Purification and structural investigation of a water-soluble polysaccharide from Flammulina velutipes

Article abstract of DOI:10.1016/j.carbpol.2011.10.061

A novel water-soluble heteropolysaccharide FVP60-B was extracted from the fruiting bodies of Flammulina velutipes with boiling water and purified by Sephacryl S-300 and S-400, which molecular weight was estimated to be 1.3 × 10⁴ Da by HPLC. It

Full text of DOI:10.1016/j.carbpol.2011.10.061

Carbohydrate Polymers 87 (2012) 2279–2283
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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Purification and structural investigation of a water-soluble polysaccharide from
Flammulina velutipes
An-qiang Zhang, Nan-nan Xiao, Ying-lin Deng, Peng-fei He, Pei-long Sun∗
College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310014, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 August 2011
Received in revised form 21 October 2011
Accepted 26 October 2011
Available online 3 November 2011
A novel water-soluble heteropolysaccharide FVP60-B was extracted from the fruiting bodies of Flam-
mulina velutipes with boiling water and purified by Sephacryl S-300 and S-400, which molecular weight
was estimated to be 1.3 × 104 Da by HPLC. It is composed of l-fucose, d-mannose, d-glucose and d-
galactose in a ratio of 1.16:0.82:1.00:3.08. Sugar analysis, methylation analysis together with 1H, 13C and
2D NMR spectroscopy disclosed that FVP60-B is consisted of a ␣-(1 → 6)-d-galactopyranan backbone with
a terminal fucosyl, terminal glucosyl and ␣-(1 → 6)-d-mannopyranan units on O-2 of 2,6-O-substituted-
d-galactosyl units.
Keywords:
Flammulina velutipes
Heteropolysaccharide
Structural analysis
Crown Copyright © 2011 Published by Elsevier Ltd. All rights reserved.
1. Instruction
Dextrans, trifluoroaceticacids (TFA) and monosaccharidestandards
(d-galactose, d-arabinose, l-fucose, l-rhamnose, d-manonose, d-
Flammulina velutipes is a commercially important edible mush-
room cultivated widely in many countries, particularly in China
and Japan. It has attracted considerable attention in the fields of
biochemistry and pharmacology due to its biological activities (Fu,
Shieh, & Ho, 2002; Wasser, 2002). It is reported that both the fruit-
ing bodies and the fungal mycelia have bioactive polysaccharides
of F. velutipes have been proved to be beneficial in immunomodula-
tory, anti-tumor, and biological activity on hepatocytes, antioxidant
activity (Leung, Fung, & Choy, 1997; Ohkuma, Tanaka, & Ikekawa,
1983; Pang et al., 2007). In order to identify correlations between
structure and functionality, we have conducted structural studies
on polysaccharides from F. velutipes. In this paper, the structural
elucidation of FVP60-B, a novel neutral polysaccharide purified
from the fruiting body of F. velutipes, is described by methylation
analyses and 1H and 13C NMR spectroscopic analyses, 2D NMR
including COSY, TOCSY, HSQC, HMBC and NOESY experiments.
xylose, d-glucose) were from Sigma. All the other reagents were of
A.R. grade and made in China. HPLC was carried out on a waters
1525 HPLC system (1525 HPLC pump, 2414 refractive index detec-
tor). GC was carried out using an Agilent 7890N instrument. GC–MS
was carried out using a ThermoFinnigan TRACE MS, and NMR spec-
tra were determined with a Varian INOVA 500.
2.2. Isolation and purification
The dried fruiting bodies of F. velutipes were cut into small
pieces and extracted with boiling water thrice (2 h for each). The
supernatant was combined and concentrated into one-tenth of the
original volume, and 95% EtOH was added slowly to final alco-
hol concentration reached 40%. The precipitated was obtained
by centrifugation (10,000 rpm, 10 min, 4 ◦C), and 95% EtOH was
continually added slowly to final concentration of 60%. The result-
ing precipitate was collected, dissolved in distilled water and
lyophilized, and defined as FVP60. A portion of FVP60 was dissolved
in water and insoluble residue was removed by centrifugation.
The supernatant was injected to a DEAE Sepharose Fast Flow
column (XK 26 mm × 100 cm), eluted first with water, and then
followed stepwise by 0.1–2 M gradient of NaCl, respectively. The
fractions were collected by a fraction collector and compounds
were detected by means of the phenol-sulfuric acid assay (Zhang,
1999), FVP60-B was obtained by elution with water, and then fur-
ther purified using High-Resolution Sephacryl S-300 and S-400 (XK
26 mm×100 cm) gel-permeation chromatography. The main frac-
tion was collected, concentrated and lyophilized to get a white
2. Experimental
2.1. Materials
Fruiting bodies were purchased from Jiangshan of Zhejiang
Province, PR China. DEAE Sepharose Fast Flow and High-Resolution
Sephacryl S-300 and S-400 were purchased from GE Healthcare.
∗
Corresponding author. Tel.: +86 571 883 20779; fax: +86 571 883 20345.
E-mail addresses: sun pl@zjut.edu.cn, sun pl@126.com (P.-l. Sun).
0144-8617/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2011.10.061

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A.-q. Zhang et al. / Carbohydrate Polymers 87 (2012) 2279–2283
standard. 13C chemical shifts were determined in relation to DSS
(ı 0.00 ppm) calibrated externally. 1H–1H correlated spectroscopy
(COSY), total correlation spectroscopy (TOCSY), heteronuclear sin-
gle quantum correlation spectroscopy (HSQC) were used to assign
signals. Two-dimensional heteronuclear multiple-bond correlation
spectroscopy (HMBC) and two-dimensional overhauser effect spec-
troscopy (NOESY) were used to assign inter-residue linkages and
sequences.
purified F. velutipes polysaccharide (FVP60-B), whose molecular
weight range was detected firstly on a column SN of TSK-gel PWXL
G4000.
2.3. Determination of purity and molecular weight
The homogeneity and molecular weight of FVP60-B were deter-
mined by high performance liquid chromatography (HPLC) on an
Agilent 1525 system equipped with a TSK-gel PWXL G4000 col-
umn and a refractive index detector (RID). 10 ␮L of sample solution
(2.0 mg/mL) was injected in each run, with distilled water as the
mobile phase at a flow rate of 1. 0 mL/min. The column was kept
at 30.0 0.1 ◦C. The linear regression was calibrated with T-series
Dextrans standards (Mw 1000, 5000, 12,000, 80 k, 150 k, 270 k and
670 kDa).
3. Results and discussion
3.1. Isolation, purification, and composition of FVP60-B
The water-soluble polysaccharide termed FVP60-B was puri-
fied by High Resolution Sephacryl S-300 and S-400 gel-
permeation chromatography. High-performance liquid chro-
matography (HPLC) of FVP60-B produced a single symmetrical
peak, indicating it was a homogeneous polysaccharide (Fig. 1). Cor-
relation with the calibration curve of Dextran standards indicated
that its molecular weight was about 1.3 × 104 Da. Lack of absorp-
tion at 280 nm by UV scanning indicated that FVP60-B contained
no protein. The infrared spectrum of FVP60-B displayed a broad
stretching intense characteristic peak at around 3428.4 cm−1 for
the hydroxyl group of the polysaccharide, and a C–H stretching
band at 2923 cm−1. The relatively absorption peak at 1634 cm−1
and the weak one at 1382 cm−1 also indicated the characteris-
tic IR absorption of polysaccharides (Ge, Zhang, & Sun, 2009). No
absorption peaks at 1730 cm−1 indicated that there were no uronic
acids. Sugar compositional analysis of FVP60-B determined by GC
indicated that it consists of l-fucose, d-mannose, d-glucose and
D-galactose in a ratio of 1.16:0.82:1.00:3.08.
2.4. Spectroscopic methods
The ultraviolet spectrum of FVP60-B was recorded with a UV-
2450 UV–visible spectrophotometer (Shimadzu Co., Kyoto, Japan).
The Fourier-transform infrared spectrum of FVP60-B was measured
on a Nicolet 6700 FT-IR spectrometer (Madison, WI, USA) using the
KBr disk method.
2.5. Sugar analysis
The identification and quantification of the monosaccha-
ride of FVP60-B was achieved by GC analysis. FVP60-B (2 mg)
was hydrolyzed with 2 M trifluoroacetic acid (TFA, 4 mL) at
110 ◦C for 2 h. Then the hydrolyzed products were reduced with
NaBH4 (20 mg) and acetylated with acetic anhydride (Albersheim,
Nevins, English, & Karr, 1967). The resulting alditol acetates
were analyzed by gas chromatography (GC) using an Agilent
7890N instrument equipped with an HP-5 capillary column
(30 m × 0.32 mm × 0.25 ␮m) and a flame-ionization detector (FID)
at a temperature program as follows: the oven temperature was
initially set at 120 ◦C, increasing to 240 ◦C at a rate of 10 ◦C/min
and then held at 240 ◦C for 6.5 min. The heater temperatures of the
injector and detector were both at 250 ◦C. Nitrogen was used as the
carrier gas. Quantitation was carried out from the peak area, using
response factors of instrument.
3.2. Structural characterization of FVP60-B
The inter-glycosidic linkages between monosaccharide residues
of FVP60-B were investigated by methylation analysis. The polysac-
charide was methylated twice and after acid hydrolysis, methylated
sugars were converted to partially methylated alditol acetates.
Total ion chromatogram (Fig. 2) showed that mainly six peaks.
Major mass fragment showed that it isn’t sugar residue at 5.80 min.
Other peaks all possess major mass fragment of methylated
sugar.
2.6. Methylation analysis
The results of methylation analysis (Table 1) of FVP60-B showed
that the galactosyl residues are mainly (1 → 6)-linked with a
number of 2,6-O-substituted Galp units. The fucosyl residues are
completely distributed at non-reducing terminals. The glucosyl
residues are mainly terminal residues.
FVP60-B (2 mg) was dissolved in DMSO (2 mL) and methy-
lated by treatment with NaOH/DMSO (0.2 mL) suspension and
iodomethane (0.2 mL) by the method (Kalyan & Paul, 1992). The
reaction mixture was extracted with CHCl3, and the solvent was
then removed by evaporation. Complete methylation was con-
The 1H NMR spectrum (Fig. 3) of the polysaccharide mainly
contained signals for five anomeric protons at ı 5.00–5.17, one
CH3–C group (H-6 of Fuc) at ı 1.28 (J5,6 = 5.0 Hz). Other sugar
protons were in the region of ı 3.30–4.20. The 13C NMR spec-
trum (Fig. 4) of the polysaccharide mainly contained signal for
anomeric carbons at ı 5.03–5.17, one CH3–C group (C-6 of Fuc) at
ı 18.34, and sugar ring carbons linked to oxygen in the region of ı
63.89–80.50.
firmed by the disappearance of the OH band (3200–3700 cm−1
)
in the IR spectrum. The permethylated polysaccharide was
hydrolyzed by treatment with HCO2H (88%, 3 mL) at 100 ◦C for
3 h, evaporated to dryness and further hydrolyzed with 2 M TFA
(4 mL) at 100 ◦C for 6 h. The partially methylated sugar in the
hydrolysate were reacted with NaBH4 and acetylated with AC2O,
and the resulting mixture of methylated alditol acetates were ana-
lyzed by GC–MS.
1H resonances for H-1, H-2, H-3 and H-4 residue a were assigned
from the cross-peaks in the 1H–1H COSY and TOCSY spectra. H-
5, H-6a and H-6b were assigned from the 1H–1H COSY, TOCSY
and HMBC spectra. The carbon chemical shifts from the C-1 to C-
6 were assigned from the HSQC spectrum (Table 2). The manno
configuration for residue a was supported from a relatively small
coupling constant value of J1,2 < 3 Hz and a large coupling con-
stant value of J4,5 = 9.5 Hz. A small J1,2 values for the d-mannosyl
residue did not give information about the anomeric configu-
ration (Paramonov et al., 2001). A NOESY experiment revealed
2.7. NMR analysis
FVP60-B (10 mg) was dried in a vacuum over P2O5 for 72 h, and
then exchanged with deuterium by lyophilizing with D2O (0.5 mL)
for three times. 1H NMR (25 ◦C, 60 ◦C) and 13C NMR (60 ◦C) spec-
tra were determined in 5-mm tubes using a Varian INOVA 500
NMR spectrometer (TOSO, Tokyo, Japan). 1H Chemical shifts were
referenced to residual HDO at ı 4.78 ppm (25 ◦C) as the internal

A.-q. Zhang et al. / Carbohydrate Polymers 87 (2012) 2279–2283
2281
Fig. 1. HPLC of FVP60-B on TSK-gel PWXL G4000 column.
RT: 0.00 - 14.50
100
NL:
1.10E7
8.13
TIC F: MS
90
80
70
60
50
40
30
20
10
0
110323004
7.06
8.75
5.80
6.26
7.93
13.17
7.20
8.83
9
13.01
12
3.88
4
9.60
10
5.00
5
10.40
11
0
1
2
3
6
7
8
13
14
Time (min)
Fig. 2. Total ion chromatogram of FVP60-B by GC–MS.
Table 1
The methylation analysis of FVP60-B isolated from the fruiting bodies of F. velutipes.
Retent time
Methylated sugar
Substituted sugar unit
Molar ratios
Major mass fragment (m/z)
6.26
7.06
7.93
8.13
8.75
2,3,4-Me3-Fucp
2,3,4,6-Me4-Glcp
2,3,4-Me3-Manp
2,3,4-Me3-Galp
3,4-Me2-Galp
Reducing end Fucp unit
Reducing end Glcp unit
6-O-Substituted Manp unit
6-O-Substituted Galp unit
2,6-di-O-Substituted Galp unit
0.24
1.00
0.48
3.10
1.11
43, 72, 89, 101, 115, 117, 131, 161, 175
43, 71, 87, 101, 117, 129, 145, 161, 205
43, 71, 87, 101, 117, 129, 161, 173, 189, 233
43, 71, 87, 101, 117, 129, 161, 173, 189, 233
43, 71, 87, 99, 129, 159, 173, 189, 233, 305
Fig. 3. 500-MHz 1H NMR spectrum of FVP60-B in D2O at 60 ◦C verified by partial of HSQC. The anomeric protons are labeled (a)–(e).

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A.-q. Zhang et al. / Carbohydrate Polymers 87 (2012) 2279–2283
Fig. 4. 500-MHz 13C NMR spectrum of FVP60-B in D2O at 60 ◦C.
Table 2
Chemical shifts data for FVP60-B isolated from the fruiting bodies of F. velutipes.
Residue
Proton or carbon
1
2
3
4
5
6a
3.97
6b
→6)-␣-d-Manp (a)
␣-d-Glcp (b)
H
C
H
C
H
C
H
C
5.17
104.95
5.16
103.98
5.12
103.98
5.08
100.70
5.03
4.12
3.95
3.83
3.74
4.21
72.65
3.98
72.20
3.87
71.15
3.88
80.50
3.88
69.83
3.95
73.25
3.92
72.30
4.10
71.21
4.07
76.00
4.10
71.21
3.85
71.21
4.21
69.80
3.91
69.39
4.01
74.20
4.21
69.80
4.18
71.90
4.23
69.90
3.80
63.89
1.28
18.34
3.70
69.99
3.74
3.93
␣-l-Fucp (c)
→2,6)-␣-d-Galp(d)
→6)-␣-d-Galp(e)
4.01
3.97
H
C
100.70
74.45
72.40
72.30
71.55
69.39
inter-residue correlations between H-1 and H-2, indicating that
residue a was ␣-configuration (Senchenkova et al., 2003). The
downfield shifts of the C-6 (ı 69.90) carbon signals with respect
to standard values for glycopyrannoses indicate that residue a was
→6)-␣-d-Manp-(1→.
d has an ␣-configuration. The downfield shifts of the C-2 (ı 80.50)
and C-6 (ı 69.99) carbon signals with respect to standard values
for glycopyrannoses indicate that residue d was →2,6)-␣-d-Galp-
(1→.
For residue e the 1H resonances for H-1, H-2, H-3 and H-4 were
assigned from the crosspeaks in the 1H–1H COSY and TOCSY spec-
tra. The H-5 resonance was assigned from the H-3/H-4 and H-4/H-5
crosspeaks in the NOESY spectrum (Reddy et al., 1998). The H-5, H-6
resonances were then obtained from the TOCSY spectrum. The cor-
responding 13C resonances were assigned from the HSQC spectrum
(Table 2). H-4 displays strong NOEs to both H-3 and H-5, which
indicated that residue e is a Gal-type residue. Residue e had an
␣-configuration at its anomeric center, which is evident from the
H-1/H-2 intra-residue correlations in the NOESY spectrum and the
crosspeaks of H-1 and C-3, C-5 in the HMBC spectrum (Stroop, Xu,
Retzlaff, Abeygunawardana, & Bush, 2001). The downfield shifts of
the C-6 (ı 69.39) carbon signals with respect to standard values
for glycopyrannoses indicate that residue e was →6)-␣-d-Galp-
(1→.
Chemical shifts of residue b were assigned according to 1H–1H
COSY and TOCSY spectra. Large coupling constants was JH-2, H-3
(8–10 Hz) and JH-3, H-4 (8–10 Hz) indicated that residue b was
a gluco-configuration. 13C resonances were assigned from the
HSQC spectrum (Table 2). H-1 appears as a singlet (J1,2 < 3 Hz)
in the 1H NMR spectrum and H-1/H-2 intra residue correla-
tions in the NOESY spectrum showing that residue b has an
␣-configuration. The combination of these data identified residue
b as ␣-d-Glcp.
The 1H resonances for H-1, 2, 3 of residue c were assigned
from 1H–1H COSY spectrum, and the assignment of H-4 was based
on the TOCSY spectrum. H-5 and H-6 were assigned from 1H–1H
COSY spectrum. Both H-3 and H-4 correlated with a signal ı 4.21,
and H-4 gave a <!– no-mfc –>NOE<!– /no-mfc –> signal at ı 1.28
were located on residue c. On basis of the proton assignments, the
chemical shifts of C-1 to C-6 were readily obtained from the HSQC
spectrum (Table 2). Both the carbon and chemical shifts are typi-
cal of 6-deoxyhexopyranose, since l-Fuc was the only such sugar
identified by GC–MS analysis. H-1 appears as a singlet (J1,2 < 3 Hz)
in the 1H NMR spectrum and H-1/H-2 intra residue correlations in
the NOESY spectrum. Both values indicated an ␣-configuration at
the anomeric center. Thus, residue c was identified as ␣-l-Fucp.
1H resonances for H-1 to H-4 of residue d were assigned from
the 1H–1H COSY and TOCSY spectra. H-5 and H-6 were assigned
from the TOCSY spectrum. The crosspeaks of H-1 and C-3, C-5
of the HMBC spectrum showed that H-5 and H-6 are located on
residue d. The chemical shifts of C-1 to C-6 were readily obtained
from the HSQC spectrum (Table 2). The H-4/5 coupling constant
was small in the 1H–1H COSY spectrum, as expected for a Gal-type
residue (Staaf, Urbina, Weintraub, & Widmalml, 1999). H-1 appears
as a singlet (J1,2 < 3 Hz) in the 1H NMR spectrum and H-1/H-2 intra
residue correlations in the NOESY spectrum indicted that residue
The sequence of glycosyl residues was determined from NOESY
studies followed by confirmation with HMBC experiments. Inter-
residue <!– no-mfc –>NOE<!– /no-mfc –> correlations (Table 3)
Table 3
Interglycosidic correlations from NOESY spectra of FVP60-B isolated from the fruit-
ing bodies of F. velutipes.
Residue
Proton
Intra-correlation
→6)-␣-d-Manp (a)
␣-d-Glcp (b)
␣-l-Fucp (c)
→2,6)-␣-d-Galp(d)
H-1
H-1
H-1
H-1
4.12 (a; H-2), 4.21 (a; H6b)
3.98 (b; H-2), 3.88 (d; H-2)
3.87 (c; H-2), 3.88 (d; H-2)
3.88 (d; H-2), 4.10 (d; H-3), 4.21 (d; H-4),
3.70 (d; H-6a), 4.01 (d; H-6b)
4.10 (d; H-3), 4.18 (d; H-5)
3.88 (e; H-2), 4.23 (e; H-5), 3.74 (e; H-6a),
4.01(d; H-6b)
H-4
H-1
→6)-␣-d-Galp(e)
H-4
4.07 (e; H-3), 4.23 (e; H-5)
a
Inter-residue NOEs are shown in bold font.

A.-q. Zhang et al. / Carbohydrate Polymers 87 (2012) 2279–2283
2283
Table 4
Acknowledgements
Two-and three-bond 1H–13C correlations for the FVP60-B isolated from the fruiting
bodies of F. velutipes.
This work was supported by Natural Science Foundation of Zhe-
jiang Province of China (No. Y3110349) and Science and Technology
Department of Zhejiang Province of China (No. 2009R50029).
Residue
Proton
H-1
Proton correlation
→6)-␣-d-Manp (a)
73.25 (a; C-2), 76.09 (a; C-4), 69.90 (a;
C-6), 80.50(d; C-2)
␣-d-Glcp (b)
␣-l-Fucp (c)
H-1
H-1
H-1
H-1
69.83 (b; C-3), 80.50(d; C-2)
References
72.30 (c; C-3), 69.80 (b; C-5), 80.50(d; C-2)
71.21 (d; C-3), 71.90 (d; C-5), 69.99(d; C-6)
72.40 (e; C-3), 71.55 (e; C-5), 63.89(e; C-6)
→2,6)-␣-d-Galp(d)
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were observed between H-1 of residue a and H-6 of residue a,
between H-1 of residue b and H-2 of residue d, between H-1 of
residue c and H-2 of residue d, between H-1 of residue d and H-6
of residue d, between H-1 of residue e and H-6a of residue d and
H-6b of residue e. HMBC experiments (Table 4) showed clear cor-
relations between H-1 of residue a and C-6 of residue a and C-2 of
residue d, between H-1 of residue b and C-2 of residue d, between
H-1 of residue c and C-2 of residue d, between H-1 of residue d and
C-6 of residue d, between H-1 of residue e and C-6 of residue e.
Based on the data presented above, it demonstrated that
FVB60-B consisted of a ␣-(1 → 6)-d-galactopyranan backbone
with
a
terminal fucosyl, terminal glucosyl and ␣-(1 → 6)-d-
mannopyranan units on O-2 of 2,6-O-substituted-d-galactosyl
units.
4. Conclusion
According to the results above, FVP60-B was isolated from the
fruiting bodies of F. velutipes with boiling water extraction and puri-
fied by DEAE Sepharose Fast Flow column and High-Resolution
Sephacryl S-300, 400. Its molecular weight was measured to be
1.3 × 104 Da by using HPLC. Gas chromatography analysis sug-
gested that FVP60-B is composed of l-fucose, d-mannose, d-glucose
and d-galactose in a ratio of 1.16:0.82:1.00:3.08. Sugar analysis,
methylation analysis together with 1H and 13C NMR spectroscopy
and 2D NMR established that FVP60-B is consisted of a ␣-(1 → 6)-
d-galactopyranan backbone with a terminal fucosyl, terminal
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