Inhibitory effects of terpenoids from the fermented broth of the ascomycete stilbohypoxylon elaeicola YMJ173 on nitric oxide production in RAW264.7 macrophages

Article abstract of DOI:10.1002/cbdv.201100025

A series of six isopimarane-type diterpene glycosides, along with an eremophilane-type sesquiterpene, i.e., elaeicolasides A-C (1-3, resp.), 16-(α-D-mannopyranosyloxy)isopimar-7-en-19-oic acid (4), hymatoxin K (5), hymatoxin L (6), and elaeicolalactone (7), were isolated from the AcOEt extract of the fermented broth of Stilbohypoxylon elaeicola YMJ173. Among these, 1-3 and 7 are new compounds based on their spectroscopic data and sugar composition analysis. The effects of 1-7 on the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells were evaluated. All these compounds inhibited NO production, detected as nitrite in the culture medium, in activated macrophages without any cytotoxicity at a concentration of 100μM. Among these compounds, 2 showed a significant activity with the average maximum inhibition (E_max) and median inhibitory concentration (IC₅₀) values of 93.3±0.5% and 79.3±0.4μM, respectively. Copyright

Full text of DOI:10.1002/cbdv.201100025

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
131
Inhibitory Effects of Terpenoids from the Fermented Broth of the Ascomycete
Stilbohypoxylon elaeicola YMJ173 on Nitric Oxide Production in RAW264.7
Macrophages
by Guei-Jane Wanga), Wen-Li Liangb), Yu-Ming Juc), Wen-Bin Yangd), Ya-Wen Change), and
Tzong-Huei Lee*e)
a) L5 Research Center, China Medical University Hospital, Taichung 404, Taiwan
b) School of Pharmacy, Taipei Medical University, Taipei 110, Taiwan
c) Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan
d) Genomics Research Center, Academia Sin, Taipei 115, Taiwan
e) Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan
(phone: þ886-2-27361661 ext. 6156; e-mail: thlee@tmu.edu.tw)
A series of six isopimarane-type diterpene glycosides, along with an eremophilane-type sesquiter-
pene, i.e., elaeicolasides A–C (1–3, resp.), 16-(a-d-mannopyranosyloxy)isopimar-7-en-19-oic acid (4),
hymatoxin K (5), hymatoxin L (6), and elaeicolalactone (7), were isolated from the AcOEt extract of the
fermented broth of Stilbohypoxylon elaeicola YMJ173. Among these, 1–3 and 7 are new compounds
based on their spectroscopic data and sugar composition analysis. The effects of 1–7 on the inhibition of
nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW264.7
cells were evaluated. All these compounds inhibited NO production, detected as nitrite in the culture
medium, in activated macrophages without any cytotoxicity at a concentration of 100 mm. Among these
compounds, 2 showed a significant activity with the average maximum inhibition (Emax) and median
inhibitory concentration (IC50) values of 93.3ꢀ0.5% and 79.3ꢀ0.4 mm, respectively.
Introduction. – Stilbohypoxylon elaeicola (Henn.) L. E. Petrini is geographically
distributed in tropical and subtropical areas, and belongs to the ascomycete family
¨
Xylariaceae [1][2]. Petrini listed Astrocystis cocoes (Henn.) Lꢀssꢁe & Spooner,
¨
Rosellinia elaeicola Henn., R. samoensis Henn., R. cocoes Henn., and S. moelleri as
synonyms of S. elaeicola [2]. So far, no report regarding the chemical constituents of
this fungus has been found in the literature. During a preliminary human disease-based
biological screening, it was found that the AcOEt extract from the fermented broth of
S. elaeicola strain No. YMJ173 exhibited a potent inducible nitric oxide synthase
(iNOS) inhibitory activity (Emax ¼98%) without any cytotoxicity against RAW264.7
cells at a concentration of 100 mg/ml [3]. We thus set out to isolate the active principles
from the fermented broth of this strain and to assess the bioactivities of the pure
isolates.
Results and Discussion. – The AcOEt extract of the fermented broth of S. elaeicola
YMJ173 was fractionated by a Sephadex LH-20 column into five portions. Portions II
and III were then subjected to HPLC to give four new compounds 1–3 and 7 (Fig. 1),
together with three known ones. Compound 4 was identified as 16-(a-d-mannopyr-
anosyloxy)isopimar-7-en-19-oic acid [4], obtained previously from Xylaria polymor-
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 1. Chemical structures of compounds 1–7
pha, a fungus of the family Xylariaceae to which S. elaeicola belongs. Compounds 5 and
6 were identified as hymatoxins K and L, respectively, two rare isopimarane
mannopyranosides with a g-lactone moiety between C(4) and C(6), which were
previously isolated from the fungus Hypoxylon mammatum [5].
Compound 1, obtained as amorphous white powder, was determined to have the
molecular formula C25H42O9 deduced from the HR-ESI-MS and 13C-NMR data. The IR
spectrum of 1 revealed the presence of a C¼C bond (1643 cmꢁ1) and a OH group
(3348 cmꢁ1). The 13C-NMR spectrum of 1 exhibited 25 C-atom signals attributable to a
C6 sugar unit and a C19 aglycone (Table 1). The 13C-NMR data of 1 were quite similar to
those of 4 except that conspicuous differences, including the disappearance of a COOH
signal at d(C) 180.2 (C(19)), and the signal of an olefinic C-atom at d(C) 137.2 (C(14)),
a lower field-shifted signal at d(C) 84.9 (C(4)), and the signal of a carbinol C-atom at
d(C) 74.0 (C(7)) were observed (Table 1). Thus, the COOH group at C(4) in 4 was
replaced by a OOH group in 1 as evidenced by the lower-field shift of the signal of C(4)
at d(C) 84.9, quite similar to that of the previously reported norabietane analogs [6].
The differences observed in the 13C-NMR spectra of 4 and 1 were also reflected in the
1H-NMR data of 1 (Table 2), in which a higher-field shift of the Me(18) signal from
d(H) 1.48 to 1.12, an olefinic HꢁC(7) signal from d(H) 5.44 (d, J¼4.4) to 4.11 (t, J¼
2.7), and CH2(14) signal at d(H) 1.95–1.99 (m) to 5.55 (s) were observed. These data
unambiguously revealed that the aglycone of 1 had a norisopimarane skeleton with a
OOH group at C(4), a OH group at C(7), and a C(8)¼C(14) bond as evidenced from
key HMBCs Me(18)/C(4), HꢁC(7)/C(5), C(8), C(9), and C(14), and Me(17)/C(14)
(Fig. 2). In the NOESY spectrum of 1 (Fig. 2), cross-peaks Me(20)/Me(17) and
Me(18), HaxꢁC(1)/HꢁC(5) and HꢁC(9), and HꢁC(7)/HꢁC(14) indicated that Me(17),
Me(18), Me(20), and HꢁC(7) were b-oriented, and HOOꢁC(4), HꢁC(5), and HꢁC(9)
were a-oriented. The sugar moiety was identified as a-d-mannopyranose by comparing
the 1H- and 13C-NMR data with those in the literature [5][7], and this was supported by
the optical-rotation measurement and sugar composition analysis of the acid hydro-
lysate of 1. The sugar was at C(16) as corroborated by correlations HꢁC(1’)/C(16) and

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
133
Table 1. 13C-NMR Data (125 MHz) of Compounds 1–3 and 7
1a)
2a)
3b)
7b)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
39.3
40.4
38.6
34.8
25.5
75.6
73.9
43.2
29.4
76.1
84.9
49.5
34.8
141.5
172.1
122.9
18.9
26.6
20.7
36.7
84.9
43.0
29.6
74.0
139.2
47.5
40.1
19.7
35.2
34.5
137.2
43.6
65.4
26.5
19.4
21.3
43.8
73.1
53.2
23.5
122.2
137.2
53.7
37.4
21.7
38.3
34.1
49.0
45.2
65.1
22.2
23.5
20.7
38.7
44.3
56.5
129.0
128.8
136.3
50.6
38.7
20.1
35.3
35.4
134.7
43.8
65.7
28.4
28.8
181.0
12.3
101.7
72.3
72.7
68.6
74.6
62.9
14.6
101.6
72.3
72.7
68.8
74.7
63.0
15.1
101.7
72.3
72.7
68.7
74.7
63.0
1’
2’
3’
4’
5’
6’
a) Recorded in C5D5N. b) Recorded in CD3OD.
Fig. 2. Key NOESY correlations ($) and HMBCs (H!C) of 1
CH2(16)/C(1’) in the HMBC experiment. Accordingly, 1 was conclusively characterized
as 7a-hydroxy-16-(a-d-mannopyranosyloxy)norisopimar-8(14)-ene 4-hydroperoxide,
and named elaeicolaside A.
The molecular formula of 2, C25H42O7, was established by analysis of the 13C-NMR
and HR-ESI-MS data. The IR spectrum revealed the presence of a OH group
1
(3402 cmꢁ1) and a C¼C bond (1680 cmꢁ1). The H- and 13C-NMR data of 2 closely
resemble those of 1 except for a carbinol C-atom resonance at d(C) 73.1 (C(4)), an

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Table 2. 1H-NMR Data (500 MHz) for Compounds 1–3 and 7. d in ppm, J in Hz.
1a)
2a)
3b)
7b)
1
2
3
1.08–1.12 (m),
1.63–1.69 (m)
1.46–1.49 (m),
1.60–1.64 (m)
1.60–1.64 (m),
1.84–1.90 (m)
2.18–2.21 (m)
1.50–1.53 (m),
1.80–1.85 (m)
4.11 (t, J¼2.7)
1.08 (dt, J¼13.1, 3.3),
1.80 (d, J¼13.1)
1.41–1.45 (m),
1.53–1.59 (m)
1.37–1.41 (m),
1.71–1.77 (m)
1.40–1.44 (m)
1.83–1.87 (m),
2.11–1.15 (m)
1.10–1.14 (m),
1.73 (d, J¼12.7)
1.50–1.54 (m),
1.87–1.91 (m)
1.06–1.10 (m),
2.14–2.18 (m)
2.13–2.16 (m)
6.07 (dd, J¼1.6, 10.0)
1.13–1.17 (m),
1.46–1.50 (m)
1.40–1.44 (m),
2.08–2.12 (m)
3.44 (br. s)
5
6
1.30 (dd, J¼3.3, 14.0)
2.01 (t, J¼14.0),
2.28 (dd, J¼3.3, 14.0)
7
8
9
5.37 (br. d, J¼4.4)
5.89 (dd, J¼2.9, 10.0)
1.95–1.98 (m)
4.59 (dd, J¼6.6, 12.3)
0.78 (t, J¼12.3),
2.20–2.23 (m)
1.69–1.73 (m)
1.87 (dd, J¼12.3, 6.6)
11
12
1.38–1.42 (m),
1.53–1.57 (m)
1.39–1.42 (m)
1.37–1.41 (m),
1.72–1.76 (m)
1.27–1.31 (m),
1.51–1.55 (m)
1.39–1.43 (m),
1.60–1.64 (m)
1.50–1.54 (m)
13
14
15
16
5.79 (s), 6.25 (s)
1.13 (s)
1.23 (s)
5.55 (s)
1.89–1.93 (m)
1.49–1.53 (m)
3.47–3.51 (m),
3.81–3.85 (m)
0.80 (s)
5.28 (s)
1.58–1.62 (m)
3.47–3.51 (m),
3.73–3.77 (m)
1.01 (s)
1.12 (s)
0.85 (s)
4.71 (d, J¼1.6)
3.76 (dd, J¼3.4, 1.6)
3.63–3.69 (m)
3.57 (t, J¼9.5)
3.50–3.54 (m)
3.67–3.71 (m),
3.81–3.85 (m)
1.58–1.62 (m)
3.42 (dt, J¼9.9, 6.7),
3.79–3.83 (m)
0.99 (s)
1.27 (s)
0.64 (s)
17
18
20
1’
2’
3’
4’
5’
6’
1.21 (s)
0.83 (s)
4.72 (d, J¼1.5)
3.75 (dd, J¼3.3, 1.5)
3.64–3.68 (m)
3.58 (t, J¼9.5)
3.49–3.53 (m)
3.67–3.71 (m),
3.81–3.85 (m)
4.68 (s)
3.73–3.77 (m)
3.65–3.69 (m)
3.61 (t, J¼9.6)
3.48–3.52 (m)
3.69–3.73 (m),
3.77–3.81 (m)
a) Recorded in C5D5N. b) Recorded in CD3OD.
olefinic resonance at d(H) 5.37 (d, J¼4.4) and d(C) 122.2 (HꢁC(7)), and a CH2 signal
at d(H) 1.91 (s) and d(C) 49.0 (CH2(14)). These data indicated that 2 was also a
norisopimarane a-d-mannopyranoside with a OH group at C(4) and a C(7)¼C(8)
bond, as confirmed by key HMBCs Me(18)/C(4), and HꢁC(7)/C(5), C(6), C(9), and
C(14). After assignment of the full 2D-NMR data, the structure of compound 2, named
elaeicolaside B, was established as 16-(a-d-mannopyranosyloxy)norisopimar-7-en-4-ol.
Compound 3 was assigned to have a molecular formula of C26H40O8 on the basis of
the 13C-NMR and HR-ESI-MS data. By comparison of the 13C-NMR data of 3
(Table 1) and 4, major differences of the resonances at d(C) 28.4 (C(17)), 56.5 (C(5)),
128.8 (C(7)), 129.0 (C(6)), and 134.7 (C(14)) indicated conjugated C(6)¼C(7) and
C(8)¼C(14) bonds in 3. The location of the olefinic bonds was further supported by key

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
135
HMBCs HꢁC(7)/C(6), C(8), and C(14), and Me(17)/C(14). These assignments were
in accordance with the UV spectrum, in which an absorption maximum at 241 nm
confirmed the presence of conjugated double bonds in 3. After further analysis of the
HMBC and NOESY of 3, the locations of all functionalities and relative configuration
were determined. Thus, 3 was deduced as 16-(a-d-mannopyranosyloxy)isopimar-
6,8(14)-dien-19-oic acid, and was named elaeicolaside C.
Compound 7, a minor component, was obtained as an amorphous white powder,
and had a molecular formula of C15H22O5, as determined by HR-ESI-MS. Its IR
spectrum exhibited absorption bands at 3402, 1753, and 1666 cmꢁ1, indicating the
1
presence of a OH and a g-lactone CO group, and a C¼C bond, respectively. The H-
(Table 2), 13C-NMR (Table 1), and HSQC spectra revealed that 7 contained a CO
group (d(C) 172.1 (C(12))), an exocyclic C¼CH2 moiety (d(C) 122.9 (C(13)) and 141.5
(C(11))), two O-bearing quaternary C-atoms (d(C) 73.9 (C(4)) and 76.1 (C(7))), two
O-bearing CH groups (d(C) 75.6 (C(3)) and 84.9 (C(8))), an aliphatic quaternary C-
atom (d(C) 34.8 (C(10))), a CH group (d(C) 43.2 (C(5))), four CH2 groups (d(C) 25.5
(C(2)), 29.4 (C(6)), 34.8 (C(1)), and 49.5 (C(9))), and two Me groups (d(C) 18.9
(C(14)) and 26.6 (C(15))). In the HMBC spectrum of 7 (Fig. 3), key cross-peaks
HꢁC(8)/C(12), CH2(13)/C(7), C(11), and C(12), Me(14)/C(1), C(5), C(9), and C(10),
and Me(15)/C(3), C(4), and C(5) established the locations of all the functionalities.
The presence of a decalin moiety fused with a g-lactone ring to form a linear 6/6/5 ring
system was deduced from the HMBC and COSY correlations HꢁC(1)/HꢁC(2),
HꢁC(2)/HꢁC(3), HꢁC(5)/HꢁC(6), and HꢁC(8)/HꢁC(9) (Fig. 3). The relative con-
figuration of 7 was determined by means of the NOESY experiment (Fig. 4), in which
key cross-peaks HꢁC(3)/Me(15), Me(15)/HeqꢁC(6), HeqꢁC(6)/HaꢁC(13), HaxꢁC(6)/
Me(14), and Me(14)/HꢁC(8) indicated that HOꢁC(3), HOꢁC(4), HOꢁC(7), Me(14),
and HꢁC(8) were a-oriented, and Me(15) and HꢁC(5) were b-oriented. Thus, 7 was
determined as 3a,4a,7a-trihydroxyeremophil-11(13)-en-12,8b-olide and named elaei-
colalactone.
Fig. 3. Key COSY (—) correlations and HMBCs (H!C) of 7
Fig. 4. Key NOESY ($) correlations of 7
The effects of 1–7 on the inhibition of NO production in lipopolysaccharide (LPS)-
activated murine macrophage RAW264.7 cells were evaluated. All these compounds
inhibited NO production, detected as nitrite in the culture medium, in activated

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
macrophages to various degrees without affecting the cellular viability. As shown in
Fig. 5,a, the Emax, the mean maximum inhibitory effect, was evaluated at a concen-
tration of 100 mm and expressed as a percentage inhibition of nitrite production induced
by LPS (200 ng/ml) in the presence of vehicle for 24 h. Of all compounds tested, 2
possessed a significant activity with the Emax and median inhibitory concentration (IC50)
values of 93.3ꢀ0.5% and 79.3ꢀ0.4 mm, respectively. The inhibitory effects were
compared with those of the reference compounds, aminoguanidine, a selective iNOS
inhibitor, and Nw-nitro-l-arginine (l-NNA), a non-selective NOS inhibitor. Under the
same conditions, aminoguanidine exhibited an Emax of 85.7ꢀ0.2% and an IC50 value of
27.5ꢀ0.4 mm. l-NNA revealed an Emax value of 47.3ꢀ0.7% and an IC50 value of 145.2ꢀ
16.7 mm, similar to our previous report [8]. The other compounds are only marginally
active. It is noteworthy that compounds 1 and 2 exhibit strongly different activities,
although they possessed the same C-atom skeleton.
Fig. 5. The Emax on NO production (a) and cell viability (b) of compounds 1–7, and two positive controls,
aminoguanidine (AG) and Nw-nitro-l-arginine (l-NNA) in LPS-activated RAW 264.7 cells. Cells were
co-incubated with LPS (200 ng/ml) and various compounds (100 mm) or vehicle (0.1%, DMSO) for 24 h.
n¼5–6 in each group. *: P<0.05 when compared with vehicle-treated cells.
This research was supported by a grant from the National Science Council of Taiwan. We are grateful
to Ms. Shwu-Huey Wang and Ms. Shou-Ling Huang for the NMR data acquisition in the Instrument
Center of Taipei Medical University and Instrumental Center of the College of Science, National Taiwan
University, respectively.
Experimental Part
General. Optical rotation: JASCO P-1020 polarimeter (Tokyo, Japan). UV Spectra: Thermo Helios
a spectrophotometer (Waltham, USA). IR Spectra: JASCO FT/IR 4100 spectrometer (Tokyo, Japan).
1H- and 13C-NMR: Bruker DRX-500 SB spectrometer (D-Ettlingen). LR- and HR-MS: VG Platform
Electrospray ESI/MS (VG, England) and Waters Synapt high-definition mass spectrometer (Miford, MA,
USA), resp.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Fermentation of Stilbohypoxylon elaeicola. Stilbohypoxylon elaeicola (Henn.) L. E. Petrini strain
No. YMJ173 was isolated by one of us (Y.-M. J.) from a specimen collected by Dr. Sabine Huhndorf,
Field Museum, Chicago, USA, in French Guiana. This strain was deposited with the Institute of Plant and
Microbial Biology, Academia Sinica, Taipei, Taiwan. The mycelium of S. elaeicola YMJ173 was
inoculated into 1-l Erlenmeyer flasks, each containing 10 g of BactoTM malt extract (Becton, Dickinson
and Company, Sparks, USA) and 500 ml of deionized H2O. The fermentation was conducted at 25–308
for 30 d.
Extraction and Isolation. The filtered fermented broth (90 l) of S. elaeicola YMJ173 was partitioned
three times with 50 l of recycled AcOEt, then concentrated in vacuum to dryness (13.0 g). Subsequently,
this residue was re-dissolved in 25 ml of MeOH and applied onto a Sephadex LH-20 column (3 cm i.d.ꢂ
65 cm) eluted by MeOH with a flow rate of 2.5 ml/min. Each subfraction (23 ml) collected was checked
for its compositions by TLC with AcOEt/AcOH/H2O 85 :10 :10 (v/v/v) for development. Compounds
were detected by dipping the plate in vanillinꢁH2SO4 and heating [8]. Subsequently, subfractions were
combined into five portions I–V. Portion II (Subfrs. 11 and 12; 560 mg) was purified by HPLC on a semi-
prep. reversed-phase (RP) column (Biosil PRO-ODS-U, 10ꢂ250 mm, 5 mm, Biotic Chemical Co., Taipei,
Taiwan) with MeOH/H2O 7:3 (v/v); 2 ml/min, to afford 5 (52.8 mg; tR 11.7 min), 6 (7.2 mg; tR 16.0 min),
and 7 (4.9 mg; tR 8.5 min). Portion III (Subfrs. 13–17, 875 mg) was purified by HPLC on the same column
with MeOH/H2O 4 :1 (v/v); 2 ml/min to give 1 (9.6 mg; tR 18.5 min), 2 (5.0 mg; tR 20.9 min), 3 (4.9 mg; tR
24.1 min), and 4 (24.0 mg; tR 22.5 min).
Monosaccharide Composition Analysis of 1–6. Compounds 1–6 (each 2 mg) were treated with 8m
CF3COOH (TFA) at 1108 for 4 h to give an acid hydrolysate. The acid hydrolysate was then vacuum-
evaporated to remove the residual TFA. Subsequently, the hydrolysate was added to 2 ml of 16 mm
NaOH, and this soln. was analyzed by high pH anion-exchange chromatographyꢁpulsed amperometric
detection (HPAEC-PAD, DIONEX ICS 3000, Sunyvale, CA, USA) [9]. HPAEC-PAD Analysis was
performed on a Carbo Pac PA10 column (2 i.d.ꢂ250 mm) with 16 mm NaOH as the eluent at a flow rate
of 0.25 ml/min. Three authentic monosaccharides including glucose, mannose, and galactose were used as
standards. When compared with the standards, the monosaccharide composition of 1–6 was confirmed to
be mannopyranose. Under above conditions, the tR value of the mannopyranose was 11.3 min.
Optical Rotation of the Mannopyranose in 1. Compound 1 (2 mg) was hydrolyzed with 1m H2SO4
(2 ml) at r.t. overnight. The mixture was then partitioned with AcOEt (2ꢂ2 ml). The lower layer was
neutralized with 2 ml of 1m Ba(OH)2 and filtered through glass wool, and the filtrate was evaporated to
give d-mannose: [a]2D3 ¼ þ11.1 (c¼0.1, H2O).
Nitrite Determination and Cell Viability Assay. The methods were essentially the same as reported in
[10]. To assess the effects on LPS-induced NO production, compounds 1–7 (purity >98% as checked by
their 1H-NMR), both reference inhibitors aminoguanidine (a specific inhibitor of iNOS) and Nw-nitro-l-
arginine (l-NNA, a non-selective NOS inhibitor), or vehicle (0.1%, DMSO) were added in the presence
of LPS (200 ng/ml) to the RAW 264.7 cells for 24 h. The nitrite concentration in the culture medium was
determined spectrophotometrically as an index of NO production. Maximum inhibition (Emax) is
expressed as the percentage inhibition at 100 mm calculated vs. vehicle plus LPS-treated cells. Both
reference inhibitors were purchased from SigmaꢁAldrich Chemical Co., and the purity of each
compound was more than 98%. A redox indicator, alamarBlue, was used to determine cytotoxicity as
reported in [11]. After the removal of culture supernatant for nitrite measurement as described above, a
soln. of 10% alamarBlue in culture medium was added to each well containing RAW264.7 cells. The
plates were incubated at 378 in a humidified 5% CO2 atmosphere for 3 h. Following incubation, the
absorbance of the alamarBlue was read spectrophotometrically at dual wavelengths of 570 and 600 nm.
The absorbance in cultures treated with LPS plus vehicle was regarded as 100% cell viability.
Statistical Analyses. Comparisons of the concentration and treatment effects were conducted with
ANOVA, followed by post hoc comparisons by NewmanꢁKeuls test as appropriate. The average IC50
value was determined by data fitting with GraFit (Erithacus Software, UK).
7a-Hydroxy-16-(a-d-mannopyranosyloxy)norisopimar-8(14)-ene 4-Hydroperoxide (¼2-[(2R,4aR,
4bR,8R,8aR,10R)-2,3,4,4a,4b,5,6,7,8,8a,9,10-Dodecahydro-8-hydroperoxy-10-hydroxy-2,4b,8-trimethyl-
phenanthren-2-yl]ethyl a-d-Mannopyranoside; 1). Amorphous white powder. [a]2D4 ¼ ꢁ10.8 (c¼0.5,
MeOH). IR (KBr): 3348, 2933, 2868, 1643, 1445, 1377, 1126, 1084, 1038, 974, 868, 807. 1H-NMR

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
(500 MHz): see Table 2. 13C-NMR (125 MHz): see Table 1. ESI-MS: 509 ([MþNa]þ ). HR-ESI-MS:
509.2721 ([MþNa]þ , C25H42NaOþ9 ; calc. 509.2727).
16-(a-d-Mannopyranosyloxy)norisopimar-7-en-4-ol (¼2-[(2S,4aS,4bR,8R,8aR)-1,2,3,4,4a,4b,5,6,
7,8,8a,9-Dodecahydro-8-hydroxy-2,4b,8-trimethylphenanthren-2-yl]ethyl a-d-Mannopyranoside; 2).
Amorphous white powder. [a]2D4 ¼ þ47.2 (c¼0.5, MeOH). IR (KBr): 3402, 2925, 1681, 1523, 1456,
1198, 1131, 1079, 821, 685. 1H-NMR (500 MHz): see Table 2. 13C-NMR: see Table 1. ESI-MS: 453 ([Mꢁ
H]ꢁ ). HR-ESI-MS: 453.2853 ([MꢁH]ꢁ , C25H41Oꢁ7 ; calc. 453.2852).
16-(a-d-Mannopyranosyloxy)isopimar-6,8(14)-dien-19-oic Acid (¼(13a)-16-(a-d-Mannopyranosyl-
oxy)pimara-6,8(14)-dien-19-oic Acid; 3). Amorphous white powder. [a]2D1 ¼ þ12.2 (c¼0.5, MeOH). UV
(MeOH): 241 (4.3). IR (KBr): 3375, 2934, 1689, 1461, 1381, 1203, 1187, 1134, 1086, 1058, 1027, 974, 799.
1H-NMR (500 MHz): see Table 2. 13C-NMR (125 MHz): see Table 1. ESI-MS: 479 ([MꢁH]ꢁ ). HR-ESI-
MS: 479.2655 ([MꢁH]ꢁ , C26H39O8ꢁ ; calc. 479.2645).
3a,4a,7a-Trihydroxyeremophil-11(13)-en-12,8b-olide (¼(3aS,4aS,7R,8S,8aS,9aR)-Dodecahydro-
7,8,9a-trihydroxy-4a,8-dimethyl-1-methylidene-2H-cyclopenta[b]naphthalen-2-one; 7). Amorphous white
powder. [a]2D4 ¼ ꢁ36.0 (c¼0.55, MeOH). UV (MeCN): 198 (3.8). IR (KBr): 3402, 2925, 1753, 1666,
1453, 1394, 1300, 1277, 1228, 1124, 1070, 1032, 1001, 908, 623. 1H-NMR (500 MHz): see Table 2. 13C-NMR
(125 MHz): see Table 1. ESI-MS: 281 ([MꢁH]ꢁ ). HR-ESI-MS: 281.1394 ([MꢁH]ꢁ , C15H21Oꢁ5 ; calc.
281.1389).
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Received January 20, 2011