Isolation, synthesis and biological activity of grifolic acid dervivatives from the inedible mushroom Albatrellus dispansus

Article abstract of DOI:10.3987/COM-05-10501

Grifolin, grifolic acid, grifolic acid methyl ester and a new compound grifolinol were isolated from methanolic extract of the inedible mushroom Albatrellus dispansus (Scutigeraceae). Their structures were established by a combination of 2D NMR, MS spectrum and chemical reaction. In addition, (+)- and (-)-daurichromenic acids were obtained by cyclization of grifolic acid. The inhibitory activity of TNF-α, anti HIV and antimicrobial activity of grifolic acid derivatives were examined.{A figure is presented}.

Full text of DOI:10.3987/COM-05-10501

HETEROCYCLES, Vol. 65, No. 10, 2005
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HETEROCYCLES, Vol. 65, No. 10, 2005, pp. 2431 - 2439
Received, 4th July, 2005, Accepted, 10th August, 2005, Published online, 12th August, 2005
ISOLATION, SYNTHESIS AND BIOLOGICAL ACTIVITY OF GRIFOLIC
ACID DERIVATIVES FROM THE INEDIBLE MUSHROOM
ALBATRELLUS DISPANSUS
Toshihiro Hashimotoa, Dang Ngoc Quang a,c,
Makiko Nukadab, and Yoshinori Asakawaa,*
aFaculty of Pharmaceutical Sciences, Tokushima Bunri University,
Yamashiro-cho, Tokushima 770-8514, Japan.
bFaculty of Food Culture, Kurashiki Sakuyo University, Kurashiki 710-0290 Japan.
cFaculty of Chemistry, Hanoi University of Education, 136 Xuan Thuy Road, Cau
Giay, Hanoi, Vietnam.
*Corresponding author; Tel.: +88-622-9611; Fax: +88-655-3051
E-mail: asakawa@ph.bunri-u.ac.jp
Abstract-Grifolin, grifolic acid, grifolic acid methyl ester and a new compound grifolinol
were isolated from methanolic extract of the inedible mushroom Albatrellus dispansus
(Scutigeraceae). Their structures were established by a combination of 2D NMR, MS
spectrum and chemical reaction. In addition, (+)- and (−)-daurichromenic acids were
obtained by cyclization of grifolic acid. The inhibitory activity of TNF-α, anti HIV and
antimicrobial activity of grifolic acid derivatives were examined.
1. INTRODUCTION
Since grifolin and neogrifolin were isolated from the inedible mushroom Albatrellus ovinus,1 not
only the chemical constitutions of other Albatrellus sp. but also their useful biological activities
have been reported. The previous publications showed that grifolin derivatives possess
broad-spectrum of biological activity, such as antimicrobial activity,2,3 plant growth inhibitory,4
tyrosinase inhibitory,5 anticholesteremic activities level in blood and liver,6 promotion melanin
synthesis by B16 melanoma cells,7 activity on human and rat vanilloid receptor 1 (VR1).8 In the
course of our investigation on the biological active substances from inedible mushrooms, we also
reported the antioxidant activity of neogrifolin derivatives from A. ovinus.9 In continuation, we
could obtain a large amount of the fruit bodies of A. dispansus, which allowed us to study its

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HETEROCYCLES, Vol. 65, No. 10, 2005
chemical constitutions, biological activity and also chemical conversions. The outcome of these
efforts is the subject of the current paper.
OH
OH
R
HOOC
Me
OMe
Me
OH
3
1. R = COOH
2. R = H
OR
OH
15'
14'
7'
13'
12'
ROOC
HOOC
Me
1'
9'
11'
3'
5'
7
1
5
OH
3
O
Me
OR
8
4. R = H
7. R = Me
5
OH
HOOC
Me
O
6
Figure 1. Structures of 1-7.
2. RESULTS AND DISCUSSION
2.1. Isolation and characterization of grifolin derivatives:
The methanolic extract of the fruit bodies of A. dispansus was partitioned between EtOAc and
water, then the EtOAc layer was concentrated and subjected repeatedly to silica gel,
reverse-phase (C-18) column to give grifolic acid (1),10 grifolin (2),10 grifolic acid methyl ester
(3)11 and a new compound, grifolinol (4) as described in EXPERIMENTAL.
The HRFABMS spectrum of grifolinol (4) showed the molecular ion peak at m/z 413.2318
corresponding to the molecular formula C23H34O5Na (calcd for C23H34O5Na, m/z 413.2304). The
IR spectrum revealed the absorption band of a carboxyl group attached to aromatic ring
(2660-3400 and 1615 cm-1), confirmed in the UV spectrum with maxima at 260 and 300 nm and
the chemical shift at δC 175.9. Interpretation of 2D NMR spectrum of 4 allowed to the assignment
of three vinylic methyls, two olefinic protons, one aromatic methyl, and two phenolic carbons. In
comparison of its NMR spectral data (Table 1) with those of grifolic acid10 suggested that 4 is formed

HETEROCYCLES, Vol. 65, No. 10, 2005
2433
from grifolic acid (1) by addition of one water molecule to the double bond C6’-7’ and the hydroxyl
group linked to a tertiary carbon C-7’ (δC 73.7). This is proved by the HMBC correlations between
H-14’ and C-6’, C-7’ and C-8’. Methylation of 4 (MeI/K2CO3/reflux) afforded trimethylgrifolinol (7)
with the molecular formula of C26H40O5 established by HRFABMS. Investigation of its NMR
spectrum revealed the presence of three methoxyl groups, which were located at C-4, C-6 and
C-7 due to the HMBC correlations between the protons of methoxyl groups and C-4, C-6 and C-7.
Consequently, 4 was determined to be 6’,7’-dihydro-7’-hydroxygrifolic acid as shown in figure 1
and named grifolinol.
2.2. Preparation of (±)-daurichromenic acids:
Since (+)-daurichromenic acid (5) was reported to show highly potent anti-HIV activity with an
EC50 value of 0.00567 µg/mL,12 it has been totally synthesized in five steps with 49% overall
yield13 and even better in four steps.14 However, none of them mentioned about
(-)-daurichromenic acid (6) and how to purified two isomers so far. Thus, (±)-daurichromenic
acids (5,6) were successfully prepared from grifolic acid (1), a major component from this fungus
with only one step in the presence of dichlorodicyanobenzoquinone (DDQ) in benzene and
stirred for 30 min at 90oC, in good yield (73 %). Then, (+) and (-)-daurichromenic acids were
separated by HPLC equipped with chiral column (Figure 2). Nevertheless, their spectral data are
identical, except for their optical rotations. In comparison their spectral data with those of
previous publication,12 compounds (5) and (6) were determined to be (+) and (-)-daurichromenic
acids with the optical rotation values of +30.9o and –29.9o, respectively. This is the first report of
(-)-daurichromenic acid (6).
(+)-daurichromenic acid
(5)
(-)-daurichromenic acid
(6)
Time (min)
Figure 2. Isolation of (+) and (−)−daurichromenic acids by HPLC with chiral column (flow rate
1mL/min, UV detection at 254 nm).

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HETEROCYCLES, Vol. 65, No. 10, 2005
Table 1. 1H and 13C NMR data of compounds (4) and (7) (CDCl3).
Compound (4)
δH (J in Hz)
Compound (7)
δH (J in Hz)
position
δC
δC
120.7
1
103.9
142.3
111.6
160.0
111.9
163.6
175.9
24.2
2
135.3
108.2
159.3
121.1
156.6
169.0
19.9
3
6.23 s
6.48 s
4
5
6
7
8
2.50 s
2.30 brs
1’
3.39 d (6.9)
5.25 t (6.9)
21.9
3.31 d (6.6)
5.15 tq (6.9, 1.1)
22.6
2’
121.9
137.6
39.9
123.0
134.8
40.1
3’
4’
2.01 m
1.46 m
1.46 m
1.95 t (7.4)
1.38 m
5’
21.9
22.2
6’
41.4
1.38 m
41.5
7’
73.7
72.8
8’
1.46 m
41.0
1.45 m
41.5
9’
2.01 m
22.6
2.00 q (7.4)
5.11 tq (7.1, 1.1)
22.6
10’
11’
12’
13’
14’
15’
6-OH
4-OMe
6-OMe
7-OMe
5.10 t (7.1)
124.3
131.8
25.7
124.5
131.7
25.7
1.67 s
1.60 s
1.17 s
1.79 s
11.93 s
1.61 brs
17.6
1.68 d (1.1)
1.14 s
17.6
26.6
26.8
16.1
1.74 d (1.1)
16.0
3.82 s
3.75 s
3.90 s
55.7
62.6
52.1

HETEROCYCLES, Vol. 65, No. 10, 2005
2435
2.3. Biological activity of grifolic acid derivatives:
Biological testing against human immuno-deficiency virus type 1 (HIV-1) were carried out with
MT-4 cells (1x104 cells/ml). The MT-4 cells were infected with HIV-1 and cultured in the presence
of various concentrations of 1-3. After four days incubation at 37oC, the number of viable cells
was determined.15 Accordingly, compounds (1-3) were devoid of anti-HIV activity (Table 2).
Table 2: Anti-HIV effects of grifolic acid derivatives.
Samples
EC50 (µM)
CC50 (µM)
Grifolic acid (1)
> 40
40
38
53
Grifolin (2)
> 38
Grifolic acid methyl ester (3)
(+)-Daurichromenic (5)*
> 53
0.00567 µg/ml (0.15 µM)
* Kashiwada et al., 2001.
Tumor necrosis factor-alpha (TNF-α) is one of the most important proinflammatory cytokines and
is produced mainly by activated monocytes and marcrophages.16 It induced various biological
responses including tissue injury, shock and apoptosis.16,17 TNF-α also induces the secretion of
cytokines such as interleukin-1 (IL-1), IL-6, and IL-10 and activates T-cells and other
inflammatory cells.18 Thus, inhibition of production of TNF-α by using drugs might be useful for
treatment of inflammatory diseases. The suppression of the production of TNF-α of grifolic acid
derivatives (1-3, 5 and 6) was tested and their results were illustrated in table 3. Grifolin (2)
strongly suppressed TNF-α production with IC50 values of 3.98 µM (Table 3), while others
compounds exhibited weak and similar inhibitory activity. The finding suggested that the
presence of the carboxylic group at C-1 in 1, 3 and 5, 6 decreased the inhibitory activity.
Table 3: Inhibitory activity of TNF-α of grifolic acid derivatives.
Samples
IC50 (µM)
20.3
Grifolic acid (1)
Grifolin (2)
3.98
Grifolic acid methyl ester (3)
(+)-Daurichromenic (5)
(−)-Daurichromenic (6)
28.0
27.8
28.4

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HETEROCYCLES, Vol. 65, No. 10, 2005
Previous studies repeatedly pointed out that grifolic acid derivatives possessed an interesting
antimicrobial activity.2,3 However, only a few microorganisms have been tested. In this time,
grifolic acid derivatives were tested against seven microorganisms, together with gentamicin
(standard for anti-bacteria) and nystatin (standard for anti-fungi). Their antimicrobial activities
were shown in Table 4. These samples have exceedingly good activity, in some cases three
times stronger than that of standards. Especially, K. pneumoniae, an important medicinal
pathogen was the most susceptible to the samples.
Table 4: Antimicrobial activity of grifolic acid derivatives (diameter of the zone of growth inhibition,
bactericidal or fungicidal zone in mm, including the diameter of disc, 6 mm).
Microorganism/Sample
E. coli
1
2
3
5
6
Gentamicin Nystatin
30
44
27
27
28
29
32
27
40
20
19
18
18
20
20
42
16
17
26
23
26
32
43
24
24
26
28
30
32
44
22
25
38
29
28
16
14
16
15
18
nt
nt
nt
K. pneumoniae
P. aeruginosa
S. aureus
nt
nt
S. enteritidis
A. niger
nt
18
17
C. albicans
nt
nt = not tested.
In conclusion, grifolic acid derivatives from Albatrellus dispansus possess potent inhibitory
activity of TNF-α and antimicrobial activity. (+)-Daurichromentic acids (5) demonstrated potent
anti-HIV activity could be synthesized from grifolic acid (1) with only one step. Further studies on
these biological activities of these compounds should be done, since this fungus produces a
large amount of grifolic acid derivatives.
3. EXPERIMENTAL
3.1. General
TLC was performed on silica gel plates (Kieselgel 60 F254, Merck) and reverse-phase C18 silica
gel plates (Merck). Column chromatography was carried out on silica gel 60 (0.2-0.5 mm,
0.04-0.063 mm, Merck) and reverse-phase C-18 (Cosmosil 75C18-OPN, Nacalai Tesque). HPLC
was performed on a Shimadzu liquid chromatograph LC-10AS with RID-10A and SPD-10A

HETEROCYCLES, Vol. 65, No. 10, 2005
2437
detectors. IR spectra were measured on a Perkin Elmer Spectrum One FT-IR spectrometer. UV
spectra were obtained on a Shimadzu UV-1650PC in MeOH solution. Optical rotations were
measured on a JASCO DIP-1000 polarimeter with CHCl3 as a solvent. NMR spectra were
recorded on a Varian Unity 600 (600 MHz for 1H NMR and 150 MHz for 13C NMR) using CDCl3
as solvent. Mass spectra including high-resolution FAB mass spectra were recorded on a JEOL
JMS AX-500 spectrometer.
3.2. Material
Fruit bodies of Albatrellus dispansus were collected in October 2002 in Okutama, Tokyo, Japan,
and identified by Mr. Yasuhiko Gotoh. A voucher specimen (No.GY020101) has been deposited
at the Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Japan.
3.3. Extraction and isolation
Fresh fruit bodies of Albatrellus dispansus (715.4 g) were extracted with MeOH (2 x 2L) at room
temperature for two weeks and the methanolic extract was partitioned between EtOAc and water.
The EtOAc layer was concentrated to give a residue (23.17 g), which was chromatographed on
SiO2 column, using a linear gradient of 5% EtOAc-hexane to 100% EtOAc to afford fifteen
fractions (Fr. 1-15). Fr-2 (5.5 g) was purified by reverse-phase column, eluting by H2O-CH3CN
(1:4) to afford grifolic acid (1) (2.35 g) and grifolic acid methyl ester (3) (0.65 g). Fr. 3-4 (10.4 g)
was recrystallized in n-hexane-Et2O to give grifolic acid (1) (7.92 g) as a white crystal (mp
93-94oC) and a mother liquid. The mother liquid (3.3 g) was further subjected to reverse-phase
column, H2O-CH3CN (1:4) as eluent, to obtain grifolic acid methyl ester (3) (0.17 g) and a mixture
(1.47 g) which was purified by silica gel column using hexane-EtOAc gradient to yield grifolic acid
(1) (0.61 g) and grifolin (2) (0.63 g). Fr-8 (0.31 g) was purified by reverse-phase column,
H2O-CH3CN (1:9) to give grifolinol (4) (40.1 mg).
Grifolinol (4): [α]2D0 – 4.5o (c 0.62, CHCl3); HRFABMS: m/z 413.2318 [M+Na]+, C23H34O5Na,
requires 413.2304; IR (KBr) cm-1: 2660-3400, 1615, 1267, 1087; UV λmax (EtOH) nm (logε): 210
(4.3), 221 (4.4), 260 (3.9), 300 (3.5); 1H and 13C NMR (CDCl3) (Table 1).
3.3.1. Preparation of (+)- and (−)-daurichromenic acids (5,6):
A solution of grifolic acid (600 mg) in dry benzene (60 mL) was added DDQ (550 mg). The

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HETEROCYCLES, Vol. 65, No. 10, 2005
reaction mixture was stirred at 90oC for 30 min, filtered and evaporated to give a residue (994
mg), which was subjected to reverse-phase column, using H2O-CH3CN (1:9) to afford a mixture
of 5 and 6 (463 mg). The isomer mixture (436 mg) was further separated by HPLC with chiral
column (Chiralpak AD-H, 1x25 cm, Daicel Chemical Industries, LTD, Japan), mobile phase
MeOH-AcOH (100:0.1), flow rate 1 mL/min, and UV detection at 254 nm to give
(-)-daurichromenic acid (6) (203 mg), [α]2D0 – 29.9o (c 1.03, CHCl3) and (+)-daurichromenic
(5)(208 mg), [α]2D0 + 30.9o (c 1.0, CHCl3), Rt 5.33 and 6.96 min, respectively.
3.3.2. Preparation of trimethylgrifolinol (7):
A solution of grifolinol (8.4 mg) in dry Me2CO (10 mL) was added K2CO3 (1.363 g) and MeI (1.2
mL). The reaction mixture was refluxed for 10 h and filtered over celite. The filtrate was
concentrated in vacuo to obtain a residue (30.6 mg), which was purified by silica gel column,
hexane-EtOAc gradient to yield 7 (3.4 mg) as oil. HRFABMS: m/z 455.2768 [M+Na]+,
C26H40O5Na, requires 455.2773; 1H and 13C NMR (CDCl3) (Table 1).
3.3.3. Biological tests:
Biological testing against human immuno-deficiency virus type 1 (HIV-1) were carried out with
MT-4 cells in culture as described previously.15
Inhibitory activity of TNF-α of grifolic acid derivatives was tested according to the method
reported in Hashimoto et al. (2003).19
Antimicrobial activity was assayed in analogy to a conventional plate diffusion assay as
described in reference.20
ACKNOWLEDGEMENTS
We thank Dr. M. Tanaka and Miss Y. Okamoto (TBU, Japan) for recording NMR and mass
spectra, respectively. Thanks are due to Prof. M. Baba (Faculty of Medicine, Kagoshima
University, Kagoshima, Japan), Mr. N. Radulovic (University of Niš, Serbia and Montenegro) and
Dr. M. Yamada and Dr. T. Kohno (Meiji Milk Products Co. LTD, Japan) for anti-HIV, antimicrobial
and inhibition of TNF-α assays, respectively. Our special acknowledgments are also due to Mr. Y.
Gotoh and Miss Y. Nomura for their collection and identification of the mushroom and technical
assistance.
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HETEROCYCLES, Vol. 65, No. 10, 2005
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