Lanostane triterpenoids from the mushroom Naematoloma fasciculare

Article abstract of DOI:10.1021/np300801x

In our continuing search for structurally interesting and bioactive metabolites from Korean wild mushrooms, bioassay-guided fractionation and a chemical investigation of the MeOH extracts of the fruiting bodies of the mushroom Naematoloma fasciculare resulted in the isolation of four new lanostane triterpenoids (1-4), together with 11 known compounds (5-15). The structures of 1-5 were determined by a combination of 1D and 2D NMR and HRMS. The absolute configuration of the 3-hydroxy-3-methylglutaryl group as a side chain in 1 and 2 was determined by the alkaline methanolysis method. The full NMR data assignment of the known compound fasciculol G (5) is reported for the first time. Compounds 1-15 were tested for their antiproliferative activities against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15) and evaluated for their inhibitory effects on nitric oxide production in a lipopolysaccharide-activated murine microglial cell line.

Full text of DOI:10.1021/np300801x

Article
pubs.acs.org/jnp
Lanostane Triterpenoids from the Mushroom Naematoloma
fasciculare
†
‡
§
⊥
,†
Ki Hyun Kim, Eunjung Moon, Sang Un Choi, Sun Yeou Kim, and Kang Ro Lee*
†Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea
‡Graduate School of East-West Medical Science, Kyung Hee University, Yongin 446-701, Korea
§Korea Research Institute of Chemical Technology, Teajeon 305-600, Korea
⊥College of Pharmacy, Gachon University, Incheon, 406-799, Korea
*
S Supporting Information
ABSTRACT: In our continuing search for structurally interesting
and bioactive metabolites from Korean wild mushrooms, bioassay-
guided fractionation and a chemical investigation of the MeOH
extracts of the fruiting bodies of the mushroom Naematoloma
fasciculare resulted in the isolation of four new lanostane
triterpenoids (1−4), together with 11 known compounds (5−15).
The structures of 1−5 were determined by a combination of 1D and
2
3
D NMR and HRMS. The absolute configuration of the 3-hydroxy-
-methylglutaryl group as a side chain in 1 and 2 was determined by
the alkaline methanolysis method. The full NMR data assignment of
the known compound fasciculol G (5) is reported for the first time.
Compounds 1−15 were tested for their antiproliferative activities
against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2,
and HCT-15) and evaluated for their inhibitory effects on nitric
oxide production in a lipopolysaccharide-activated murine microglial cell line.
n our continuing search for structurally interesting and
lipopolysaccharide (LPS)-activated BV-2 cells, a microglial
cell line.
1
−5
I
bioactive metabolites from Korean wild mushrooms,
we
have collected scores of endemic mushroom species in the
mountainous areas during the hot humid summer and prepared
MeOH extracts of the mushrooms for antitumor-activity
screening tests. Among the collected wild mushrooms, the
extract of the mushroom Naematoloma fasciculare (Strophar-
iaceae) showed significant cytotoxicity against A549, SK-OV-3,
SK-MEL-2, and HCT-15 cell lines. The bitter poisonous
mushroom N. fasciculare (Japanese name: Nigakuritake), which
is widely distributed worldwide, grows on the stumps of old
RESULTS AND DISCUSSION
■
Fresh fruiting bodies of N. fasciculare were collected at
Donggureung, Guri of GyeongGi-do, Korea, and extracted
with 80% aqueous MeOH to give an extract that showed
cytotoxic activity against A549, SK-OV-3, SK-MEL-2, and
HCT-15 cells. A chemical investigation of the MeOH extract
led to the isolation of 15 lanostane triterpenoids (1−15). The
1
2,14
6
known compounds were identified as fasciculol E (6),
trees in tufts. This mushroom is well known for its diverse
12
15
16
fasciculic acid B (7), fasciculic acid F (8), fasciculol D (9),
phytochemicals (ergosterols, triterpenoids, and sesquiterpe-
16
16
16
6
−13
fasciculol F (10), fasciculol B (11), fasciculol C (12),
17 17
noids),
and particularly for the presence of lanostane
8
−12
fasciculol H (13), fasciculol I (14), and sublateriol C
18
triterpenoids as the toxic components.
Lanostane triterpe-
(
15), by comparison of their spectroscopic and physical data
noids from this mushroom inhibit the growth of Chinese
with reported values.
Fasciculol J (1) was obtained as a colorless gum. The
cabbage seedlings and have antimicrobial and calmodulin
8
−12
inhibitory activities.
molecular formula was determined to be C H O from the
In the course of the search for new bioactive metabolites of
the poisonous mushroom N. fasciculare, we isolated four new
lanostane triterpenoids (1−4), together with 11 known
compounds (5−15), from the MeOH extracts of the fruiting
bodies using a bioassay-guided fractionation technique. The
isolated compounds (1−15) were evaluated for their
antiproliferative activities against four human cancer cell lines
36 60
9
+
molecular ion peak [M + Na] at m/z 659.4132 (calcd for
C H O Na, 659.4135) in the positive-ion HRESIMS. The IR
36 60
9
−1
spectrum exhibited absorptions of hydroxy (3357 cm ) and
carbonyl (1683 cm ) groups. The H NMR spectrum (Table
) showed the presence of signals due to eight tertiary methyl
−1
1
1
(
A549, SK-OV-3, SK-MEL-2, and HCT-15) and for their
Received: November 15, 2012
inhibitory effects on nitric oxide (NO) production in
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Table 1. H (500 MHz) and 13C NMR (125 MHz) Data of
1
1
2,16
Fasciculols J (1) and K (2)
1
2
δC
δC
a
a
position (CD OD)
δH (J in Hz)
(CD OD)
δH (J in Hz)
3
3
1
2
40.6 t
66.4 d
1.26 m; 2.04 m
3.83 td (10.5,
40.3 t
66.5 d
1.25 m; 2.06 m
3.82 td (10.0,
4.5)
4
.0)
3
4
5
6
7
8
9
1
1
84.4 d
38.6 s
50.3 d
17.9 t
4.55 d (10.5)
84.3 d
38.5 s
50.3 d
17.8 t
27.4 t
135.1 s
132.4 s
37.8 s
32.5 t
4.56 d (10.0)
1.27 m
1.26 m
1.53 m; 1.71 m
2.07 m
1.55 m; 1.74 m
2.06 m
27.6 t
135.3 s
132.2 s
37.8 s
33.2 t
0
1
2.09 br s
2.08 br s
2
.65 br d (8.0)
2.68 br d (8.0)
4.02 br d (8.0)
1
1
1
1
1
1
1
1
2
2
2
3
4
5
6
7
8
9
0
1
72.1 d
49.2 s
49.1 s
31.8 t
27.6 t
42.6 d
15.5 q
19.0 q
36.1 d
16.5 q
4.01 br d (8.0)
72.8 d
49.7 s
49.6 s
31.3 t
26.0 t
37.7 d
15.9 q
18.7 q
42.8 d
60.5 t
1.28 m; 1.71 m
1.53 m; 2.08 m
2.23 q (9.5)
0.66 s
1.22 m; 1.74 m
1.56 m; 2.09 m
2.45 q (9.5)
0.64 s
1.11 s
1.11 s
1.44 m
1.40 m
1.04 d (6.5)
3.72 dd (11.0,
2.5)
3
.78 dd (11.0,
.5)
4
2
2
2
2
2
2
2
2
3
1
2
2
3
4
5
6
7
8
9
0
′
33.1 t
27.6 t
78.3 d
72.5 s
24.2 q
23.5 q
27.6 q
15.8 q
23.8 q
171.3 s
46.3 t
1.36 m; 1.55 m
1.33 m; 1.53 m
3.23 br d (9.5)
27.4 t
27.9 t
77.9 d
72.4 s
24.2 q
23.4 q
27.5 q
16.5 q
22.7 q
171.6 s
46.0 t
1.45 m; 1.64 m
1.41 m; 1.58 m
3.24 br d (10.0)
1.16 s
1.13 s
0.91 s
0.91 s
1.08 s
1.16 s
1.13 s
0.91 s
0.91 s
1.08 s
′
2.64 d (15.5)
2.68 d (15.5)
2.74 d (15.5)
2
.72 d (15.5)
3′
4′
5′
69.9 s
26.7 q
46.1 t
70.1 s
26.6 q
45.8 t
1.39 s
1.40 s
2.42 d (15.5)
2.60 d (15.5)
2.54 d (15.5)
2
.57 d (15.5)
6′
173.8 s
173.9 s
a
The assignments were based on DEPT, HMQC, and HMBC
experiments.
5
45.3457 (calcd for C H O Na, 545.3454), consistent with a
30 50 7
1
13
[
δH 0.66, 0.91, 0.91, 1.08, 1.11, 1.13, 1.16, and 1.39], a
molecular formula of C H O . The H and C NMR data
30 50 7
18
secondary methyl [δH 1.04 (d, J = 6.5 Hz)], and four
(Table 2) were similar to those of sublateriol C (15), with the
oxygenated methine [δ 3.23 (br d, J = 9.5 Hz), 3.83 (td, J =
added presence of signals for an epoxy group [δ 3.10 (1H, d, J
H
H
2
1,22
1
0.5, 4.0 Hz), 4.01 (br d, J = 8.0 Hz), and 4.55 (d, J = 10.5
= 6.5 Hz, H-7); δ 55.8 (C-7) and δ 61.4 (C-8)]
in 3
C
C
1
3
Hz)] groups. The C NMR spectrum (Table 1) showed 36
carbon signals, which were attributed to nine methyl, 10
methylene, and seven methine groups, as well as 10 quaternary
instead of signals for a double bond at δ 5.66 (br d, J = 5.9 Hz,
H
18
H-7); δ 124.6 (C-7) and δ 143.6 (C-8) in 15. The location
C
C
1
1
of the epoxy group was confirmed to be C-7/C-8 by H− H
COSY correlations starting at H-5 via H-6 and ending at H-7 in
combination with HMQC and HMBC correlations of H-7/C-5,
H-11/C-8, and H-30/C-8 (Figure 1). This result suggested that
carbons, including two olefinic carbons [δ 132.2 and 135.3],
C
two oxygenated quaternary carbons [δ 69.9 and 72.5], and
C
two carbonyl groups [δC 171.3 and 174.7] determined by
analysis of the DEPT and HMQC spectra. These data
suggested that compound 1 is a lanostane triterpenoid, which
7
,8
the Δ double bond in 15 was oxidized as an epoxy group in 3.
The 7α,8α-configuration of the epoxide ring was deduced from
B
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1
1
Figure 1. H− H COSY (bold lines) and key HMBC (arrows) correlations for 1 and 3.
1
13
the correlations of H-7/H-5 and H-7/H-30 in the NOSEY
spectrum (Figure 2). The remaining relative configuration of 3
was established to be identical to 15 based on the coupling
constants and a NOESY experiment and by comparison of the
H and C NMR data with those of 15.
Fasciculol M (4) was obtained as a colorless gum with a
and by comparison of the H and C NMR data with those of
12.
Fasciculol G (5) is a known compound reported by Ikeda et
al. from this mushroom. The NMR data of fasciculol G (5)
have not been reported, although its melting point, MS, IR, and
specific rotation were published by Ikeda et al. We performed
full NMR data assignments (Table 2) of 5 by analysis of 2D
NMR data (including DEPT, HMQC, HMBC, and NOESY).
To the best of our knowledge, the NMR data of fasciculol G are
reported for the first time in this study.
Compounds 1−15 were evaluated for their antiproliferative
activities against the A549, SK-OV-3, SK-MEL-2, and HCT-15
human cancer cell lines using the SRB bioassay. Doxorubicin
was used as a positive control. All isolated triterpenoids except
for compounds 1 and 2 showed cytotoxic activity against the
four human cancer cell lines with IC values of 2.29−28.48 μM
(Table 3). The newly isolated compounds 3 and 4 exhibited
significant cytotoxic activity against A549, SK-OV-3, SK-MEL-
2, and HCT-15 cell lines (IC50 (3): 6.59, 7.08, 8.26, and 8.53
23
1
13
23
molecular formula of C H O based on the molecular ion
3
0
50
7
+
peak [M + Na] at m/z 545.3457 (calcd for C H O Na,
45.3454) by HRESIMS. The H and C NMR data (Table 2)
were similar to those of fasciculol C (12), with a noticeable
difference being the presence of a signal for a ketone group (δC
30
50
7
1
13
5
16
1
99.7), which was determined to be at C-7 by the HMBC
24
correlations from H-5 (δ 1.71) to C-7, from H-6 (δ 2.35 and
H
H
2
.53) to C-7, and from H-30 (δ 1.11) to C-7. Full assignments
H
1
13
of the H and C NMR data of 4 were performed by DEPT,
HMQC, HMBC, and NOESY spectra. The relative config-
uration of 4 was elucidated to be identical to 12 on the basis of
the coupling constants and a NOESY experiment (Figure 2)
5
0
C
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Journal of Natural Products
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Figure 2. Key NOESY (dashed arrow) correlations for 1, 3, and 4.
μM, and IC (4): 3.99, 7.36, 4.77, and 8.50 μM, respectively).
Moreover, compounds 11, 12, and 15 displayed considerable
cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15
cell lines (IC50 (11): 7.85, 8.53, 5.17, and 8.22 μM, IC50 (12):
Based on the expanded understanding that cancer pro-
gression is associated with inflammatory responses, the anti-
inflammatory activities of compounds 1−15 were also
evaluated by examining their effects on LPS-induced NO
production in murine microglia BV-2 cells. Among the tested
compounds, compounds 4 and 7 moderately inhibited NO
production, with IC50 values of 49.9 and 93.1 μM without cell
toxicity, respectively (Supporting Information, Table 1S). The
remaining compounds did not show any significant effects.
Some cell toxicity was observed in cells treated with
compounds 5, 9−12, 14, and 15, whereas other compounds
had no influence on cell viability.
50
2.37, 2.82, 2.29, and 3.06 μM, and IC50 (15): 4.47, 3.29, 4.54,
and 7.71 μM, respectively). Interestingly, compounds 5, 6, 9,
and 10, with an N-glycyl-3-hydroxy-3-methylglutaryl group as a
side chain, showed significant selective cytotoxicity against the
SK-MEL-2 cell line (IC (5): 8.60 μM, IC (6): 9.06 μM, IC
5
0
50
50
(9): 9.16 μM, and IC50 (10): 5.73 μM). The presence of a 3-
hydroxy-3-methylglutaryl group linked at C-3 in compounds 1
and 2 markedly reduced the activity when compared to
compounds 7 and 8, with the side chain connected to C-2.
Compounds 13 and 14, with a ketone functionality at C-12,
had good selective cytotoxicity against the SK-OV-3 cell line
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO P-1020 polarimeter. IR spectra were recorded
on a Bruker IFS-66/S FT-IR spectrometer. UV spectra were recorded
using an Agilent 8453 UV−visible spectrophotometer. NMR spectra
were recorded on a Varian UNITY INOVA 500 NMR spectrometer
(
IC50 (13): 9.86 and IC50 (14): 7.98 μM). A number of
lanostane-type triterpenes isolated from fungi have recently
been reported to have potential as anticancer agents, and these
compounds were found to arrest the cell cycle in either the G1
1
13
operating at 500 MHz ( H) and 125 MHz ( C), respectively. ESIMS
and HRESIMS spectra were recorded on a Micromass QTOF2-MS.
Semipreparative HPLC was performed using a Gilson 306 pump with
a Shodex refractive index detector. Chromatographic separation was
performed on an Econosil RP-18 10 μ column (250 × 10 mm i.d.).
Silica gel 60 (Merck, 230−400 mesh) and RP-C silica gel (Merck,
or the G /M, affect the transporting proteins MDR and MRP,
2
increase levels of p53 and Bax, decrease MMP expression,
induce apoptosis, or inhibit not only the phosphorylation of
25
Erk1/2 but also the activation of NF-κB and AP-1. Thus, it
appears that the active compounds 3−15 isolated from the
mushroom N. fasciculare may be valuable in further studies
addressing their cytotoxic mechanism and for in vivo
experiments.
18
2
30−400 mesh) were used for column chromatography. Silica Waters
Sep-Pak Vac 6 cc and C18 Waters Sep-Pak Vac 6 cc cartridges were
also used for column chromatography. TLC was performed using
Merck precoated silica gel F254 plates and RP-18 F254s plates. Spots
D
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Table 2. H (500 MHz) and 13C NMR (125 MHz) Data of Fasciculols L (3), M (4), and G (5)
1
3
4
5
a
a
a
position
1
δC (CD OD)
δH (J in Hz)
1.46 m
.18 dd (17.5, 4.5)
δC (CD OD)
δH (J in Hz)
1.42 m
δC (CD OD)
δH (J in Hz)
3
3
3
45.2 t
42.6 t
40.3 t
1.26 m; 2.00 m
2
2.18 dd (17.5, 4.5)
3.70 td (10.0, 4.5)
2.97 d (10.0)
2
3
4
5
6
67.8 d
82.3 d
39.1 s
48.5 d
21.2 t
3.66 td (10.0, 4.5)
2.88 d (10.0)
67.9 d
81.9 d
38.9 s
50.2 d
36.1 t
65.8 d
84.1 d
38.6 s
50.4 d
17.9 t
3.82 td (10.0, 4.0)
4.57 d (10.0)
0.89 m
1.71 dd (14.5, 3.0)
2.35 dd (14.5, 3.0)
1.28 m
1.86 m; 2.09 m
1.53 m; 1.72 m
2
.53 t (14.5)
7
8
9
55.8 d
61.4 s
3.10 d (6.5)
199.7 s
138.2 s
164.0 s
40.5 s
27.5 t
135.2 s
132.2 s
37.6 s
2.08 m
145.8 s
37.8 s
1
1
0
1
125.2 d
5.91 br d (5.5)
4.22 br d (5.5)
33.7 t
2.33 br d (21.0)
.05 dd (21.0, 8.0)
32.9 t
2.08 br s
3
2.68 br d (8.0)
4.01 br d (8.0)
1
1
1
1
1
1
1
1
2
2
2
3
4
5
6
7
8
9
0
1
73.6 d
48.2 s
47.8 s
26.5 t
26.2 t
39.2 d
16.0 q
22.7 q
42.3 d
60.5 t
72.1 d
49.9 s
47.6 s
33.1 t
27.7 t
36.5 d
15.9 q
17.5 q
42.5 d
60.6 t
4.10 br d (8.0)
72.1 d
49.3 s
49.2 s
31.7 t
27.5 t
42.6 d
15.5 q
18.8 q
36.1 d
16.5 q
1.30 m; 1.72 m
1.57 m; 2.03 m
2.43 q (10.0)
0.81 s
1.38 m; 1.98 m
1.53 m; 2.12 m
2.38 q (9.5)
1.28 m; 1.69 m
1.55 m; 2.10 m
2.23 q (9.5)
0.66 s
0.61 s
1.14 s
1.24 s
1.11 s
1.46 m
1.36 m
1.41 m
3.72 dd (11.0, 0.5)
3.71 dd (11.0, 1.5)
3.79 dd (11.0, 3.5)
1.44 m; 1.64 m
1.42 m; 1.58 m
3.25 br d (10.5)
1.05 d (6.5)
3
.76 dd (11.0, 2.0)
2
2
2
2
2
2
2
2
3
1
2
3
4
5
6
7
8
2
3
4
5
6
7
8
9
0
′
′
′
′
′
′
′
′
26.7 t
27.8 t
77.9 d
72.4 s
24.2 q
23.4 q
27.5 q
15.4 q
23.0 q
1.44 m; 1.67 m
1.42 m; 1.57 m
3.26 br d (10.5)
26.7 t
27.9 t
77.9 d
72.4 s
24.3 q
23.4 q
27.0 q
15.5 q
23.8 q
34.2 t
27.5 t
78.3 d
72.4 s
24.2 q
23.5 q
27.5 q
15.7 q
23.5 q
171.7 s
46.8 t
70.1 s
27.5 q
46.7 t
172.7 s
40.3 t
172.6 s
53.9 q
1.37 m; 1.58 m
1.33 m; 1.55 m
3.23 d (11.0)
1.16 s
1.13 s
1.02 s
0.84 s
1.09 s
1.16 s
1.13 s
1.01 s
0.90 s
1.11 s
1.16 s
1.13 s
0.91 s
0.91 s
1.08 s
2.73 s
1.41 s
2.59 s
3.95 br s
3.72 s
OMe
a
The assignments were based on DEPT, HMQC, and HMBC experiments.
were detected on TLC under UV light or by heating after spraying
with anisaldehyde−sulfuric acid.
Mushroom Material. Fresh fruiting bodies of N. fasciculare were
collected at Donggureung, Guri of GyeongGi-do, Korea, in August
cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines
using the SRB bioassay. We selected the CHCl -soluble fraction for
3
the current phytochemical study, as the CHCl -soluble fraction was
3
the most cytotoxic against the tested tumor cell lines. The CHCl3-
2
009. A voucher specimen (SKKU-2009-08) of the mushroom was
soluble fraction (1.8 g) was separated by silica gel (230−400 mesh)
authenticated by one of the authors (K.R.L.) and was deposited at the
herbarium of the School of Pharmacy, Sungkyunkwan University,
Korea.
Extraction and Isolation. Air-dried and powdered N. fasciculare
fruiting bodies (88 g) were extracted twice with 80% aqueous MeOH
column chromatography [65 g, 1.5 × 32 cm, eluted with CHCl −
3
MeOH, 20:1 (1.8 L) and 4:1 (1.0 L)] to yield 10 fractions (Fr. A, 20:1,
0.3 L; Fr. B, 20:1, 0.3 L; Fr. C, 20:1, 0.3 L; Fr. D, 20:1, 0.3 L; Fr. E,
20:1, 0.3 L; Fr. F, 20:1, 0.3 L; Fr. G, 4:1, 0.2 L; Fr. H, 4:1, 0.2 L; Fr. I,
4:1, 0.2 L; and Fr. J, 4:1, 0.4 L). Fraction F (115 mg) was subjected to
fractionation with passage over a C18 Waters Sep-Pak Vac 6 cc (80%
aqueous MeOH) to give 13 subfractions (F1−F13). Subfraction F4 (8
mg) was purified by semipreparative HPLC (80% MeOH(aq)) with a
Shodex refractive index detector, using an Econosil RP-18 10 μ
column (250 × 10 mm i.d.), to yield compound 14 (3 mg).
(
each 2.0 L × 3 days) at room temperature and filtered. The filtrate
was evaporated under vacuum to obtain a crude MeOH extract (9.4
g), which was suspended in distilled water and then successively
partitioned with n-hexane, CHCl , and n-BuOH, yielding 900 mg, 1.8
g, and 2.8 g of residues, respectively. Each fraction was evaluated for
3
E
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Journal of Natural Products
Article
Table 2; positive HRESIMS m/z 545.3457 [M + Na]+ (calcd for
Table 3. Cytotoxicity of Compounds 1−15 against Four
C H O Na, 545.3454).
Fasciculol M (4): colorless gum; [α]D +21.9 (c 0.15, MeOH); IR
Human Cancer Cell Lines Using the SRB Bioassay
30 50
7
25
a
IC50 (μM)
(KBr) ν 3357, 2945, 2832, 2495, 2235, 1667, 1451, 1121, 1031, 674
max
−
1
1
cm ; UV (MeOH) λ (log ε) 250 (2.72) nm; H (CD OD, 500
max
3
compound
A549
SK-OV-3
SK-MEL-2
HCT-15
MHz) and 13C NMR (CD OD, 125 MHz) data, see Table 2; positive
3
1
>30
>30
6.59
3.99
>30
>30
27.20
25.52
>30
>30
7.85
2.37
>30
>30
4.47
0.02
>30
>30
7.08
7.36
>30
>30
22.17
16.17
>30
>30
8.53
2.82
9.86
7.98
3.29
0.01
>30
>30
>30
8.53
8.50
>30
>30
28.48
27.96
>30
>30
8.22
3.06
>30
>30
7.71
0.13
+
HRESIMS m/z 545.3457 [M + Na] (calcd for C H O Na,
3
0
50
7
2
3
4
5
6
7
8
9
1
1
1
1
1
1
>30
5
45.3454).
Fasciculol G (5): colorless gum; [α]25 +6.4 (c 0.30, MeOH); IR
8.26
4.77
8.60
9.06
16.60
25.23
9.16
5.73
5.17
2.29
>30
D
(KBr) ν 3355, 2944, 2833, 2495, 2235, 1687, 1451, 1121, 1032, 674
max
−
1
1
cm ; UV (MeOH) λ (log ε) 213 (3.84) nm; H (CD OD, 500
max
3
MHz) and 13C NMR (CD OD, 125 MHz) data, see Table 2; positive
3
+
HRESIMS m/z 730.4508 [M + Na] (calcd for C H NO Na,
39 65
10
7
30.4506).
Alkaline Methanolysis of 1 and 2. Compounds 1 and 2 (each
3
.0 mg) were treated with 3% NaOMe in MeOH at room temperature
0
1
2
3
4
5
for 2 h. Each reaction mixture was then passed through an Amberlite
IRA-67 column (Rohm and Haas) for neutralization and was
evaporated under vacuum to obtain a residue, which was purified on
a Sephadex LH-20 column (Pharmacia) using MeOH to give the main
triterpene body [fasciculol B (1a; 0.7 mg) from 1 and fasciculol C (2a;
>30
0
.6 mg) from 2] and methyl (S)-3-hydroxy-3-methylglutarate [1b (0.8
4.54
0.01
16,19,20
b
mg) from 1 and 2b (0.5 mg) from 2] as a viscous syrup.
doxorubicin
1
a: colorless gum; [α]2 +75.4 (c 0.07, MeOH); H NMR and
5
1
a
D
IC50 value of compounds against each cancer cell line, which was
16
ESIMS data were identical to those of fasciculol B. 1b: viscous syrup;
defined as the concentration (μM) that caused 50% inhibition of cell
25
1
[
α] +6.5 (c 0.08, CHCl ); H NMR (CDCl , 500 MHz) δ 3.73 (3H,
b
D 3 3
growth in vitro. Doxorubicin as a positive control.
s, OMe), 2.72 (1H, d, J = 16.5 Hz, H-2), 2.67 (2H, s, H-4), 2.58 (1H,
+
d, J = 16.5 Hz, H-2), 1.40 (3H, s, H-6); ESIMS m/z 177 [M + H] . 2a:
colorless gum; [α]2 +63.5 (c 0.06, MeOH); H NMR and ESIMS data
5
1
Subfraction F7 (7 mg) was purified by separation with semipreparative
HPLC (83% MeOH(aq)) to give compound 13 (3 mg). Compound
D
1
6
25
were identical to those of fasciculol C. 2b: viscous syrup; [α]D +6.0
c 0.05, CHCl ); H NMR and ESIMS data were identical to those of
1
(
1
0 (3 mg) was obtained from subfraction F8 (6 mg) by separation
3
1
b.
Cytotoxicity Testing. The cell lines used were A549 (non-small-
with semipreparative HPLC (85% MeOH(aq)). Subfraction F9 (25
mg) was purified by semipreparative HPLC (90% MeOH(aq)) to
afford compounds 5 (8 mg) and 9 (6 mg). Fraction G (58 mg) was
subjected to purification by passage over a Silica Waters Sep-Pak Vac 6
cell lung adenocarcinoma), SK-OV-3 (ovary malignant ascites), SK-
MEL-2 (skin melanoma), and HCT-15 (colon adenocarcinoma).
These cancer cell lines were provided by the National Cancer Institute
cc (CHCl −MeOH, 4:1) to give compound 6 (6 mg). Fraction H
3
(
NCI). An SRB bioassay was used to determine the cytotoxicity of
(
150 mg) was subjected to fractionation by passage over a C Waters
18
24
each compound against the cell lines mentioned above. The assays
were performed at the Korea Research Institute of Chemical
Technology. Doxorubicin was used as a positive control. Doxorubicin
cytotoxicity against the A549, SK-OV-3, SK-MEL-2, and HCT-15 cell
lines was IC50 0.02, 0.01, 0.01, and 0.13 μM, respectively.
Sep-Pak Vac 6 cc (80% MeOH(aq)) to afford 10 subfractions (H1−
H10). Subfraction H3 (7 mg) was purified by separation with
semipreparative HPLC (73% MeOH(aq)) to furnish compound 4 (4
mg). Subfraction H6 (8 mg) was purified by semipreparative HPLC
(
75% MeOH(aq)) to give compound 3 (3 mg). Compound 15 (5
Measurement of NO Production and Cell Viability. Murine
mg) was isolated from subfraction H7 (43 mg) by separation with
semipreparative HPLC (75% MeOH(aq)). Fraction I (108 mg) was
subjected to fractionation by passage over a C18 Waters Sep-Pak Vac 6
cc (80% MeOH(aq)) to afford eight subfractions (I1−I8). Subfraction
I5 (18 mg) was subjected to purification with passage over a Silica
4
microglia BV-2 cells were plated into a 96-well plate (3 × 10 cells/
well). After 24 h, cells were pretreated with samples for 30 min and
then stimulated with 100 ng/mL of LPS for another 24 h. Nitrite, a
soluble oxidation product of NO, was measured in the culture media
using the Griess reaction. The supernatant (50 μL) was harvested and
mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1%
N-1-napthylethylenediamine dihydrochloride in 5% phosphoric acid).
Absorbance at 540 nm was measured after 10 min using a microplat−e
Waters Sep-Pak Vac 6 cc (CHCl −MeOH, 8:1) to obtain compound
3
1
2 (10 mg). Compound 11 (5 mg) was isolated from subfraction I6
(
11 mg) by passage over a silica Waters Sep-Pak Vac 6 cc (CHCl −
3
MeOH, 8:1). Subfraction I7 (35 mg) was purified by separation with
semipreparative HPLC (85% MeOH(aq)) to give compounds 1 (7
mg), 7 (8 mg), and 8 (4 mg). Fraction J (52 mg) was subjected to
fractionation with passage over a C18 Waters Sep-Pak Vac 6 cc (80%
MeOH(aq)) to give two subfractions (J1 and J2). Finally, subfraction
J2 (10 mg) was purified by separation with semipreparative HPLC
reader. Sodium nitrite was used as the standard to calculate the NO2
concentration. Cell viability was assessed by a 3-[4,5-dimethylthiazol-
G
2
-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. N -Monometh-
yl-L-arginine (L-NMMA, Sigma, USA) was tested as a positive control.
L-NMMA is a nonspecific NO synthase inhibitor.
(
70% MeOH(aq)) to furnish compound 2 (6 mg).
Fasciculol J (1): colorless gum; [α]2 +15.8 (c 0.35, MeOH); IR
5
ASSOCIATED CONTENT
D
■
(
KBr) ν 3357, 2946, 2833, 2496, 2235, 1683, 1451, 1121, 1030, 674
max
*
S
Supporting Information
−1
1
13
cm ; H (CD OD, 500 MHz) and C NMR (CD OD, 125 MHz)
data, see Table 1; positive HRESIMS m/z 659.4132 [M + Na] (calcd
for C H O Na, 659.4135).
3
3
+
1D and 2D NMR data of 1−5 and inhibitory effects on NO
production of compounds 1−15 in LPS-activated BV-2 cells
(Table 1S). These materials are available free of charge via the
Internet at http://pubs.acs.org.
3
6
60
9
25
Fasciculol K (2): colorless gum; [α]D +17.1 (c 0.30, MeOH); IR
(
KBr) ν 3357, 2946, 2833, 2495, 2236, 1685, 1451, 1121, 1032, 674
max
−1
1
13
cm ; H (CD OD, 500 MHz) and C NMR (CD OD, 125 MHz)
data, see Table 1; positive HRESIMS m/z 675.4082 [M + Na] (calcd
for C H O Na, 675.4084).
3
3
+
AUTHOR INFORMATION
■
3
6
60 10
25
Corresponding Author
*Tel: +82-31-290-7710. Fax: +82-31-290-7730. E-mail: krlee@
skku.ac.kr.
Fasciculol L (3): colorless gum; [α]D +13.0 (c 0.15, MeOH); IR
−1
(
KBr) νmax 3358, 2945, 2832, 2494, 2235, 1451, 1121, 1030, 674 cm ;
1
13
H (CD OD, 500 MHz) and C NMR (CD OD, 125 MHz) data, see
3
3
F
dx.doi.org/10.1021/np300801x | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research was supported by Basic Science Research
Program through the National Research Foundation of Korea
■
(
NRF) funded by the Ministry of Education, Science and
Technology (2012-0002237). The authors thank Drs. E. J.
Bang, S. G. Kim, and J. J. Seo at the Korea Basic Science
Institute for the NMR and MS spectra measurements.
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dx.doi.org/10.1021/np300801x | J. Nat. Prod. XXXX, XXX, XXX−XXX