New lanostanoids of Ganoderma tsugae

Article abstract of DOI:10.1021/np990367l

Three new compounds, (24R,S)-3α-acetoxy-24-hydroxy-5α-lanosta-8,25- dien-21-oic acid, named tsugaric acid C (1); 3α-acetoxy-5α-lanosta-8,24- diene-21-O-β-D-xyloside, named tsugarioside B (2); and 3α-acetoxy(Z)-24- methyl-5α-lanosta-8,23,25-trien-21-oic acid ester b-D-xyloside, named tsugarioside C (3), and a mixture of two known steroids were isolated from the fruit bodies of Ganoderma tsugae. The structures of 1-3 were determined by spectral and chemical methods. The cytotoxic activity of the lanostanoid constituents of this fungus was evaluated against several different cancer cell lines.

Full text of DOI:10.1021/np990367l

514
J . Nat. Prod. 2000, 63, 514-516
New La n osta n oid s of Ga n od er m a tsu ga e
Huey-J en Su,† Yih-Fen Fann,† Mei-Ing Chung,† Shen-J eu Won,‡ and Chun-Nan Lin*,†
School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan 807, Republic of China, and Department of
Microbiology, Medical College, National Cheng Kung University, Tainan, Taiwan 701, Republic of China
Received J uly 23, 1999
Three new compounds, (24R,S)-3R-acetoxy-24-hydroxy-5R-lanosta-8,25-dien-21-oic acid, named tsugaric
acid C (1); 3R-acetoxy-5R-lanosta-8,24-diene-21-O-â-D-xyloside, named tsugarioside B (2); and 3R-acetoxy-
(Z)-24-methyl-5R-lanosta-8,23,25-trien-21-oic acid ester â-D-xyloside, named tsugarioside C (3), and a
mixture of two known steroids were isolated from the fruit bodies of Ganoderma tsugae. The structures
of 1-3 were determined by spectral and chemical methods. The cytotoxic activity of the lanostanoid
constituents of this fungus was evaluated against several different cancer cell lines.
Ta ble 1. 13C NMR Data for 1-4a
Previously, we isolated and characterized three new
lanostanoids, tsugaric acid A (3R-acetoxy-5R-lanosta-8,24-
dien-21-oic acid) (5), tsugaric acid B (3R-acetoxy-16R-
hydroxy-24ê-methyl-5R-lanosta-8,25-dien-21-oic acid) (6),
and tsugarioside A, 3R-acetoxy-5R-lanosta-8,24-dien-21-oic
acid ester â-D-glucoside (7), along with four known com-
pounds, 3â-hydroxy-5R-lanosta-8,24-dien-21-oic acid (8),
3-oxo-5R-lanosta-8,24-dien-21-oic acid (10), ergosta-7,22-
dien-3â-ol, and 2â,3R,9R-trihydroxy-5R-ergosta-7,22-diene
from the fruit bodies of Ganoderma tsugae Murr. (Polypo-
raceae).1,2 In continued studies on the consitutents of this
fungus, three new lanostanoids were obtained from a
MeOH extract of the fruit body: tsugaric acid C (1),
tsugariosides B (2) and C (3), and a mixture of the already
known compounds 5R,8R-epidioxyergosta-6,22-dien-3â-ol
and 5R,8R-epidioxyergosta-6,9(11),22-trien-3â-ol. Recently,
we reported that 7 and 2â,3R,9R-trihydroxy-5R-ergosta-
7,22-diene mediate their cytotoxicity through apoptosis and
cell-cycle inhibition,2 respectively. In the present paper, the
structural characterization of 1-3 and the cytotoxic effects
against a small cancer-line panel of several of the G. tsugae
constituents and their derivatives are reported.
The HREIMS of 1 indicated a molecular ion peak at m/z
514.3662, which corresponded to a molecular formula
C32H50O5. IR absorptions were indicative of a hydroxyl
group (3465 cm-1), an ester (1730 cm-1), and a CdC double
bond (1656 cm-1). The EIMS of 1 showed significant
peaks at m/z 496 [M - H2O]+, 481 [496 - Me]+, 421 [481
- CH3COOH]+, and 281 [421 - (side chain) - H2O)]+. The
1H NMR spectrum of 1 showed signals for six tertiary
methyl groups, an acetyl proton signal, two oxygen-bearing
methine proton signals at δ 4.01 (m) and 4.61 (1H, br s,
H-3â), and two olefinic proton signals a δ 4.75, 4.89 (1H,
br s, H-27 of 24R,24S) and 4.75, 4.92 (1H, br s, H-27 of
24R,24S).1-3 In addition to the above evidence, the presence
of correlations between the proton signal at δ 4.01 and C-24
and between H-27 and C-27 (Table 1) in the HMQC
spectrum, and the interactions between the proton signal
at δ 4.01 and H-27 and between Me-26 and H-27 in the
NOESY spectrum suggested that 1 is a 24-epimeric pair
of 3R-acetoxy-24-hydroxy-lanostanoids with a ∆25 double
bond. The proton signal at δ 4.01 was assigned to H-24.3
The ratio of these epimers was assumed to be 1:1 based on
the signal intensity of H-27. The information obtained from
carbon
1 [(CD3)2CO]b 2 (CDCl3)b 3 (CDCl3) 4 (CDCl3)b
1
2
3
4
5
6
7
8
30.5
24.6
78.8
38.1
46.9
19.4
27.3
135.6
136.3
38.4
22.2
30.8
45.8
51.0
28.3
28.7
48.6
17.0
30.2
23.3
78.1
36.7
45.3
18.0
26.0
134.0
134.6
36.9
21.0
30.8
44.3
49.9
27.5
29.7
40.6
16.1
19.0
44.9
70.2
30.7
24.7
30.4
23.3
78.1
36.7
45.3
18.0
25.9
133.7
134.7
36.9
20.8
30.8
44.3
49.5
27.0
28.9
47.0
16.3
19.0
47.8
175.2
32.9
123.5
30.1
25.7
76.0
36.9
44.3
18.1
28.0
134.0
135.0
37.5
21.0
30.7
44.2
50.8
28.0
29.8
42.9
16.0
19.0
49.9
62.6
30.5
24.3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
20.0
49.0
178.1
34.3
32.3 (24R)
31.8 (24S)
76.5 (24R)
75.6 (24S)
24
124.8
132.3
124.0
241
25
25.7
150.0 (24R)
149.8 (24S)
110.8 (24R)
111.5 (24S)
18.7 (24R)
18.1 (24S)
28.7
131.4
17.7
25.7
155.4
132.0
17.8
26.0
26
27
106.7
17.7
28
29
30
1′
2′
3′
4′
5′
27.6
21.8
24.4
102.7
71.9
73.7
69.7
63.7
170.9
21.4
27.6
21.8
24.4
94.5
72.3
75.9
69.5
65.8
170.9
21.4
27.7
22.2
25.0
22.9
25.3
COCH3
COCH3
171.3
21.8
a
The number of protons directly attached to each carbon was
verified by DEPT experiments. Signals obtained by 1H-1H
COSY, HMBC, NOESY, or ROESY techniques.
b
the 2D NMR and comparison of 13C NMR data with those
of lanostanoids allowed the assignment of the 13C NMR
signals of 1 as shown in Table 1. The signals of C-23 to
C-27 appeared as a doublet (Table 1), supporting the fact
that 1 is a mixture of C-24 epimers.3,4 Therefore, 1 was
* To whom correspondence should be addressed. Tel.: +886 7 3121101,
ext. 2163. Fax:+886 7 5562365. E-mail: lincna@cc.kmu.edu.tw.
† Kaohsiung Medical University.
‡ Medical College, National Cheng Kung University.
10.1021/np990367l CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/18/2000

Notes
J ournal of Natural Products, 2000, Vol. 63, No. 4 515
Ta ble 2. Cytotoxicity of Lanostanoids Isolated from G. tsugae (ED50 values in µG/mL)
cell linea
compound
PLC/PRF/5
T-24
212
HT-3
7.2
c
c
SiHa
9.5
c
c
CaSKi
3
5
6
7
8
9
6.5
6.8
NS
NS
8.6
3.1
NS
NSb
c
10.3
c
c
NS
NS
1.73
6.8
8.4
c
c
9.2
12.1
4.4
5.8
8.2
c
NS
c
3.5
c
6.9
5.5
c
5.1
6.2
3.9
7.2
10
11
c
NS
c
c
NS
NS
cisplatin
actinomycin D
5.3
c
1.3
c
c
c
c
1.4 × 10-3
1.5 × 10-3
5.6 × 10-4
8.1 × 10-4
1.9 × 10-3
a
b
For significant activity of the pure compounds, an ED50<4.0 µg/mL is required. NS, no significant activity of the pure compounds.
c Not determined.
characterized as a 1:1 mixture of the C-24 epimers of 3R-
acetoxy-24-hydroxy-5R-lanosta-8,25-dien-21-oic acid (1).
Compound 2, colorless needles, showed [R]27D +7.6°(c 0.1,
CHCl3). It also gave a positive Liebermann-Burchard
reaction and possesses the molecular formula C37H60O7 as
determined from positive FABMS ([M + 1]+ at m/z 617)
and 1H and 13C NMR data. It failed to show a molecular
ion peak under high-resolution conditions. IR absorptions
were indicative of a hydroxyl group (3425 cm-1), an ester
spectrum, and an interaction between Me-241 and H-23 in
the ROESY spectrum, clearly indicated that 3 is a 3R-
acetoxy-(Z)-24-methyl-5R-lanosta-8,23,25-trien-21-oic acid
ester â-D-xyloside (3).2,6-8 Therefore, the tertiary methyl
and olefinic proton signals at δ 1.67 and 5.08 were assigned
to Me-241 and H-23, respectively. The information obtained
from 2D NMR and 13C NMR data further supported the
characterization of 3 and allowed the assignment of 13C
NMR signals of 3 as shown in Table 1.1,2,6-8
The cytotoxic activity of the constituents isolated from
this fungus against PLC/PRF/5, T-24, 212, HT-3, SiHa, and
CaSKi cells was studied in vitro. The results are listed in
Table 2. Compound 7 showed significant activity against
T-24 cells, while 5, 8, and 9 showed significant activity
against T-24, HT-3, and CaSKi cells, respectively. Cisplatin
and actinomycin D were used as positive controls.
1
(1745 cm-1), and a CdC double bond (1645 cm-1).The H
NMR spectrum of 2 showed signals for seven tertiary
methyl groups, an acetyl proton signal, an oxygen-bearing
methine proton signal at δ 4.66 (1H, br s, H-3â), and an
olefinic proton signal at δ 5.09 (1H, t, J ) 6.8 Hz, H-24).2,5
In addition to the above evidence, the presence of an
anomeric proton signal at δ 4.39 (1H, d, J ) 7.2 Hz) and
signals corresponding to a secondary carbon (δ 70.2), a
tertiary carbon (δ 124.8), three quaternary carbons (δ
131.4, 134.0, and 134.6), and five xylosyl carbons (δ 102.7,
73.7, 71.9, 69.7, and 63.7) in the 1H and 13C NMR spec-
tra of 2, as well as significant peaks at m/z 391 [M -
CH3COOH - CH3 - xylose]+ and 149 [xylose - H]+ in the
FABMS of 2, clearly indicated that 2 is 3R-acetoxy-5R-
lanosta-8,24-diene-21-O-â-D-xyloside (2).2,6,7
Acidic methanolysis of 2 with HCl/MeOH gave the
3R-O-acetate of 4 and methyl R- and â-D-xylopyranoside.
On alkaline hydrolysis, the 3R-O-acetate of 4 gave 4, which
was further identified by comparison with the product
obtained from the reduction of 7 with LiAlH4. The informa-
tion obtained from 2D NMR and 13C NMR data further
supported the characterization of 2 and allowed the as-
signment of 13C NMR signals of 2 as shown in Table 1.1,2,6,7
The HRFABMS of 3 indicated a molecular ion peak at
m/z 643.3791 ([M + 1], +42 mmu error), which cor-
responded to a molecular formula of C38H58O8. IR absorp-
tions were indicative of a hydroxyl group (3445 cm-1), an
ester (1745 and 1720 cm-1), and a CdC double bond (1645
cm-1). The EIMS of 3 showed significant peaks at m/z 512
[M - (xylose-H2O) - 2H]+, 483 [512 - CO - H]+, and 423
1
[483 - CH3COOH]+. The H NMR spectrum of 3 showed
signals for seven tertiary methyl groups, an acetyl proton
signal, an oxygen-bearing methine proton signal at δ 4.66
(1H, t, J ) 5.6 Hz, H-3â), and three olefinic proton signals
at δ 4.65 (1H, s, H-26), 4.75 (1H, s, H-26), and 5.08 (1H, t,
J ) 6.8 Hz).2,5 In addition to the above evidence, the
presence of an anomeric signal at δ 5.56 (1H, d, J ) 7.2
1
Exp er im en ta l Section
Hz) in the H NMR spectrum of 3, signals corresponding
to a tertiary carbon (δ 125.3), five quaternary carbons (δ
132.3, 133.7, 134.7, 155.4, and 175.2), and five xylosyl
carbons (δ 94.5, 75.9, 72.3, 69.5, and 65.8) in the 13C NMR
spectrum of 3, as well as correlations between Me-241 and
C-23 and C-24, and between H-27 and C-24 in the HMBC
Gen er a l Exp er im en ta l P r oced u r es. Melting points are
reported uncorrected. The optical rotations were obtained on
a J ASCO model DIP-370 digital polarimeter. IR spectra were
recorded on a Hitachi model 260-30 spectrophotometer. 1H (400
MHz) and 13C (100 MHz) NMR spectra were recorded on a

516 J ournal of Natural Products, 2000, Vol. 63, No. 4
Notes
Varian Unity-400 spectrometer, and MS were obtained on a
J MS-HX 100 mass spectrometer.
5.6 Hz, H-3â), 4.75 (1H, s, H-26), 5.08 (1H, t, J ) 6.8 Hz, H-23),
5.56 (1H, d, J ) 7.2 Hz, H-1′); 13C NMR (CDCl3, 100 MHz),
see Table 1; FABMS (positive) m/z 665 [M + Na]+ (14), 551
(12), 543 (10), 311 (9), 237 (16), 193 (11), 108 (100); HRFABMS
(positive) m/z 643.3791 [M + 1]+ (calcd for C38H58O8, 643.4210).
F u n ga l Ma ter ia l. G. tsugae was collected at Liu-Kuei
Shian, Kaohsiung Hsien, Taiwan, Republic of China, during
J uly 1995. A voucher specimen (9501) is deposited in the
laboratory of Medicinal Chemistry. It was identified by Dr.
Ming-Hong Yen, School of Pharmacy, Kaohsiung Medical
University. Compounds 5-8 and 10 were isolated and identi-
fied as reported previously.1 Compound 8 was acetylated using
Ac2O in pyridine, and 7 was hydrolyzed in methanolic 5% KOH
to give 9 and 11, respectively.
Extr a ction a n d Isola tion . Air-dried fruit bodies (10 kg)
were extracted with CHCl3 and MeOH, respectively. The
MeOH extract was chromatographed on a Si gel column, and
elution with cyclohexane-CH2Cl2 (1:4) yielded a mixture of
5R,8R-epidioxyergosta-6,22-dien-3â-ol and 5R,8R-epidioxyer-
gosta-6,9(11),22-trien-3â-ol (25 mg, 0.0005%). Elution with
CH2Cl2-EtOAc (3:2) afforded 1 (20 mg, 0.0004%), 2 (10 mg,
0.0002%), and 4 (15 mg, 0.0003%). The known compounds were
identified by spectroscopic methods and comparison with
reported data.9,10
Tu m or Cell Gr ow th In h ibition Assa ys. A microassay for
cytotoxicity was performed using a MTT (3-[4,5-dimethyl-
thiazo-2-yl]-5-[3-carboxymethoxy-methoxyphenyl]- 2[4-sul-
fophenyl]-2H-tetrazolium bromide) assay.11,12 Briefly, 1-3 ×
103 cells/100 µL were seeded in 96-well microplates (Nunck,
Roskilde, Denmark) and preincubated for 6 h to allow cell
attachment. This medium was then aspirated, and 100 µL of
fresh medium containing various concentrations of test drug
were added to the cultures. The cells were incubated with each
drug for 6 days. Cell survival was evaluated by adding 10 µL
of tetrazolium salt solution (1 mg MTT/mL in PBS). After 4 h
incubation at 37 °C, 100 µL DMSO were added to dissolve the
precipitate of reduced MTT. Microplates were then shaken for
15 min, and the absorbance was determined at 550 nm with
a multiwell scanning spectrophotometer (Dynex MR 5000,
Chantilly, VA).
Tsu ga r ic a cid C (1): colorless needles (MeOH); mp 213-
215 °C; IR (KBr) νmax 3465, 1730, 1656 cm-1; 1H NMR [(CD3)2-
CO, 400 MHz] δ 0.80 (3H, s, Me-18), 0.86 (3H, s, Me-28), 0.91
(3H, s, Me-29), 0.94 (3H, s, Me-30), 1.03 (3H, s, Me-19), 1.67
(3H, s, Me-27), 2.00 (3H, s, OAc), 2.26 (1H, m, H-20), 3.75 (1H,
br s, OH-24), 4.01 (1H, m, H-24), 4.61 (1H, br s, H-3), 4.75,
4.89 (1H, br s, H-27 of 24R,24S), 4.75, 4.92 (1H, br s, H-27 of
24R,24S); 13C NMR [(CD3)2CO, 100 MHz], see Table 1; EIMS
m/z 514 [M]+ (8), 496 (3), 481 (9), 421 (54), 281 (17), 187 (39),
81 (66), 69 (58), 43 (100); HREIMS m/z 514.3662 (calcd for
PLC/PRF/5 cells were established from a human hepatoma
and known to produce HBs Ag continuously in culture fluids.13
Human hepatoma PLC/PRF/5, T24 cells, human cervical
carcinoma, HT-3, SiHa, and CaSki cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL,
Grand Island, NY)11,12 containing 10% fetal bovine serum (FBS;
Gibco BRL), 2 mM L-glutamine, 100 u/mL penicillin, and 100
µg/mL streptomycin. The 212 cells (an inducible Ha-ras onco-
gene transformed NIH/3T3 cell line) were maintained in
minimum essential alpha medium (MEM; Gibco BRL), con-
taining 10% calf serum (Gibco BRL), and antibiotics.14 For the
microassay, the growth medium was supplemented with 10
mM HEPES buffer pH 7.3 and incubated at 37 °C in a CO2
incubator.
C
32H50O5, 514.3658).
Tsu ga r iosid e B (2): colorless needles (CHCl3); mp 135-
137 °C; [R]27 +7.6° (c 0.1, CHCl3); IR (KBr) νmax 3425, 1745,
D
1
1645 cm-1; H NMR (CDCl3, 400 MHz) δ 0.71 (3H, s, Me-18),
0.86 (3H, s, Me-28), 0.91 (3H, s, Me-29), 0.92 (3H, s, Me-30),
0.99 (3H, s, Me-19), 1.60 (3H, s, Me-26), 1.67 (3H, s, Me-27),
2.07 (3H, s, OAc), 2.33 (1H, m, H-20), 4.39 (1H, d, J ) 7.2 Hz,
Ack n ow led gm en t. This work was supported by a grant
from the National Science Council of the Republic of China
(NSC 85-2331-B037-021)
H-1′), 4.66 (1H, br s, H-3â), 5.09 (1H, t, J ) 6.8 Hz, H-24); 13
C
NMR (CDCl3, 100 MHz), see Table 1; FABMS (positive) m/z
617 [M + 1]+ (0.2), 413 (3), 391 (12), 154 (39), 149 (100).
Red u ction of 7 w ith LiAlH 4. Compound 7 (0.06 mmol)
was dissolved in anhydrous THF (5 mL). LiAlH4 (0.6 mmol)
was added to this solution, and the mixture was refluxed for
24 h. The excess LiAlH4 was decomposed with wet diethyl
ether, and the mixture was extracted with diethyl ether. The
diethyl ether layer was washed with H2O, dried over MgSO4,
and evaporated to dryness. The residue was chromatographed
on a Si gel column, and by elution with CHCl3, afforded 4 (0.02
Refer en ces a n d Notes
(1) Lin, C. N.; Fann, Y. F.; Chung, M. I. Phytochemistry 1997, 46, 1143-
1146.
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Prod. 1998, 61, 485-487.
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Y. J . Nat. Prod. 1993, 56, 1437-1443.
mmol) as colorless needles (CHCl3): mp 71-72 °C; [R]27
D
-12.7° (c 0.3, CHCl3); IR (KBr) νmax 3965 (OH), 1656 (CdC);
1H NMR (CDCl3, 400 MHz) δ 0.71 (3H, s, Me-18), 0.87 (3H, s,
Me-28), 0.89 (3H, s, Me-29), 0.90 (3H, s, Me-30), 0.98 (3H, s,
Me-19), 1.61 (3H, s, Me-26), 1.68 (3H, s, Me-27), 3.43 (1H, br
s, H-3), 3.71 (2H, m, H-21), 5.11 (1H, m, H-24); 13C NMR
(CDCl3, 100 MHz), see Table 1; EIMS m/z 442 [M]+ (3), 427
[M - CH3]+ (4), 409 [427 - H2O]+ (7), 281 [m/z 409 - side
chain - H]+ (4), 255 (5), 187 (13), 109 (96), 69 (100); HREIMS
m/z 442.3789 (calcd for C30H50O2, 442.3811).
(7) Agrawal, P. K. Phytochemistry 1992, 31, 3307-3330.
(8) Houghton, P. J .; Lian, L. M. Phytochemistry 1986, 25, 1939-1944.
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Tsu ga r iosid e C (3): colorless needles (CHCl3); mp 181-
183 °C; [R]27 +10° (c 0.1, CHCl3); IR (KBr) νmax 3445, 1745,
D
(13) Nakajima, Y.; Kuwata, T.; Nomita, Y.; Okuda, K. Microbiol. Immunol.
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1720, 1656 cm-1
;
1H NMR (CDCl3, 400 MHz) δ 0.74 (3H, s,
Me-18), 0.86 (3H, s, Me-28), 0.91 (3H, s, Me-29), 0.93 (3H, s,
Me-30), 0.97 (3H, s, Me-19), 1.57 (3H, s, Me-27), 1.67 (3H, s,
Me-241), 2.06 (3H, s, OAc), 4.65 (1H, s, H-26), 4.66, (1H, t, J )
NP990367L