Anti-HIV-1 and anti-HIV-1-protease substances from Ganoderma Lucidum

Abstract

A new highly oxygenated triterpene named ganoderic acid α has been isolated from a methanol extract of the fruiting bodies of Ganoderma lucidum together with twelve known compounds. The structures of the isolated compounds were determined by spectroscopic means including 2D-NMR. Ganoderiol F and ganodermanontriol were found to be active as anti-HIV-1 agents with an inhibitory concentration of 7.8 μg ml−1 for both, and ganoderic acid B, ganoderiol B, ganoderic acid C1, 3β-5α-dihydroxy-6β-methoxyergosta-7,22-diene, ganoderic acid α, ganoderic acid H and ganoderiol A were moderately active inhibitors against HIV-1 PR with a 50% inhibitory concentration of 0.17–0.23 mM.

Full text

ANTI-HIV-1 AND ANTI-HIV-1-PROTEASE SUBSTANCES
FROM GANODERMA LUCIDUM
SAHAR EL-MEKKAWY, MESELHY R. MESELHY, NORIO NAKAMURA, YASUHIRO TEZUKA,
MASAO HATTORI,*NOBUKO KAKIUCHI,$KUNITADA SHIMOTOHNO,$
TAKUYA KAWAHATA%and TORU OTAKE%
Research Institute for Traditional Sino-Japanese Medicines, Toyama Medical and Pharmaceutical
University, 2630 Sugitani, Toyama, 930-0194, Japan, $Institute for Virus Research, Kyoto University,
53 Kawara-machi, Showgoin, Sakyo-ku, Kyoto, 606-8397, Japan and %Osaka Prefectural Institute of
Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku, Osaka, 537-0025, Japan
(Received 20 October 1997)
Key Word IndexÐGanoderma lucidum; Polyporaceae; bioactive plant products; anti-HIV-1;
HIV-1-protease; triterpene; ganoderiol F; ganodermanontriol.
AbstractÐA new highly oxygenated triterpene named ganoderic acid ahas been isolated from a methanol
extract of the fruiting bodies of Ganoderma lucidum together with twelve known compounds. The structures
of the isolated compounds were determined by spectroscopic means including 2D-NMR. Ganoderiol F and
ganodermanontriol were found to be active as anti-HIV-1 agents with an inhibitory concentration of
7.8 mgml
ÿ1
for both, and ganoderic acid B, ganoderiol B, ganoderic acid C1, 3b-5a-dihydroxy-6b-methoxy-
ergosta-7,22-diene, ganoderic acid a, ganoderic acid H and ganoderiol A were moderately active inhibitors
against HIV-1 PR with a 50% inhibitory concentration of 0.17±0.23 mM. #1998 Elsevier Science Ltd. All
rights reserved
INTRODUCTION
Over the past decade, substantial progress has been
made in de®ning strategies for the treatment of
human immunode®ciency virus (HIV)-infected dis-
ease, acquired immunode®ciency syndrome (AIDS),
where natural products can serve as a source of
structurally novel chemicals that are worth investi-
gating as speci®c inhibitors of HIV as well as its
essential enzymes, protease (PR) and reverse tran-
scriptase (RT).
The fruiting body of Ganoderma lucidum
(Japanese name: Reishi) is one of the valuable
crude drugs, which has long been used in China
and Japan as a traditional Chinese medicine or a
folk medicine for the treatment of various kinds of
diseases [1]. Several biologically active triterpenes
and sterols have been isolated from this mushroom
and proved eective as cytotoxic [2, 3], antiviral [4]
and antiin¯ammatory agents [5, 6]. Polysaccharides
and glycoproteins possessing hypoglycemic [7, 8]
and immunostimulant [9±13] activities have also
been isolated from a water extract. In the course of
our continuing search for natural products as anti-
HIV agents, a methanol extract of the fruiting
bodies was found to be moderately active for inhi-
bition of HIV-1-induced cytopathogenicity (com-
plete inhibition at 31.3 mgml
ÿ1
in MT-4 cells) and
of its essential enzyme, protease (PR) (ca. 50% inhi-
bition at 100 mgml
ÿ1
). Therefore, this extract was
selected for further fractionation. When subjected
to bioassay-guided fractionation, the extract yielded
several active compounds. This paper describes the
isolation of thirteen compounds, and their inhibi-
tory eects against HIV-1 and its enzyme PR.
RESULTS AND DISCUSSION
Isolation and structure determination of compounds
isolated from the fruiting bodies of Ganoderma luci-
dum
Bioactivity-guided fractionation of the methanol
extract enriched the anti-HIV-1 and HIV-1-PR in-
hibitory eects in two fractions B and C. Final
puri®cation of the active compounds was achieved
by repeated column chromatography and HPLC to
give thirteen compounds, 4,5,8and 9from frac-
Phytochemistry Vol. 49, No. 6, pp. 1651±1657, 1998
#1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
0031-9422/98/$ - see front matter
PII: S0031-9422(98)00254-4
*Author to whom correspondence should be addressed.
Fax +81-764-34-5060, E-mail: saibo421@ms.toyama-
mpu.ac.jp.
1651

tion B, and 1±3,6,7and 12 from fraction C. Three
compounds 10,11 and 13 were also obtained from
fraction A. The structures of the known compounds
were identi®ed on the basis of their spectroscopic
properties when compared with those reported for
ganoderic acids A (2) [14±16], B (3) [14±16], C1
(4) [16, 17] and H (5) [18, 19], ganoderiols A (6) [20],
B(7) [20] and F (8) [21], and ganodermanontriol
(9) [20], (all were previously isolated from the same
mushroom). In addition, ergosterol (10), ergosterol
peroxide (11, previously isolated from the sponge
Ascidia nigra) [22], cerevisterol (12) [23, 24] and
3b-5a-dihydroxy-6b-methoxyergosta-7,22-diene (13)
(both were previously isolated from the mushroom
Agaricus blazei) [24] were identi®ed (Fig. 1).
Compound 1was obtained as an amorphous
powder, [a]
D
+55.58(CHCl
3
). A molecular formula
of C
32
H
46
O
9
was estimated from a molecular ion at
m/z 574 [M]
+
in its EI-MS. The UV absorption at
254 nm and the IR band at 1650 cm
ÿ1
suggested the
presence of a conjugated ketone group. The
1
H NMR spectrum of 1analyzed by the aid of
1
H
Fig. 1. Structures of compounds isolated from a methanol extract of the fruiting bodies of Ganoderma
lucidum.
S. EL-MEKKAWY et al.1652

1
H COSY and HMQC experiments showed signals
for seven methyls (including two as doublet at d
0.97 and 1.17), and three methine protons at d3.27
(dd,J= 11.0 and 4.8 Hz), 4.80 (t,J= 8.5 Hz) and
5.62 (s) (Table 1). In addition, a singlet at d2.23
for an ester methyl was also seen. The
13
C NMR
and DEPT spectra demonstrated signals character-
istic for eight methyls, seven methines (including
three oxymethines at d66.2, 77.3 and 79.1), and ele-
ven quaternary carbons (including ®ve carbonyls at
d170.2, 181.0, 193.9, 199.0 and 206.1) (Table 1).
These data suggested a highly oxygenated lanos-
tane-type triterpene close to the structures of 3,5
and ganoderic acids G (14) [18] and K (15) [25].
However, the dierence in chemical shift between
C-8 and C-9 (6.1 ppm) in 1suggested that its substi-
tution pattern in rings B and C was similar to that
of 5, when compared to that reported for 5(about
6.0 ppm) and 3/14 (about 16.5 ppm). The EI-MS
(Fig. 2a) showed prominent fragment ions at m/z
514 [M-HOAc]
+
corresponding to the loss of an
acetoxyl group (as acetic acid) from the molecule,
and successive losses of 18 mass units (m/z 496 and
478) indicating the presence of two hydroxyl
groups. The fragment ions m/z 417 [a]
+
and 115
[e]
+
(resulted from the cleavage between C-22 and
C-23) suggested the same side chain as in related
ganoderic acids. Acetylation of 1with Ac
2
O-pyri-
dine yielded a triacetate (1a) (EI-MS m/z 658 [M]
+
;
two additional ester methyl proton signals at d2.05
and 2.10), thereby providing evidence for the pre-
sence of two hydroxyl groups in 1.
The precise connectivities of 1were established
by interpretation of the HMBC data summarized in
Table 1. Long-range correlations between H-5 and
C-7/C-9; H-30 and C-8; H-19 and C-9; and H-12
and C-11 con®rmed the diketone substitution at C-
7 and C-11. Correlations between H-18 and C-12,
and between H-12 and a carbonyl carbon signal at
d170.2 (IR, 1730 cm
ÿ1
) revealed the connectivity of
the acetoxyl group at C-12. Since H-28 and H-29
were shift-correlated with C-3, a hydroxyl group
was concluded to be located at C-3. On the other
hand, the
1
H-
1
H correlations between H-15 and H-
16a and H-16b led to the presence of the other hy-
droxyl group at C-15.
The relative stereochemistry of 1was con®rmed
by measuring the NOESY and NOE dierence
spectra of 1a as shown in Fig. 2b. The NOE
observed between H-3 (d4.48, dd,J= 11.3,
4.9 Hz), H-29 (d0.91) and H-5 (d1.65), con®rmed
the b-con®guration of the acetoxyl group at C-3,
which was equatorially oriented. Similarly, the b-
con®guration of the acetoxyl group at C-12 was
inferred from the correlations observed between H-
12 (d5.67) and the proton signal at d1.42 (H-30).
Appreciable enhancement of H-15 (d5.89) upon ir-
radiation of H-30, and vice versa, together with no
evidence of spatial correlations between H-15 and
H-18 (d0.89), con®rmed the b-con®guration of an
acetoxyl group at C-15 (d68.9). The NOE between
H-17 (d2.54) and both of H-12 and H-30 (in the
NOESY spectrum of 1) con®rmed the con®guration
at C-17. As regards the stereochemistry at C-25, the
absolute con®guration was suggested to be R, when
compared with that of ganoderic acid H (3, given
the name ganoderic acid C by Hirotani et al. [26])
having the same side chain which was con®rmed by
X-ray analysis. On the basis of the above ®ndings,
compound 1was determined as 12b-acetoxy-
3b,15b-dihydroxy-7,11,23-trioxo-5a-lanosta-8-en-26-
oic acid and named ganoderic acid a.
Inhibitory eects of isolated compounds on HIV-1-
induced cytopathogenicity, HIV-1-protease and HIV-
1-reverse transcriptase
Investigation of anti-HIV-1 and PR-inhibitory ac-
tivities of compounds 1-13 showed that some com-
pounds had moderate inhibitory activity (Table 2).
In the primary screening test for anti-HIV-1 ac-
tivity, compounds 8and 9were found to inhibit
HIV-1 induced cytopathic eect (CPE) in MT-4
cells with a 100% inhibitory concentration (IC)
value of 7.8 mgml
ÿ1
for both compounds, and the
IC value for both was a half of the respective cyto-
toxic concentration (CC) value.
As for HIV-1 PR inhibitory eects, the activity
was determined by analyzing the hydrolysates of a
Table 1. NMR Spectral Data of Compound 1(in CDCl
3
)
Atom
13
C
1
H HMBC
1 33.1 t2.70, 1.18
2 27.2 t1.68 (2H)
3 77.3 d3.27 dd (11.0, 4.8) C-28, C-29
4 39.0 s
5 51.2 d1.56 d(14.5) C-4, C-6, C-7, C-9, C-10,
C-19, C-28, C-29
6 36.6 t2.65, 2.54 C-5, C-7, C-8, C-10
7 199.0 s
8 145.6 s
9 151.7 s
10 40.3 s
11 193.9 s
12 79.1 d5.62 sC-11, C-13, C-14, C-18,
CH
3
CO
13 47.9 s
14 58.5 s
15 66.2 d4.80 t(8.5)
16 38.0 t2.75, 1.92
17 44.6 d2.54
18 12.1 q0.81 sC-12, C-13, C-14, C-17
19 17.9 q1.33 sC-1, C-5, C-9, C-10
20 29.4 d2.24
21 21.5 q0.97 d(6.5) C-17, C-20, C-22
22 48.5 t2.46, 2.30
23 206.1 s
24 46.6 t2.40, 2.80
25 35.1 d2.91
26 181.0 s
27 17.1 q1.17 d(6.5) C-24, C-25, C-26
28 27.8 q1.02 sC-3, C-4, C-5, C-29
29 15.5 q0.88 sC-3, C-4, C-5, C-28
30 21.2 q1.72 sC-8, C-13, C-14
CH
3
CO 170.2 s
CH
3
CO 20.9 q2.23 sCH
3
CO
Anti-HIV agents from Ganoderma lucidum 1653

synthetic substrate in the presence or absence of the
isolated compounds using a HPLC method. Of the
tested compounds, 3and 7were found to be
the most active against HIV-1 PR with an IC
50
of
0.17 mM for both compounds. Other compounds
such as ganoderiol F (8), ganoderic acid C1 (4), 3b,
5a-dihydroxy-6b-methoxyergosta-7,22-diene (13),
ganoderic acid a(1), ganoderic acid H (5) and
ganoderiol A (6) inhibited the enzyme activity to a
similar extent (IC
50
=0.18±0.32 mM).
However, all compounds examined did not show
any inhibitory activity against another essential
enzyme, HIV-1-RT, at concentrations below
0.25 mM.
In the present experiment, we found that D
7(8)
,
D
9(11)
-lanostadiene-type triterpenes had relatively
strong anti-HIV-1 activity. On the other hand,
D
8(9)
-lanostene-type triterpenes and ergostane-type
compounds 10±12 had no inhibition of HIV-1-
induced cytopathic eects.
As to HIV-1-protease, we could not obtain any
conclusive ®ndings on the structure-activity re-
lationship. Lanostane-type triterpenes showed IC
50
of 0.17±0.32 mM, while ergosterol derivatives had
no inhibitory activity. However, it was reported
that synthetic cosalane and its derivatives had an
anti-HIV-1 eect as well as inhibitory eects on RT
and PR [27]. Several triterpenes have been described
Fig. 2. (a) Proposed mass fragmentation pattern of 1. (b) Stereostructure for 1and 1a as indicated by
dierence NOE and NOESY spectra.
S. EL-MEKKAWY et al.1654

as antiviral compounds. Glycyrrhizin shows some
limited activity against a whole range of viruses
including HIV-1 [28]. Salaspermic acid [29] and sub-
erol (a lanostane-type) [30] inhibit HIV-1 in H9
cells in the upper micromolar range. Bile acid de-
rivatives are slightly active at 10
ÿ4
M against HIV-1
in MT-4 cells [31]. Betulinic acid derivatives
(lupane-type) have been described as potent inhibi-
tors of the cytopathogenicity of HIV-1 in CEM 4
and MT-4 cells without aecting HIV-1 RT or PR
activity [32]. When compared with other triterpenes
reported, compounds 8and 9can be used as leads
to develop other related compounds with potential
anti-HIV-1 activity.
EXPERIMENTAL
General
Mps: uncorr. Optical rotations: in CHCl
3
soln at
20±268C. UV: in MeOH. IR: in KBr. EI-MS: ioniz-
ation voltage 70 eV.
1
H and
13
C NMR: 500 MHz
and 125 MHz, respectively.
Material
Fruiting bodies of Ganoderma lucidum (Leyss. ex
Fr.) Karst. (Polyporaceae) were obtained from Alps
Chemical Industries Co. (Takayama, Japan) and
Linzhi General Institute of Co. Ltd. (Tokyo,
Japan), and their specimens were deposited at the
Museum of Materia Medica, Toyama Medical and
Pharmaceutical University (Toyama, Japan).
Enzymes and chemicals
Recombinant HIV-1 PR (purity 96% by SDS-
PAGE) was purchased from Bachem
Feinchemikalien AG (Bubendort, Switzerland).
Recombinant HIV-1-RT was obtained from Eiken
Chemicals Co. Ltd. (Osaka, Japan). A template-pri-
mer, (rA)
n
Ç(dT)
12±18
for the RT assay was obtained
from Pharmacia (Uppsala, Sweden). [Methyl-H
3
]-
thymidine 5'-triphosphate (dTTP) (speci®c activity,
1.70 TBq mmol
ÿ1
) was obtained from Amersham-
Japan (Tokyo, Japan).
Isolation procedure
The chipped fruiting bodies of G. lucidum (5 kg)
was extracted with MeOH (3 20 l) at room temp.
to give 182 g of a solid extract. The MeOH extract
was suspended in 50% aq. MeOH (2 l), the organic
solvent was removed and ®ltered through a column
of Diaion HP-20 (2 l), washed with H
2
O and then
with MeOH. The MeOH eluate was evaporated in
vacuo to give 163 g of a solid extract. The MeOH
eluate (100 g) was chromatographed on silica gel,
and elution was started with hexane, CHCl
3
and
30% MeOH in CHCl
3
to give three fractions, fr. A
(18 g), fr. B (40 g) and fr. C (26 g). CC/silica gel
(C
6
H
6
±EtOAc, 6:4) of fr. A (3 g) followed by
MPLC/silica gel (C
6
H
6
±Me
2
CO, 7:3) aorded 10
(365 mg), 11 (62 mg) and 13 (10 mg). Repeated CC
of fr. B (5 g) using silica gel (C
6
H
6
±Me
2
CO, 7:3),
RP-2 (40% aq. MeOH), MPLC/Si gel (C
6
H
6
±
Me
2
CO, 7:3), and ®nally MPLC/silica gel (5%
MeOH in CHCl
3
) gave 4(8 mg), 5(9 mg), 8
(36 mg) and 9(16 mg). Similarly, repeated CC of fr.
C (4 g) yielded 1(40 mg), 2(43 mg), 3(47 mg), 6
(6 mg), 7(15 mg) and 12 (14 mg).
12b-Acetoxy-3b,15b-dihydroxy-7,11,23-trioxo-5a-
lanosta-8-en-26-oic acid (ganoderic acid a;1)
Amorphous powder, [a]
D
+558(CHCl
3
,c1.0). IR
KBr
max cm
ÿ1
: 3450 (OH), 1750 (carbonic acid C.O),
1730 (ester C.O), 1700 (C.O) and 1650sh (conju-
gated C.O). UV l
max
(log E) nm: 254 (3.8). EI-MS
m/z 574 [M]
+
, 532 [M ÿCH
2
CO]
+
, 514
[M ÿHOAc]
+
, 496 [M ÿHOAc ÿH
2
O]
+
, 478
Table 2. Inhibitory Activities of Compounds from Ganoderma lucidum against
Protease and Proliferation of HIV-1
HIV-1
HIV-PR
Compound IC (mg/ml) CC (mg/ml) IC
50
(mM)
Ganoderic acid a(1) NE >1000 0.19
Ganoderic acid A (2) (1000) >1000 >1.0
Ganoderic acid B (3) NE >1000 0.17
Ganoderic acid C1 (4) NE >1000 0.18
Ganoderic acid H (5) NE >1000 0.20
Ganoderiol A (6) NE >1000 0.23
Ganoderiol B (7) (7.8) 500 0.17
Ganoderiol F (8) 7.8 15.6 0.32
Ganodermanontriol (9) 7.8 15.6 >1.0
Ergosterol (10) NE 1000 >1.0
Ergosterol peroxide (11) NE 15.6 >1.0
Cerevisterol (12) NE 31.3 >1.0
3b-5a-Dihydroxy-6b-methoxy ergosta-7,22-diene (13). NE 15.6 0.18
IC, the minimum concentration for complete inhibition of HIV-1 induced CEP in MT-4 cells by
microscopic observation. CC, the minimum concentration for appearance of MT-4 cell toxicity by
microscopic observation. NE, not eective. (), concentration at which weak anti-HIV-1 activity
was observed.
Anti-HIV agents from Ganoderma lucidum 1655

[M ÿHOAc ÿ2H
2
O]
+
, 417 [a]
+
, 306 [g + H]
+
and
115 [e]
+
.
3b,15b-Diacetylganoderic acid a(1a)
Amorphous powder,
1
H NMR (CDCl
3
): d0.89
(3H, s,H
3
-18), 0.91 (3H, s,H
3
-29), 0.96 (3H, d,
J= 6.5 Hz, H
3
-21), 1.00 (3H, s,H
3
-28), 1.24 (3H,
s,H
3
-19), 1.24 (3H, d,J= 7.0 Hz, H
3
-27), 1.42
(3H, s,H
3
-30), 1.65 (1H, H-5), 2.05 (3H, s,CH
3
-
CO), 2.10 (3H, s,C
H
3
-CO), 2.25 (3H, s, CH
3
-CO),
4.48 (1H, dd,J= 11.3, 4.9 Hz, H-3), 5.67 (1H, s,
H-12), 5.89 (1H, t,J= 8.4 Hz, H-15).
13
C NMR: d
68.9 (C-15), 79.9 (C-3), 80.0 (C-12), 146.1 (C-8),
154.4 (C-9), 170.0, 170.5 and 170.9 (3 acetoxyl
C1O), 179.6 (C-26), 191.9 (C-11), 207.5 (C-7) and
210.3 (C-23). EI-MS m/z 658 [M]
+
, 616
[M ÿCH
2
CO]
+
, 598 [M ÿHOAc]
+
, 538
[M ÿ2HOAc]
+
, 478 [M ÿ3HOAc]
+
, 390.
Reverse transcriptase assay
The assay was performed as previously
reported [33].
Protease assay
25 ml of HIV-1-PR assay buer (Bachem HIV-1
protease assay Kit S-100) containing 2.5 mgofa
substrate, His-Lys-Ala-Arg-Val-Leu-(pNO
2
-Phe)-
Glu-Ala-NLe-Ser-NH
2
, were mixed with 2.5 mlofa
DMSO soln of test compound, then 6.25 mlof
recHIV-1-PR (0.25 mg protein) was added to the
mixture. The reaction mixture was incubated for
15 min at 378C and then stopped by addition of
2.5 ml of 10% TFA. The hydolysate and remained
substrate were quantitatively analyzed by HPLC
under the following conditions: injection volume,
5ml; column, RP-18 (4.6 150 mm, Merck), elution,
a linear gradient of acetonitrile (20±40%) in 0.1%
TFA; ¯ow rate, 1.0 ml min
ÿ1
; detection, 280 nm.
The hydrolysate and substrate were eluted at 9.44
and 4.35 min, respectively. The inhibitory activity
of the compound in the HIV-1-PR reaction
was calculated as follows: %inhibition = 100
(A
control
ÿA
sample
)/(A
control
); where Ais a relative
peak area of the hydrolysate. Under the conditions,
acetylpepstatin was used as a positive control, its
IC
50
being 0.30 mM.
Cells
The HTLV-I-carrying cell line MT-4 cells were
used. They were maintained at 378C under 5% CO
2
in RPMI-1640 medium (Flow Laboratories, Irvine,
Scotland), supplemented with 10% fetal calf serum
(FCS, Flow laboratories, North Ryde, Australia),
100 mgml
ÿ1
of streptomycin (Meiji Seika, Tokyo,
Japan) and 100 U ml
ÿ1
of penicillin G (Banyu
Pharmaceutical, Tokyo, Japan).
Virus
The LAV-1 strain of HIV-1 was obtained from
culture supernatant of MOLT-4 cells that had been
persistently infected with LAV-1.
Primary screening for anti-HIV-1activity
MT-4 cells were infected for 1 hr with HIV-1 at
TCID
50
of 0.001 per cell. Then, the cells were
washed and resuspended at 1 10
5
cells ml
ÿ1
in
RPMI-1640 medium. A 200 ml per well of the cell
suspension was cultured for 5 days in a 96-well cul-
ture plate containing various concentrations (12
doses, maximum 1000 mgml
ÿ1
and minimum
0.49 mgml
ÿ1
) of the isolated compounds. Control
assays were performed, without these compounds,
with HIV-1-infected and uninfected cultures. On
day 5, the IC of the test compound required to pre-
vent HIV-1-induced CPE completely was deter-
mined through an optical microscope and the cell
growth was examined to give the CC that reduces
the viability of MT-4 cells.
AcknowledgementsÐA part of this study was ®nan-
cially supported by Japan Health Science
Foundation (Tokyo, Japan) and Lingzhi General
Institute Co. (Tokyo, Japan).
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