Huangshan Mountain and immunomodulatory activity. International
Journal of Biological Macromolecules, 72, 1272-1276.
Gao, C., Wang, Y., Wang, C., & Wang, Z. (2013). Antioxidant and immunological activity in
vitro of polysaccharides from Gomphidius rutilus mycelium. Carbohydrate Polymers,
92(2), 2187-2192.
Kalač, P. (2013). A review of chemical composition and nutritional value of wild-growing
and cultivated mushrooms. Journal of the Science of Food and Agriculture, 93(2),
209-218.
Liu, Y., Du, Y. Q., Wang, J. H., Zha, X. Q., & Zhang, J. B. (2014). Structural analysis and
antioxidant activities of polysaccharide isolated from Jinqian mushroom. International
Journal of Biological Macromolecules, 64, 63-68.
Luo, Q., Sun, Q., Wu, L., & Yang, Z. (2012). Structural characterization of an
immunoregulatory polysaccharide from the fruiting bodies of Lepista
sordida. Carbohydrate Polymers, 88(3), 820-824.
Manivasagan, P., Sivasankar, P., Venkatesan, J., Senthilkumar, K., Sivakumar, K., & Kim, S.
K. (2013). Production and characterization of an extracellular polysaccharide from
Streptomyces violaceus MM72. International Journal of Biological Macromolecules, 59,
29-38.
27
Page 27 of 31

435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
Needs, P. W., & Selvendran, R. R. (1993). Avoiding oxidative degradation during sodium
hydroxide/methyl iodide-mediated carbohydrate methylation in dimethyl
sulfoxide. Carbohydrate Research, 245(1), 1-10.
Nitschke, J., Modick, H., Busch, E., Rekowski, R. W. V., Altenbach, H., & Mölleken, H.
(2011). A new colorimetric method to quantify β-1, 3-1, 6-glucans in comparison with
total β-1, 3-glucans in edible mushrooms. Food Chemistry, 127(2), 791–796.
Palacios, I., García-Lafuente, A., Guillamón, E., & Villares, A. (2012). Novel isolation of
water-soluble polysaccharides from the fruiting bodies of Pleurotus ostreatus
mushrooms. Carbohydrate Research, 358, 72-77.
Popov, S. V., Ovodova, R. G., Golovchenko, V. V., Khramova, D. S., Markov, P. A., Smirnov,
V. V., Shashkov, A. S., & Ovodov, Y. S. (2014). Pectic polysaccharides of the fresh plum
Prunus domestica L. isolated with a simulated gastric fluid and their anti-inflammatory
and antioxidant activities. Food Chemistry, 143, 106-113.
Qi, H., Zhang, Q., Zhao, T., Chen, R., Zhang, H., Niu, X., & Li, Z. (2005). Antioxidant
activity of different sulfate content derivatives of polysaccharide extracted from Ulva
pertusa
(Chlorophyta)
in
vitro.
International
Journal
of
Biological
Macromolecules, 37(4), 195-199.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999).
Antioxidant activity applying an improved ABTS radical cation decolorization
assay. Free Radical Biology and Medicine, 26(9), 1231-1237.
Shao, P., Chen, X., & Sun, P. (2013). In vitro antioxidant and antitumor activities of different
sulfated polysaccharides isolated from three algae. International Journal of Biological
28
Page 28 of 31

457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
Macromolecules, 62, 155-161.
Suvakanta, D., Narsimha, M. P., Pulak, D., Joshabir, C., & Biswajit, D. (2014). Optimization
and characterization of purified polysaccharide from Musa sapientum L. as a
pharmaceutical excipient. Food Chemistry, 149, 76-83.
Villares, A. (2013). Polysaccharides from the edible mushroom Calocybe gambosa: structure
and chain conformation of a (1→4), (1→6)-linked glucan. Carbohydrate Research, 375,
153-157.
Villares, A., Mateo-Vivaracho, L., & Guillamón, E. (2012). Structural features and healthy
properties of polysaccharides occurring in mushrooms. Agriculture, 2(4), 452-471.
Wang, F., Hou, Y., Ding, X., Hou, W., Song, B., Wang, T., Li, J., & Zeng, Y. (2013).
Structure elucidation and antioxidant effect of a polysaccharide from Lactarius
camphoratum (Bull.) Fr.. International Journal of Biological Macromolecules, 62,
131-136.
Wang, J. H., Luo, J. P., & Zha, X. Q. (2010). Structural features of a pectic polysaccharide
from the stems of Dendrobium nobile Lindl. Carbohydrate Polymers, 81(1), 1-7.
Wang, J., Xu, X., Zheng, H., Li, J., Deng, C., Xu, Z., & Chen, J. (2009). Structural
characterization, chain conformation, and morphology of a β-(1→ 3)-d-glucan isolated
from the fruiting body of Dictyophora indusiata. Journal of Agricultural and Food
Chemistry, 57(13), 5918-5924.
Wani, B. A., Bodha, R. H., & Wani, A. H. (2010). Nutritional and medicinal importance of
mushrooms. Journal of Medicinal Plants Research, 4(24), 2598-2604.
Wasser, S. P., & Weis, A. L. (1999). Medicinal properties of substances occurring in higher
29
Page 29 of 31