Structure characterization of polysaccharide isolated from the fruiting bodies of Tricholoma matsutake

Article abstract of DOI:10.1016/j.carbpol.2010.04.010

A novel heteropolysaccharide was isolated from the fruiting bodies of Tricholoma matsutake through DEAE-cellulose column and Sephadex G-100 column. The T. matsutake polysaccharide (TMP-A) had a molecular weight of 8.89 × 10⁴ Da and was mainly composed of 4-D-Glc:4,6-D-Glc:3-D-Gal:T-D-Xyl and with the ratio of 7:1:1:1. The structural features of TMP-A were investigated by a combination of total hydrolysis, methylation analysis, gas chromatography-mass spectrometry (GC-MS), scanning electron microscope (SEM), infrared (IR) spectra, nuclear magnetic resonance (NMR) spectroscopy and dynamical analysis of the atomic force microscope (AFM) studies. The results indicated that TMP-A had a backbone of 1,4-β-glucopyranos which branches at O-6 composed of an (1 → 3)-α-galactopyranose residue and terminated with α-xylopyranose residue. Chain conformation study showed that the polysaccharide took random coil compact conformation.

Full text of DOI:10.1016/j.carbpol.2010.04.010

Carbohydrate Polymers 81 (2010) 942–947
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Structure characterization of polysaccharide isolated from the fruiting bodies of
Tricholoma matsutake
Xiang Dinga,1, Su Fenga,1, Mei Caob,1, Mao-tao Lia, Jie Tanga, Chun-xiao Guoa, Jie Zhanga,
Qun Suna, Zhi-rong Yanga, Jian Zhaoa,b,∗
a Key Laboratory of Biological Resource and Ecological Environment of the Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, PR China
b Key Laboratory of Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, Chengdu 610072, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel heteropolysaccharide was isolated from the fruiting bodies of Tricholoma matsutake through
DEAE–cellulose column and Sephadex G-100 column. The T. matsutake polysaccharide (TMP-A) had a
molecular weight of 8.89 × 104 Da and was mainly composed of 4-D-Glc:4,6-D-Glc:3-D-Gal:T-D-Xyl and
with the ratio of 7:1:1:1. The structural features of TMP-A were investigated by a combination of total
hydrolysis, methylation analysis, gas chromatography–mass spectrometry (GC–MS), scanning electron
microscope (SEM), infrared (IR) spectra, nuclear magnetic resonance (NMR) spectroscopy and dynamical
analysis of the atomic force microscope (AFM) studies. The results indicated that TMP-A had a backbone
of 1,4-␤-glucopyranos which branches at O-6 composed of an (1 → 3)-␣-galactopyranose residue and
terminated with ␣-xylopyranose residue. Chain conformation study showed that the polysaccharide
took random coil compact conformation.
Received 26 December 2009
Received in revised form 2 March 2010
Accepted 7 April 2010
Available online 14 April 2010
Keywords:
Monosaccharide component
Polysaccharide structure
GC–MS
13C and 1H NMR
© 2010 Elsevier Ltd. All rights reserved.
Tricholoma matsutake
1. Introduction
has been used for the prevention and treatment of diseases for sev-
eral thousand years (Gong, Su, Chen, Feng, & Cao, 2002; Gurein et al.,
Oxidation is essential to many organisms for the production
of energy to fuel biological processes. However, the uncontrolled
production of superoxide anion free radicals is involved in onset
of many diseases such as cancer, atherosclerosis and degenerative
processes (Mau, Tsai, Tseng, & Huang, 2005). Plant polysaccharides
have shown strong antioxidant properties and can be used as a
novel potential antioxidant (Hu, Xu, & Hu, 2003; Ramarahnam,
Osawa, Ochi, & Kawaishi, 1995; Wang & Luo, 2007). In addi-
tion, polysaccharides extracted from mushrooms, such as Lentinus
edodes, Ganoderma tsugae and Cordyceps sinensis, have also exhib-
ited antioxidant properties by their free radical scavenging ability
(Chen, Xie, Nie, Li, & Wang, 2008; Chen, Zhong, Zeng, & Ge, 2008;
Dong & Yao, 2008; Tseng, Yang, & Mau, 2008; Yu, Yin, Yang, &
Liu, 2008). Such natural antioxidants may be effective to protect
humans from free radicals damage and many chronic diseases
(Kinsella, Frankel, German, & Kanner, 1993).
2003; Hur, Park, Kang, & Joo, 2004). As an extract from the fruiting
bodies of T. matsutake, polysaccharides (TMP) have shown strong
anti-tumor bioactive properties (Gao, 1997; Liu, 2001; Mikio et al.,
1999; Shung, 2004). The determination of the structure of polysac-
charides is necessary to establish a fundamental guide for assessing
the bioactivity and pharmacological mechanism in vivo/in vitro.
In this work, we reported a novel water-soluble polysaccharide
was extracted and purified from the fruiting bodies of T. matsutake
using a DEAE-cellulose column chromatography and a Sephadex G-
100 column chromatography. Its chemical structures were further
characterized and our results provided a basis for future pharma-
cological studies.
2. Materials and methods
2.1. Chemicals
Tricholoma matsutake is a kind of fungi belonging to Subgenus
Tricholoma. As a traditional edible fungus in oriental countries, it
The fruiting bodies of T. matsutake were collected in Xiaojing
country of Sichuan province, China, and were authenticated by Prof
Sao-rong Ge (College of Life Sciences, Sichuan University, Chengdu,
China). At the same time, a voucher specimen had been preserved in
Key Laboratory for Biological Resource and Ecological Environment
of Education Ministry, College of Life Sciences, Sichuan Univer-
sity. DEAE-Cellulose 52 and Sephadex G-100 were purchased from
Sigma–Aldrich (Mainland, China). Monosaccharide standards, Dex-
∗
Corresponding author at: Key Laboratory of Biological Resource and Ecolog-
ical Environment of the Ministry of Education, College of Life Sciences, Sichuan
University, Chengdu 610064, PR China. Tel.: +86 28 85460487; fax: +86 28 85460487.
E-mail address: zj804@163.com (J. Zhao).
1
These authors contributed equally to this research.
0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.04.010

X. Ding et al. / Carbohydrate Polymers 81 (2010) 942–947
943
2.3. Purity and fractionation of polysaccharides
The resulting polysaccharide solution was dialyzed, concen-
trated and lyophilized. TMP (8 g) was subjected to a DEAE–cellulose
column (Tris–HCl, pH 7.0, 4.5 cm × 50 cm, Cl−) and eluted stepwise
with 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 1.0 M NaCl. The eluate was moni-
tored by the phenol–sulfuric acid method (Dubois, Gillis, Hamilton,
Rebers, & Smith, 1956). The 0 M NaCl eluation was concentrated,
dialyzed, and lyophilized. Then the resulting polysaccharide was
purified on a Sephadex G-100 column (2.6 cm × 60 cm) and eluted
with 0 M NaCl. The resulting T. matsutake polysaccharide, named
TMP-A, was obtained by the above processes and the yield rate of
TMP-A was 0.22% (0.432 g) for the starting material.
2.4. Measurement of molecular weight of TMP-A
Fig. 1. Elution curve of TMP-A in Sephadex G-100 column chromatography.
High performance gel permeation chromatography (HPGPC)
was carried out to measure molecular weight. The column was cali-
brated with standard T-series Dextran (T-500, T-110, T-70, T-40 and
T-10). The data were processed with Waters GPC (Millennium32
software).
tran T-500, T-110, T-70, T-40, and T-10, were purchased from
Beijing Biodee Biotechnology Co., Ltd. (Beijing, China). All other
reagents used were of analytical grade.
2.5. Monosaccharide composition analysis
2.2. Extraction of polysaccharides from T. matsutake
The polysaccharide TMP-A (5.0 mg) was hydrolyzed with 2 M
trifluoroacetic acid at 110 ◦C for 6 h on the mechanism of acid-
catalyzed hydrolysis (Nelson & Cox, 2004; Yu et al., 2009). Excess
acid was removed by co-distillation with Methanol after the hydrol-
ysis was completed. One part of the hydrolysate (1.0 mg) was used
for thin layer chromatography analysis as described previously (Liu,
Dong, & Fang, 2001; Partridge, 1949; Zhang, 1999), and the other
(1.0 mg) was dissolved in pyridine (0.2 mL). The derivatization reac-
tion was initiated by addition of hexamethyl-disilazane (0.2 mL)
and trimethyl chloro-silicane (0.2 mL) according to the method
described by Dong, Zhang, Lin, and Fang (1995) and Guentas et al.
(2001). The resulting supernatant was examined by GC–MS at a
temperature program of 50–230 ◦C with a rate of 2 ◦C/min (Chen,
Xie, et al., 2008; Chen, Zhong, et al., 2008; Needs & Selvendran,
1993).
The fruiting bodies (200 g) of T. matsutake were soaked with
95% EtOH for 6 h to remove lipids by filtration. Then the residue
was dried and extracted with boiling water for three times
(6 h for each). The filtrate was concentrated, dialyzed (MWCO
5000, Sigma), and centrifuged to remove insoluble material and
small molecular compounds. Then the supernatant was added
with 3 volumes of 95% EtOH to precipitate crude polysaccha-
rides. The precipitate was recovered by centrifugation and washed
successively with absolute EtOH, followed by drying in vacuo
at 45 ◦C, yielding the crude T. matsutake polysaccharide (TMP)
(32.8 g, recovery 16.4%). Sevag method (Staub, 1965) was used
for the deproteination of TMP, 30% H2O2 for decoloration, respec-
tively.
Fig. 2. The purified polysaccharide TMP-A were imaged by SEM.

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X. Ding et al. / Carbohydrate Polymers 81 (2010) 942–947
2.9. Atomic force micrograph (AFM)
The normal sample preparation procedure consisted of spread-
ing of a dilute (25 ␮g/mL) polymer solution onto a freshly cleaved
mica surface and successive air-drying under ambient pressure,
temperature, and humidity (Kirby, Gunning, & Morris, 1995;
Michela, Meredith, Paola, Neil, & Rizzo, 2009). The atomic force
microscopy was operated in the tapping-mode (Gunning et al.,
2003; Morri, Gunning, Kirby, Round, & Waldron, 1997).
3. Results and discussion
Fig. 3. FTIR spectra of polysaccharide TMP-A.
3.1. Extraction, purity and composition of polysaccharides
The crude polysaccharide, named TMP, was obtained from
the fruiting bodies of T. matsutake with a yield of 16.4%. After
fractionation on DEAE-Cellulose 52 and Sephadex G-100 column
chromatography, 432 mg of TMP-A was obtained from the 0 M NaCl
eluate and detected by the phenol–sulfuric acid assay (Dubois et
al., 1956). The homogeneity of the polysaccharide was elucidated
by the following tests: TMP-A was eluted from gel-filtration chro-
matography on Sephadex G-100 column and was also detected
by the phenol–sulfuric acid assay as a single peak (Fig. 1). The
amber-coloured crystal TMP-A were shown by SEM in Fig. 2. No
absorption at 280 nm and 260 nm in UV absorption spectra of
TMP-A demonstrated the absence of protein and nucleic acid in
2.6. Methylation analysis
The polysaccharide, TMP-A (10 mg), was methylated according
to the Hakomori method (Hakomori, 1964). After being methylated
completely, the permethylated polysaccharide was depolymerized
with 90% aqueous formic acid (3 mL) for 10 h at 100 ◦C in a sealed
tube. The methylated sugars were derivatized using the method
described by Dong et al. (1995) and analyzed by GC–MS.
2.7. UV and infrared (IR) spectra analysis
20
this polysaccharide and it had the same optical rotation: [˛]D
TMP-A was tested in UV from 200 nm to 600 nm. And Infrared
analysis of the samples was obtained by grinding a mixture of
polysaccharide with dry KBr and then pressing in a mold. Spec-
tra were run in the 4000–400 cm−1 region (Kumar, Joo, Choi, Koo,
& Chang, 2004).
– 1.648◦ (c0.5, water), in different low concentration of ethanol
using HK7-SGW-1 automatic optical polarimeter at room temper-
ature. Weight-average molecular weight was around 8.89 × 104 Da.
The Rf values of hydrolyzed sample monosaccharide using TLC
were 0.272, 0.538, 0.156, respectively, which were the same as the
standard monosaccharide (Glucose, Galactose, Xylose). The color
of monosaccharides (Glc, Gal) were shown clearly as blue and Xyl
as brown-green. The three monosaccharides, D-Glc, D-Xyl and D-
Gal were also identified using the hydrolysate of TMP-A by GC–MS
and their ratios were 8:1:1. TMP-A was supposed to contain the
D-configuration monosaccharide according to GC–MS analysis.
2.8. Nuclear magnetic resonance (NMR) experiment
1H NMR spectra and 13C NMR spectra were recorded on a Var-
ian Unity INOVA 400/45 in D2O with tetramethylsilane as internal
standard.
Fig. 4. The 13C NMR spectra of polysaccharide TMP-A.

X. Ding et al. / Carbohydrate Polymers 81 (2010) 942–947
945
Fig. 5. Predicted chemical structure of polysaccharide TMP-A.
Fig. 6. Atomic force microscopy (AFM) planar (a) and cubic images (b) of molecular structure of TMP-A from the fruiting bodies of Tricholoma matsutake.
3.2. Structure elucidation of TMP-A
After methylation by the modified method (Hakomori, 1964)
for four times, the methylated polysaccharide was depolymer-
ized and converted into partially methylated ramifications. The
analysis of the methylated monosaccharide was conducted by
GC–MS. The information in MS showed that fragment ion peaks
were consistent with data of D-configuration monosaccharide
fragment ions peaks which can be concluded that the glucose,
galactose and xylose residues were D configuration. Methylation
analysis for TMP-A proved that the glucose residues were 2,3-
bis-substituted and 2,3,6-tri-subsituted, respectively, the galactose
residues were 2,4,6-tri-subsituted, and the xylose residue was
2,3,4-tri-subsituted (see Table 2 and Fig. 5). Both results of
monosaccharide composition analysis and methylated linkage
analysis of TMP-A indicated that (1 → 4)-linked-glucose was the
largest amounts residue of the polysaccharide structure, and
the branched residue was (1 → 4,6)-linked-glucose revealing that
(1 → 4)-linked-glucose should be possible to form the backbone
structure. The relative amounts of (1 → 4,6)-linked-glucose indi-
cated that approximate branch ratios could theoretically be 12.5%,
The intensity of bands around 3408.22 cm−1 in the IR spec-
trum (Fig. 3) was due to the hydroxyl stretching vibration of the
polysaccharide and as expected they were broad. The bands in the
region of 2923.62 cm−1 were due to C–H stretching vibration and
the bands in the region of 1643.29 cm−1 were due to associated
water (Cao et al., 2006; Park, 1971). Two strong absorption bands
at 1075.03 cm−1, 1041.64 cm−1 in the range of 1200–1000 cm−1
suggested that the monosaccharide of TMP-A had a pyrnaose-
ring (Barker, Bourne, Stacey, & Whiffen, 1954; Cui et al., 2008)
(Fig. 3). The absorption at 875.43 cm−1 indicated that TMP-A had
␤-glycopyranosidic linkages, which was also indicated by the
anomeric proton signals at ı 4.660 in the 1H NMR (400 MHz)
(Chakraborty, Mondal, Pramanik, Rout, & Islam, 2004; Kim et al.,
2000). Moreover, the characteristic absorptions at 799.73 cm−1
indicated ␣-configurations existing in the polysaccharide (Wu &
Tu, 2005), which was in good agreement with the anomeric proton
signals at ı5.263, ı5.182 and ı5.107 in the 1H NMR (400 MHz) spec-
trum. The resonances in the region of 98–106 ppm in the 13C NMR
(200 MHz) spectrum of TMP-A were attributed to the anomeric
carbon atoms of glucopyranose (Glcp), galactopyranose (Galp) and
xylopyranose (Xylp) (Wang, Liang, & Zhang, 2001; Wang, Luo, &
Liang, 2004; Zhao, Kan, Li, & Chen, 2005). In the anomeric carbon
region, signals at ı105.2 could be attributed to C-1 of →4)-␤-D-
Glcp-(1→; ı105.4 to C-1 of →4,6)-␤-D-Glcp-(1→; ı100.7 to C-1 of
→3)-␣-D-Galp-(1→; ı104.1 to C-1 of ␣.-D-Xylp-(1→, respectively
(Fig. 4). All the assignment of the carbon atoms signals was shown
in Table 1.
Table 1
13C NMR chemical shift data (ı, ppm) for polysaccharide TMP-A.
Sugar residues
Chemical shifts, ı (ppm)
C1 C2 C3
105.239 69.370 78.160 72.941 78.750 63.636
C4
C5
C6
→4)-␤-D-Glcp-(1→
→4,6)-␤-D-Glcp-(1→ 105.423 69.370 78.426 72.121 79.598 63.636
→3)-␣-D-Galp-(1→
␣.-D-Xylp-(1→
100.762 70.820 77.388 75.439 80.717 63.258
104.194 71.047 75.638 74.305 72.121

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X. Ding et al. / Carbohydrate Polymers 81 (2010) 942–947
Table 2
GC–MS results of methylation analysis of TMP-A.
Methylated sugar
Linkage
Mass fragments (m/z) (relative abundance, %)
2,3,6-Me3-Glc
2,3-Me2-Glc
2,4,6-Me3-Gal
2,3,4-Me3-Xyl
1,4–
1,4,6–
1,3–
T–
45, 59, 73, 88, 101, 116, 133, 146, 159, 232 (100, 62, 53, 38, 21, 14, 20, 3, 7, 2.3)
45, 59, 73, 88, 101, 116, 133, 146, 174, 232 (100, 56, 43, 36, 23, 15, 20, 4, 5, 2.4)
45, 59, 73, 89, 116, 146, 159, 191, 204, 233 (100, 63, 46, 29, 19, 2.7, 3, 4.3, 2.1, 1.7)
45, 59, 73, 88, 101, 116, 133, 146, 174 (100, 47, 35, 31, 29, 18, 12, 4.7, 1.9)
namely on average one branching point for each eight residues
of backbone. Residues of branch structure were (1 → 3)-linked-
galactose and terminated with ␣-xylopyranose residue, which also
get from those data of monosaccharide composition analysis and
methylated linkage analysis. It is concluded that a repeating unit
of TMP-A has a backbone of (1 → 4)-␤-d-glucose residues which
branches at O-6 based and the branch was supposed to be the
composition of an (1 → 3)-␣-d-galactose residue and one termi-
nated with ␣-d-xylose residue. The predicted structure of the novel
polysaccharide TMP-A was shown in Fig. 5.
The topographical AFM planar image of TMP-A deposited from
a 25 ␮g/mL water solution was shown in Fig. 6a. Only spherical
lumps can be seen which was in good agreement with the mea-
surement performed by SEM (Fig. 2b) and the diameter and height
of the lumps ranged from 20 nm to 56 nm (Fig. 6a). The heights of
the spherical structures were much higher than that of a single
polysaccharide chain (about 0.1–1 nm), suggesting that molecu-
lar aggregation was involved (Fig. 6b). We can see that irregularly
shaped large structures were formed together like worm from AFM
cubic image of TMP-A (Fig. 6b), which suggested that TMP-A could
take random coil compact conformation. The conformation might
be related to its side chains (Marit, Gjertrud, Pawel, Berit, & Bj␾rn,
2003; Wang et al., 2010).
Chakraborty, I., Mondal, S., Pramanik, M., Rout, D., & Islam, S. S. (2004). Structural
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Gurein, L., Vaario, L.-M., Matsushita, N., Shindo, K., Suzuki, K., & Lapeyrie, F. (2003).
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According to the results above, it was concluded that the
highly purified novel polysaccharide obtained from T. matsutake
is a heteropolysaccharide namely TMP-A. The present study also
showed that TMP-A consisted of three monosaccharides, namely
D-Glc, D-Xyl and D-Gal and their ratios were 8:1:1 by GC–MS.
Structure study demonstrated that TMP-A had a backbone of
(1 → 4)-␤-glucopyranose residues which branches at O-6 and the
branches were composed of an (1 → 3)-␣-galactopyranose residue
and terminated with ␣-xylopyranose residue. Chain conformation
study showed that the polysaccharide took random coil compact
conformation. Further work is necessary to reveal detailed phar-
macological effects of TMP-A including antioxidant, anticancer
properties and degenerative processes with aging, etc.
Acknowledgements
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Liu, P. (2001). Anticancer and mechanism research of glucoprotein MTSGS1
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The authors thank Pr. Pu Su (Department of Chemistry of Medic-
inal Natural Products and Key Laboratory of Drug Targeting of
Education Ministry PRC, West China College of Pharmacy, Sichuan
University) for helpful assistance in NMR experiments. This project
was supported by Chinese National Programs of High Technology
Research and Development (2007AA021506) and the 11th Five
Years Key Programs for Science and Technology Development of
China (2007BAD81B04).
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Research, 338, 2459–2475.
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water extracts from Ganoderma tsugae Murrill. LWT-Food Science and Technology,
38, 589–597.
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