Cytotoxic triterpenoids from Ganoderma lucidum

Article abstract of DOI:10.1016/j.phytochem.2010.06.005

A systematic study of the metabolites in Ganoderma lucidum led to isolation of 43 triterpenoids, six of them (1-6) are hitherto unknown. The structures of the latter were elucidated on the basis of spectroscopic studies and comparison with the known related compounds. All of the compounds were assayed for their inhibitory activities against human HeLa cervical cancer cell lines. Some compounds exhibit significant cytotoxicity, and their structure-activity relationships are discussed.

Full text of DOI:10.1016/j.phytochem.2010.06.005

Phytochemistry 71 (2010) 1579–1585
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Cytotoxic triterpenoids from Ganoderma lucidum
Chun-Ru Cheng, Qing-Xi Yue, Zhi-Yuan Wu, Xiao-Yi Song, Si-Jia Tao, Xiao-Hui Wu, Ping-Ping Xu,
*
**
Xuan Liu, Shu-Hong Guan , De-An Guo
Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology,
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Guo Shoujing Road 199, Zhangjiang Hi-Tech Park,
Shanghai 201203, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A systematic study of the metabolites in Ganoderma lucidum led to isolation of 43 triterpenoids, six of
them (1–6) are hitherto unknown. The structures of the latter were elucidated on the basis of spectro-
scopic studies and comparison with the known related compounds. All of the compounds were assayed
for their inhibitory activities against human HeLa cervical cancer cell lines. Some compounds exhibit sig-
nificant cytotoxicity, and their structure–activity relationships are discussed.
Received 28 November 2009
Received in revised form 28 February 2010
Available online 6 July 2010
Keywords:
Ganoderma lucidum
Triterpenoids
Ó 2010 Elsevier Ltd. All rights reserved.
Cytotoxicity
Structure and activity relationship
1. Introduction
launched a systematic study to investigate the chemical constitu-
ents in the CH2Cl2-soluble extract from G. lucidum. In the present
Ganoderma lucidum (Fr.) P. Karst. belongs to the family of Gano-
dermataceae (Basidiomyetes) (Ziegenbein et al., 2006), and whose
clinical use can be traced back to 100 BC (Leung and Lin, 2002).
With its beautiful legends, G. lucidum was believed by the ancient
people to cure many kinds of diseases, and it was considered as an
elixir that could revive the dead. In modern times, the mystery of G.
lucidum arose the interest of scientists all over the world, and the
publications and patents of G. lucidum have increased every year
(Boh et al., 2007). It was reported that G. lucidum possessed activ-
ities including anti-tumor (Liu et al., 2009; Yue et al., 2007, 2008;
Stanley et al., 2005; Sliva, 2006; Müller et al., 2006), antimicrobial
(Yoon et al., 1994; Wang and Ng, 2006), antiviral (especially anti-
HIV activities) (Min et al., 1998), and antiaging activities (Shieh
et al., 2001).
Triterpenoids are typical chemical constituents in G. lucidum,
and have an important role in the pharmacological effects de-
scribed above. Furthermore, due to the unique structures, they
were very important in the chemotaxonomy of genus Ganoderma.
Since the first triterpenoid of ganoderic acid A was reported by
Kubota et al. (1982), more than 150 compounds had been sepa-
rated from Ganoderma spp. (Boh et al., 2007). Even today, the
number of the new compounds identified from it seemed unend-
ingly to increase. In order to search for bioactive metabolites, we
study, 43 triterpenoids were isolated, including six new com-
pounds. Herein, we described the structural elucidation and cyto-
toxic assay of these compounds.
2. Results and discussion
Phytochemical study of the CH2Cl2 extract of G. lucidum led to
the isolation of 43 triterpenoids, including 6 new triterpenoids
(Fig. 1) and 37 known compounds (Table 3). The known com-
pounds were identified as ganodermadiol (7) (Arisawa et al.,
1986), ganoderic acid DM (8) (Wang et al., 1997), ganoderenic acid
F (9) (Nishitoba et al., 1989), ganodermanondiol (10) (Fujita et al.,
1986), lucidadiol (11) (González et al., 1999), 15
-lanosta-7,9,24(E)-triene-26-oic acid (12) (Li et al., 2006),
,26-dihydroxy-5 -lanosta-7,9,24(E)-trien-3-one (13) (Gon-
zález et al., 2002), lucidumol A (14) (Min et al., 1998), 3b-hydro-
xy-5 -lanosta-7,9,24(E)-trien-26-oic acid (15) (Li et al., 2006),
3b-hydroxy-7-oxo-5 -lanosta-8,24(E)-dien-26-oic acid (16) (Li
a-hydroxy-3-oxo-
5a
15a
a
a
a
et al., 2006), ganodermanontriol (17) (Fujita et al., 1986), ganode-
riol F (18) (Nishitoba et al., 1988a), lucideric acid A (19) (Kikuchi
et al., 1986a), ganoderic acid D (20) (Kohda et al., 1985), lucidone
A (21) (Nishitoba et al., 1988b), ganolucidic acid E (22) (Nishitoba
et al., 1988a), ganoderic acid F (23) (Komoda et al., 1985), gano-
derenic acid D (24) (Komoda et al., 1985), ganoderic acid E (25)
(Kikuchi et al., 1985a), ganoderic acid J (26) (Nishitoba et al.,
1985c), ganoderic acid B (27) (Kubota et al., 1982), ganoderic acid
* Corresponding author. Tel.: +86 21 50271516; fax: +86 21 50272789.
** Corresponding author. Tel.: +86 21 50271516; fax: +86 21 50272789.
E-mail addresses: shuhong_guan@163.com (S.-H. Guan), gda5958@163.com
(D.-A. Guo).
A
(28) (Kubota et al., 1982), 7b,12b-dihydroxy-3,11,15,23-
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.06.005

1580
C.-R. Cheng et al. / Phytochemistry 71 (2010) 1579–1585
O
O
H
HO
HO
H
O
O
O
O
OH
OH
O
OH
O
O
O
O
O
O
O
H
H
O
OH
H
2
1
3
O
O
O
O
O
OH
O
O
R3
5 R1 = O
R2 = O
R3 = CH2CH3
R3 = CH3
OH
H
OH
H
6 R1=
R2=
O
H
R1
R2
H
4
Fig. 1. Structures of compounds 1–6.
tetraoxo-5
a
-lanosta-8-en-26-oic acid (29) (Nishitoba et al.,
and H-19, H-18, indicating that H-11 was on the b-orientation
same as H-19 and H-18. Thus, the structure of 1 was assigned as
1985b), 12b-hydroxy-3,7,11,15,23-pentaoxo-5
a-lanosta-8-en-26-
oic acid (30) (Komoda et al., 1985), ganoderenic acid B (31) (Komo-
da et al., 1985), methyl ganoderate H (32) (Kikuchi et al., 1985a),
methyl ganoderate B (33) (Kohda et al., 1985), 12b-acetoxy-
11a-hydroxy-3,7-dioxo-5a-lanosta-8,24(E)-dien-26-oic acid.
Compound 2 was isolated as a white powder, with ½a D23
ꢁ 14:0
ꢀ
(c 0.1, CHCl3). Its HREIMS spectrum showed the molecular ion peak
at m/z 484.3187 corresponding to the molecular formula C30H44O5.
3b,7b-dihydroxy-11,15,23-trioxo-5a-lanosta-8,20-dien-26-oic
acid (34) (Yang et al., 2007), ganolucidic acid A (35) (Kikuchi et al.,
1985b), methyl lucidenate C (36) (Nishitoba et al., 1985a), ganod-
eric acid H (37) (Kikuchi et al., 1985a), ganoderic acid AM (38)
(Lin et al., 1993), ganoderic acid T-Q (39) (Lin et al., 1988),
The UV spectrum of 2 indicated the presence of an a, b-unsaturated
carbonyl group [kmax (CHCl3) 248 nm]. The 1H NMR spectrum of 2
showed the presence of a vinyl proton [dH 6.89 (t, J = 7.0 Hz)], a sec-
ondary methyl group [dH 0.99 (d, J = 6.0 Hz)], a vinyl methyl (dH
1.84) and five tertiary methyls (dH 0.87, 0.92, 1.09, 1.15, 1.59).
The 13C NMR spectrum of 2 suggested the presence of two carbonyl
groups (dC 214.9 and dC 199.8) and a tetra-substituted double bond
(dC 139.7 and 199.8). The 1H and 13C NMR spectra of 2 were very
similar to those of 1 except for a minor difference. The HMBC
and HSQC spectra suggested that compound 2 had the same overall
skeleton as compound 1. Analysis of the ROESY spectrum estab-
lished that the stereochemistry of these two compounds was dif-
ferent at position 11. There was no correlation observed between
3b,7b,15
a
-trihydroxy-11,23-dioxo-5
a
-lanosta-8-en-26-oic
acid
(40) (Hirotani and Furuya, 1986), ganoderic acid K (41) (Morigiwa
et al., 1986), ganoderic acid G (42) (Kikuchi et al., 1985b), gano-
derenic acid A (43) (Komoda et al., 1985). The structures of the
known compounds were identified by comparison of their spectro-
scopic data with those reported in the literature.
Compound 1 was isolated as a white powder, with ½a D23
þ 13:3
ꢀ
(c 0.33, CHCl3). Its HREIMS spectrum gave a molecular ion peak
at m/z 484.3187 corresponding to the molecular formula
C
30H44O5. The 1H NMR spectrum of 1 was indicative of five tertiary
H-11 and H-18, H-19, indicating that the H-11 might be in an a-
methyls (dH 0.69, 1.11, 1.12, 1.14, 1.41), a vinyl methyl (dH 1.84)
and a secondary methyl [dH 0.96 (d, J = 6.0 Hz)] group, an oxyme-
thine proton [dH 4.53 (dd, J = 5.2, 9.2 Hz)] and one vinyl proton
[dH 6.89 (t, J = 7.2 Hz)], respectively. The 13C NMR and DEPT spectra
exhibited the presence of 23 sp3 carbons due to seven methyls,
eight methylenes, four methines including an oxymethine. Com-
parison of these spectroscopic data with those of ganoderic acid
DM (Wang et al., 1997), suggested that the skeleton and side-chain
moiety of 1 should be the same except for the presence of an extra
hydroxy group. The UV spectrum of 1 exhibited an absorption
orientation. Fortunately, we had established compound 1 had a
b-orientation at position H-11, so H-11 in compound 2 must be
in the
a-orientation. The relative configuration at H-11 of com-
pound 2 was also supported by comparing the 1H NMR spectra
of the two compounds. Since the hydroxyl group in compound 2
was on the same face as Me-18 and Me-19, the proton signals of
H-18 and H-19 moved downfield. Therefore, compound 2 was
established as 11b-hydroxy-3,7-dioxo-5a-lanosta-8,24(E)-dien-
26-oic acid.
Compound 3 was isolated as a colorless gum, ½a D22
þ 85:0 (c
ꢀ
maximum at 248 nm, suggesting the presence of an
a, b-unsatu-
0.14, CHCl3). Its HREIMS spectrum displayed a molecular ion peak
at m/z 570.2829 corresponding to the molecular formula C32H42O9.
Its UV spectrum exhibited an absorption maximum at 246 nm,
rated carbonyl group. The location of the carbonyl group was con-
firmed by the analysis of its HMBC spectrum. In the HMBC
spectrum, there were correlations between the proton signals at
dH 2.59 (H-6, dd, J = 14.8, 15.2 Hz), 2.40 (H-6, dd, J = 3.2, 15.6 Hz)
and the carbon resonance at dC 199.4 (C-7), and between the pro-
ton signals at dH 1.41 (H-19, 3H, s), 2.50 (H-12) and the carbon sig-
nal at dC 158.7 (C-9), and between the proton resonance at dH 1.14
(H-30, 3H, s) and the carbon signal at dC 142.1 (C-8). The above evi-
dence indicated the presence of a 7-ketone-8-ene structural frag-
ment. The attachment of the hydroxy group to C-11 was
confirmed by the HMBC cross-peaks between the proton signal
at dH 4.53 (H-11, dd, J = 5.2, 9.2 Hz) and the carbon resonances at
dC 158.7 (C-9), dC 142.1 (C-8), dC 44.6 (C-12), dC 40.1 (C-10). The rel-
ative configuration of 1 was determined by analyzing the ROESY
spectrum. Key ROESY correlations were observed between H-11
suggesting the presence of
a, b-unsaturated carbonyl group. The
1H and 13C NMR spectra resembled ganoderenic acid D (Komoda
et al., 1985), except for the proton (dH 5.71(s), 2.11(s)) and carbon
(dC 78.5, 170.6, 20.6) resonances which was a characteristic of an
acetoxyl group substituted on a carbon atom where two adjacent
carbon atoms bear no proton (Komoda et al., 1985). The position
of the acetoxyl group was further confirmed by the correlation be-
tween H-12 and C-31 in the HMBC spectrum. The position of the
alkene in the side-chain was confirmed by analysis of the HMBC
spectrum. In the HMBC spectrum, there were correlations between
the proton signals at dH 3.25 (H-17, t, J = 10.4 Hz), 2.15 (H-21, s)
and the carbon resonances at dC 154.1 (C-20), dC 126.1 (C-22),
and between the proton signal at dH 6.12 (H-22, s) and the carbon

C.-R. Cheng et al. / Phytochemistry 71 (2010) 1579–1585
1581
resonances at dC 154.1 (C-20), dC 197.8 (C-23). Structure 3 was fur-
ther confirmed by the reaction of 3 with NaOH solution (1%) in
aqueous methanol to give the deacetylated product 29 (a known
compound). While the absolute stereochemistry at position C-25
was uncertain, all of the natural products (confirmed by X-ray)
with this kind of side-chain have been reported to possess R config-
uration in position C-25. Thus, compound 3 was provisionally as-
the HREIMS. The UV spectrum exhibited maximum absorption at
251 nm. Comparing the 1H NMR and 13C NMR spectra with known
compounds, compound 5 had almost the same chemical shifts as
those of ganoderic acid F (Komoda et al., 1985; Kikuchi et al.,
1986b), except for an additional O-CH2CH3 group and upfielded
esterified carboxylic carbon at dC 175.6. This evidence indicated
that 5 was the ethyl esterified derivative of ganoderic acid F. Thus,
compound 5 was characterized as 12b-acetoxy-3,7,11,15,23-pen-
signed to be 12b-acetoxy-7b-hydroxy-3,11,15,23-tetraoxo-5a-
lanosta-8,20-dien-26-oic acid.
taoxo-5a-lanosta-8-en-26-oic acid ethyl ester.
Compound 4 was isolated as a white powder with a ½a D22
ꢀ
þ 23:0
Compound 6 was obtained as a white powder, with ½a D25
þ 68:0
ꢀ
(c 0.14, CHCl3). Its molecular formula of C27H40O4 was established
from HREIMS ([M]+ at m/z 428.2923, calcd. 428.2927) with 8 de-
grees of unsaturation. The UV spectrum exhibited an absorption
(c 0.32, CHCl3). Its molecular formula was determined as C33H48O9
by HREIMS (m/z, 588.3347, calcd. 588.3298). It showed an absorp-
tion band at 252 nm due to an a, b-unsaturated carbonyl group in
maximum at 251 nm, suggesting the presence of an
a, b-unsatu-
the UV spectrum. The 1H and 13C NMR spectra of 6 were similar to
those of ganoderic acid K (Kikuchi et al., 1986b; Morigiwa et al.,
1986), except that an additional oxygenated methyl was present
and the carboxylic carbon moved upfield. Thus evidence indicated
that 6 was the methyl esterified derivative of ganoderic acid K.
Thus, structure 6 was determined as 3b,7b-dihydroxy-12b-acet-
rated double bond. The 1H NMR spectrum of 4 showed the pres-
ence of five tertiary methyl groups (dH 0.68, 0.93, 1.09, 1.12,
1.33) and a secondary methyl group [dH 0.93 (d, J = 6.0 Hz)]. The
13C NMR spectrum exhibited two carbonyl groups (dC 214.7,
198.1), a tetra-substituted double bond (dC 139.5, 162.7) and a car-
boxylic acid group (dC 178.1). Comparing the NMR spectroscopic
data with the known compounds, compound 4 had the same skel-
eton with that of ganoderic acid DM (Wang et al., 1997) and the
same side-chain as that of lucideric acid A (Kikuchi et al., 1986a).
The two parts of the structures were connected at C-20 and C-17,
this being supported by the correlation between H-17 and C-20
in the HMBC spectrum. Therefore, compound 4 was assigned as
oxy-11,15,23-trioxo-5a-lanosta-8-en-26-oic acid methyl ester.
Compounds 1–43 were found only in the genus Ganoderma.
Therefore, they could be used as marker compounds to distinguish
genus Ganoderma from other genus, which is significant for the
quality control of these herbal medicines.
The cytotoxicity of the isolated triterpenoids against human
HeLa cervical cancer cells is summarized in Table 3. Among the
4,4,14
a
-trimethyl-3,7-dioxo-5
a
-chol-8-en-24-oic acid.
compounds examined, 15
en-3-one (13), lucidadiol (11) and ganoderiol F (18) showed strong
cytotoxic activities (IC50 1, 5 and 8 M, respectively). The new
compounds (2, 4 and 5) and the known compounds (10, 12, 14,
a,26-dihydroxy-5a-lanosta-7,9,24(E)-tri-
Compound 5 was separated as a white powder, with ½a D22
ꢀ þ
136:0 (c, CHCl3). It possessed the molecular formula of C34H46O9
(m/z 598.3145, calcd. 598.3142) as evidenced from analysis of
l
Table 1
1H NMR spectroscopic data for compounds 1–6.a
Position
1
1
2
3
4
5
6
2.18m, 2.30m
1.88m, 2.80m
1.38m,
2.80m
2.42m,
2.58m
1.82m, 2.14m
1.72ddd(5.6, 8.8, 14.8),
2.73ddd(4.0, 9.6, 14.4)
2.46m, 2.47m
0.92m, 2.61ddd(3.6, 7.2,
13.6)
1.65m
2
2.46m, 2.70ddd(6.4, 14.4, 2.42m, 2.82m
14.8)
2.50m, 2.70ddd(6.8,
10.4, 15.8)
3
5
6
3.19dd(5.2, 10.8)
0.89m
2.24m, 1.68m
2.26dd(3.2, 14.4)
2.40dd(3.2, 15.6),
2.59dd(14.8, 15.2)
2.07dd(3.2, 14.0)
2.37dd(3.6, 16.8),
2.67dd(14.4, 16.8)
1.52m
1.76m,
2.18m
2.14dd(14.0, 3.2)
2.38m, 2.54dd(16.0,
14.4)
2.31m
2.78m, 2.49m
7
11
12
4.88t(8.8)
4.79t(8.8)
5.60s
4.53dd(5.2, 9.2)
1.86dd(5.2, 13.6),
2.50dd(4.0, 8.4)
1.61m, 2.08m
4.61d(7.2)
2.36m
1.28m, 1.82m
1.91d(14.8), 2.30dd(15.2, 5.71s
5.66s
7.6)
1.78m, 2.10m
1.98m, 1.41m
15
16
1.42m, 2.02m
1.72m, 2.10m
1.98m, 1.28m
2.67dd(8.4,
11.2)
3.25t(10.4)
1.02s
1.34s
1.90dd(8.0, 18.0), 2.79dd(8.4,
18.8)
2.57m
0.85s
1.32s
2.30m
0.98d(6.0)
2.31m, 2.41m
2.12dd(9.6, 19.6),
2.68dd(8.0, 19.2)
2.47m
0.95s
1.25s
2.44m
0.96d(5.6)
2.28dd(8.4, 16.0), 2.43m
17
18
19
20
21
22
23
24
1.58m
0.69s
1.41s
1.40m
0.96d(6.0)
1.18m, 1.56m
2.10m, 2.23m
6.89t(7.2)
1.46m
0.92s
1.59s
1.46m
0.99d(6.0)
1.19m, 1.58m
2.12m, 2.24m
6.89t(7.0)
1.48m
0.68s
1.33s
1.46m
0.93d(6.0)
1.36m, 1.88m
2.30m, 2.41m
2.15s
6.12s
2.55m,
2.92m
2.98m
1.23d(6.8)
1.12s
2.42m, 2.82dd(8.4, 17.6)
2.45m, 2.83dd(8.8, 17.6)
25
27
28
29
2.92m
1.17d(7.2)
1.13s
1.11s
1.80s
2.95m
1.17d(7.2)
1.02s
0.84s
1.48s
1.84s
1.12s
1.11s
1.14s
1.84s
1.09s
1.15s
0.87s
1.12s
1.09s
0.93s
1.11s
1.54s
30
CH3CO
–
2.11s
2.24s
2.25s
4.12q(4.2)
OCH2CH3
OCH2CH3
–
1.24t(7.2)
3.66s
–OCH3
a
Recorded at 400 MHz in CDCl3, dH in ppm, J in Hz.

1582
C.-R. Cheng et al. / Phytochemistry 71 (2010) 1579–1585
15, 17, 20, 22 and 32) exhibited moderate activities. The other
compounds showed weak inhibition against the human HeLa cer-
vical cancer cells.
4.3. Extraction and isolation
The air-dried and powdered fruit bodies of G. lucidum (2 kg)
were extracted with EtOH-H2O (10 L ꢂ 3, 95:5 v/v). The crude ex-
tract (924 g) was washed with petroleum ether (60–90 C) to re-
move fatty acids, then extracted with CH2Cl2 (1.5 L ꢂ 5) to give
the total triterpenoids fraction (54 g). An aliquot of the CH2Cl2 ex-
tract (35 g) was applied to a silica gel column (400–600 mesh)
eluted successively with CHCl3–MeOH (200:1–1:1 gradient sys-
tem) to obtain 5 fractions. Fraction 2 was subjected to a silica gel
column eluted with petroleum ether–EtOAc (10:1–1:1 gradient
system) to afford six fractions, F21–F26. F21 was subjected to
Sephadex LH-20 CC (petroleum ether–CHCl3–MeOH, 2:1:1), then
recrystallized to afford ganoderic acid DM (100 mg). F22 was ap-
After the summary of the cytotoxic assay results, it was found
that the ganoderic alcohols showed stronger activities than ganod-
eric acids, hence an hydroxy group substituted in position 26 may
be a very important structural feature for cytotoxic activity. How-
ever, the increase in number of the hydroxy groups in the side-
chain, could result in a decrease of activity. This tendency also oc-
curred in the ring skeleton. The more hydroxy groups, the lower
the inhibitory activity.
Comparing the activities of compound 32 with 37, compound
33 with 27, compound 5 with 23, it was found that esterified form
of the carboxylic acid group in the side-chain would slightly in-
crease the activity.
plied to
a Sephadex LH-20 (petroleum ether–CHCl3–MeOH,
As shown in the Table 3, most of the active compounds contained
a carbonyl group in position 3; hence, we might conclude that C-3
carbonyl group was crucial for the cytotoxic property. Generally,
compounds with double bonds placed at positions 7 and 9 exhibited
higher activities than that harboring the 7-ketone-8-ene structure.
One strange phenomenon was that acetylation of 15-OH would re-
sult in a decrease in cytotoxicity (comparing compound 12 with 39).
2:1:1), and then further purified by semipreparative HPLC
(MeOH–H2O, 90:10) to afford ganodermanondiol (20 mg) and gan-
oderic acid T-Q (25 mg). F23 was recrystallized to obtain lucidadiol
(9 mg), F24 was extensively subjected to silica gel CC (petroleum
ether–CHCl3–Me2CO, 8:1:1), Sephadex LH-20, and then further
purified by semipreparative HPLC to give ganoderol B (18 mg),
lucidumol
A
(30 mg) and 15a-hydroxy-3-oxo-5a-lanosta-
7,9,24(E)-trien-26-oic acid (23 mg). F25 was purified by semipre-
parative HPLC (MeOH-H2O, 70:30, detection wavelength, 252 nm)
3. Conclusions
to give 3b-hydroxy-5a-lanosta-7,9,24(E)-triene-26-oic acid (3 mg)
and 15 ,26-dihydroxy-5
a
a
-lanosta-7,9,24(E)-trien-3-one (6 mg).
In conclusion, 43 triterpenoids were separated from G. lucidum,
including 6 new compounds. The chemical structures of the new
compounds were elucidated on the basis of spectroscopic studies.
All of the compounds were assayed for their cytotoxicity against
human HeLa cervical cancer cell lines, and the structure–activity
F26 was applied to a succession silica gel column (400–600 mesh,
CHCl3-MeOH, 100:1-10:1 gradient system) and Sephadex LH-20
(PE-CHCl3-MeOH, 2:1:1) chromatography to afford 3b-hydroxy-7-
oxo-5a-lanosta-8,24(E)-dien-26-oic acid (8 mg), ganodermanont-
riol (25 mg), 6 (10 mg), ganoderiol F (7 mg) and lucideric acid A
relationships were discussed. The C-3 carbonyl group, the double
8
bonds (D7,
,
D9, 11), the type of the side-chain and the number
Table 2
of hydroxy group played an important role in the structure–activ-
ity relationships.
13C NMR spectroscopic data for compounds 1–6.a
Position
1
2
3
4
5
6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
34.8t
34.6t
214.4s
47.5s
50.8d
37.5t
199.4s
142.1s
158.7s
40.1s
65.8d
44.6t
47.6s
48.1s
32.6t
27.9t
49.7d
16.9q
19.2q
36.0d
18.4q
34.6t
25.8t
145.3d
126.8s
172.7s
11.9q
25.1q
21.6q
25.3q
34.5t
34.4t
214.9s
47.4s
51.6d
37.2t
199.8s
139.7s
160.6s
39.7s
67.2d
42.7t
43.4s
48.9s
31.9t
28.7t
48.1d
17.3q
19.5q
36.2d
18.6q
34.6t
25.8t
145.3d
126.7s
172.7s
11.9q
25.1q
21.8q
24.9q
35.1t
34.1t
35.3t
34.3t
214.7s
47.2s
50.4d
37.1t
198.1s
139.5s
162.7s
39.4s
23.8t
30.1t
44.9s
47.8s
28.5t
31.8t
48.9d
15.9q
17.9q
35.9d
18.3q
30.8t
30.9t
178.1s
33.9t
33.6t
215.1s
46.8s
50.8d
37.4t
34.4t
27.4t
78.2d
38.6s
49.1d
36.7t
66.2d
155.8s
142.9s
38.5s
192.0s
79.5d
49.6s
60.6s
216.2s
37.9t
45.2d
12.0q
18.6q
28.2d
21.9q
47.9t
207.4s
46.6t
34.6d
176.1s
17.1q
28.0q
15.4q
24.0q
170.4s
20.9q
4. Experimental
215.9s
46.8s
49.2d
27.6t
65.8d
156.7s
141.6s
38.2s
191.8s
78.5d
50.3s
4.1. General experimental procedures
Optical rotations were determined on a Perkin–Elmer 341
polarimeter, whereas UV spectra were recorded on a Shimadzu
UV-2450 spectrometer. IR spectra were recorded on a Perkin–El-
mer 577 spectrometer, whereas NMR spectra were measured on
a Bruker AM-400 spectrometer for 1H, 13C, HSQC and HMBC spec-
tra, and a Varian Inova-600 spectrometer for ROESY. EIMS (70 eV)
analyses were carried out on a Finnigan-MAT 95 mass spectrome-
ter. Analytical and semipreparative HPLC was performed on an Agi-
lent 1100 with an Agilent DAD spectrophotometer and a SB-C18
198.6s
149.8s
145.9s
39.2s
194.1s
78.9d
47.6s
58.7s
205.6s
37.8t
44.3d
12.0q
18.7q
29.4d
21.6q
48.4t
59.9s
215.1s
38.4t
50.1d
14.4q
18.1q
154.1s
20.3q
126.1d
197.8s
47.5t
34.4d
180.0s
17.0q
26.6q
21.0q
24.0q
170.6s
20.6q
column (4.6 ꢂ 250 mm, 5
lm) or Eclipse XDB-C18 column (10 ꢂ
250 mm, 5 m). All solvents used were of analytical grade (Shang-
l
hai Chemical Reagents Company, Ltd.). Silica gel (400–600 mesh,
Qingdao Haiyang Chemical Co., Ltd.) and Sephadex LH-20 gel
(Amersham Biosciences) were also used for column chromatogra-
phy (CC). Fractions were monitored by TLC and spots were visual-
ized by heating silica gel plates sprayed with 10% H2SO4 in EtOH.
207.6s
46.6t
34.7d
175.6s
17.1q
27.6q
20.3q
20.7q
170.2s
20.9q
60.7t
25.3q
21.4q
24.9q
4.2. Plant material
The fruit bodies of G. lucidum were purchased in Huang Moun-
tain (Anhui Province), People’s Republic of China. The voucher
specimen (No. 200508006) was deposited in Shanghai Research
Center for TCM Modernization, National Engineering Laboratory
for TCM Standardization Technology, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences.
CH3CO
CH3CO
OCH2CH3
OCH2CH3
OCH3
14.1q
51.9q
a
Recorded at 400 MHz in CDCl3, dC in ppm.

C.-R. Cheng et al. / Phytochemistry 71 (2010) 1579–1585
1583
(30 mg). In the same manner, F3 (5.6 g) was applied to a silica gel
column (400–600 mesh) eluted with PE (60–90 C)–Me2CO (7:1–
3:1) to afford 6 fractions, F31–F36. Each fraction was subjected
to silica gel CC and repeated semipreparative HPLC to afford
ganoderic acid D (50 mg), 1 (9 mg), 2 (5 mg), lucidone A (4 mg),
ganolucidic acid E (9 mg), 4 (4 mg), ganoderic acid F (40 mg), gano-
derenic acid D (35 mg), ganoderic acid E (22 mg), ganoderic acid J
(20 mg), ganoderenic acid F (10 mg). F4 (8.9 g) was separated by
repeated column chromatography (CHCl3-MeOH, 100:1-5:1 gradi-
ent system) and semipreparative HPLC (MeOH-H2O, 40:60, detec-
tion wavelength, 252 nm) to afford ganoderic acid B (35 mg),
ganoderic acid A (40 mg), 7b,12b-dihydroxy-3,11,15,23-tetraoxo-
5
a
-lanosta-8-en-26-oic acid (23 mg), 12b-hydroxy-3,7,11,15,23-
pentaoxo-5 -lanosta-8-en-26-oic acid, ganoderenic acid B (20 mg),
methyl ganoderate H (5 mg), 3 (5 mg), methyl ganoderic acid B
(25 mg), 12b-acetoxy-3b,7b-dihydroxy-11,15,23-trioxo-5 -lano-
a
a
sta-8,20-dien-26-oic acid (6 mg), ganolucidic acid A (5 mg), methyl
lucidenate C (4 mg), 5 (8 mg), ganoderic acid H (10 mg) and
Table 3
Cytotoxicity of compounds 1–43 against human HeLa cervical cancer cell lines.a
R7
COOR9
O
R8
R61: R7=CH2OH, R8=CH3
R62: R7=COOH, R8=CH3
R63: R7=CH2OH, R8=CH2OH
R64: R9=H
R4
R6
R5
R3
R65: R9=CH2 CH3
R66: R9=CH3
O
OH
R10
OH
R1
R2
OR11
H
R69: R11=H
R67: R10=CH3
R68: R10=CH2OH
R60: R11=CH3
R1
R2
R3
R4
R5
R6
Double bond
IC50 (uM)
9
9
9
9
9
9
8
9
9
8
9
8
8
9
8
9
1
2
3
4
5
6
7
8
@O
@O
@O
@O
@O
@O
b-OH
@O
@O
b-OH
H
@O
@O
H
@O
H
H
@O
H
@O
H
H
a
-OH
H
H
b-OAc
H
b-OAc
b-OAc
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
b-OAc
H
H
H
H
R62
R62
R64
R69
R65
R66
R61
R62
R64
R67
R61
R62
R61
R67
R62
R62
R68
R63
R69
R64
b-Ac
R62
R64
R64
R64
R64
R64
R64
R64
R64
R64
R66
R66
R64
R64
R60
R64
R64
R62
R64
R64
R64
R64
D8,
D8,
D8,
D8,
D8,
D8,
D7,
D8,
D8,
D7,
D8,
D7,
D7,
D8,
D7,
D8,
123
51
>300
48
b-OH
@O
H
@O
@O
H
H
@O
H
H
H
H
H
22
@O
H
@O
@O
H
H
@O
H
,
D20,
@O
63
b-OH
b-OH
@O
@O
@O
b-OH
@O
@O
166
147
167
258
32
5
58
1
21
59
>300
33
8
>300
22
11
,
D9,
22
9
,
,
D20,
D9,
11
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
H
11
a
a
-OH
-OH
,
,
D9,
D9,
11
@O
H
H
H
H
11
b-OH
b-OH
@O
@O
@O
@O
b-OH
@O
@O
@O
@O
@O
b-OH
@O
@O
H
H
H
H
,
D9,
D7, 8 D9,
11
8
11
H
D7,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D8,
D7,
D8,
D8,
D8,
D8,
,
D9,
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
8
9
9
9
9
b-OH
b-OH
b-OH
H
@O
b-OH
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
H
@O
@O
@O
114
31
a-OH
@O
@O
@O
>300
>300
108
>300
202
>300
>300
>300
204
64
114
137
169
101
>300
213
>300
149
>300
143
>300
22
,
D20,
@O
H
H
H
a-OH
b-OH
b-OH
b-OH
@O
b-OH
@O
b-OH
b-OH
H
b-OH
@O
@O
H
b-OH
b-OH
b-OH
b-OH
@O
a-OH
b-OH
b-OH
H
b-OAc
H
b-OAc
H
H
@O
@O
@O
@O
@O
@O
@O
22
22
b-OH
b-OH
b-OH
b-OH
@O
b-OH
b-OH
b-OH
@O
b-OH
b-OH
b-OH
@O
,
,
D20,
D20,
a-OH
@O
@O
@O
b-OAc
H
H
11
a
a
-OAc
-OH
,
,
D9,
@O
@O
@O
@O
H
b-OAc
b-OH
H
@O
@O
22
a-OH
D20,
a
The activity was shown as IC50 value, which was the concentration (uM) of tested compound that resulted in 50% inhibition of cell growth. Results were expressed as the
mean value of triplicate data points. Adriamycin was used as a positive control (IC50 = 0.58um).

1584
C.-R. Cheng et al. / Phytochemistry 71 (2010) 1579–1585
ganoderic acid AM1 (7 mg). Whereas fraction F5 (11.4 g) was sep-
arated by repeated column chromatography (CHCl3-MeOH, 100:1-
1:1 gradient system) and semipreparative HPLC (MeOH-H2O,
30:70, detection wavelength, 252 nm) to give compounds
4.4. Cytotoxicity assay
Cytotoxicity against the HeLa cells was evaluated by using the
MTT (methyl thiazole tetrazolium) method according to the proto-
cols described (Alley et al., 1988) with adriamycin as a positive
3b,7b,15
a
-trihydroxy-11,23-dioxo-5
a
-lanosta-8-en-26-oic
acid
(10 mg), ganoderic acid K (20 mg), ganoderic acid G (15 mg), gano-
derenic acid A (18 mg).
control (IC50 = 0.58 lM against HeLa cells).
Acknowledgments
4.3.1. 11
a
-Hydroxy-3,7-dioxo-5
a
-lanosta-8,24(E)-dien-26-oic acid
(1)
This work was financially supported by the National Natural
Science Foundation of China (NNSFC; Grant No. 30701077), the Na-
tional Supporting Program for Traditional Chinese Medicine from
the ministry of Sciences and Technology of China (No.
2006BAI08B03-03), the National Science and Technology Major
Project ‘‘Key New Drug Creation and Manufacturing Program”, Chi-
na (Nos. 2009ZX09308-005, 2009ZX09311-001, 2009ZX09502-
020), and The Ministry of Science and Technology of the People’s
Republic of China.
White powder; ½a D23
ꢀ
þ 13:3 (c 0.33, CHCl3); UV (CHCl3) kmax
(log e) 248 (2.33) nm; IR (dry film) mmax 3323, 2962, 2919, 1711,
1645, 1587, 1383, 1261, 1099, 1022, 955, 800 cmꢁ1; for 1H and
13C NMR spectroscopic data, see Tables 1 and 2; positive EIMS m/
z 484 [M]+ (90), 466 (16), 332 (22), 258 (22), 125 (100); HREIMS
m/z 484.3187 [M]+ (calcd. for C30H44O5, 484.3189).
4.3.2. 11b-Hydroxy-3,7-dioxo-5
a-lanosta-8,24(E)-dien-26-oic acid (2)
White powder; ½a D23
ꢀ
ꢁ 14:0 (c 0.10, CHCl3); UV (CHCl3) kmax
(log e) 248 (2.36), nm; IR (KBr) mmax 3523, 3421, 2962, 2925,
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2852, 1709, 1658, 1645, 1574, 1458, 1419, 1380, 1261, 1097,
1022, 802 cmꢁ1; for 1H and 13C NMR spectroscopic data, see Tables
1 and 2; EIMS m/z 484 [M]+ (100), 469 (26), 451 (42), 423 (27), 261
(18), 125 (28); HREIMS m/z 484.3187 [M]+ (calcd. for C30H44O5,
484.3189).
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Colorless gum; ½a D22
þ 85:0 (c 0.14, CHCl3); UV (CHCl3) kmax
ꢀ
(log e) 246 (2.39) nm; IR (dry film) mmax 3446, 2966, 2928, 1732,
1705, 1684, 1610, 1462, 1373, 1230, 1049, 951, 754 cmꢁ1; for 1H
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570 [M]+ (8), 552 (10), 493 (32), 492 (92), 464 (24), 343 (32),
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[M]+ (calcd. for C32H42O9, 570.2829).
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4.3.4. 4,4,14
a
-Trimethyl-3,7-dioxo-5
a
-chol-8-en-24-oic acid (4)
White powder; ½a D22
ꢀ
þ 23:0 (c 0.14, CHCl3); UV (CHCl3) kmax
(log e) 252 (1.79) nm; IR (KBr) mmax 3392, 2962, 2918, 1709,
1668, 1459, 1417, 1379, 1261, 1097, 1020, 800 cmꢁ1; for 1H and
13C NMR spectroscopic data, see Tables 1 and 2; EIMS m/z 428
[M]+ (68), 413 (100), 395 (42), 377 (56), 327 (24), 285 (16), 271
(16), 195 (17), 135 (17), 55 (20); HREIMS m/z 428.2923 [M]+ (calcd.
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a-lanosta-8-en-26-oic
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White powder; ½a D22
ꢀ
þ 136:0 (c 0.18, CHCl3); UV (CHCl3) kmax
acid
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556 (45), 538 (20), 510 (32), 495 (16), 441 (31), 398 (19), 380
(30), 353 (30), 318 (16), 316 (52), 302 (36), 288 (10), 255 (25),
237 (28), 209 (34), 191 (72), 158 (20), 143 (100), 115 (49), 95
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a-lanosta-8-
en-26-oic acid methyl ester (6)
Yellow gum; ½a 2D5
ꢀ
þ 68:0 (c 0.32, CHCl3); UV (CHCl3) kmax (log
e)
254 (2.04) nm; IR (dry film) mmax 3435, 2968, 2933, 2875, 1732,
1679, 1583, 1462, 1373, 1232, 1043, 758 cmꢁ1; for 1H and 13C
NMR spectroscopic data, see Tables 1 and 2; EIMS m/z 588 [M]+
(6), 560 (14), 417 (15), 306 (100), 241 (10), 191 (14), 139 (14),
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