全 文 ： 菌物学报
jwxt@im.ac.cn 15 September 2013, 32(5): 876-882
Http://journals.im.ac.cn Mycosystema ISSN1672-6472 CN11-5180/Q © 2013 IMCAS, all rights reserved.
Supported by National Natural Science Foundation of China (No. 31000036 and No. 21072219) and grants from Chinese Academy Sciences
(KSCX2-YW-G-074-04).
*Corresponding author. E-mail: heranli@suda.edu.cn, baol@im.ac.cn
Received: 14-06-2012, accepted: 16-10-2012
Bioactive composition of the solid culture of the edible
mushroom Pleurotus citrinopileatus on rice and the antioxidant
effect evaluation
LI Yong-Xia1, 2 HAN Jun-Jie2 YANG Xiao-Li2 LI He-Ran1* WANG Ya-Qi2
WANG Shao-Juan2 BAO Li2*
1College of Pharmacy, Soochow University, Suzhou, Jiangsu 215123, China
2State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
Abstract: The edible mushroom Pleurotus citrinopileatus was fermented on cooked rice by the solid-state fermentation
technique. The ethyl acetate extract of the P. citrinopileatus-fermented rice showed the strong reducing ability in antioxidant
bioassay. Indole-3-carboxaldehyde (1), 5, 7-dimethoxyisobenzofuran-1(3H)-one (2), 3, 5-dihydroxybenzyl acetate (3),
4-hydroxybenzaldehyde (4), 4-hydroxybenzaldehyde (5), and ergosterol (6) were isolated from the ethyl acetate extract.
Their structures were elucidated on the basis of MS and NMR spectroscopic data. Their antioxidant effects of the DPPH
scavenging activity, the chelating ability, and the reducing ability, were screened. The isolation of indole-3-carboxaldehyde as
a secondary metabolite from edible mushroom and its structural assignment were reported for the first time. The P.
citrinopileatus-fermented rice might be useful as a new functional food.
Key words: Pleurotus citrinopileatus Singer, antioxidat, indole-3-carboxaldehyde, phthalide
金顶侧耳次级代谢产物及抗氧化活性研究
李永霞 1,2 韩俊杰 2 杨晓莉 2 李贺然 1* 王雅琪 2 王少娟 2 宝丽 2*
1苏州大学药学院 江苏 苏州 215123
2中国科学院微生物研究所真菌学国家重点实验室 北京 100101
摘 要：采用硅胶柱色谱、ODS柱色谱、Sephadex LH-20柱色谱、HPLC等分离方法，对金顶侧耳大米发酵乙酸乙
酯提取物进行分离；通过 HR-ESI-MS、1H-NMR和 13C-NMR等光谱学方法对其结构进行鉴定。并检测铁氰化钾还原
能力、1,1-二苯基-2-苦基苯肼（DPPH）自由基清除能力和亚铁离子螯合能力。从金顶侧耳大米发酵粗提物中分离鉴
DOI:10.13346/j.mycosystema.2013.05.014
LI Yong-Xia et al. / Bioactive composition of the solid culture of the edible mushroom Pleurotus citrinopileatus on rice and the antioxidant effect evaluation
菌物学报
877
定了吲哚甲醛、苯肽等 6 个化合物，吲哚甲醛为首次从该菌中分离得到，活性测试表明金顶侧耳粗提取物具有一定
的还原能力，其大米发酵产物有可能开发成为一种新型功能性食品。
关键词：金顶侧耳，抗氧化，吲哚甲醛，苯肽
Mushrooms have captured the attention of
some researchers on account of their exploitable
bioactive constituents. Many edible mushrooms, in
particular those rich in polysaccharides or
polysaccharide-protein complex, not only their good
taste but also the important source of bioactive
metabolites (Ferreira et al. 2010).
The technique of solid-stated fermentation
(SSF) involves the growth and metabolism of
microorganism on moist solid substrates in the
absence of free flowing water. The SSF technique
has become a useful method to produce some
traditional food in Asia for centuries. In China, the
traditional food Monascus-fermented rice becomes
very popular functional food due to its variety in
medicinal properties including antihypertensive,
hypocholesterolaemic, antimicrobial, antioxidant,
and anticarcinogenetic bioactivity (Wang & Lin
2007). For the purpose of adding more nutritional
and medicinal value to edible mushroom, we try to
ferment the edible mushrooms by SSF technique.
The solid cultures of edible mushrooms on rice were
extracted with ethyl acetate, and the organic solvent
extracts were assayed for antioxidant activity. As a
result, an organic solvent extract of the solid culture
of the edible mushroom Pleurotus citrinopileatus
was found to have good antioxidant activity. To
evaluate the potential of the P. citrinopileatus-
fermented rice as functional food or complementary
formulations, a detailed chemical investigation was
conducted on its organic solvent extract.
P. citrinopileatus belonging to the family
Pleurotaceae is a delicious edible and medicinal
mushroom, rich in protein, amino acids and
vitamins, and has a wide range of pharmacological
activities. These include hypolipidemic, antioxidant,
hypoglycemic, immunostimulant, antitumor
activities (Chan et al. 2009; Lee et al. 2007; Sun
2011; Sun et al. 2010). A novel agglutination factors
with anti-tumor and HIV-1 inhibitory activity (Li et
al. 2008) has been isolated from the fruiting bodies
of P. citrinopileatus. In the current research, indole-
3-carboxaldehyde (1), 5, 7-dimethoxyisobenzofuran-
1(3H)-one (2), 3, 5-dihydroxybenzyl acetate (3),
4-hydroxybenzaldehyde (4), 4-hydroxybenzaldehyde
(5), and ergosterol (6) were isolated from the solid
culture of P. citrinopileatus. Their chemical
structures (Fig. 1) were elucidated by their
physicochemical properties and spectral data,
including 1D, 2D-NMR and HR-ESI-MS. The
plausible biosynthetic pathway for phthalide in P.
citrinopileatus was also discussed.
Fig. 1 Structures of compounds 1–6 isolated from Pleurotus
citrinopileatus.
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1 MATERIALS AND METHODS
1.1 General
UV data were recorded on a Shimadzu
Biospec-1601 spectrophotometer. 1H and 13C-NMR
data were acquired with Bruker Avance-500
spectrometer using solvent signals as references.
HR-ESI-MS data were obtained using a Bruker
APEX III 7.0T spectrometer. TLC was carried out on
Silica gel HSGF254 and the spots were visualized by
spraying with 10% H2SO4 and heating. Silica gel
(Qingdao Haiyang Chemical Co., Ltd., People’s
Republic of China), LH-20 (Amersham Biosciences),
and ODS (Lobar, 40–63m, Merck) were used for
column chromatography. Preparative HPLC was
performed on an Agilent 1,200 HPLC system using an
ODS column (RP-8, 250×10mmol/L, YMC Pak, 5m)
and a UV detector with a flow rate of 2.5mL/min.
1.2 Fungal material and cultivation
The strain of P. citrinopileatus used in this
work is kept in the culture collection at the Institute
of Microbiology, Chinese Academy of Sciences,
Beijing (CGMCC 5.597). The fungal strain was
cultured on slants of potato dextrose agar at 25℃ for
10 days. Agar plugs were inoculated in 500mL
Erlenmeyer flask containing 120mL of media (0.4%
glucose, 1% malt extract, and 0.4% yeast extract; the
final pH of the media was adjusted to 6.5) before
sterilization, and incubated at 25℃ on a rotary
shaker at 180r/min for 3 days. Large scale
fermentation was carried out in 500mL Erlenmeyer
flask each containing 80g of rice and 120mL of
distilled water, and 121℃, 30min autoclaved. Each
flask was inoculated with 5.0mL of the culture
medium and incubated at 25℃ for 40 days.
1.3 Extraction and isolation
The fermented rice substrate was extracted with
ethyl acetate by exhaustive maceration and the
organic solvent was evaporated to dryness under
vacuum to afford the crude extract (5g). The ethyl
acetate extract was subjected to a silica gel column
using a dichloromethane-acetone gradient elution to
afford 15 fractions (PC-1-PC-15). The fraction PC-2
(300mg) was separated by ODS column
chromatography (CC) using a gradient of increasing
methanol (20%–100%) in water to afford 6
subfractions (2-1-2-6). The subfraction 2–5 (30mg)
was followed by RP-HPLC using 50% acetonitrile in
water to afford 2 (3.2mg, tR 18.8min) and 3 (3.8mg, tR
17.1min). The subfraction 2–6 (30mg) was further
separated by preparative TLC using petroleum
ether-ethyl acetate (6:1, v/v) to afford 6 (17.8mg). The
fraction PC-4 (625mg) was separated by ODS CC
using a gradient of increasing methanol (20%–100%)
in water to afford 6 subfractions (4-1-4-6). The
subfraction 4-4 was separated by Sephadex LH-20
CC eluting with methanol, and then purified by
RP-HPLC using 40% methanol in water to afford 1
(14.5mg, tR 37.7min) and 4 (2.6mg, tR 23.2min). The
subfraction 4–5 was separated by Sephadex LH-20
CC eluting with methanol, and then purified by
RP-HPLC using 20% methanol in water to afford 4
(5.4mg, tR 23.2min) and 5 (2.5mg, tR 13.0min).
1.4 Reducing power
The reducing power was determined according
to the method of Oyaizu (1986). Each sample
(2.5mL) in deionised water was mixed with 2.5mL
of 1% potassium ferricyanide (Sigma), and the
mixtures were incubated at 50℃, for 20min. After
2.5mL of 10% trichloroacetic acid (w/v, Wako) were
added, the mixture was centrifuged at 200g for
10min. The upper layer (5mL) was mixed with 5mL
of deionised water and 1mL of 0.1% ferric chloride
(Wako) and the absorbance was measured at 700nm
against a blank. A higher absorbance indicated a
higher reducing power. Ascorbic acid was used for
comparison (Oyaizu 1986; Yen & Chen 1995).
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1.5 Chelating ability on ferrous ions
Chelating ability was determined according to
the methods of Dinis et al. (1994). Each sample
(1.0mL) in deionised water was mixed with 3.7mL
of methanol and 0.1mL of 2mmol/L ferrous chloride
(Merck). The reaction was initiated by the addition
of 5mmol/L ferrozine (0.2mL, Sigma). After 10min
at room temperature, the absorbance of the mixture
was determined at 562nm against a blank. A lower
absorbance indicates a higher chelating power.
Ethylenediaminetetraacetic acid (EDTA) was used
for comparison (Oyaizu 1986; Yen & Chen 1995).
1.6 Scavenging ability on 1,1-diphenyl-2-
picrylhydrazuyl (DPPH) radical
Each sample (4.0mL) in deionised water, was
mixed with 1.0mL of methanolic solution containing
DPPH (Sigma) radicals, resulting in a final
concentration of 0.2mmol/L DPPH. The mixture was
shaken vigorously and left to stand for 30min in the
dark, and the absorbance was then measured at
517nm, against a blank (Shimada et al. 1992). The
scavenging ability was calculated as follows:
scavenging ability (%) = [(A517 of control–A517 of
sample)/A517 of control]×100. Ascorbic acid was
used for comparison.
2 RESULTS AND DISCUSSION
2.1 Structure determination
Compound 1 was isolated as colorless needles,
and its molecular formula was determined to be
C9H6NO on the basis of HR-ESI-MS (found
146.0613, calcd 146.0600 m/z, [M+H]+) and NMR
spectral data (Table 1). The UV spectrum showed
absorptions at 200 and 300nm. The 1H-NMR
spectrum of 1 showed signals ascribable for an
aldehyde proton at δ 9.91 (s, H-9), five aromatic
proton at δ 8.18 (d, J = 7.6, H-1), 7.29 (m, H-2 and
H-3), 7.50 (d, J = 7.6, H-4), 8.12 (s, H-6). The
13C-NMR spectrum of 1 displayed signals for 9
carbons including one carbonyl carbons at δ 187.6,
eight olefinic at δ 139.8, 139.1, 125.9, 125.1, 123.7,
122.7, 120.5, 113.7. Moreover, the HMBC spectrum
of 1 (Fig. 2) showing the correlations between H-9
and C-6 (δ 139.8) and C-8 (δ 125.1), between H-6
and C-7 (δ 122.7), C-8 (δ 125.1) and C-9 (δ 187.6),
between H-4 and C-2 (δ 123.7), C-5 (δ 120.5) and
C-7 (δ 122.7), and between H-1 and C-3 (δ 139.1)
and C-8 (δ 125.1) confirmed the structure of 1 as
indole-3-carboxaldehyde (Fig. 2). Compound 1 is a
well-known reactant intermediate for the synthesis
of analgesic agent, antibacterial and antifungal
agent, and antiamoebic and cytotoxic agent. It is the
first time to report indole-3-carboxaldehyde as a
secondary metabolites produced by mushroom and
its structural assignment.
Compound 2 was isolated as colorless needles,
and its molecular formula was determined to be
C10H10O4 on the basis of its HR-ESI-MS (found
196.0659, calcd 196.0652 m/z, [M+H]+) and NMR
spectral data. The 1H-NMR spectrum of 2 was
indicative of the presence of two aromatic protons at
δ 6.93 (H-6, s) and 6.86 (H-4, s), one oxymethylene
at δ 5.25 (2H, s), and two methoxy groups at δ 3.91
Table 1 1H-NMR (500MHz, in CD3OD) and 13C-NMR
(125MHz, in CD3OD) data for compound 1
1
Position
δ(H) δ(C) HMBC
1
2
3
4
5
6
7
8
9
8.18 (d, J= 7.6Hz)
7.29 (m)
7.29 (m)
7.50 (d, J=7.6Hz)
8.12 (s)
9.91 (s)
125.9
123.78
139.0
113.7
120.5
139.8
122.7
125.2
187.6
3, 8
4, 7
1, 8
2, 7, 5
7, 8, 9
6, 8
ISSN1672-6472 CN11-5180/Q Mycosystema September 15, 2013 Vol.32 No.5
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880
Fig. 2 Key HMBC correlations of compound 1.
(3H, s) and 3.88 (3H, s). Finally it was determined
as 5, 7-dimethoxyisobenzofuran-1(3H)-one by the
comparison of their spectroscopic data with
literature values (Combes & Finet 1997).
Compounds 3–6 were identified as 3,5-
dihydroxybenzyl acetate, 4-hydroxybenzaldehyde,
2,4-dihydroxy-6-hydroxymethylbenzaldehyde, and
24-methylcholest-5-en-3-ol by HR-ESI-MS and
1H-NMR data analysis, as well as the comparisons
with the data previously published (Combes & Finet
1997; Li et al. 2008; Nobosu et al. 1996).
The edible mushrooms have been found to
contain non-hallucinogenic indole derivatives with a
variety of bioactivities including antioxidant,
anticancer, anti-aging activity, anti-inflammatory
and analgesic effects (Bozena & Katarzyna
2012). Phthalide compounds including
5,7-dimethoxyisobenzofuran-1(3H)-one (2) have
been isolated from the edible mushroom Sparassis
crispa, possessing superoxide dismutase-like,
suppression of LPS-induced PGE2 and NO
Production in RAW264 cells (Kazuko et al. 2010).
Ergosterol is a principal sterol in fungi (Czub &
Baginski 2006). Ergosterol and its derivatives
possess significant pharmacological activities (Bok
et al. 1999). Compounds 1–5 were isolated from P.
citrinopileatus for the first time. The solid-state
fermentation of edible mushrooms on cornmeal can
increase the biosynthesis of bioactive components in
mushroom, which is a useful method to increase the
production of bioactive secondary metabolites from
edible mushrooms.
2.2 Antioxidant bioassay
The ethyl acetate extract of P. citrinopileatus-
fermented rice and the isolated compounds 1–6 were
assayed for the scavenging activity on DPPH
radicals, the chelating ability on ferrous ions, and the
reducing ability.
With regard to DPPH scavenging activity, the
ethyl acetate extract of the P. citrinopileatus- fermented
rice showed moderate effect at the concentration of
1mg/mL with the percent inhibition value of
(39.88±1.28)%. The scavenging ability on DPPH
radicals of the ethyl acetate extracts of the solid culture
and its concentration was positive dependencies.
Compounds 1–6 did not show free radical scavenging
activity at a concentration of 200μmol/L. The positive
control vitamin C showed strong scavenging activity
with the EC50 value of (30.22±2.21)μmol/L.
Iron is known as the most important lipid
oxidation prooxidant due to its high reactivity. The
ferrous state of iron accelerates lipid oxidation by
breaking down hydrogen and lipid peroxides into
reactive free radicals via the Fenton reaction. The
chelating activities on ferrous ion of the crude extract
of the solid culture of P. citrinopileatus and compounds
1–6 were shown in Table 2 and Table 3. The crude
extract of the P. citrinopileatus-fermented rice showed
no chelating activity within the range of
0.125–1.0mg/mL, and compounds 1–6 showed
moderate chelating capacity with percent chelating
value of 34.17, 28.77, 29.72, 39.47, 37.54, 26.94% at a
concentration of 200μmol/L, respectively. The positive
control of EDTA exhibited chelating activity on ferrous
ion with the EC50 value of (0.13±0.00)mg/mL. The
LI Yong-Xia et al. / Bioactive composition of the solid culture of the edible mushroom Pleurotus citrinopileatus on rice and the antioxidant effect evaluation
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weakened chelating ability is likely the results of
mutual influence of compounds in the ethyl acetate
extracts in solid-culture.
The reducing capacity of compounds can be
regarded as a significant indicator of the antioxidant
activity. The reducing power was determined using
the potassium ferricyanide reduction method. In the
range of 0.125–1mg/mL, the ethyl acetate extracts of
the solid culture of P. citrinopileatus showed strong
reducing ability (Table 2). Compounds 1, 2, and 6
showed weak reducing ability with percent reducing
value of (23.70±10.32)%, (22.22±5.44)%,
(29.63±2.10)% at the concentration of 200μmol/L,
respectively (Table 4). The positive control vitamin C
showed strong reducing activity with the EC50 value
of (2.25±0.21)μmol/L. Probably, the strong reducing
ability of the ethyl acetate extracts of the solid culture
may be the synergistic effect of the components or
due to compounds not obtained in current
investigation. The antioxidant activities of the crude
extract and purified compounds of P. citrinopileatus
provide the evidence for the application of the P.
citrinopileatus-fermented rice as new functional food.
2.3 Biosynthetic pathway of phthalides
On the basis of the structural characters of the
metabolites isolated from P. citrinopileatus, the possible
biosynthetic pathway of 5, 7-dimethoxyisobenzofuran-
1(3H)-one (2) in P. citrinopileatus was proposed.
Compound 2 should be biosynthesized by the
acetate-malonate pathway. Compound A can be
formed by cycloaddition from acetyl coenzyme A and
malonyl coenzyme A. Compound 3 and compound 4
can be generated by acetylation and oxidation
respectively from compound A. Compound 4 was the
key intermediate product for the biosynthesis of 2 in
the edible mushroom P. citrinopileatus. Compound
2 can be obtained via esterification and oxidation
from compound 4 (Fig. 3).
Table 2 Antioxidant capacity of the ethyl acetate extract of the solid culture of P. citrinopileatus (%)
Concentration DPPH scavenging ability Reducing ability Chelating ability
1mg/mL 39.88±1.28 107.41±2.77 9.91±0.05
0.5mg/mL 7.03±1.87 102.22±1.81 8.81±0.05
0.25mg/mL -4.17±2.59 104.44±3.63 7.81±0.89
0.13mg/mL -27.52±1.33 108.89±3.63 7.60±0.15
Table 3 Chelating ability of compounds 1–6 (%)
Concentration 1 2 3 4 5 6
200μmol/L 34.17±0.21 28.77±0.64 29.72±0.10 39.47±0.32 37.54±0.25 26.94±0.19
100μmol/L 18.07±0.10 19.00±0.13 23.94±0.73 30.25±0.21 19.03±0.27 24.57±0.43
50μmol/L 15.45±0.08 11.08±0.98 9.51±0.29 27.15±0.18 14.76±0.27 21.99±0.46
25μmol/L 3.79±0.17 8.91±0.89 6.55±0.15 13.52±0.13 8.40±0.48 14.62±0.29
Table 4 Reducing ability of compounds 1–6 (%)
Concentration 1 2 3 4 5 6
200μmol/L 23.70±10.32 22.22±5.44 4.44±3.14 9.63±4.19 7.41±3.78 29.63±2.10
100μmol/L 2.22±4.80 10.37±2.77 2.96±5.83 4.44±3.14 -14.81±1.05 16.30±1.05
50μmol/L -4.44±1.81 4.44±3.63 -1.48±5.54 3.70±1.05 -12.59±4.19 8.15±1.05
25μmol/L -11.85±2.10 -10.37±2.77 -8.15±1.05 -2.22±7.26 -11.11±6.29 3.70±2.77
ISSN1672-6472 CN11-5180/Q Mycosystema September 15, 2013 Vol.32 No.5
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Fig. 3 The possible biosynthetic pathway of phthalide (2).
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