Lanostane Triterpenes from the Tibetan Medicinal Mushroom Ganoderma leucocontextum and Their Inhibitory Effects on HMG-CoA Reductase and α-Glucosidase

Article abstract of DOI:10.1021/acs.jnatprod.5b00331

(Chemical Equqation Presented). Sixteen new lanostane triterpenes, ganoleucoins A-P (1-16), together with 10 known tripterpenes (17-26), were isolated from the cultivated fruiting bodies of Ganoderma leucocontextum, a new member of the Ganoderma lucidum complex. The structures of the new compounds were elucidated by extensive spectroscopic analysis and chemical transformation. The inhibitory effects of 1-26 on HMG-CoA reductase and α-glucosidase were tested in vitro. Compounds 1, 3, 6, 10-14, 17, 18, 23, 25, and 26 showed much stronger inhibitory activity against HMG-CoA reductase than the positive control atorvastatin. Compounds 13, 14, and 16 presented potent inhibitory activity against α-glucosidase from yeast with IC₅₀ values of 13.6, 2.5, and 5.9 μM, respectively. In addition, the cytotoxicity of 1-26 was evaluated against the K562 and PC-3 cell lines by the MTT assay. Compounds 1, 2, 6, 7, 10, 12, 16, 18, and 25 exhibited cytotoxicity against K562 cells with IC₅₀ values in the range 10-20 μM. Paclitaxel was used as the positive control with an IC₅₀ value of 0.9 μM. This is the first report of secondary metabolites from this medicinal mushroom.

Full text of DOI:10.1021/acs.jnatprod.5b00331

This is an open access article published under an ACS AuthorChoice License, which permits
copying and redistribution of the article or any adaptations for non-commercial purposes.
Article
pubs.acs.org/jnp
Lanostane Triterpenes from the Tibetan Medicinal Mushroom
Ganoderma leucocontextum and Their Inhibitory Effects on HMG-CoA
Reductase and α‑Glucosidase
Kai Wang,†,‡,⊥ Li Bao,†,⊥ Weiping Xiong,§,⊥ Ke Ma,† Junjie Han,† Wenzhao Wang,† Wenbing Yin,†
and Hongwei Liu*,†
†State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichenxi Road,
Chaoyang District, Beijing 100101, People’s Republic of China
‡University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
§Institute of Vegetable Research, Tibet Academy of Agriculture and Animal Science, No. 157 Jinzhuxi Road, Lasa 850000,
People’s Republic of China
S
* Supporting Information
ABSTRACT: Sixteen new lanostane triterpenes, ganoleucoins A−P (1−16), together with 10 known tripterpenes (17−26),
were isolated from the cultivated fruiting bodies of Ganoderma leucocontextum, a new member of the Ganoderma lucidum
complex. The structures of the new compounds were elucidated by extensive spectroscopic analysis and chemical transformation.
The inhibitory effects of 1−26 on HMG-CoA reductase and α-glucosidase were tested in vitro. Compounds 1, 3, 6, 10−14, 17,
18, 23, 25, and 26 showed much stronger inhibitory activity against HMG-CoA reductase than the positive control atorvastatin.
Compounds 13, 14, and 16 presented potent inhibitory activity against α-glucosidase from yeast with IC50 values of 13.6, 2.5, and
5.9 μM, respectively. In addition, the cytotoxicity of 1−26 was evaluated against the K562 and PC-3 cell lines by the MTT assay.
Compounds 1, 2, 6, 7, 10, 12, 16, 18, and 25 exhibited cytotoxicity against K562 cells with IC50 values in the range 10−20 μM.
Paclitaxel was used as the positive control with an IC50 value of 0.9 μM. This is the first report of secondary metabolites from this
medicinal mushroom.
Ganoderma species (Ganodermataceae, Polyporales) have a long
history of being used as folk medicine for the treatment and
prevention of various diseases in China and Japan.1 They contain
lanostane triterpenes, meroterpenes, alkaloids, and polysacchar-
ides as bioactive constituents. The lanostane triterpenes from
Ganoderma species presented various bioactivities, including
cytotoxicity,2 anti-inflammatory activity,3 hepatoprotective
effect,4 and inhibitory activities against aldose reductase,5 5α-
reductase,6 HMG-CoA reductase,7 and α-glucosidase.8
The Tibet plateau is usually called the “Roof of the World” with
an average altitude of 4500 m. The unique ecosystems of the Tibet
plateau, such as high-cold bush and meadows, and forests, produce a
great biodiversity of edible and medicinal mushrooms.9 People living
in Tibet have a long history of using wild mushrooms as food and folk
medicine. Among them, Ophiocordyceps sinensis is the most famous
medicinal mushroom due to its wide range of pharmacological
effects.10,11 Ganoderma leucocontextum was discovered as a new
species of Ganoderma in Tibet in 2014. G. leucocontextum is called
Zanglingzhi in the Tibet Autonomous Region and believed to possess
beneficial effects on human health.12 There have been no reports of
chemical investigation of G. leucocontextum due to its scarcity in
nature. We cultivated this new Ganoderma species in house for the
first time and conducted chemical research on the fruiting bodies
obtained. As a result, 16 new lanostane triterpenes, ganoleucoins
A−P (1−16), together with 10 known tripterpenes (17−26) were
isolated. The structures of the new compounds were elucidated by
extensive spectroscopic analysis and chemical derivation (Figure 1).
The inhibitory activity of compounds 1−26 against HMG-CoA
reductase and α-glucosidase and their cytotoxicity against K562 and
PC-3 cell lines were evaluated in vitro.
Received: April 14, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Structures of compounds 1−26.
and C-26, confirmed the planar structure of 1 (Figure 2). The
relative configuration of 1 was determined by ROESY experi-
ments. The ROESY correlations of H-5 with H-7 and H2-28,
H-17 with H3-21, H3-18 with H-12β and H-20, H3-19 with
H-12β and H3-29, H3-27 with H2‑23, and H3-30 with H-7 and
H-17 assigned the α-orientation for H-5, H-7, H-17, CH3-21,
HOCH2-28, and CH3-30, the β-orientation for CH3-18, CH3-19,
and CH3-29, and the E-geometry for the C-24/C-25 double
bond (Figure 3). The absolute configurations of 13R and 14R
were assigned on the basis of the positive and negative Cotton
effects at 245 nm (Δε = +4.2) for a π → π* transition and 354 nm
(Δε = −0.5) for a n → π* transition by the octant rule for the α,β-
unsaturated ketone group.4 Combining the relative configuration
established that the absolute configuration of 1 was determined
as 4R, 5R, 7S, 10S, 13R, 14R, 17R, and 20R.
RESULTS AND DISCUSSION
■
The ethanol extract of G. leucocontexum was partitioned between
water and ethyl acetate. The ethyl acetate-soluble fraction was
subjected to chromatographic separation using silica gel, ODS,
Sephadex LH-20, and preparative HPLC to yield 26
triterpenoids. Ten known compounds (17−26) were identified
as ganoderiol J (17),13 ganoderic acid DM (18),14 ganoderone
A (19),15 11-hydroxy-3,7-dioxo-5-lanosta-8,24(E)-dien-26-oic
acid (20),16 3,7-dioxo-8,24(Z)-tirucalladien-26-oic acid (21),17
ganoderiol F (22),18 ganoderic acid SZ (23),19 ganoderiol B
(24),20 ganoderic acid Y (25),21 and ganodermanontriol (26)22
by comparison of their spectroscopic data with the literature data.
Ganoleucoin A (1) was isolated as a colorless powder. The
molecular formula of 1 was determined to be C30H44O6 on the
basis of HRTOFMS data. The 1H NMR spectrum of 1 showed
resonances for six methyl signals [δH 0.87 (s), 0.89 (d, J =
6.6 Hz), 1.10 (s), 1.18 (s), 1.34 (s), and 1.85 (s)], one oxygenated
methylene [δH 4.10 (d, J = 11.3 Hz) and 3.39 (d, J = 11.3 Hz)],
one oxygenated methine [δH 4.38 (dd, J = 9.2, 5.6 Hz)], and
one olefinic proton [δH 6.86 (t, J = 7.3 Hz)]. The 13C NMR
spectrum of 1 exhibited 30 carbon resonances corresponding to
six methyls, nine methylenes (one oxygenated at δC 66.0), four
methines (one oxygenated carbon at δC 73.1), four quaternary
carbons, two pairs of olefinic carbons (δC 127.1, 138.0, 145.1, and
163.6), and three carbonyl carbons (δC 172.5, 198.6, and 222.8,
respectively). A lanostane tripterpenoid skeleton was deduced
for 1 based on the above NMR data (Table 1 and Table 4). The
1H−1H COSY correlations of H2-1−H2-2, H-5−H2-6−H-7 and
H2-15−H2-16−H-17−H-20/(H3-21)−H2-22−H2-23−H-24, to-
gether with the key HMBC correlations from H3-18 to C-12,
C-13, C-14, and C-17, from H3-19 to C-1, C-5, C-9, and C-10,
from H2-28 to C-3, C-4, C-5, and C-29, from H3-29 to C-3, C-4,
C-5, and C-28, from H-5 to C-3, C-7, C-28, and C-29, from H-7
to C-5, C-6, C-8, and C-9, from H2-12 to C-9, C-11, C-13, C-14,
C-17, and C-18, from H3-21 to C-17, C-20, and C-22, from H-24
to C-22, C-25, C-26, and C-27, and from H3-27 to C-24, C-25,
The molecular formula of ganoleucoin B (2) was established as
1
C30H44O7 by HRTOFMS data. Its H and 13C NMR spectros-
copic data revealed a similar structure to 1 except for an extra
oxygenated methine [δH 4.29 (s); δC 78.8] (Table 1 and
Table 4). The location of a hydroxy group at C-12 was confirmed
by the HMBC correlations from H3-18 to C-12, C-13, C-14,
and C-17 and from H-12 to C-9 and C-11 (Figure 2). The
relative configurations of H-7 and H-12 were assigned as α- and
β-orientation, respectively, on the basis of NOE correlations
of H-12 with H3-18 and H3-19 and of H-7 with H-5 and H3-30
(Figure 3). The structure of 2 was established by detailed
interpretation of its 2D NMR data. As described in 1, the positive
and negative Cotton effects at 237 and 317 nm indicated the 13R
and 14R configurations.
Ganoleucoin C (3) was assigned the molecular formula
1
C32H46O8 by HRTOFMS data. A detailed comparison of H
and 13C NMR spectral data between 3 and 2 indicated that
compound 3 was an acetylated derivative of 2 (Table 1 and
Table 4). The acetyl moiety was deduced from signals due to one
methyl group (δH 2.18, s; δC 21.2) and one carbonyl carbon (δC
170.7). The HMBC correlations from the H-12 to the carbonyl
B
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
a
Table 1. 1H NMR Spectral Data of Compounds 1−6
no.
1
1
2
3
4
5
6
b
1.71 m
2.66 m
1.59 m
2.64 m
1.61 m
2.63 m
1.49 m
2.54 m
1.45 m
2.54 m
1.58 m
2.67 m
2
2.99 dt (14.6, 8.9)
2.38 m
2.84 dt (14.8, 9.0)
2.46 m
2.85 dt (14.6, 8.9)
2.38 dd (15.8, 8.1)
1.86 m
2.77 dt (14.6, 8.9)
2.47 dd (15.9, 7.9)
1.74 m
2.77 dt (14.8, 9.0)
2.54 m
2.80 dt (14.6, 8.9)
2.45 dd (16.1, 7.9)
2.47 m
5
6
1.84 m
1.84 m
1.70 m
2.50 m
2.52 m
2.51 m
2.19 m
2.32 m
2.71 m
1.26 m
1.25 m
1.25 m
1.65 m
1.70 m
2.05 m
7
4.38 dd (9.2, 5.6)
2.45 m
4.33 dd (9.2, 5.6)
4.29 s
4.38 t (7.0)
5.62 s
4.84 t (8.4)
5.65 s
4.81 dd (9.0, 5.7)
4.37 s
12
5.70 s
2.73 m
15
16
1.76 m
1.80 m
1.99 m
2.13 m
2.10 m
2.27 m
2.68 m
2.70 m
1.77 m
1.80 m
1.78 dd (18.8, 8.1)
2.16 m
2.68 dd (19.0, 8.2)
2.41 m
2.26 m
2.05 dd (19.0, 8.2)
2.50 m
17
18
19
20
21
22
1.91 m
2.25 m
2.47 m
0.87 s
0.68 s
0.88 s
0.99 s
0.81 s
0.85 s
1.10 s
1.21 s
1.34 s
1.30 s
1.38 s
1.33 s
1.39 m
1.54 m
1.44 m
1.60m
1.99 m
1.66 m
0.89 d (6.6)
2.27 m
1.10 d (6.6)
2.27 m
0.93 d (6.5)
2.25 m
1.02 d (6.5)
2.30 m
1.16 d (6.6)
2.24 m
1.04 d (6.6)
2.31 m
2.14 m
2.15 m
2.13 dt (16.1, 7.9)
1.19 m
2.17 m
2.19 m
2.18 dt (16.1, 8.0)
1.22 m
23
24
27
28
1.17 m
1.16 m
1.17 m
1.15 m
6.86 t (7.3)
1.85 s
6.88 t (7.3)
1.84 s
6.86 t (7.0)
1.83 s
6.86 t (7.1)
1.85 s
6.86 t (7.3)
1.83 s
6.88 t (7.1)
1.85 s
4.10 d (11.3)
3.39 d (11.3)
1.34 s
4.05 d (11.4)
3.45 d (11.4)
1.32 s
4.04 d (11.2)
3.42 d (11.2)
1.32 s
4.05 d(11.3)
3.48 d (11.3)
1.30 s
4.05 d (11.4)
3.52 d (11.3)
1.29 s
4.03 d (11.1)
3.45 d (11.1)
1.31 s
29
30
2′
1.18 s
1.28 s
1.15 s
1.50 s
1.29 s
1.82 s
2.18 s
2.22 s
2.23 s
a
b
Recorded at 500 MHz in CDCl3, δH in ppm, J in Hz. “m” means multiplet or overlapped with other signals.
carbon at δC 170.7 confirmed the acylation of the hydroxy group
at C-12 (Figure 2). The NMR signal assignment was made for 3
by 2D NMR data interpretation. Similar Cotton effects observed
at 250 and 347 nm between 3 and 2 confirmed the 13R and 14R
configurations.
Ganoleucoin D (4) had a molecular formula of C32H44O9, as
determined by HRTOFMS data. The NMR data of 4 were
similar to those of 3, except for the presence of an additional
ketone moiety (δC 216.7) (Table 1 and Table 4). HMBC
correlations from H-12 to C-9, C-11, C-13, C-14, C-17, and
C-18, from H3-18 to C-12, C-13, C-14, and C-17, and from H3-30
to C-8, C-13, C-14, and C-15 indicated a carbonyl group at C-15
in 4. The structure of 4 was fully assigned by detailed elucidation
of its 2D NMR spectra (Figure 2 and Figure 3). The CD
spectrum of 4 showed similar Cotton effects to those of 1−3,
indicative of the same 13R and 14R configurations.
Ganoleucoin F (6) was determined to have a molecular
formula of C32H42O9 on the basis of HRTOFMS data. The 1H
and 13C NMR data of 6 were similar to those of 4, except for the
presence of an additional ketone group (δC 198.2) and the
absence of one oxygenated methine (δC 65.6) in 4 (Table 1 and
Table 4). The HMBC correlations from H-5 to C-3, C-4, C-6,
C-7, C-9, C-10, C-19, C-28, and C-29 and from H2-6 to C-4, C-5,
C-7, C-8, and C-10, as well as the 1H−1H COSY correlation of
H-5−H2-6, confirmed the location of a carbonyl group at C-7
(Figure 2). The relative configuration of H-12 was assigned as
β-orientation by NOE correlations of H-12 with H3-18 and
H3-19 (Figure 3). Accordingly, the structure of 6 was identified as
described in Figure 1.
Ganoleucoin G (7) possessed the molecular formula
C30H46O5 on the basis of HRTOFMS data. The NMR data of
7 were similar to those of ganoderiol F (22), except for an addi-
tional carbonyl moiety (δC 200.6) and an oxygenated methine
replacing two olefinic methines (Table 2 and Table 5). HMBC
correlations (Figure 2) from H-5 to C-3, C-4, C-6, C-7, C-9,
C-10, C-19, C-28, and C-29, from H-7 to C-5, C-6, C-8, C-9, and
C-14, from H2-12 to C-9, C-11, C-13, C-14, C-17, and C-18,
and from H3-30 to C-8, C-13, C-14, and C-15, together with the
1H−1H COSY correlations of H-5−H2-6−H-7, determined the
location of a hydroxy group at C-7 and a carbonyl group at C-11,
respectively. The relative configuration of 7 was assigned by
ROESY experiment (Figure 3). As described in 1, the absolute
configuration at C-13 and C-14 in 7 was determined to be 13R
and 14R by CD data.
The molecular formula of ganoleucoin E (5) was determined
as C30H42O8 on the basis of HRTOFMS data. The 1H and 13
C
NMR spectra of 5 resembled those of 4, except for the loss
of the acetyl group (Table 1 and Table 4). HMBC correlations
(Figure 2) from H-5 to C-3, C-7, and C-28, from H-7 to C-5, C-6,
C-8, C-9, and C-14, from H-12 to C-9, C-11, C-13, C-14, C-17,
and C-18, from H3-18 to C-12, C-13, C-14, and C-17, from H3-19
to C-1, C-5, C-9, and C-10, from H2-28 to C-3, C-4, C-5, and
C-29, and from H3-29 to C-3, C-4, C-5, and C-28 confirmed the
substitution of the hydroxy groups at C-7 and C-12 and the
carbonyl groups at C-3, C-11, and C-15 in 5, respectively. NOE
correlations of H-12 with H3-18 and H3-19 and of H-7 with H-5
and H30-30 determined the α- and β-orientation for H-7 and
H-12, respectively.
The molecular formula of ganoleucoin H (8) was determined
to be C30H44O5 on the basis of HRTOFMS data. A comparison
C
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
a
Table 2. 1H NMR Spectral Data of Compounds 7−12
no.
1
7
8
9
10
11
12
b
1.63 m
2.51 m
1.75 m
2.61 m
1.49 m
2.48 m
1.61 m
2.71 m
1.61 m
2.68 m
1.23 m
2.61m
2
2.89 dt(14.8, 9.0)
2.36 m
2.96 dt (14.8,9.0)
2.54 m
2.72 dt (15.0, 8.9)
2.31 m
2.88 dt (14.6, 8.9)
2.43 m
2.91 dt (14.6, 8.9)
2.58 m
3.01dt (14.6, 8.9)
1.69 m
5
6
1.66 m
2.25 m
2.19 m
1.58 m
2.28 m
2.33 m
1.97 m
2.53 m
2.08 m
2.32 m
2.64 m
2.78 m
1.34 m
2.37 m
1.61 m
1.80 m
2.05 m
1.88 m
7
4.37 dd (9.0, 5.6)
1.99 m
4.89 t (8.2)
1.37 m
12
2.58 m
2.40 m
1.62 m
1.83 m
1.60 m
1.67 m
0.81 s
2.72 m
2.64 m
2.00 m
2.15 m
1.64 m
1.69 m
0.82 s
2.60 t (15.1)
2.42 m
5.65 s
4.53 s
2.79 m
2.84 m
15
16
4.52 m
2.52 m
2.70 m
2.30 m
2.42 m
0.98 s
2.64 m
2.63 m
1.87 m
2.75 dd (18.3, 8.0)
2.57 m
2.71 dd (18.2, 7.9)
2.13 m
17
18
19
20
21
22
1.57 m
0.68 s
0.64 s
0.86 s
0.94 s
1.28 s
1.13 s
1.44 s
1.47 s
1.38 s
1.33 m
0.81 d (6.6)
2.09 m
1.92 m
1.05 m
5.46 t (6.6)
4.25 s
1.41 m
0.92 d (6.6)
2.17 m
2.04 m
1.14 m
5.54 t (6.6)
4.23 s
1.39 m
1.80 m
1.03 d (6.5)
2.32 m
2.20 m
1.19 m
6.90 t (7.1)
1.73 m
2.62m
0.94 d (6.6)
2.16 m
1.12 d (6.5)
2.27 m
0.99 d (6.6)
2.28 m
1.99 m
2.14 dt (15.1, 7.8)
1.16 m
2.14 m
23
24
26
27
28
1.12 m
1.23 m
5.54 t (6.6)
4.24 s
6.84 t (7.1)
6.84 t (7.1)
4.37 s
4.33 s
4.35 s
1.86 s
1.80 s
1.82 s
1.05 s
1.12 s
1.11 s
4.65 d (11.2)
4.08 d (11.2)
1.21 s
4.53 d (11.7)
4.11 d (11.7)
1.20 s
4.54 d (11.5)
4.14 d (11.5)
1.22 s
29
30
2′
0.98 s
1.17 s
1.11 s
1.21 s
1.10 s
1.40 s
1.52 s
1.70 s
1.61 s
2.68 m
2.69 m
2.71 m
4′
2.63 m
2.64 m
2.63 m
6′
1.39 s
1.36 s
1.38 s
2″
2.23 s
a
b
Recorded at 500 MHz in CDCl3, δH in ppm, J in Hz. “m” means multiplet or overlapped with other signals.
of NMR data between 8 and 7 indicated the loss of an oxygenated
methine and the presence of an additional carbonyl moiety (δC
201.4) in 8 (Table 2 and Table 5). Interpretation of its HSQC,
HMBC, and ROESY data assigned the structure of 8 (Figure 2).
NOE correlations of Hβ-12 with H3-18 and H3-19, H3-19 with
H3-29, H-17 with H3-21 and H3-30, and H-5 with H3-28 and
H3-30 determined the β-orientation for CH3-18, CH3-19, and
CH3-29 and the α-orientation for H-5, H-17, CH3-28, and
CH3-30 (Figure 3), respectively.
Compound 10 was isolated as a yellow powder, and its molec-
ular formula was determined to be C38H52O13 by HRTOFMS
data. The 1H NMR data of 10 exhibited signals due to five tertiary
methyls (δH 0.98, 1.21, 1.39, 1.44, and 1.52), one acetyl methyl
(δH 2.23), a vinyl methyl (δH 1.86), a secondary methyl [δH 1.03
(d, J = 6.5 Hz)], and three oxymethines [δH 6.90 (t, J = 7.1 Hz);
δH 4.89 (t, J = 8.2 Hz); δH 5.65 (s)] (Table 2). The 13C NMR and
HSQC spectra showed 38 carbon resonances including seven
methyls, nine methylenes (one oxygenated at δC 66.3), six
methines (two oxygenated carbons at δC 66.2 and 79.9), four
quaternary carbons, one oxygenated tetrasubstituted carbon
at δC 70.3, four olefinic carbons at δC 141.4, 127.5, 144.9, and
156.2, and seven carbonyl carbons (δC 170.7, 171.6, 173.0, 174.6,
192.2, 213.1, and 216.8) (Table 5). Furthermore, a lanostane
triterpenoid structure identical with that of ganoleucoin D (4)
was supported by HMBC and ROESY experiments (Figure 2).
The remaining signals in the NMR spectra of 10 were elucidated
as a 3-hydroxy-3-methylglutaryl (HMG) moiety by HMBC cor-
relations from H-2′ (δH 2.68, 2.63) to C-1′ (δC 171.6), from H-4′
(δH 2.68, 2.63) to C-5′ (δC 174.6), and from H-6′ (δH 1.39) to
C-2′ (δC 44.8), C-3′ (δC 70.3), and C-4′ (δC 44.6). The HMG
moiety was attached at C-28 of the triterpenoid skeleton on the
basis of HMBC correlations from H2-28 [δH 4.08 (d, J = 11.2
Hz), 4.65 (d, J = 11.2 Hz)] to C-1′ (δC 171.6). The absolute
configuration of the chiral carbon in the HMG group was
Ganoleucoin I (9) possessed a molecular formula of
C30H46O5, as determined by HRTOFMS data. The 1H and 13
C
NMR data of 9 resembled those of 7, including five singlet
methyls, one doublet methyl, 10 methylenes (two oxygenated),
four methines (one oxygenated), four quaternary carbons, two
pairs of olefinic carbons, and two carbonyl carbons (Table 2 and
Table 5). HMBC correlations from H3-30 to C-8, C-13, C-14,
and C-15 (δC 65.9) and from H-15 (δH 4.52, m) to C-8, C-13,
C-14, C-16, and C-17, as well as the 1H−1H COSY correlations
of H-15−H2-16−H-17, indicated the substitution of a hydroxy
group at C-15 in 9 (Figure 2). The planar structure of 9 was
established by detailed analysis of its 2D NMR spectra. The
relative configuration of H-15 was assigned as α-orientation by
the NOE correlations of H-15 with H-17 and H3-30 (Figure 3).
The CD spectrum of 9 was similar to that of 1, indicating 13R and
14R configurations.
D
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
a
a
Table 3. 1H NMR Spectral Data of Compounds 13−16
Table 4. 13C NMR Spectral Data of Compounds 1−6
no.
1
13
14
15
16
b
no.
1
2
3
4
5
6
2.26 m
1.78 m
2.27 m
1.75 m
2.27 m
1.76 m
2.12 m
1.79 m
1
35.0
34.6
222.8
49.9
52.3
29.6
73.1
163.6
138.0
36.7
198.6
51.8
46.8
53.6
18.0
38.8
49.1
17.4
20.2
36.0
18.1
34.5
25.9
145.1
127.1
172.5
12.2
66.0
22.7
19.0
34.4
34.0
221.4
50.2
52.1
29.6
72.9
165.4
136.7
36.5
199.7
78.8
53.4
54.4
18.0
36.9
49.1
11.3
20.1
34.1
20.0
34.2
26.7
145.6
126.8
172.3
12.2
65.8
22.6
18.0
34.4
34.3
221.9
50.1
52.1
29.5
72.5
163.5
138.0
36.7
192.5
80.4
51.5
54.8
29.8
37.1
48.7
12.6
18.8
34.1
20.1
34.4
26.5
144.8
127.3
172.5
12.2
65.9
22.7
19.9
170.7
21.2
34.9
34.5
219.1
50.3
49.5
27.0
65.6
157.0
140.9
37.5
192.4
79.7
60.5
49.7
216.7
37.7
46.1
13.6
19.1
31.9
20.9
33.1
26.5
143.8
127.6
172.2
12.1
65.5
22.0
24.5
170.4
20.7
35.0
34.8
218.3
50.9
49.9
27.3
65.8
158.3
140.0
37.6
199.9
78.6
60.3
51.7
217.4
37.6
46.7
12.4
19.3
31.9
21.0
33.4
26.9
144.5
127.4
172.6
12.2
65.6
21.9
23.5
34.2
33.8
2
2
2.80 dt (14.5, 9.0) 2.79 dt (14.5, 9.0) 2.77 dt (14.5, 9.0) 2.69 dt (14.6,
8.9)
3
218.8
50.1
4
2.34 m
2.35 m
2.33 m
2.46 m
1.45 m
2.33 m
2.53 m
5
51.5
5
6
1.55 m
1.54 m
1.55 m
6
39.0
2.05 m
2.07 m
2.06 m
7
198.2
146.2
149.7
36.8
2.23 m
2.21 m
2.20 m
8
7
5.39 d (5.8)
5.39 d (5.8)
5.50 d (6.5)
5.39 d (5.8)
5.50 d (6.5)
9
11 5.50 d (6.5)
2.11 m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1′
2′
1.99 m
194.2
79.3
12 2.21 m
2.10 m
2.21 m
2.22 m
1.79 m
2.12 m
2.19 m
1.67 m
58.7
15 1.93 m
1.29 m
1.95 m
1.95 m
1.99 m
47.7
1.26 m
1.29 m
1.21 m
206.0
37.6
16 1.65 m
1.35 m
1.63 m
1.63 m
2.32 m
1.38 m
1.38 m
2.70 m
45.3
17 1.54 m
18 0.58 s
19 1.21 s
20 1.48 m
21 0.91 d (6.5)
22 1.63 m
1.05 m
1.54 m
1.54 m
2.15 m
12.3
0.59 s
0.55 s
0.67 s
19.6
1.20 s
1.21 s
0.94 s
33.4
1.45 m
1.49 m
1.80 m
20.4
0.91 d (6.5)
1.63 m
0.91 d (6.5)
1.63 m
0.95 d (6.5)
1.68 m
34.2
1.04 m
1.05 m
1.13 m
26.6
23 1.62 m
1.46 m
1.62 m
1.58 m
1.60 m
144.4
127.4
172.0
12.3
1.46 m
1.46 m
1.45 m
24 3.44 d (10.2)
26 4.42 d (11.1)
4.19 d (11.1)
27 1.15 s
28 0.86 s
29 1.13 s
30 1.09 s
4.99 d (9.9)
3.49 d (12.1)
3.32 d (12.1)
1.13 s
4.77 d (9.5)
3.39 d (11.5)
3.22 d (11.5)
1.15 s
3.43 d (10.2)
4.41 d (11.4)
4.19 d (11.4)
1.11 s
65.5
22.8
0.86 s
0.85 s
1.08 s
21.0
1.13.s
1.13 s
1.12 s
170.3
21.0
1.08 s
1.07 s
1.34 s
1′ 3.65 dd (16.1,8.5) 3.65 dd (16.1, 8.6) 3.50 d (7.3)
3.65 dd (16.1,
8.5)
a
Recorded at 125 MHz in CDCl3, δC in ppm.
3.59 dd (16.1,8.5) 3.60 dd (16.1, 8.6)
3.60 dd (16.1,
8.5)
group and the presence of an additional carbonyl group (δC
197.8) instead of an oxygenated methine (δC 66.3) in 10 (Table 2
and Table 5). The HMBC correlations from H-5 (δH 2.28, m)
and H2-6 (δH 2.64, m) to C-7 (δC 197.8), as well as the 1H−1H
COSY correlation of H-5−H2-6, confirmed the location of a
carbonyl moiety at C-7. A detailed examination of its 2D NMR
spectroscopic data confirmed the structure of 11 (Figure 2). The
relative configuration of H-12 was assigned as β-orientation by
NOE correlations of H-12 with H3-18 and H3-19.
2′ 5.97 t (8.5)
4′ 2.25 m
5′ 2.08 m
6′ 5.04 m
8′ 1.97 m
9′ 2.14 m
10′ 5.04 m
12′ 1.68 s
6.01 t (8.5)
2.27 m
2.07 m
5.05 m
1.98 m
2.14 m
5.05 m
1.68 s
6.95 t (7.3)
2.44 t (7.6)
2.16 m
5.96 t (8.5)
2.25 m
2.07 m
5.04 m
1.98 m
2.11 m
5.05 m
1.66 s
5.18 t (7.0)
1.96 t (7.0)
2.08 m
5.09 t (6.3)
1.67 s
13′ 1.61 s
1.60 s
1.60 s
1.57 s
Ganoleucoin L (12) was obtained as a yellow powder. The
molecular formula of C36H48O11 was confirmed on the basis of
HRTOFMS data. The 1H and 13C NMR data of 12 were similar
to those of 11, except for the absence of an oxygenated methine
(δC 77.8) in 11 and the presence of an extra methylene (δC 44.1)
(Table 2 and Table 5). The HMBC correlations from H3-18
(0.86, s) to C-12, C-13, C-14, and C-17 and from H2-12 to C-9,
C-11, C-13, C-14, C-17, and C-18 confirmed the structural
variation at C-12 between 11 and 12. Structural determination
and full signal assignment of 12 were solved on the basis of
2D NMR spectral analysis (Figure 2). The absolute configuration
of the HMG group in compounds 11 and 12 was determined
as S by using the same method as described for 10. The HMG
group has been found to be conjugated in flavonoids from
Oxytropis falcata,23 triterpenoids from Piptoporus betulinus24 and
Ganoderma resinaceum,27 and phenylpropanoids from Edge-
worthia chrysantha.28
14′ 1.53 s
3″ 6.64 d (1.5)
1.53 s
1.60 s
1.53 s
6.63 d (1.5)
6. 89 d (1.5)
6.63 d (1.5)
5″ 6.61 dd (8.2, 1.5) 6.60 dd (8.2, 1.5) 6.88 dd (8.2, 1.5) 6.60 dd (8.2, 1.5)
6″ 6.73 d (8.2) 6.73 d (8.2) 6.63 d (8.2) 6.71 d (8.2)
a
b
Recorded at 500 MHz in CDCl3, δH in ppm, J in Hz. “m” means
multiplet or overlapped with other signals
determined as S by using the reference method (Scheme 1).23−26
Compound 10a was obtained by amidation of 10 with (S)-1-
phenylethylamine. Reduction of 10a with LiBH4, followed by
acetylation, gave 5-O-acetyl-1-[(S)-phenylethyl]mevalonamide
(10b), which had the same 1H NMR data as those of (3R)-5-O-
acetyl-1-[(S)-phenylethyl]mevalonamide.24 Compound 10 was
designated as ganoleucoin J.
Ganoleucoin K (11) had a molecular formula of C36H48O12, as
determined by HRTOFMS data. The 1H and 13C NMR data of
11 resembled those of 10, except for the absence of the acetyl
E
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 2. Key 1H−1H COSY and HMBC correlations of 1−16.
Ganoleuconin M (13) was assigned the molecular formula
13C NMR spectra of 13 exhibited signals due to nine singlet
methyl groups, two doublet methyl groups, 14 methylenes
1
C51H74O7 on the basis of HRTOFMS data. The H NMR and
F
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 3. Key ROESY correlations of 1, 2, 4, 6, 7, 9, 13, and 16.
including one oxygenated methylene [δH/δC 4.42 (d, J = 11.1),
4.19 (d, J = 11.1 Hz)/69.2], four methines including one
oxymethine (δC 77.2), four quaternary carbons, one oxygenated
tetrasubstituted carbon (δC 74.1), five pairs of double bonds in-
cluding five protonated olefinic carbons (δH/δC 5.96 (t, J = 8.5 Hz)/
140.3, 5.50 (d, J = 6.5)/117.3, 5.39 (d, J = 5.8 Hz)/120.1,
5.04 (m)/124.3, and 5.05 (m)/122.5), a 1,2,4-trisubstituted
aromatic ring [δH 6.61 (dd, J = 8.2, 1.5 Hz), 6.64 (d, J = 1.5 Hz),
6.73 (d, J = 8.2 Hz); δC 115.1, 117.1, 117.4, 124.3, 149.0, and
149.4], and two carbonyl carbons (δC 170.4 and 217.2) (Table 3
and Table 6). A detailed analysis of its 2D NMR spectra revealed
the presence of ganodermanontriol or its side-chain triol
diastereomers and a 12-deoxylganomycin A unit in 13. The
HMBC correlations of H2-26 (δH 4.42 and 4.19) with C-15′ (δC
170.4) indicated that the ganodermanontriol moiety was linked
with a 12-deoxylganomycin A unit by an ester linkage (C-26 to
C-15′). In its ROESY spectrum, the NOE correlations of H-2′
with H-4′, H-5′ with H3-14′, and H-6′ with H2-8′ assigned the Z
and E configurations for the C-2′−C-3′ and C-6′−C-7′ double
bonds, respectively. Ganodermanontriol (26) and its three
stereoisomeric triols have been synthesized in an early report and
have been reported to have different chemical shifts of C-24,
C-25, and C-26 in the side chain.29 To confirm the absolute
configuration in the side chain of the tripterpenoid moiety in 13,
compound 13 was hydrolyzed in 2% sodium methoxide−
methanol to afford ganodermanontriol (26). Thus, the abso-
lute configuration of C-24 and C-25 in 13 was assigned as 24S
and 25R.
The molecular formula of ganoleuconin N (14) was
determined as C51H74O7 by HRTOFMS data. The NMR data
of 14 were similar to those of 13, including the NMR signals due
to a ganodermanontriol moeity and a farnesyl hydroquinone unit
(Table 3 and Table 6). The structure of 14 was confirmed and
fully assigned by detailed analysis of its 2D NMR data. In the
HMBC spectrum, the correlation of H-24 at δH 4.99 (d, J =
9.9 Hz) with the carbonyl group at δC 171.3 confirmed the linkage
of a 12-deoxylganomycin A unit (A) with ganodermanontriol
through an ester bond between C-24 and C-15′. The absolute
G
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
a
Table 5. 13C NMR Spectral Data of Compounds 7−12
Ganoleuconin P (16) was determined to have a molecular
formula of C51H74O8 on the basis of HRTOFMS data. The IR
spectrum showed the presence of carbonyl (1719 and
1700 cm−1) groups. A comparison of NMR data between 13
and 16 indicated that 16 was a triterpene-farnesyl hydroquinone
conjugate. The 1H and 13C NMR spectra of 16 exhibited signals
corresponding to dihydroxy-3,7-dioxo-8-ene-lanosta-26-ol30 and
12-deoxylganomycin A units (Table 3 and Table 6). Detailed
analysis of the 2D NMR spectra of 13 established its structure.
The HMBC correlations of H2-26 (δH 4.19 and 4.41) with C-15′
(δC 170.3) indicated that the dihydroxy-3,7-dioxo-8-ene-lanosta-
26-ol moiety was conjugated with the 12-deoxylganomycin A
unit by an ester linkage (C-26 to C-15′). In its ROESY spectrum,
the NOE correlations of H-2′ with H2-4′, H2-5′ with H3-14′, and
H-6′ with H2-8′ assigned the Z and E configurations for the
C-2′−C-3′ and C-6′−C-7′ double bonds, respectively. Due to
the structural similarity in the side chain between 13 and 16, the
absolute configurations of C-24 and C-25 in 16 were deduced
by NMR data comparison. The 13C NMR data of C-22, C-23,
C-24, C-25, and C-26 in 16 were almost the same as those of 13,
indicating the same absolute configuration of 24S and 25R. To
date, only three triterpene-farnesyl hydroquinone conjugates
(ganosinensins A−C) have been reported from the fruiting body
of G. sinense.31
The medicinal mushroom G. lucidum was reported to have
hypolipidemic and antidiabetic effects. The intake of powdered
mycelium of G. lucidum was able to decrease total plasma
cholesterol, total liver triglyceride, and total liver cholesterol
levels in animals.32 The water extract of G. lucidum was confirmed
to have beneficial effects in treating type 2 diabetes mellitus
through the suppression of hepatic PEPCK gene expression.33
7-Oxo-ganoderic acid Z and 15-hydroxy-ganoderic acid S from
G. lucidum exhibited inhibitory activities against HMG Co-A
reductase with IC50 values of 22.3 and 21.7 μM, respectively.7
Ganoderol B was found to possess α-glucosidase inhibition with
an IC50 of 119.8 μM.8 Glucosidase and HMG Co-A reductase
inhibitors have been considered to be valuable compounds for
the development of hypolipidemic and antidiabetic drugs.34−36
Herein, we used in vitro HMG-CoA reductase and
α-glucosidase inhibition assays to test the hypolipidemic and
antidiabetic effects of compounds isolated from the fruiting
bodies of G. leucocontextum. Ganoleuconins A−D (1-4), F (6), H
(8), and J−N (10−14), ganoderiol J (17), ganoderic acid DM
(18), 3,7-dioxo-8,24(Z)-tirucalladien-26-oic acid (21), ganode-
ric acid S (23), ganoderic acid Y (25), and ganodermanontriol
(26) exhibited HMG-CoA reductase inhibition with IC50 values
in the range 8−100 μM (Table 7). Compounds 1, 3, 6, 10−14,
17, 18, 23, 25, and 26 showed much stronger inhibitory activity
no.
7
8
9
10
11
12
1
35.0
34.2
218.4
45.1
51.0
27.4
67.3
160.3
140.0
37.9
200.6
51.6
47.2
46.5
29.3
30.2
50.2
17.8
17.2
36.0
18.4
36.2
24.5
131.9
136.7
68.0
60.4
27.8
20.6
27.6
35.0
34.0
215.8
46.8
49.7
37.1
201.4
151.6
149.7
38.8
202.1
51.2
46.7
48.7
27.4
31.9
49.0
16.9
18.0
35.9
18.4
35.8
24.3
131.4
136.8
67.8
60.5
27.5
20.3
25.9
35.0
34.8
214.8
47.6
50.9
32.8
28.1
159.1
142.2
40.2
199.7
37.7
48.3
47.7
65.9
44.7
49.8
17.1
25.2
36.1
18.6
36.0
24.4
132.1
136.4
67.9
60.5
25.4
21.7
19.4
34.9
33.2
213.1
50.9
50.6
27.2
66.3
156.2
141.4
37.4
192.2
79.9
60.8
49.9
216.8
38.1
46.1
13.6
18.6
32.0
21.0
34.9
26.7
144.9
127.5
173.0
12.2
66.2
21.7
24.4
171.6
44.8
70.3
44.6
174.6
27.4
170.7
20.9
34.4
33.8
211.2
50.6
51.7
39.3
197.8
147.5
148.8
37.4
201.7
77.8
57.9
49.5
207.0
37.3
45.7
11.0
18.5
32.9
20.4
33.7
26.7
144.8
127.3
173.2
12.1
65.7
22.3
20.2
171.5
44.8
69.8
44.7
175.7
27.3
34.3
34.2
2
3
211.8
51.6
50.5
39.5
198.7
147.0
149.9
40.2
199.6
44.1
57.3
49.1
208.1
37.1
45.3
16.2
18.6
35.7
18.5
34.5
25.8
144.5
127.5
173.0
12.2
65.8
22.5
21.2
171.6
44.8
69.9
44.7
175.2
27.3
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
1′
2′
3′
4′
5′
6′
1″
2″
a
Recorded at 125 MHz in CDCl3, δC in ppm.
configuration of the triterpene moiety in 14 was determined by
the same method as described in 13.
Ganoleuconin O (15) was obtained as a yellow powder. It had
the same molecular formula of C51H74O7, as determined by
HRTOFMS data. The NMR data of 15 were similar to those
of 13 (Table 3 and Table 6). A significant difference observed
than the positive control atorvastatin. Compound 10 (IC50
=
in the H and 13C spectra of 13 and 15 lies in the farnesyl
1
26.4 μM) showed much stronger inhibitory activity than
compound 4 (IC50 = 97.5 μM), which indicated the significance
of the HMG moiety linking at C-28 for the HMG-CoA reductase
inhibitory activity. Ganoleuconins E (5), M (13), N (14), and P
(16) and ganoderone A (19) showed strong inhibitory activity
against α-glucosidase from yeast with IC50 values of 12.7, 13.6,
2.5, 5.9, and 13.7 μM (Table 7). All other compounds isolated
presented relatively weak inhibitory activity with IC50 values
greater than 50 μM. The inhibitory effects of compounds 13−16
were further investigated using small intestinal mucosa from rats.
Compounds 13, 14, and 16 showed much stronger inhibitory
activity against α-glucosidase, sucrase, and maltase from rats
(Table 7) than the positive control acarbose. Preliminary
structure−activity relationship analysis showed that the farnesyl
hydroquinone unit, especially for signals due to the olefins and
1,2,4-trisubstituted benzene. A detailed examination of its
2D NMR spectral data (1H−1H COSY, HSQC, HMBC, and
ROESY) confirmed the presence of ganodermanontriol and
a farnesyl hydroquinone unit (B). The HMBC correlation of
H-24 at δH 4.77 (d, J = 9.5 Hz) with the carbonyl group at
δC 169.6 concluded the attachment of the moiety B at C-24.
The alkaline hydrolysis of 15 also afforded ganodermanontriol
(26). The NOE correlations of H2-1′ with H2-4′, H2-5′ with
H3-14′, and H-6′ with H2-8′ suggested the E and E con-
figurations for the C-2′−C-3′ and C-6′−C-7′ double bonds,
respectively. Thus, the structure of 15 was assigned as shown
in Figure 1.
H
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
a
Scheme 1. Determination of the Absolute Configuration in the HMG Moiety of 10
a
Key: (a) (S)-1-phenylethylamine, DMF, Et3N, PyBOP, HOBt; (b) LiBH4, THF; (c) Ac2O, pyridine.
a
Table 6. 13C NMR Spectral Data of Compounds 13−16
no.
13
14
15
16
no.
13
14
15
16
1
36.8
35.0
217.2
47.6
51.1
23.8
120.1
143.0
144.6
37.9
117.3
37.3
43.9
50.4
28.0
31.6
50.8
15.9
22.6
36.6
18.8
33.7
26.8
77.2
74.1
69.2
36.8
35.0
217.2
47.6
51.0
23.8
120.1
142.9
144.6
37.9
117.3
37.3
43.9
50.4
28.1
31.6
50.8
15.9
22.6
35.8
18.5
32.9
26.8
76.9
73.4
66.9
36.6
35.0
217.1
47.6
51.1
23.8
120.0
143.0
144.6
37.9
117.4
37.3
43.9
50.4
28.0
31.6
50.8
15.8
22.6
36.7
18.7
33.4
26.8
76.9
73.5
67.0
35.5
34.5
215.0
47.4
50.5
37.3
198.5
163.3
139.7
39.6
25.1
30.2
47.9
45.1
26.6
32.0
49.1
16.0
24.0
36.6
19.0
33.7
26.7
77.4
74.2
69.2
27
28
29
30
1′
2′
3′
4′
5′
6′
7′
8′
9′
10′
11′
12′
13′
14′
15′
1″
2″
3″
4″
5″
6″
22.2
25.5
22.2
25.5
22.2
25.5
21.0
25.9
2
3
25.6
25.6
25.6
21.5
4
21.0
18.3
17.9
18.0
5
31.9
31.9
29.8
31.9
6
140.2
131.5
34.5
141.3
131.6
34.3
142.0
132.4
27.9
140.3
131.5
34.5
7
8
9
28.2
25.0
25.3
28.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
124.4
136.8
39.8
124.3
136.7
39.7
123.2
136.4
39.8
124.3
136.7
39.7
27.6
27.9
27.2
27.6
122.5
131.4
25.9
122.5
131.0
25.9
124.4
131.7
25.9
122.5
131.4
25.5
17.9
17.9
17.9
17.9
16.3
16.3
16.2
16.3
170.4
149.4
124.0
117.4
149.0
115.1
117.1
171.3
149.4
124.0
117.2
149.0
115.2
117.2
169.6
149.7
126.5
117.0
147.5
114.3
116.3
170.3
149.3
124.3
117.2
149.1
115.1
117.2
a
Recorded at 125 MHz in CDCl3, δC in ppm.
hydroquinone moiety in 13, 14, and 16 contributed greatly to
their inhibitory activity against α-glucosidase.
myelogenous leukemia) and PC-3 (human prostate cancer). As
shown in Table 8, compounds 1, 2, 6, 7, 8, 10−14, 16−19, and
25 exhibited cytotoxicity against K562 cells with IC50 values in
the range 10 to 50 μM. For the PC-3 cell line, compounds 8, 14,
16, and 19 showed cytotoxicity with IC50 values of 24.2, 14.4,
10.8, and 28.5 μM, respectively.
In summary, 26 cytotoxic lanostane triterpenes (1−26)
including 16 new compounds were obtained from the fruiting
body of the medicinal mushroom G. leucocontextum. The inhibitory
activity of compounds 1−26 against HMG-CoA reductase and
α-glucosidase and cytotoxicity against K562 and PC-3 cell lines were
evaluated. This is the first report of bioactive secondary metabolites
from this medicinal mushroom, which expanded the chemistry of
Triterpenes are considered major pharmacologically active
compounds contributing to the antitumor efficacy of Ganoderma.
Ganoderic acid D induced apoptosis in HeLa cell.37 Ganoderiol
F exhibited cytotoxicity against Lewis lung carcinoma (LLC),
sarcoma-180, and T-47D cell lines in vitro38 and induced
senescence in hepatoma HepG2 cells.39 Ganoderic acid T
showed significant antitumor effects in vivo.40 Ganoderic acid X
inhibited topoisomerases and induced apoptosis of cancer
cells.41 To evaluate the cytotxicity of triterpenes isolated from
G. leucocontexum, compounds 1−26 were tested for their
antiproliferative activity against tumor cell lines K562 (human
I
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 7. HMG-CoA Reductase and α-Glucosidase Inhibitory Activity of 1−26
small intestinal mucosa from rat
HMG-CoA reductase
inhibition (IC50, μM)
α-glucosidase inhibition
(from baker’s yeast) (IC50, μM)
α-glucosidase inhibition
sucrase inhibition
(IC50, μM)
maltase inhibition
(IC50, μM)
(IC50, μM)
a
1
17.1 3.5
66.9 10.3
26.6 4.3
97.5 9.1
>100
>50
NT
NT
NT
2
>50
NT
NT
NT
3
>50
NT
NT
NT
4
>50
NT
NT
NT
5
12.7 2.1
>50
NT
NT
NT
6
20.5 4.4
>100
NT
NT
NT
7
>50
NT
NT
NT
8
32.9 4.0
>100
>50
NT
NT
NT
9
>50
NT
NT
NT
10
26.4 10.2
10.7 2.9
13.3 2.7
18.9 2.0
24.1 6.2
>100
>50
NT
NT
NT
11
>50
NT
NT
NT
12
>50
NT
NT
NT
13
13.6 3.1
2.5 0.7
>50
1.5 0.2
8.1 2.9
>50
8.4 1.8
22.5 5.7
>50
8.1 1.7
10.1 1.9
>50
14
15
16
>100
5.9 2.2
>50
9.3 1.1
NT
18.5 2.3
NT
16.2 3.0
NT
17
12.6 2.7
9.5 1.5
>100
18
> 50
NT
NT
NT
19
13.7 2.3
>50
NT
NT
NT
20
>100
NT
NT
NT
21
56.1 10.4
>100
>50
NT
NT
NT
22
>50
NT
NT
NT
23
12.5 2.3
>100
>50
NT
NT
NT
24
>50
NT
NT
NT
25
8.6 2.0
16.8 5.8
atorvastatin
32.1 7.7
>50
NT
NT
NT
26
>50
NT
NT
NT
positive control
acarbose
273.1 30.5
acarbose
38.1 6.0
acarbose
20.2 4.5
acarbose
16.1 4.1
a
“NT” means “not tested”.
Table 8. Cytotoxicity of Compounds 1−26
K562 (IC50, μM)
PC-3 (IC50, μM)
K562 (IC50, μM)
PC-3 (IC50, μM)
1
17.8 4.6
19.7 4.6
>200
>200
14
15
16
17
18
19
20
21
22
23
24
25
26
48.4 9.7
103.6 7.9
10.7 1.8
41.6 4.1
18.8 3.5
27.7 4.5
>200
14.4 4.1
>200
2
>200
3
>200
10.8 3.5
>200
4
147.22 13.2
>200
88.3 17.6
>200
5
81.6 13.4
28.5 3.1
>200
6
18.2 3.3
11.4 4.3
115.4 12.8
>200
>200
7
132.4 25.7
24.2 6.6
>200
8
55.4 3.7
54.9 4.75
65.2 9.9
76.6 11.9
17.5 4.5
80.9 8.5
paclitaxel
148.6 19.9
79.5 11.3
75.2 17.9
102.9 25.7
179.8 22.5
105.8 25.4
doxorubicin
2.0 0.4
9
10
12.3 3.9
22.6 5.2
14.4 1.8
48.6 10.3
paclitaxel
0.9 0.1
110.5 14.1
>200
11
12
>200
13
58.4 11.4
doxorubicin
2.0 0.4
positive control
positive control
0.9 0.1
with 10% H2SO4 and heating. Silica gel (Qingdao Haiyang Chemical
Co., Ltd., People’s Republic of China) and Sephadex LH-20 (Amersham
Biosciences) were used for column chromatography. HPLC separation
was performed on an Agilent 1200 HPLC system using an ODS column
(C18, 250 × 9.4 mm, YMC Pak, 5 μm; detector: UV) with a flow rate
of 2.0 mL/min. UV and IR spectral data were acquired using a
ThermoGenesys-10S UV−vis and Nicolet IS5 FT-IR spectrophotom-
eter, respectively. Optical rotations were measured on an Anton Paar
MCP 200 automatic polarimeter. NMR spectral data were obtained
with a Bruker Avance-500 spectrometer (CDCl3, δH 7.26/δC 77.16). The
HSQC and HMBC experiments were optimized for 145.0 and 8.0 Hz,
the Ganoderma species and provided evidence for the usage of
G. leucocontexum as an herbal medicine and a promising source of
new bioactive agents. The discovery of new bioactive lanostane
triterpenes from G. leucocontextum also supports its phylogenetic
taxonomy as a new member of the G. lucidum complex.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Solvent used for extraction
and chromatographic separation was analytical grade. TLC was carried
out on silica gel HSGF254, and compounds were visualized by spraying
J
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
respectively. HRTOFMS data were measured using an Agilent Accurate-
Mass-Q-TOF LC/MS 6520 instrument.
(c 1.95 × 10−3 M, MeOH) λmax (Δε) 237 (+3.1), 317 (−0.5) nm; IR
(neat) νmax 3319, 2948, 2925, 2840, 2820, 1723, 1702, 1625, 1586, 1425,
1
Fungal Material. The strain of G. leucocontexum was isolated
from its fruiting bodies collected in the Linzhi region of Tibet.
G. leucocontexum was cultured on slants of potato dextrose agar at 25 °C
for 10 days. Agar plugs were inoculated into a bag (18 × 35 cm) with the
culture medium composed of 140 g of cotton seed hulls, 36 g of wood
powders, 22 g of rice bran, 2 g of CaSO4, and 120 mL of distilled water.
Two hundred bags were inoculated and incubated at 25 °C for 90 days in
a greenhouse. The fruiting bodies obtained were identified by W.-P.X.
(one of the coauthors) by comparing their morphological characteristics
with those published for G. leucocontexum.12
Extraction and Isolation. The air-dried and powdered fruiting
bodies of G. leucocontexum (3 kg) were extracted three times with ethyl
alcohol (3 × 10 L), and organic solvent was evaporated to dryness under
vacuum to afford the crude extract (179 g). The ethanol extract (170 g)
was partitioned between ethyl acetate and water. The EtOAc extract
(43.5 g) was subjected to silica gel column chromatography (CC) using
hexane−ethyl acetate in a gradient elution (v/v, 100:0, 100:2, 100:5,
100:8, 100:10, 100:15, 100:20, 100:40, 100:50), followed by dichloro-
methane−methanol elution (v/v, 100:1, 100:2, 100:3, 100:5, 100:10,
100:15, 100:20, 0:100) to give 23 fractions (GL-1−GL-23).
Fraction 16 (1.42 g) eluted with hexane−ethyl acetate (v/v, 100:50)
was subjected to silica gel column chromatography with a CHCl3−
MeOH gradient solvent system (100:0−0:100) to yield 10 fractions.
Compound 26 (121.1 mg) was obtained from fraction GL-16-3 by
recrystallization in MeOH. Compounds 16 (7.2 mg, tR 27.2 min), 13
(5.1 mg, tR 28.1 min), 14 (3.5 mg, tR 25.1 min), and 15 (4.5 mg, tR
34.2 min) were isolated from fraction GL-16-8 (130.0 mg) by RP-HPLC
using 81% methanol in water.
Fraction 17 (3.45 g) eluted with dichloromethane−methanol (v/v,
100:1) was further separated on Sephadex LH-20 CC eluted with 50%
methanol in water to give 12 subfractions (GL-17-1−GL-17-12).
Fraction GL-17-3 (123 mg) was further purified by Sephadex LH-20 CC
eluted with 50% methanol in water to afford compounds 18 (24 mg), 19
(28.1 mg), and 20 (20 mg). Compounds 23 (31.2 mg, tR 32.2 min), 24
(20.7 mg, tR 36.5 min), and 25 (50.1 mg, tR 40 min) were obtained from
fraction GL-17-12 (201.2 mg) by RP-HPLC using 75% methanol in
water.
1369, 1364, 1226, 866, 754, 612 cm−1; for H and 13C NMR data see
Tables 1 and 4; positive HRTOFMS m/z [M + H]+ 517.3160 (calcd for
C30H45O7, 517.3160).
Ganoleuconin C (3): colorless powder; [α]25 +90.10 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 220 (4.10), 260 (2.00) nm; CD (c
1.85 × 10−3 M, MeOH) λmax (Δε) 250 (+3.2), 347 (−0.7) nm; IR (neat)
νmax 3320, 2979, 2952, 2925, 2835, 2820, 1725, 1702, 1622, 1588,
1441, 1368, 1374, 1236, 856, 612 cm−1; for 1H and 13C NMR data see
Tables 1 and 4; positive HRTOFMS m/z [M + H]+ 559.3266 (calcd for
C32H47O8, 559.3265).
Ganoleuconin D (4): colorless powder; [α]25 +83.00 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 216 (4.10), 262 (2.00) nm; CD (c
2.05 × 10−3 M, MeOH) λmax (Δε) 260 (+3.6), 350 (−0.4) nm; IR (neat)
νmax 3320, 2961, 2952, 2925, 2850, 2840, 1714, 1702, 1622, 1588,
1441, 1368, 1374, 1236, 856, 615 cm−1; for 1H and 13C NMR data see
Tables 1 and 4; positive HRTOFMS m/z [M + H]+ 573.3060 (calcd for
C32H45O9, 573.3058).
Ganoleuconin E (5): colorless powder; [α]25 +93.50 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 225 (4.10), 260 (2.00) nm; CD
(c 1.96 × 10−3 M, MeOH) λmax (Δε) 260 (+4.5), 355 (−0.3) nm; IR
(neat) νmax 3325, 2979, 2954, 2910, 2832, 2812, 1725, 1710, 1703, 1622,
1
1588, 1441, 1368, 1254, 1372, 1236, 836, 612 cm−1; for H and 13C
NMR data see Tables 1 and 4; positive HRTOFMS m/z [M + H]+
531.2952 (calcd for C30H43O8, 531.2952).
Ganoleuconin F (6): colorless powder, [α]25 +106.00 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 216 (4.10), 262 (2.00) nm; CD
(c 1.55 × 10−3 M, MeOH) λmax (Δε) 226 (+3.2), 267 (+2.4), 310 (−0.9)
nm; IR (neat) νmax 3315, 3210, 2961, 2952, 2918, 2849, 2828, 1723,
1713, 1700, 1619, 1588, 1425, 1375, 1365, 1230, 856, 736, 615 cm−1; for
1H and 13C NMR data see Tables 1 and 4; positive HRTOFMS m/z
[M + H]+ 571.2902 (calcd for C32H43O9, 571.2902).
Ganoleuconin G (7): colorless powder; [α]25 +50.30 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 200 (3.50), 254 (2.10) nm; CD (c
1.86 × 10−3 M, MeOH) λmax (Δε) 260 (+3.6), 355 (−0.7) nm; IR (neat)
νmax 3310, 2979, 2960, 2920, 2855, 2832, 1713, 1701, 1639, 1579, 1440,
1390, 1360, 870, 725, 620 cm−1; for 1H and 13C NMR data see Tables 2
and 5; positive HRTOFMS m/z [M + H]+ 487.3418 (calcd for C30H47O5,
487.3418).
Fraction 19 (5.32 g) eluted with dichloromethane−methanol (v/v,
100:2) was further separated on ODS CC using a gradient of methanol−
water (30−100%) to give 23 subfractions (GL-19−1−GL-19−23).
Compounds 1 (23.2 mg, tR 32.2 min), 2 (15.2 mg, tR 34.5 min), and 3
(12.4 mg, tR 36.2 min) were obtained from fraction GL-19-13 (150.5
mg) by RP-HPLC using 56% acetonitrile in water. Fraction GL-19-12
(352.3 mg) was subjected to Sephadex LH-20 CC eluted with 70%
methanol in water to afford five subfractions (GL-19-12-1−GL-19-12-5).
Compounds 4 (5.2 mg, tR 32.3 min) and 5 (7.2 mg, tR 36.2 min) were
purified from subfraction GL-19-12-3 (35.2 mg) by RP-HPLC using 54%
acetonitrile in water. Compounds 6 (6.1 mg, tR 32.1 min), 8 (5.4 mg, tR
35.2 min), and 21 (7.1 mg, tR 37.1 min) were isolated from GL-19-12-4
(20.5 mg) by RP-HPLC using 56% acetonitrile in water. Subfraction
GL-19-22 (102.5 mg) was separated by RP-HPLC using 60%
acetonitrile in water to afford compound 9 (43.1 mg, tR 39.2 min).
Compounds 10 (23.1 mg, tR 39.8 min), 7 (13.2 mg, tR 35.1 min), and 22
(3.6 mg, tR 38.2 min) were purified from fraction GL-19-16 (90.2 mg) by
RP-HPLC using 71% methanol in water. Fraction GL-19-15 (123 mg)
was further separated by Sephadex LH-20 CC eluted with 50% methanol
in water to afford compounds 11 (45 mg), 12 (43.1 mg), and 17 (7.1 mg).
The physical properties and spectroscopic data of the new compounds are
as follows.
Ganoleuconin H (8): colorless powder; [α]25 +36.00 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 200 (3.50), 254 (2.10) nm; CD (c
2.05 × 10−3 M, MeOH) λmax (Δε) 225 (+3.2), 267 (+2.3), 305 (−0.5)
nm; IR (neat) νmax 3310, 2979, 2960, 2920, 2855, 2825, 1723, 1715,
1700, 1639, 1574, 1430, 1380, 1360, 870, 724, 618 cm−1; for 1H and 13
C
NMR data see Tables 2 and 5; positive HRTOFMS m/z [M + H]+
485.3263 (calcd for C30H45O5, 485.3262).
Ganoleuconin I (9): colorless powder; [α]25D +50.20 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 200 (3.50), 254 (2.10) nm; CD (c 1.88 × 10−3
M, MeOH) λmax (Δε) 255 (+3.9), 345 (−0.8) nm; IR (neat) νmax 3325,
2980, 2948, 2910, 2845, 2835, 1702, 1685, 1619, 1557, 1424, 1378,
1360, 870, 724, 620 cm−1; for 1H and 13C NMR data see Tables 2 and 5;
positive HRTOFMS m/z [M + H]+ 487.3419 (calcd for C30H47O5,
487.3418).
Ganoleucoin J (10): yellow powder; [α]25D +70.50 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 218 (4.29), 260 (2.12) nm; CD (c 1.70 × 10−3
M, MeOH) λmax (Δε) 250 (+4.1), 352 (−0.7) nm; IR (neat) νmax
3310, 2977, 2969, 2922, 2875, 2860, 1723, 1706, 1685, 1641, 1577,
1
1457, 1379, 1372, 875, 740, 615 cm−1; for H and 13C NMR data see
Tables 2 and 5; positive HRTOFMS m/z [M + H]+ 717.3480 (calcd for
C38H53O13, 717.3481).
Ganoleuconin A (1): colorless powder; [α]25 +72.40 (c 0.1,
Ganoleucoin K (11): yellow powder, [α]25D +42.40 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 218 (4.10), 260 (2.12) nm; CD (c 1.92 × 10−3
M, MeOH) λmax (Δε) 225 (+4.2), 280 (+2.4), 310 (−0.8) nm; IR (neat)
νmax 3308, 2975, 2960, 2912, 2885, 2854, 1715, 1701, 1683, 1625, 1450,
1380, 1372, 826, 739, 615 cm−1; for 1H and 13C NMR data see Tables 2
and 5; positive HRTOFMS m/z [M + H]+ 673.3217 (calcd for
C36H49O12, 673.3219).
D
MeOH); UV (MeOH) λmax (log ε) 218 (4.10), 258 (2.00) nm; CD (c
2.01 × 10−3 M, MeOH) λmax (Δε) 245 (+4.2), 354 (−0.5) nm; IR (neat)
νmax 3315, 2980, 2948, 2840, 2820, 1702, 1696, 1618, 1586, 1429, 1360,
1347, 1236, 856, 612 cm−1; for 1H and 13C NMR data see Tables 1 and
4; positive HRTOFMS m/z [M + H]+ 501.3216 (calcd for C30H45O6,
501.3211).
Ganoleuconin B (2): colorless powder; [α]25 +53.00 (c 0.1,
Ganoleucoin L (12): yellow powder; [α]25D +86.75 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 218 (4.20), 262 (2.00) nm; CD (c 2.01 × 10−3
D
MeOH); UV (MeOH) λmax (log ε) 225 (4.04), 260 (1.98) nm; CD
K
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
M, MeOH) λmax (Δε) 224 (+3.9), 282 (+2.5), 305 (−0.9) nm; IR (neat)
νmax 3325, 2960, 2949, 2910, 2870, 2860, 1716, 1703, 1684, 1625, 1590,
1447, 1380, 1372, 1232, 872, 740, 615 cm−1; for 1H and 13C NMR data
see Tables 2 and 5; positive HRTOFMS m/z [M + H]+ 657.3270 (calcd.
for C36H49O11, 657.3269).
of the α-glucosidase. The inhibition rates (%) = [(OD control − OD
control blank) − (OD test − OD test blank)]/(OD control − OD
control blank) × 100%. Acarbose was utilized as the positive control
with an IC50 of 273.1 μM.
Inhibition Assay against α-Glucosidase, Sucrase, and Maltase
from Rat’s Small Intestinal Mucosa. A slightly modified method of
the rat intestinal mucosa assay developed by Kwon et al. was used.44,45 A
total of 1.0 g of rat-intestinal mucosa powder was suspended in 3 mL of
0.9% saline, and the suspension was sonicated at 4 °C 12 times (each
30 s). After centrifugation (10000g) at 4 °C for 30 min, the resulting
supernatant was used as the enzyme solution for the assay. Each
compound dissolved in DMSO (10 μL) was mixed with 20 μL of
substrate (maltose, sucrose, p-nitrophenyl-α-D-glucopyranoside, respec-
tively), 10 μL of enzyme solution, and 60 μL of 0.1 M phosphate buffer
(pH 6.9). After preincubation for 40 min, a commercial kit (GOD assay,
Jiancheng Biological Engineering Institue, Najing, China) was used to
test the production of glucose. The absorbance was read at 550 nm
under a Spectra Max 190 microplate reader (Molecular Devices Inc.).
The blank was prepared by adding potassium phosphate buffer instead
of enzyme. The control was prepared by adding potassium phos-
phate buffer instead of test compounds. The inhibition rates (%) =
[(OD control − OD control blank) − (OD test − OD test blank)]/
(OD control − OD control blank) × 100%.
Inhibition Assay against HMG-CoA Reductase. The HMG-CoA
reductase prepared from pig liver microsomes was assayed by the
previously described colorimetric method with a slight modification.46,47
In brief, a mixture containing 10 μL of test compounds (final
concentrations of 2.5, 5, 10, 20, 40, 80 μM), 20 μL of prepared enzyme
(final concentration of about 4 U/mL), 40 μL of HMG-CoA (final
concentration of 200 μM), and 10 μL of 0.1 mol/L potassium phosphate
buffer (pH 7.0) (containing 3.5 mmol/L EDTA, 10 mmol/L
dithiothreitol, and 0.1 g/L boving serum albumin) was incubated at
37 °C for 5 min. After that, 20 μL of NADPH solution (final con-
centration of 100 μM) was added to the reaction solution and incubated
for 15 min at 37 °C. The assay was conducted in a 96-well plate, and
the absorbance was determined at 340 nm using a Spectra Max
190 microplate reader (Molecular Devices Inc.). The blank was
prepared by adding potassium phosphate buffer instead of HMG-CoA
reductase. The control was prepared by adding potassium phos-
phate buffer instead of test compounds. The inhibition rates (%) =
[(OD test − OD test blank) − (OD control blank − OD control)]/
(OD control blank − OD control) × 100%.
Ganoleuconin M (13): yellow powder; [α]25D +23.00 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 248 (4.12), 254 (3.99),377(2.10) nm; IR
(neat) νmax 3380, 2930, 2860, 1725, 1704, 1642, 1440, 1403, 1348, 1301,
1239, 1167, 1018, 1000, 745, 649 cm−1; for 1H and 13C NMR data see
Tables 3 and 6; positive HRTOFMS m/z [M + H]+ 799.5508 (calcd for
C51H75O7, 799.5507).
Ganoleuconin N (14): yellow powder; [α]25D +19.20 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 245 (3.60), 254 (3.51), 377(1.99) nm; IR
(neat) νmax 3452, 2925, 2820, 1725, 1725, 1630, 1460, 1423, 1345, 1301,
1234, 1190, 1128, 1023, 1000, 654 cm−1; for 1H and 13C NMR data see
Tables 3 and 6; positive HRTOFMS m/z [M + H]+ 799.5510 (calcd for
C51H75O7, 799.5507).
Ganoleuconin O (15): yellow powder; [α]25D +23.20 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 248 (3.52), 254 (3.40), 377 (2.00) nm; IR
(neat) νmax 3452, 2939, 2829, 1719, 1680, 1635, 1462, 1425, 1348, 1301,
1
1234, 1190, 1128, 1023, 980 cm−1; for H and 13C NMR data see
Tables 3 and 6; positive HRTOFMS m/z [M + H]+ 799.5510 (calcd for
C51H75O7, 799.5507).
Ganoleuconin P (16): yellow powder; [α]25D +9.00 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 200 (3.50), 254 (3.40), 377 (2.00) nm; IR
(neat) νmax 3452, 2925, 2849, 1719, 1700, 1635, 1460, 1423, 1345, 1301,
1234, 1190, 1128, 1023, 1000, 649 cm−1; for 1H and 13C NMR data see
Table 3 and 6; positive HRTOFMS m/z [M + H]+ 815.5456 (calcd for
C51H75O8, 815.5456).
Determination of the Absolute Configuration in Compounds
10−12.23 (S)-1-Phenylethylamine (19 μL, 150 μmol), Et3N (32 μL,
225 μmol), PyBOP (58.5 mg, 115 μmol), and HOBt (20.0 mg,
150 μmol) were added to a solution of compound 10 (50.1 mg,
75 μmol) in 0.5 mL of DMF at 0 °C. The mixture was stirred at room
temperature for 5 h. The reaction was quenched with dilute aqueous
HCl, and the organic materials were extracted with EtOAc to afford a
yellow residue, which was purified by Sephadex LH-20 CC eluting with
MeOH to give amide 10a (54.2 mg). Compound 10a was further
transformed into 10b (5.4 mg) by the method described in an earlier
report.23 The H NMR spectrum of 10b was consistent with that of
1
(3R)-5-O-acetyl-1-[(S)-phenylethyl]mevalonamide rather than the 3S
isomer reported in the literature.39 The absolute configuration of the
HMG moiety in compounds 11 and 12 (each 10.0 mg) was determined
using the same method, and all the final products were proved to be
(3R)-5-O-acetyl-1-[(S)-phenylethyl]mevalonamide.
(3R)-5-O-Acetyl-1-[(S)-phenylethyl]mevalonamide (10b): colorless
oil; [α]25D −60.10 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 248 (3.52),
254 (3.40) nm; IR (neat) νmax 3452, 2939, 1740, 1640, 1620 cm−1; 1H
NMR (CDCl3, 500 MHz) δ 7.27−7.37 (5H, m, C6H5-1′), 6.34 (1H, d,
J = 7.3 Hz, NH), 5.14 (1H, quintet, J = 7.2 Hz, H-1′), 4.23 (2H, t, J = 6.8
Hz, H-5), 2.41, 2.28 (each 1H, d, J = 14.4 Hz, H-2), 2.04 (3H, s,
CH3COO), 1.85 (2H, m, H-4), 1.50 (3H, d, J = 6.8 Hz, H-2′), 1.23 (3H,
s, H-6); positive HRTOFMS m/z [M + H]+ 277.1623 (calcd for
C16H23NO3, 277.1622).
Alkaline Hydrolysis of Compounds 13−15. According to a
published procedure,42 compound 13 (3.0 mg) dissolved in 2% sodium
methanolate−methanol (4 mL) was stirred at room temperature for
1 h. The reaction mixture was neutralized with 10% acetic acid. After
removal of the solvent from the filtrate under reduced pressure, the
residue was dissolved in water (10 mL) and extracted with ethyl acetate
(10 mL). The organic solvents were evaporated and purified by
preparative TLC on silica gel (dichloromethane−methanol, 20:1) to
give compound 26 (1.8 mg). Compounds 14 and 15 were hydrolyzed by
the same method to give compound 26.
Inhibition Assay against α-Glucosidase from Baker’s Yeast.
As described in our earlier work,43 the bioassay was conducted using a
96-well plate, and the absorbance was determined at 405 nm using a
Spectra Max 190 microplate reader (Molecular Devices Inc.). The
control was prepared by adding a phosphate buffer instead of test
compounds. The blank was prepared by adding phosphate buffer instead
Cytotoxicity Assay. The cytotoxicity against K562 and PC-3 cell
lines of compounds 1−26 was tested using the MTT method as
previously reported.48
Statistical Analyses. The bioactivity values were expressed as
means of three independent experiments, and each was carried out in
triplicate.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jnatprod.5b00331.
NMR spectra data of compounds 1−26 and 10b (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +86 10 62566577. E-mail: liuhw@im.ac.cn (H.-W. Liu).
Author Contributions
⊥K. Wang, L. Bao, and W.-P. Xiong contributed equally to this
article.
Notes
The authors declare no competing financial interest.
L
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(30) Peng, X. R.; Liu, J. Q.; Xia, J. J.; Wang, C. F.; Li, X. Y.; Deng, Y. Y.;
Bao, N. M.; Zhang, Z. R.; Qiu, M. H. Phytochemistry 2015, 114, 137−
145.
(31) Sato, N.; Ma, C. M.; Komatsu, K.; Hattori, M. J. Nat. Prod. 2009,
72, 958−961.
(32) Rubel, R.; Santa, H. S. D.; Fernandes, L. C.; Bonatto, S. J. R.; Bello,
S.; Figueiredo, B. C.; Filho, J. H. C. L.; Santos, C. A. M.; Socol, C. R.
World J. Microbiol. Biotechnol. 2011, 27, 1083−1089.
(33) Seto, S. W.; Lam, T. Y.; Tam, H. L.; Au, A. L. S.; Chan, S. W.;
Chan, J. H.; Wu, J. H.; Yu, P. H. F.; Leung, G. P. H.; Ngai, S. M.; Yeung, J.
H. K.; Leung, P. S.; Lee, S. M. Y.; Kwan, Y. W. Phytomedicine 2009, 16,
426−436.
(34) Van de Laar, F. A.; Lucassen, P. L. B. J.; Akkermans, R. P.; Van de
Lisdonk, F. H.; Rutten, G. E. H. M.; Van Weel, C. Diabetes Care 2005,
28, 154−163.
ACKNOWLEDGMENTS
■
Financial support of the National Nature Science Foundation
(21472233) and the Ministry of Science and Technology of
China (2014CB138304) is acknowledged. Dr. J. Ren (State Key
Laboratory of Mycology, Institute of Microbiology, Chinese
Academy of Sciences) is appreciated for his help in measuring the
NMR data. We appreciated the kind help from Mr. R. Ramesh in
polishing the language.
REFERENCES
■
(1) Sanodiya, B. S.; Thakur, G. S.; Baghel, R. K.; Prasad, G. B.; Bisen, P.
S. Curr. Pharm. Biotechnol. 2009, 10, 717−742.
(2) Wu, T. S.; Shi, L. S.; Kuo, S. C. J. Nat. Prod. 2001, 64, 1121−1122.
(3) Ko, H. H.; Hung, C. F.; Wang, J. P.; Lin, C. N. Phytochemistry 2008,
69, 234−239.
(35) Genser, B.; Grammer, T. B.; Stojakovic, T.; Siekmeier, R.; Marz,
W. Int. J. Clin. Pharmacol. Ther. 2008, 46, 497−510.
(36) Sanodiya, B. S.; Thakur, G. S.; Baghel, R. K.; Prasad, G. B. K. S.;
Bisen, P. S. Curr. Pharm. Biotechnol. 2002, 5, 266−277.
(37) Yue, Q. X.; Cao, Z. W.; Guan, S. H.; Liu, X. H.; Tao, L.; Wu, W. Y.;
Li, Y. X.; Yang, P. Y.; Liu, X.; Guo, D. A. Mol. Cell. Proteomics 2008, 7,
949−961.
(4) Peng, X. R.; Liu, J. Q.; Wang, C. F.; Li, X. Y.; Shu, Y.; Qiu, M. H. J.
Nat. Prod. 2014, 77, 737−743.
(5) Fatmawati, S.; Shimizu, K.; Kondo, R. Bioorg. Med. Chem. Lett.
2011, 21, 7295−7297.
(6) Liu, J.; Kurashiki, K.; Shimizu, K.; Kondo, R. Biol. Pharm. Bull.
2006, 29, 392−395.
(38) Zhang, Q.; Zuo, F.; Nakamura, N.; Ma, C. M.; Hattori, M. J. Nat.
Med. 2009, 63, 304−310.
(7) Li, C. J.; Li, Y. M.; Sun, H. H. Nat. Prod. Res. 2006, 20, 985−991.
(8) Fatmawati, S.; Shimizu, K.; Kondo, R. Phytomedicine 2011, 18,
1053−1055.
(39) Chang, U. M.; Li, C. H.; Lin, L.; Huang, C. P.; Kan, L. S.; Lin, S. B.
Life Sci. 2006, 79, 1129−1139.
(40) Tang, W.; Liu, H. W.; Zhao, W. M.; Wei, D. Z.; Zhong, J. J. Life Sci.
2006, 80, 205−211.
(9) Mao, X. L. The Large Economic Fungi in Tibet; Beijing Science &
Technology Press: Beijing, 1993.
(41) Li, C. H.; Chen, P. Y.; Chang, U. M.; Kan, L. S.; Fang, W. H.; Tsai,
K. S.; Lin, S. B. Life Sci. 2005, 77, 252−265.
(10) Sung, G. H.; Hywel-Jones, N. L.; Sung, J. M.; Luangsa-Ard, J. J.;
Shrestha, B.; Spatafora, J. W. Stud. Mycol. 2007, 57, 5−59.
(11) Shashidhar, M. G.; Giridhar, P.; Sankar, K. U.; Manohar, B. J.
Funct. Foods 2013, 5, 1013−1030.
(12) Li, T. H.; Hu, H. P.; Deng, W. Q.; Wu, S. H.; Wang, D. M.;
Tsering, T. Mycoscience 2015, 56, 81−85.
(13) Liu, J. Q.; Wang, C. F.; Li, Y.; Luo, H. R.; Qiu, M. H. Planta Med.
2012, 78, 368−376.
(42) Ma, K.; Han, J. J.; Jin, T.; Yang, X. L.; Zhao, F.; Li, S. F.; Song, F.
H.; Liu, M. M.; Liu, H. W.; Bao, L. Food Chem. 2014, 143, 239−245.
(43) Ma, K.; Han, J. J.; Bao, L.; Wei, T. Z.; Liu, H. W. J. Nat. Prod. 2014,
77, 942−947.
(44) Kwon, Y. I.; Vattem, D. A.; Shetty, K. Asia Pac. J. Clin. Nutr. 2006,
15, 107−118.
(45) Thao, N. P.; Luyen, B. T. T.; Tai, B. H.; Yang, S. Y.; Jo, S. H.;
Cuong, N. X.; Nam, Y. I.; Kwon, Y. I.; Minh, C. V.; Kim, Y. H. Bioorg.
Med. Chem. Lett. 2014, 24, 1192−1196.
(46) Tormanen, C. D.; Redd, W. L.; Srikantaiah, M. V.; Scallen, T. J.
Biochem. Biophys. Res. Commun. 1976, 68, 754−762.
(47) Xie, W. D.; Wang, W.; Su, H.; Xing, D. M.; Cai, G. P.; Du, L. J. J.
Pharmacol. Sci. 2007, 103, 267−274.
(48) Wang, K.; Bao, L.; Qi, Q. Y.; Zhao, F.; Ma, K.; Pei, Y. F.; Liu, H. W.
J. Nat. Prod. 2015, 78, 146−154.
(14) Wang, F. S.; Cai, H.; Yang, J. S.; Zhang, Y. M.; Hou, C. Y.; Liu, J.
Q.; Zhao, M. J. Yaoxue Xuebao 1997, 32, 447−450.
(15) Niedermeyer, T. H. J.; Lindequist, U.; Mentel, R.; Gordes, D.;
Schmidt, E.; Thurow, K.; Lalk, M. J. Nat. Prod. 2005, 68, 1728−1731.
(16) Cheng, C. R.; Yue, Q. X.; Wu, Z. Y.; Song, X. Y.; Tao, S. J.; Wu, H.
X.; Xu, P. P.; Xuan, L.; Guan, S. H.; Guo, D. A. Phytochemistry 2010, 71,
1579−1585.
(17) Makino, M.; Motegi, T.; Fujimoto, Y. Phytochemistry 2004, 65,
891−896.
(18) Nishitoba, T.; Oda, K.; Sato, H.; Sakamura, S. Agric. Biol. Chem.
1988, 52, 367−372.
(19) Li, C. J.; Yin, J. H.; Guo, F. J.; Zhang, D. C.; Sun, H. H. Nat. Prod.
Res. 2005, 19, 461−465.
(20) Sato, H.; Nishitoba, T.; Shirasu, S.; ODA, K.; Sakamura, S. Agric.
Biol. Chem. 1986, 50, 2887−2890.
(21) Toth, J. O.; Luu, B.; Ourisson, G. Tetrahedron Lett. 1983, 24,
1081−1084.
(22) Fujita, A.; Arisawa, M.; Saga, M.; Hayashi, T.; Morita, N. J. Nat.
Prod. 1986, 49, 1122−1125.
(23) Wang, S. S.; Zhang, X. J.; Que, S.; Tu, G. Z.; Wan, D.; Cheng, W.;
Liang, H.; Ye, J.; Zhang, Q. Y. J. Nat. Prod. 2012, 75, 1359−1364.
(24) Hirai, N.; Koshimizu, K. Phytochemistry 1981, 20, 1867−1869.
(25) Kamo, T.; Asanoma, M.; Shibata, H.; Hirota, M. J. Nat. Prod.
2003, 66, 1104−1106.
(26) Hattori, Y.; Horikawa, K. H.; Makabe, H.; Hirai, N.; Hirota, M.;
Kamo, T. Tetrahedron: Asymmetry 2007, 18, 1183−1186.
(27) Niu, X. M.; Li, S. H.; Xiao, W. L.; Sun, H. D.; Che, C. T. J. Asian
Nat. Prod. Res. 2007, 9, 659−664.
(28) Baba, K.; Taniguti, M.; Yoneda, Y.; Kozawa, M. Phytochemistry
1990, 29, 247−249.
(29) Kennedy, E. M.; P'Pool, S. J.; Jiang, J. H.; Sliva, D.; Minto, R. E. J.
Nat. Prod. 2011, 74, 2332−2337.
M
DOI: 10.1021/acs.jnatprod.5b00331
J. Nat. Prod. XXXX, XXX, XXX−XXX