Two new aromatic compounds and a new D-arabinitol ester from the mushroom Hericium erinaceum

Article abstract of DOI:10.1016/j.tet.2011.11.068

Two new aromatic compounds (1, 2), new d-arabinitol ester (3), and two known compounds (4, 5) were isolated from mushroom Hericium erinaceum. The structures of 1-5 were elucidated on the basis of spectral data. Compounds 1, 2, 4, and 5 exhibited α-glucosidasae inhibitory activity.

Full text of DOI:10.1016/j.tet.2011.11.068

Tetrahedron 68 (2012) 2007e2010
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Two new aromatic compounds and a new -arabinitol ester from the mushroom
D
Hericium erinaceum
,
a
a
b
Mitsuo Miyazawa a , Toshiyuki Takahashi , Isao Horibe , Ryuuzou Ishikawa
*
a Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, 3-4-1 Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan
b Taiyo Togo Iryo Kaihatsu Co., Ltd., 2-6-5 Fukono, Daitou-shi, Osaka 574-0072, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new aromatic compounds (1, 2), new D-arabinitol ester (3), and two known compounds (4, 5) were
Received 7 November 2011
Received in revised form 19 November 2011
Accepted 23 November 2011
Available online 27 November 2011
isolated from mushroom Hericium erinaceum. The structures of 1e5 were elucidated on the basis of
spectral data. Compounds 1, 2, 4, and 5 exhibited -glucosidasae inhibitory activity.
Ó 2011 Elsevier Ltd. All rights reserved.
a
Keywords:
Hericium erinaceum
Isohericerine
D
-Arabinitol ester
-Glucosidase inhibitor
a
1. Introduction
crude extract. The extract was fractionated by silica gel column
chromatography. As a consequence, two new aromatic compounds
Mushrooms have been not only used as food materials with
their unique flavor and texture, but also recognized as an important
source of biologically active compound of medicinal value.1 In re-
cent years, there is increased medical and pharmaceutical interest
in mushrooms as sources of novel immunomodulators,2 antitumor
agents,3 antibiotics,4 and antihypertensives.5 Hericium erinaceum
(Yamabushitake in Japanese) is known as an edible mushroom that
belongs to the Hericiaceae family and grows on dead trunks of hard
woods such as narra, oak, and beech in Japan, China, Europe. In
regarding to chemical components of H. erinaceum, various con-
stituents such as hericene, hericerin, hericenone erinacine, erina-
cerin, and polysaccharides have been reported as the chemical
components.6e12 Its fruiting bodies have been used for the treat-
ment of dyspepsia, gastric ulcer, and enervation in China. This
mushroom has some beneficial effects, including antioxidant,13
antitumor,14e16 antimicrobial,17,18 pollen tube germination/growth
inhibitior,19 and the stimulating the synthesis of nerve growth
factor (NGF).20 These results suggest the usefulness of H. erinaceum,
thus, we investigated chemical component of H. erinaceum.
(1, 2), new
were isolated from this mushroom.
D
-arabinitol ester (3), and two known compounds (4, 5)
2. Result and discussion
The fruiting bodies of H. erinaceum were extracted with EtOAc.
The solvent was filtered and then removed in vacuo to give a brown
* Corresponding author. Tel.: þ81 6 6721 2332; fax: þ81 6 6727 2024; e-mail
address: miyazawa@apch.kindai.ac.jp (M. Miyazawa).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.11.068

2008
M. Miyazawa et al. / Tetrahedron 68 (2012) 2007e2010
Compound 1 was isolated as colorless prisms. Its molecular
including DEPT and IR spectra with those of known compound
hericerin suggested that this molecule involves isoindolinone
skeleton.
formula of 1 was determined as C27H33O3N by HREIMS [m/z
419.2470 [M]þ (calcd for C27H33O3N, 419.2480)]. The presence of
a hydroxyl group (3419 cmꢀ1), a
g
-lactam (1646 cmꢀ1), and an ar-
The partial structure was determined by interpretation of the
HMBC spectrum revealed correlations as shown in Fig. 1. The
connection between nitrogen atom and phenylethyl group was
indicated by HMBC correlations from H-100 to C-1 and C-3. Iso-
indoline-1-one substructure was disclosed by HMBC correlations
from H-7 to C-1 and H-3 to C-4. On the other hand, in the case of
hericerin, NOE was observed between the signal at aromatic
proton and methylene proton of isoindolinone skeleton.19 These
results indicated that chemical structure of compound 1 is dif-
ferent from that of hericerin, thus, we named compound 1
isohericerin.
omatic ring (1589 cmꢀ1) were confirmed by IR spectral data. Car-
bon NMR, including DEPT, indicated nine quaternary carbons (dC
169.0, 158.4, 150.4, 139.1, 138.7, 132.1, 132.0, 121.1, and 118.1). The
connectivity of carbon and hydrogen atoms was established from
the HSQC spectrum. Proton NMR spectrum indicated a methoxy
group (dH 3.83), aromatic protons (dH 7.26 (2H), 7.22 (2H), 7.19 (1H),
6.95 (1H)), three methyl groups (dH 1.80, 1.66 and 1.58), six meth-
ylenes (dH 4.17, 3.84, 3.49, 2.97, 2.10, 2.05) and, two trisubstituted
olefins (dH 5.24, 5.03). Also signal observed at dH 6.52 (1H, s), which
had no HSQC correlation with any carbon signals, was assignable to
phenolic proton (4-OH) (Table 1). Comparison of 1H, 13C NMR
Table 1
1H and 13C NMR spectra data for compounds 1,2 and 4,5 (500 MHz for 1H; 125 MHz
for 13C)
Position
1
dH (J in Hz)
dC
2
dH (J in Hz)
dC
1
169.0
170.5
2
3
3a
4
5
6
4.17 s
48.2
4.19 s
43.2
131.7
150.3
120.0
158.5
96.4
132.1
150.4
118.1
158.4
97.5
7
6.95 s
6.73 s
7a
121.1
22.8
22.8
139.1
39.6
26.3
123.7
132.0
25.7
16.1
17.7
44.2
34.8
138.7
128.6
128.6
126.4
123.4
22.5
122.5
134.2
39.5
10
3.49 d (6.8)
5.24 br t (6.8)
3.31 d (7.1)
5.12 t (7.1)
20
30
40
2.05 m
2.10 m
5.03 br t (6.8)
1.89 t (7.6)
1.99 td (7.6, 7.0)
5.02 t (7.0)
50
26.4
60
124.3
130.8
25.7
16.1
17.7
70
Fig. 1. (a) Key HMBC correlations (H/C) of 1. (b) Key HMBC correlations (H/C) of 2.
80
1.66 s
1.80 s
1.58 s
3.84 br t (7.2)
2.97 t (7.2)
1.58 s
1.72 s
1.51 s
90
100
100
200
Compound 2 was isolated as an amorphous powder. The mo-
lecular formula of 2 was determined to be C19H25O3N by HREIMS
[m/z 315.1829 [M]þ (calcd for C19H25O3N, 315.1759)].
300
400, 800
500, 700
600
7.22 dd (7.2, 1.7)
7.26 dd (7.2, 7.2)
7.19 dd (7.2, 1.7)
6.52 s
The presence of a hydroxyl group (3263 cmꢀ1), a
g-lactam
(1667 cmꢀ1), and an aromatic ring (1625, 1593 cmꢀ1) were con-
firmed by IR spectral data. The 1H and 13C NMR spectra were almost
similar to those of 1 (Table 1). It was suggested that 2 is isohericerin
analogues compound without the phenylethyl moiety. The HMBC
showed correlations for H2-3/C-1, H2-3/C-4, H-2/C-1, H-2/C-3, H-2/
C-3a, H-2/C-7a, and H-7/C-1 (Fig. 1). Compound 2 was determined
as N-De phenylethyl isohericerin by 1H, 13C NMR, HSQC, HMBC
analyses.
Compound 3 was isolated as a white powder. The IR spectrum
indicated the presence of OH (3322 cmꢀ1) and C]O (1735 cmꢀ1).
The 13C NMR spectrum including DEPT displayed 23 carbon reso-
nances, comprising 3 sp3 methines, 14 sp3 methylenes, 1 sp3
methyl, 4 sp2 methines, and a carbonyl. The 1He1H COSY spectrum
4-OH
6-OMe
NH
9.42 s
3.79 s
8.36 s
3.83 s
56.0
56.0
Position
1
2
3
4
5
6
7
4
dH (J in Hz)
dC
5
dH (J in Hz)
dC
138.4
112.9
162.9
118.1
163.5
105.6
62.9
193.1
21.3
121.2
135.7
39.8
143.3
112.6
163.7
103.5
163.0
103.5
62.7
193.6
21.3
121.5
131.5
39.8
6.52 s
5.32 s
10.11 s
3.24 d (7.0)
5.17 br t (7.0)
6.52 s
4.94 s
10.21 s
3.33 d (7.2)
5.17 t (7.2)
8
1
20
30
40
1.95 t (7.0)
2.04 m
5.05 br t (6.8)
1.95 t (7.6)
2.04 m
5.06 br t (6.8)
of
compound
3
showed
the
partial
structure
50
26.7
26.7
eOeCH2eCHOReCHOReCHOReCH2eOe. Therefore, we assumed
that compound 3 is sugar alcohol ester. Pursuant to this, we hy-
drolyzed compound 3 to obtain D-arabinitol and methyl linoleate.
These compounds were identified with NMR data, specific rotation
and MS spectrum and retention index (RI).21e23 Moreover, HMBC
spectrum revealed the correlation from methylene proton of sugar
alcohol to carbonyl carbon. The result indicated that a linoleoyl
group linked to carbon atom of C-1 or C-5. To confirm the linoleoyl
60
124.3
131.2
25.6
16.1
17.6
173.2
34.2
25.6
22.7e31.9
14.1
124.4
131.4
25.7
16.0
17.6
70
80
1.63 s
1.77 s
1.57 s
1.64 s
1.77 s
1.57 s
90
100
100
200
2.33 t (7.6)
1.61 tt (7.6, 7.6)
1.23e1.32 m
0.88 t (7.6)
12.36 s
300
400-1500
1600
3-OH
5-OMe
group connected C-1 or C-5 position, we synthesized 1-D-arabini-
12.37 s
3.92 s
tol-monolinoleate. Compound 3 was confirmed as 1-
monolinoleate by comparing the NMR data of 1-
D
-arabinitol-
-arabinitol-
3.91 s
55.9
55.8
D
In CDCl3 for 1, 4, and 5; in DMSO for 2.
monolinoleate synthesized with that of compound 3. Compounds 4

M. Miyazawa et al. / Tetrahedron 68 (2012) 2007e2010
2009
and 5 were identified as hericene A and 4-[30,70-dimethyl-20,60-
3.3. Extraction and isolation
octadienyl]-2-formyl-3-hydroxy-5-methyoxybenzylalcohol,
re-
spectively, by comparing with previous spectral data.24 Arone et al.,
obtained compound 5 by methanolysis of hericenes AeC,24 how-
ever, there is no report as the isolated compound 5 from natural
source. We isolated compound 5 as a natural compound from H.
erinaceum for the first time.
The fruiting bodies of H. erinaceum (1.2 kg) were extracted with
EtOAc (3 l, two times) for 3 h. The solution was concentrated under
reduced pressure. The EtOAc-soluble part (28.0 g) was fractionated
by silica gel column chromatography with a hexane/EtOAc (17:3,
1:1, MeOH, v/v) to obtain four fractions: 1 (10.2 g), 2 (6.2 g), 3
(1.8 g), 4 (9.8 g). Fraction 1 (10.2 g) was separated by SiO2 column
chromatography with a CH2Cl2/EtOAc (9:1, v/v) to obtain five
fractions. Compound 4 (840 mg) was obtained from fractions 1 to 4
(1.6 g) by crystallization (diethylether/ethanol). Compound 4 was
identified as hericene A. Fraction 3 (1.8 g) was separated by SiO2
column chromatography with CH2Cl2/EtOAc (9:1, 4:1, 0:1, MeOH, v/
v) to obtain four fractions. Fraction 3-2 (240 mg) was further sep-
arated by SiO2 column chromatography with a hexane/EtOAc (4:1,
v/v) to afford compound 5 (40 mg). Fraction 3-3 (780 mg) was
further separated by SiO2 column chromatography with CH2Cl2/
EtOAc (4:1, v/v) to afford compound 1 (172 mg). Fraction 4 (9.8 g)
was further separated by SiO2 column chromatography with hex-
ane/acetone (4:1, 3:2, MeOH, v/v) to obtain three fractions. Fraction
4-3 (7.8 g) was further separated by SiO2 column chromatography
with CH2Cl2/acetone (4:1, 7:3, 3:2, MeOH, v/v) to obtain four frac-
tions. Fraction 4-3-2 (426 mg) was further separated by SiO2 col-
umn chromatography with CH2Cl2/acetone (3:2, v/v) to obtain two
fractions. Compound 2 (72 mg) was obtained from fraction 4-3-2-1
(148 mg) by crystallization (hexane/acetone). Fraction 4-3-3 (1.5 g)
was further separated by SiO2 column chromatography with
CH2Cl2/acetone (3:2, v/v) to obtain three fractions. Compound 3
was obtained from fraction 4-3-3-1 (420 mg) by crystallization
(CH2Cl2/acetone).
Compounds 1e5 were evaluated in
activity assay. Compounds 1, 2, 4, and 5 exhibited
inhibitory activity. Fifty percent inhibition (IC50) values of 1, 2, 4,
and 5 were 11.7, 12.5, 12.3, and 12.3 M, respectively (Table 2). This
result indicated that chemical components of H. erinaceum effects
on -glucosidase inhibitory activity.
a
-glucosidase inhibitory
a
-glucosidase
m
a
Table 2
Inhibitory activity of compounds 1e5 against a-glucosidase
Compound
IC50
(
m
M)a
Compound
IC50 (m
M)a
1
2
3
4
12.3ꢂ1.3
12.3ꢂ6.2
>300
11.7ꢂ0.2
5
12.5ꢂ1.3
278.0ꢂ1.7
907.5ꢂ2.2
1-Deoxynojirimycinb
Acarboseb
a
Data represents means of ꢂSD of triplicate samples.
b
These compounds were used as reference compounds.
3. Experimental
3.1. General
Thin layer chromatographies (TLC) were performed on pre-
coated plates (silica gel 60F254, 0.25 mm, Merk, Darmstadt, Ger-
many). Column chromatographies were carried out using 70e230
mesh silica gel (Kieselgel 60, Merck, Germany). 1H NMR spectra
were recorded at 400 MHz on a JEOL ECA-400 spectrometer, or at
JEOL ECA-500 spectrometer or at JEOL ECA-700 spectrometer in
D2O, CDCl3, and DMSO-d6 with DSS as internal standard for D2O
and TMS for CDCl3 and DMSO-d6. 13C NMR spectra were recorded at
100 MHz on a JEOL ECA-400 spectrometer, at JEOL ECA-500 spec-
trometers or at JEOL ECA-700 spectrometer. For gas chromatogra-
phy mass spectrometry (GCeMS), an Agilent Technologies 6890N
(Agilent Technologies (Tokyo, Japan)) gas chromatograph equipped
with a split injector was directly coupled to Agilent Technologies-
5973N-MSD (Agilent Technologies (Tokyo, Japan)) mass spec-
trometer and an HP-5MS capillary column (30 m length, 0.25 mm
i.d.) (Agilent Technologies (Tokyo, Japan)); a split injection of 10:1
was used. Helium at 1.8 ml/min was used as a carrier gas. The
temperature of the ion source was 280 ꢁC, and the electron energy
was70 eV. The oven temperature was programmed from 40 to
260 ꢁC at 4 ꢁC/min. The injection temperature was 270 ꢁC and the
detector temperature 280 ꢁC. The electron impact (EI) mode was
used. The retention index (RI) was calculated using a homologous
series of n-alkane C5eC28 for HP-5MS column. High-resolution
EIMS (HREIMS) was obtained on a JEOL the Tandem MS station
JMS-700 (Japan). IR spectra were determined with a JASCO FT/
IR-470 plus Fourier transform infrared spectrometer.
3.4. Hydrolysis of compound 3
Compound 3 (132 mg, 0.32 mmol) was dissolved in 1 ml of
methanol and 28% sodium methoxide (260 ml, 1.28 mmol) was
added. The mixture was stirred at room temperature for 2 h. The
solvent was evaporated under reduced pressure to give white solid.
The white solid was separated by silica gel column chromatography
with a CH2Cl2/MeOH (9:1) repeatedly, to give
D-arabinitol (39 mg,
0.26 mmol) and methyl linoleate (88 mg, 0.30 mmol).
D-Arabinitol
was identified by comparing with NMR data and specific rotation,
while methyl linoleate was identified by comparing with MS
spectrum and retention index (RI).
3.5. Synthesis of 1-D-arabinitol-monolinoleate
A mixture of -arabinose (2.0 g, 13.3 mmol), tritylchloride (4.1 g,
D
14.7 mmol), triethylamine (3.3 ml, 24.0 mmol), dimethylamino-
pyridine (81.0 mg, 0.67 mmol), and THF (20 ml) was stirred at room
temperature for 1 h. The mixture was poured into water and
extracted with ethylacetate three times. The organic layer was
washed with water, and dried on anhydrous sodium sulfate and the
solvent was evaporated to afford 5-O-trityl-
D-arabinose (2.0 g, yield
38.0%). 5-O-Trityl- -arabinose (2.0 g, 5.1 mmol) was added to
D
a mixture of methanol (20 ml) and ethanol (7 ml). The solution was
kept in an ice bath at 0 ꢁC and NaBH4 (94 mg, 2.6 mmol) was added
in small portions over a period of 30 min with constant stirring.
After keeping it for 90 min, solvent was evaporated to afford pale
yellow solid. The obtained solid was purified by column chroma-
Melting points (mp) were measured on an MP-5000D melting-
point apparatus. Alpha-glucosidase (EC 3.2.1.20) was purchased from
Oriental Yeast Co., Ltd (Tokyo, Japan). p-Nitrophenyl-a-D-glucopyr-
anoside was purchased from Wako Pure Chemistry (Osaka, Japan).
All solvents were purchased from Kanto Chemical (Tokyo, Japan).
tography (CH2Cl2/MeOH, 9:1, v/v) on silica gel to afford 5-O-trityl-
arabinitol (1.29 g, 64.0%). To a stirred solution of 5-O-trityl- -ara-
binitol (500 mg, 1.27 mmol), linoleoyl chloride (490 l, 1.52 mmol)
D-
D
3.2. Fungus materials
m
in dichloromethane (15 ml) was added dimethylaminopyridine
(8 mg, 0.065 mmol), and stirred at room temperature for 18 h. The
solvent was evaporated under reduced pressure to give pale yellow
solid. The obtained solid was column chromatographed on SiO2
The fruiting bodies of H. erinaceum were collected in Hokkaido,
Japan, in January 2010 and identified by the biotechnology labo-
ratory at Kinki University.

2010
M. Miyazawa et al. / Tetrahedron 68 (2012) 2007e2010
with CH2Cl2/MeOH (9:1, v/v) to give 1-linoleoyl-5-O-trityl-
D
-ara-
3.10. Compound 4
binitol (396 mg, 47%). 1-Linoleoyl-5-O-trityl- -arabinitol (486 mg,
D
1 mmol) was added to a mixture of ethylacetate (4 ml) and formic
acid (2 ml) and stirred at room temperature for 30 min. The re-
action mixture was diluted with ethylacetate (10 ml), after neu-
tralization with saturated sodium hydrogencarbonate solution,
extracted with ethylacetate. The solvent was removed in vacuo and
the remaining residue subjected to chromatography (silica gel, ethyl
Hericene A: white powder; mp 41e42 ꢁC; IR nmax (KBr, cmꢀ1):
3450, 1730, 1625, 1572; HREIMS m/z: 556.4132 (calcd forC35H56O5:
556.4128, Mþ). 1H and 13C NMR shown in Table 1.
3.11. Compound 5
4-[30,70-Dimethyl-20,60-octadienyl]-2-formyl-3-hydroxy-5-
methyoxybenzylalcohol: yellow oil; IR nmax (film, cmꢀ1): 3364,
1620; HREIMS m/z 318.4149 (calcd for C19H26O4: 318.4144, Mþ). 1H
and 13C NMR shown in Table 1.
acatate/acetone¼80:20) to afford 1-
D-arabinitol-monolinoleate
(200 mg, 58%) as white solid. Furthermore, 1-D-arabitniol-mono-
linoleate was crystallized from hexane/acetone.
3.6. Bioassay
3.12.
D-Arabinitol
3.6.1.
activity was determined using the modified version of the method
according to Li et al.25
-Glucosidase (25 l, 0.2 U/ml), 25 l of var-
ious concentrations of samples, and 175 l of 50 mM sodium
phosphate buffer (pH 7.0) were mixed at room temperature for
10 min. The reaction was started by the addition of 25 l of 2.5 mM
-glucopyranoside. The reaction mixture was in-
a-Glucosidase inhibitory activity. a-Glucosidase inhibitory
[
a
]
D ꢀ6.8 (c 1.25, MeOH). 1H NMR (D2O 400 MHz): 3.45 (1H, dd,
J¼8.0, 2.0 Hz, H-3), 3.50e3.54 (3H, m, H-1a, H-1b, H-5a), 3.62 (1H,
m, H-4), 3.70 (1H, dd, J¼12.0, 2.0 Hz, H-5b), 3.80 (1H, m, H-2). 13C
NMR (D2O, 100 MHz): 65.6 (C-5), 65.7 (C-1), 72.9 (C-2), 73.1 (C-3),
73.6 (C-4).
a
m
m
m
m
p-nitrophenyl-a-D
cubated for 10 min at 37 ꢁC. The activities of glucosidase were
detected in 96-well plate, and the absorbance was determined at
405 nm (for p-nitrophenol) in a microplate reader (Corona Electric
3.13. Methyl linoleate
MS (EI) m/z (%) 294[M]þ (12), 263 (9), 164 (7), 150 (9), 136 (9),
124 (11), 123 (13), 110 (21), 109 (26), 96 (37), 95 (57), 81 (87), 67
(100), 55 (60), 41 (56). RI: 2093.
Co., Ltd). Control sample contained 25 ml DMSO in place of test
samples. Percentage of enzyme inhibition was calculated as
(1ꢀB/A)ꢃ100, where A represents absorbance of control without
test samples, and B represents absorbance in presence of test
samples. All the tests were run in duplicate. Acarbose and 1-
deoxynojirimycin were used as the positive control in this study.
References and notes
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The fifty percent inhibitory concentration (IC50) values were
expressed as meanꢂSD, (n¼3).
3.7. Compound 1
Isohericerin: colorless prisms; mp 157e158.5 ꢁC; IR nmax (KBr,
cmꢀ1): 3419, 1646, 1589; HREIMS m/z: 419.2470 (calcd for
C27H33O3N: 419.2480, Mþ). 1H and 13C NMR shown in Table 1.
3.8. Compound 2
N-De phenylethyl isohericerin: amorphous powder; mp
171e174 ꢁC; IR nmax (KBr, cmꢀ1): 3263, 1625, 1593; HREIMS m/z:
315.1829 (calcd for C19H25O3N: 315.1759, Mþ). 1H and 13C NMR
shown in Table 1.
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