全 文 ：Mycosystema
菌 物 学 报 15 July 2008, 27(4): 582-586

jwxt@im.ac.cn
ISSN1672-6472 CN11-5180Q
©2008 Institute of Microbiology, CAS, all rights reserved.






Trehalose, a non-reducing disaccharide from Ganoderma
applanatum

MA Hong-Xia ZHOU Zhong-Bo BAO Hai-Ying BAU Tolgor LI Yu*
Jilin Agricultural University, Changchun 130118, China


Abstract: Trehalose, a non-reducing disaccharide, was separated for the first time from Ganoderma applanatum
fruit body. The structure of trehalose was determined by electrospray ionization mass spectrometric and NMR
data. The content of trehalose was determined by improved anthrone-sulphuric acid colorimetric method and it
was 0.48% of dried weight of G. applanatum fruit body. Mannitol was simultaneously obtained during
separation of trehalose from G. applanatum.
Key words: fruit body, structure of trehalose, mannitol, anthrone-sulphuric acid colorimetric


1 INTRODUCTION
Ganoderma applanatum (Pers.) Pat. grows on stump, falling wood, and many kinds of stumpage
of broad leaf trees. It is used as a folk remedy to treat hepatitis (Choe et al. 1997; Shang 1998). Some
of natural products have been reported from G. applanatum. The metabolites isolated from G.
applanatum are mainly polysaccharide, but occasionally steroids, triterpenes, fat acids and other acids
(Gan et al. 1998; Shim et al. 2004; Boh et al. 2004). Some polysaccharides have antitumor and
antivirus activities. Some steroidal compounds have antibacterial activity in vitro. In the present paper,
trehalose, a non-reducing disaccharide, is reported for the first time from G. applanatum.
2 MATERIALS AND METHODS
2.1 Plant material
G. applanatum fruit body was collected at Taonan county of Jilin province, China. It was

Supported by Science Foundation of Jilin Agricultural University
*Corresponding author. E-mail: hongxia0731001@yahoo.com.cn
Received: 09-05-2007, accepted: 04-03-2008
DOI:10.13346/j.mycosystema.2008.04.022
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smashed after identified and dried.
2.2 Extraction and isolation
The smashed G. applanatum fruit body was extracted with petroleum ether for three times. The
residue was then dried and extracted with 95% ethanol for 24 hours under agitation for three times.
The supernatant of three extractions were combined and concentrated and then the brown sediment
was obtained. The macrocrystal was formed after the brown sediment was treated with 95% ethanol
at 4℃ for 48 hours and seperated from ethanol mixture. The macrocrystal was then dissolved in
ddH2O and filtered through a 0.45μm micropore filter and then purified by HPLC (C18 Microsorb
3µm, 10×250mm, 1mL/min, 90:10 H2O-MeOH, 210nm, 35℃). One single peak with the retention
time of 8.332min was collected (compound 1), the other fractions were collected, evaporated and
then purified by HPLC (C18 Microsorb 3µm, 10×250mm, 1mL/min, 75:25 (v/v) H2O-MeOH, 210nm,
40℃). Another single peak with retention time of 9.437min was collected (compound 2) and the
remainder fraction was collected, evaporated and then purified by HPLC (C18 Microsorb 3µm, 10 ×
250mm, 1mL/min, 90:105 (v/v) H2O-MeOH, 210nm, 35℃). The Compound 3 with the retention time
of 12.801min was separated from the other fraction by fraction collection. The sample was
freeze-dried with ALPHA1-4 freeze-drying machine.
2.3 Identification of compounds
The melting point and solubility of compounds were measured. The structure of compounds
were examined by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic
resonance (NMR) spectroscopy. Conditions of ESI-MS and NMR experiments were as reported
(Petzold et al. 2006; Martin et al. 1985). Identification of compounds were made by comparison with
spectra of standards and assignments reported previously. Degree of variation of the method is ±10%.
Multiplicities of 13C spectra were assigned by distortionless enhancement by polarization transfer
(DEPT). ESI-MS and NMR measurements were provided by Riverside Mass Spectrometry Facility
of the Applied Chemistry Institute of Jilin Province.
2.4 Content determination of trehalose in G. applanatum
The content of trehalose in G. applanatum fruit body was determined by using
anthrone-sulphuric acid colorimetric method (Feng 1989). The smashed G. applanatum fruit body
50g was dried after extracted three times with petroleum ether, and the dried powder was extracted
with 95% ethanol for 24 hours under agitation for three times. The EtOH-soluble fraction was
combined and decolored 30min with 2% absorbite, subsequently filtered under normal pressure, and
then 880mL sample solution was obtained. Standard curve of trehalose was prepared and sample
solutions of 0.05mL (A), 0.08mL (B), and 0.1mL (C) were measured accurately for determining the
content of trehalose respectively in G. applanatum fruit body using the method mentioned.
584 Mycosystema

3 RESULT
3.1 Compounds
Compound 1: White powder of compound 1 was obtained by freeze-drying the corresponding
peak collected from HPLC. The melting point of this powder is 155℃. It is dissoluble in H2O,
pyridine and phenylamine, and slightly soluble in MeOH and EtOH but not soluble in ether. The
molecular weight of compound 1 is 194.2 according to the EIMS (M-H:m/z) results. 1H-NMR
(DMSO-d6, 400MHz) result analysis of compound 1 shows that there are multiple resonance peaks
ranged from δ=3.312 to δ=4.398 and the comparison of 13C-NMR (DMSO-d6) chemical shift
between compound 1 and standard mannitol is shown in Table 1. 1H-NMR and 13C-NMR chemical
shift of compound 1 is similar to that of standard mannitol. DEPT result of compound 1 shows that a
is CH2OH, b is CHOH and c is CHOH.

Table 1 Comparison of 13C-NMR chemical shift between compound 1 and standard mannitol
Number of carbon Compound 1 Mannitol Carbon position of compound 1
1 63.08 64.20 a
2 69.05 71.50 b
3 70.62 72.60 c
Note: Chemical structural formula of compound 1 is as follows:
CH2－OH
∣
HO－CH
∣
HO－CH
∣
CH－OH
∣
CH－OH
∣
CH2－OH

Compound 2: White powder of compound 2 was obtained by freeze-drying of the second main
peak fraction. The quantity obtained was too scant to make identification.
Compound 3: Colorless clumpy crystal of compound 3 was obtained from freeze-drying of the
third main peak fraction. The melting point of this crystal is 140 , and the other physical and ℃
chemical characters are similar to those of trehalose. The molecular weight of compound 3 is 341.2
according to its EIMS (M-H:m/z) results. 1H-NMR (CDCl3, 600MHz) result analysis of compound 3
shows there are multiple resonance peaks ranged from δ=3.222 to δ=5.919 and the comparison of
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13C-NMR (CDCl3) chemical shift between compound 3 and that of standard trehalose is shown in
Table 2. There are a –CH2 and five –CH in compound 3 from DEPT results.

Table 2 Comparison of 13C-NMR chemical shift between compound 3 and standard trehalose
Number of carbon Trehalose Compound 3
1 94.8 93.04
2 73.9 71.99
3 74.4 72.41
4 73.5 70.91
5 72.3 69.58
6 62.5 60.46
Note: chemical structural formula of compound 3 is as follows:
O H
O
OHH
OH H
H
OH
CH2OH
O
CH2OH
H
OH
HOH
H
OHH
H
H
1
23
4
5
6


3.2 Content determination of trehalose in sample solution
The content of trehalose of Ganoderma applanatum fruit body is 0.48% (Table 3).

Table 3 Result of content determination of trehalose
Number of sample solution OD Amount of trehalose (μg) Content of trehalose (g/100g dry weight)
A 0.217 13.63 0.48
B 0.314 21.74 0.48
C 0.387 27.84 0.49

4 DISSCUSION
Trehalose can form particular protective membrane on the surface of the cell. Ganoderma
applanatum which can live on dry stumpage maybe related to the function of trehalose. Trehalose has
been used as biological agent, medicine, food additive, and cosmetic, but the supply falls shorts of
demand. The content of trehalose in G. applanatum is 0.48% of dried weight of G. applanatum fruit
body, determined by anthronesulphuric acid colorimetric method improved. It is hopeful that the
trehalose isolated from G. applanatum can make up deficiency of the product. The artificial
586 Mycosystema

cultivation of G. applanatum is in need of study.

Acknowledgment: We thank Applied Chemistry Research Institute of Jilin Province for providing high
resolution mass spectrometric data.

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