Sesquiterpenes from an Egyptian herbal medicine, pulicaria undulate, with inhibitory effects on nitric oxide production in RAW264.7 macrophage cells

Article abstract of DOI:10.1248/cpb.60.363

The methylene chloride - methanol (1 : 1) extract from the air-dried aerial parts of wild Pulicaria undulata collected in North Sinia, Egypt, showed inhibitory effects on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) in RAW264.7 macrophages. From the extract, three new sesquiterpenes named 5α-hydroperoxyivalin, 8-epi-xanthanol, and 8-epi-isoxanthanol were isolated together with four known sesquiterpenes. The structure of each new sesquiterpenes was determined on the basis of physicochemical and chemical evidence. In addition, all the sesquiterpenoids significantly inhibited the production of NO. Ivalin (IC₅₀ =-2.0 μM) and 2α- hydroxyalantolactone (1.8 μM) showed particularly strong inhibitory effects, but had strong cytotoxic effects as well. Furthermore, ivalin and 2α-hydroxyalantolactone concentration-dependently reduced inducible NO synthase (iNOS) protein levels in RAW264.7 cells.

Full text of DOI:10.1248/cpb.60.363

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March 2012
Regular Article
Chem. Pharm. Bull. 60(3) 363 370 (2012)
363
Sesquiterpenes from an Egyptian Herbal Medicine, Pulicaria undulate,
with Inhibitory Effects on Nitric Oxide Production in RAW264.7
Macrophage Cells
Mohamed-Elamir F. Hegazy,a,b Hisashi Matsuda,a Seikou Nakamura,a Mikuko Yabe,a
,a
Tomoko Matsumoto,a and Masayuki Yoshikawa*
b
a Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan: and Chemistry of Medicinal
Plants Department, and Center of Excellence for Advanced Sciences, National Research Centre; El-Tahrir St., Dokki,
Giza 12622, Egypt.
Received November 17, 2011; accepted December 20, 2011; published online January 6, 2012
The methylene chloride–methanol (1:1) extract from the air-dried aerial parts of wild Pulicaria undu-
lata collected in North Sinia, Egypt, showed inhibitory effects on lipopolysaccharide (LPS)-induced produc-
tion of nitric oxide (NO) in RAW264.7 macrophages. From the extract, three new sesquiterpenes named
5α-hydroperoxyivalin, 8-epi-xanthanol, and 8-epi-isoxanthanol were isolated together with four known ses-
quiterpenes. The structure of each new sesquiterpenes was determined on the basis of physicochemical and
chemical evidence. In addition, all the sesquiterpenoids significantly inhibited the production of NO. Ivalin
(IC50=2.0μM) and 2α-hydroxyalantolactone (1.8μM) showed particularly strong inhibitory effects, but had
strong cytotoxic effects as well. Furthermore, ivalin and 2α-hydroxyalantolactone concentration-dependently
reduced inducible NO synthase (iNOS) protein levels in RAW264.7 cells.
Key words Pulicaria undulata; sesquiterpenoid; nitric oxide production inhibitor; RAW264.7 cell;
Compositae
The genus Pulicaria, belonging to the tribe Inuleae of the injury, and chronic or acute inflammation. NO is produced by
Compositae family, consists of 100 species with a distribu- the oxidation of L-arginine catalyzed by NO synthase (NOS).
tion from Europe to North Africa and Asia, particularly In the NOS family, inducible NOS (iNOS) is particularly well
centered around the Mediterranean.1) Phytochemical studies known to be involved in the pathological overproduction of
on Pulicaria species have yielded several flavonoids and ter- NO. Chronic inflammation also contributes substantially to
penoids, including sesquiterpenoids and diterpenoids. Most environmental carcinogenesis. Various infectious diseases
importantly, a number of compounds from Pulicaria species and physical, chemical, and immunological factors participate
have been found to possess potent bioactivities, and could be in inflammation-related carcinogenesis. Under inflammatory
promising candidates for the development of potential drugs conditions, reactive oxygen and nitrogen species are generated
and value-added products.2) Pulicaria undulata (L.) KOSTEL from inflammatory and epithelial cells, and so the suppression
[syn. P. crispa (FORSSK.) BENTH. et HOOK. f., Francoeuria cris- of signaling molecules for iNOS expression may have great
pa (FORSSK.) CASS.] is an annual herb or sometimes a perennial potential for the prevention and treatment of inflammation-
—
7)
sub shrub, producing small bright yellow flower. Known in associated carcinogenesis.4
Egypt as Dethdath, it is commonly used in traditional herbal In the present study, the methylene chloride–methanol (1:1)
medicines to treat inflammation, as an insect repellent, and extract from the air-dried aerial parts of wild P. undulata col-
even as a herbal tea.3)
lected in North Sinia, Egypt, was found to have inhibitory
The inorganic free radical nitric oxide (NO) has been im- effects on LPS-induced NO production in RAW264.7 macro-
plicated in physiological and pathological processes such as phages. Furthermore, we examined the inhibitory effects of all
vasodilation, nonspecific host defense, ischemia-reperfusion isolated constituents on NO production induced by lipopoly-
Fig. 1. Structures of the Compounds Isolated from Pulicaria undulate
*To whom correspondence should be addressed. e-mail: myoshika@mb.kyoto-phu.ac.jp
© 2012 The Pharmaceutical Society of Japan

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Vol. 60, No. 3
saccharide (LPS). In this paper, we describe the isolation and (1H each, both brs, H2-13)], and a carbonyl carbon (δC 172.5,
structural elucidation of the new constituents (1, 4, 5) and the C-12). The proton and carbon signals of 1 in the 1H- and
inhibitory effects of constituents from the dried aerial parts of 13C-NMR spectra resembled those of ivalin (2),9,10) except for
P. undulate.
the signals around the 5-position. Double quantum filter cor-
relation spectroscopy (DQF COSY) indicated the presence of
partial structures (written in bold in Fig. 2), and heteronuclear
Results and Discussion
Isolation and Structural Elucidation The methylene- multiple bond connectivity spectroscopy (HMBC) revealed
chloride (CH2Cl2)–methanol (MeOH) (1:1) extract (6.77%) of long-range correlations between the following protons and
the air-dried aerial parts of P. undulate inhibited production carbons: H-1 and C-2; H-6 and C-5, 7; H-7 and C-11; H-13
of NO in LPS-simulated RAW264.7 cells (IC50=7.5μg/mL).8) and C-12; H-14 and C-1, 5, 9; H-15 and C-5. These results
To clarify its active components, the extract was subjected supported that 1 was an alantolactone derivative with a hydro-
to normal and reverse phase chromatography to afford three peroxyl moiety. Next, nuclear Overhauser enhancement spec-
new compounds named 5α-hydroperoxyivalin (1, 0.0004%), troscopy (NOESY) revealed NOE correlations between the
8-epi-xanthanol (4, 0.0013%), and 8-epi-isoxanthanol (5, following proton pairs: H-1β and H-2, H3-14; H-2 and H3-14;
0.0022%) together with four known sesquiterpenoids, ivalin H-6α and H-7; H-6β and H3-14; H-7 and H-8 (Fig. 2). Finally,
(2, 0.0012%),9,10) 2α-hydroxyalantolactone (3, 0.017%),11) xan- the stereostructure of 1 was characterized by a single crystal
thanol (6, 0.0009%),12,13) and isoxanthanol (7, 0.0036%).12,13)
X-ray crystallographic analysis. The ORTEP representation of
5α-Hydroperoxyivalin (1) was obtained as colorless needles the X-ray structure is presented in Fig. 3. On the basis of this
with a positive optical rotation ([α]D25 +22.0 in MeOH). Its evidence, the structure of 5α-hydroperoxyivalin (1) was char-
°
IR spectrum showed absorption bands at 3450, 1735 and acterized as shown.
1650cm−1 ascribable to hydroxy1, γ-lactone and olefin func-
8-epi-Xanthanol (4) and 8-epi-isoxanthanol (5) were isolated
tions. Positive-ion fast atom bombardment (FAB)-MS indi- as colorless needles with negative optical rotation (4: [α]D25
cated a quasimolecular ion peak at m/z 303 (M+Na)+, and −15.0 ; 5: [α]D −22.0 in MeOH). Their IR spectra showed
high-resolution (HR)-FAB-MS revealed the molecular formula absorption bands assignable to hydroxyl, γ-lactone, ace-
C15H20O5. The 1H-NMR (CD3OD) and 13C-NMR (Table 1) toxyl and olefin functions (4, 5: 3450, 1738, 1732, 1652cm−1).
spectra of 1, which were assigned based on NMR experi- Chemical ionization (CI)-MS revealed a common quasimolec-
ments,14) showed signals assignable to a methyl [δ 0.97 (3H, ular ion peak at m/z 309 (M+H)+ and the molecular formula
s, H3-14)], two methines with an oxygen function [δ 3.76 (1H, C17H25O5 was determined by HR-CI-MS. The proton and car-
25
°
°
m, H-2), 4.59 (1H, ddd, J=1.3, 5.5, 10.3Hz, H-8)], four olefinic bon signals in the H- and 13C-NMR spectra of 4 and 5 were
1
protons [δ 4.79, 5.09 (1H each, both brs, H2-15), 5.70, 6.07 very similar to those of xanthanol (6)12,13) and isoxanthanol
Table 1. 1H- (600MHz, 1: CD3OD, 4, 5: CDCl3) and 13C- (150MHz, 1: CD3OD, 4, 5: CDCl3) NMR Data of 1, 4, and 5
1
4
5
Position
1
δH (J in Hz)
1.43 ma)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
—
45.0
145.2
147.7
1.66 dd (11.6, 11.6)
2
3
3.76 m
67.4
42.4
5.10 dd (4.8, 8.9)
76.3
43.5
4.04 dd (5.5, 8.2)
73.4
42.1
2.44 m
2.55 dd (11.6, 11.6)
1.67 m
1.90 m
1.67 m
1.92 m
—
4
5
6
147.2
84.6
29.0
3.81 m
65.9
124.1
26.2
4.91 m
69.3
122.3
26.0
—
5.84 dd (5.5, 8.9)
5.69 dd (6.2, 8.9)
1.42 dd (2.1, 14.0)a)
2.33 dd (6.8, 14.0)
2.28 ddd (4.8, 8.9,
14.5)
2.27 ddd (4.8, 8.9,
15.5)
2.43 m
2.39 m
7
8
3.36 m
38.8
78.8
3.29 m
41.5
78.9
3.31 m
41.4
79.1
4.59 ddd (1.3, 5.5,
10.3)
4.57 ddd (2.7, 10.0,
12.3)
4.57 ddd (2.7, 11.0,
12.3)
9
1.87 dd (1.3, 14.0)
1.94 dd (5.5, 14.0)
36.7
1.81 m
2.03 m
36.9
1.79 m
2.03 ddd (2.7, 4.1,
12.3)
37.0
—
—
—
10
11
12
13
37.9
143.7
172.5
121.1
2.62 m
—
34.0
138.6
169.7
122.9
2.56 m
—
32.9
138.7
170.3
122.2
—
—
5.70 s like
6.07 s like
5.51 d (2.7)
6.25 d (2.7)
5.49 d (2.7)
6.22 d (2.7)
14
15
0.97 s
24.2
1.12 d (7.5)
1.21 d (6.2)
21.5
23.7
1.14 d (6.9)
1.23 d (6.2)
22.1
20.3
4.79 s like
5.09 s like
113.9
1′
2′
2.01 s
21.6
2.00 s
21.5
170.4
170.7
a) Overlapping signals.

March 2012
365
Fig. 2. Significant DQF COSY, HMBC, and NOE Correlations 1, 4, and 5
by NOESY experiments, which showed NOE correlations
between the following proton pairs: H-7 and H-8; H-8 and
H-9α, H-10; H-9α and H-10; H-9β and H3-14. In addition, it
was reported that the absolute stereostructures of compounds
with an α-methylene-γ-lactone moiety such as xanthanolides
were determined based on circular dichroic (CD) spectra.15,16)
Therefore, the absolute configurations of the 8-position on
4 and 5 were confirmed from CD spectra. Namely, the CD
spectra of xanthanol (6) and isoxanthanol (7) with the 7R and
8S configurations showed positive Cotton effects at 254nm
Δ
Δ
( ε +0.19 in MeOH) and 252nm ( ε +0.19 in MeOH) for the
8S configuration in the α-methylene-γ-lactone, respectively.
On the other hand, the CD data for 4 and 5 showed negative
Δ
Cotton effects at 257nm ( ε 4: −0.34, 5: −0.45 in MeOH), so
Fig. 3. ORTEP Representation of 1
that the absolute configurations of the 8-position were deter-
mined to be both R. Furthermore, the absolute configurations
(7),12,13) respectively, except for the signals around the 8-posi- of the 2- and 4-positions were characterized by the applica-
tion. The H-NMR (CDCl3) and 13C-NMR (Table 2) spectra, tion of a modified Mosher s method. Namely, treatment of 4
1
’
which were assigned based on results of various NMR ex- with (−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride
periments,14) showed signals due to three methyls [4: δ 1.12 [(−)-MTPA-Cl] in pyridine yielded a mixture of (S)-MTPA
(3H, d, J=7.5Hz, H3-14), 1.21 (3H, d, J=6.2Hz, H3-15), 2.01 esters, 8a and 9a (4:3),17) by intramolecular acetate transfer.
(3H, s, H3-1′); 5: δ 1.14 (3H, d, J=6.9Hz, H3-14), 1.23 (3H, Whereas, the (R)-MTPA esters, 8b and 9b (4:3), were de-
d, J=6.2Hz, H3-15), 2.00 (3H, s, H3-1′)], three methines with rived from 4 by treatment with (+)-MTPA-Cl in pyridine. As
an oxygen function [4: δ 3.81 (1H, m, H-4), 4.57 (1H, ddd, shown in Fig. 4, the signals due to the protons attached to the
J=2.7, 10.0, 12.3Hz, H-8), 5.10 (1H, dd, J=4.8, 8.9Hz, H-2); 4 and 15-positions were observed at higher fields in the (S)-
Δ
5: δ 4.04 (1H, dd, J=5.5, 8.2Hz, H-2), 4.57 (1H, ddd, J=2.7, MTPA ester (8a) compared with the (R)-MTPA ester (8b) [ δ:
11.0, 12.3Hz, H-8), 4.91 (1H, m, H-4)], and three olefinic negative], while the signals due to the protons of the 5 and
Δ
protons [4: δ 5.51, 6.25 (1H each, both d, J=2.7Hz, H2-13), 14-positions were observed at lower fields in 8a than 8b [ δ:
5.84 (1H, dd, J=5.5, 8.9Hz, H-5); 5: δ 5.49, 6.22 (1H each, positive]. Furthermore, the signal due to the proton attached to
both d, J=2.7Hz, H2-13), 5.69 (1H, dd, J=6.2, 8.9Hz, H-5)]. the 15-position was observed at higher fields in the (S)-MTPA
Δ
As shown in Fig. 2, the DQF COSY experiments indicated ester (9a) than (R)-MTPA ester (9b) [ δ: negative], while the
the presence of partial structures (written in bold), and in the signals due to the protons of the 2, 5 and 14-positions were
Δ
HMBC experiments, long-range correlations were observed observed at lower fields in 9a than 9b [ δ: positive]. Thus the
between the following protons and carbons: 4: H-2 and C-10, absolute configurations at the 2- and 4-positions of 4 were de-
2′; H-5 and C-2, 6; H-8 and C-12; H-10 and C-5; H-13 and C-7, termined to be both S orientations. In the same manner, treat-
11, 12; H-14 and C-1, 9, 10; H-15 and C-3, 4; H-1' and C-2′; 5: ment of 5 with (−)-MTPA-Cl in pyridine yielded a mixture of
H-2 and C-10; H-4 and C-2′; H-5 and C-2, 6; H-8 and C-12; (S)-MTPA esters, 8a and 9a (5:4). Whereas, the (R)-MTPA
H-10 and C-5; H-13 and C-7, 11, 12; H-14 and C-1, 9, 10; H-15 esters, 8b and 9b (5:4), were derived from 5. Therefore, the
and C-3, 4; H-1′ and C-2′. Next, the relative stereostructures absolute configurations of the 2- and 4-positions in 5 were
of the 7-, 8- and 10-positions in 4 and 5 were characterized clarified to be also both S. Finally, treatment of 4 and 5 with

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367
Fig. 4 Determination of the Absolute Configurations of 2- and 4-Positions on 4 and 5
effects. Furthermore, effects of 2, 3, and CAPE on the induc-
tion of iNOS were examined using a sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) Western-blot
analysis. As shown in Fig. 5, 2 and 3 concentration-depend-
ently inhibited iNOS protein production in RAW264.7 cells.
Recent studies have demonstrated that sesquiterpenes with
an α-methylene-γ-lactone moiety (e.g. parthenolide, costu-
nolide, and dhydrocostuslactone) to have inhibitory effects
on nuclear factor-kappa B (NF-κB) activation, and thereby
iNOS expression following the inhibition of NO produc-
tion in LPS-stimulated mouse peritoneal macrophages and
RAW264.7 cells, and that the α-methylene-γ-lactone moiety
—
21)
is essential for strong activity.18
In the present study using
RAW264.7 cells, eudesman-type sesquiterpenes, 2 and 3, also
showed strong inhibitory effects, but 1 having a hydroperox-
—
ide group and guaianolide-type sesquiterpenes (4 7) showed
—
moderate inhibition (IC50 of 14 24μM) and weaker cytotoxic
—
effects. The IC50 values of 1 and 4 7 were similar to that
of α-methylene-γ-lactone (IC50=22.8μM). These findings con-
firm that not only the α-methylene-γ-lactone structure but
also other structures are important for strong inhibition. The
structure–activity relationships of this type of sesquiterpene,
excluding the lactone ring, should be studied further.
Fig. 5. Inhibitory Effects of 2, 3, and CAPE on Induction of iNOS in
LPS-Stimulated RAW264.7 Cells
Experimental
General Experimental Procedures The following instru-
Ac2O and pyridine furnished the same diacetate compound,
10. Consequently, the structure of 8-epi-xanthanol (4) and ments were used to obtain physical data: specific rotations,
8-epi-isoxanthanol (5) was determined as shown. a Horiba SEPA-300 digital polarimeter (l=5cm); IR spectra,
—
Effects of 1 7 on Production of NO in RAW264.7 Cells a Shimadzu FTIR-8100 spectrometer; CD spectra, a JASCO
In murine macrophage RAW264.7 cells, LPS alone induces J-720WI spectrometer; CI-MS, electron ionization (EI)-MS
the transcription and protein synthesis of iNOS, and increased and HR-CI-MS, a JEOL JMS-GCMATE mass spectrom-
NO production. Using the Griess reaction, a spectrophotomet- eter; FAB-MS and HR-FAB-MS, a JEOL JMS-SX 102A
ric determination of nitrite (NO2−) was carried out to quantify mass spectrometer; 1H-NMR spectra, JEOL JNM-ECA600
levels in the conditioned medium of RAW264.7 cells treated (600MHz) spectrometers; 13C-NMR spectra, JEOL JNM-
with LPS. This cell-based assay system has been used for ECA600 (150MHz) spectrometers with tetramethylsilane as
drug screening and the evaluation of potential inhibitors of the an internal standard; and HPLC, a Shimadzu RID-10A re-
pathways leading to the induction of iNOS and NO produc- fractive index detector and YMC-Pack ODS-A (YMC, Inc.,
—
tion. Our results showed that all compounds (1 7) signifi- 250×4.6mm i.d.) and (250×20mm i.d.) columns for analytical
cantly inhibited NO production in LPS-activated RAW264.7 and preparative purposes, respectively. The following experi-
cells (Table 2). Ivalin (2) and 2α-hydroxyalantolactone (3) mental materials were used for chromatography: normal-phase
showed the stronger inhibitory effects with IC50 values of 2.0 silica gel column chromatography, Silica gel BW-200 (Fuji
—
and 1.8μM, but stronger cytotoxic effects also. The reference Silysia Chemical, Ltd., 150 350 mesh); reversed-phase silica
compounds, caffeic acid phenethyl ester (CAPE) and parthe- gel column chromatography, Chromatorex ODS DM1020T
—
nolide, exhibited strong inhibitory effects with IC50 values (Fuji Silysia Chemical, Ltd., 100 200 mesh); TLC, precoated
of 3.5μM and ca. 1.1μM, respectively, together with cytotoxic TLC plates with Silica gel 60F254 (Merck, 0.25mm) (ordinary

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Vol. 60, No. 3
phase) and Silica gel RP-18 F254S (Merck, 0.25mm) (reversed 600MHz) δ: 1.05 (3H, d, J=6.9Hz, H3-14), 1.21 (3H, d,
phase); and reversed-phase HPTLC, precoated TLC plates J=6.2Hz, H3-15), 1.80, 2.10 (1H each, both m, H2-3), 2.03
with Silica gel RP-18 WF254S (Merck, 0.25mm). Detection was (3H, s, CH3CO–), 4.58 (1H, m, H-8), 4.81 (1H, m, H-4), 5.23
achieved by spraying with 1% Ce(SO4)2–10% aqueous H2SO4 (1H, dd, J=6.2, 8.2Hz, H-2), 5.54, 6.28 (1H each, both d,
followed by heating.
Plant Material The air-dried aerial parts of Pulicaria
J=2.8Hz, H2-13), 5.84 (1H, dd, J=5.5, 9.6Hz, H-5).
2-(R)-MTPA Ester Derivative (8b): 1H-NMR (CDCl3,
undulata (L.) KOSTEL were collected in June 2010, from North 600MHz) δ: 0.95 (3H, d, J=6.8Hz, H3-14), 1.23 (3H, d,
Sinia, Egypt. A voucher specimen, SK-103, has been deposited J=6.2Hz, H3-15), 1.84, 2.11 (1H each, both m, H2-3), 2.03
in the Herbarium of St. Katherine protectorate, Egypt.
(3H, s, CH3CO–), 4.50 (1H, m, H-8), 4.84 (1H, m, H-4), 5.24
Extraction and Isolation The aerial parts (1.8kg) of P. (1H, dd, J=5.5, 7.6Hz, H-2), 5.51, 6.27 (1H each, both d,
undulata were powdered and extracted with CH2Cl2–MeOH J=2.8Hz, H2-13), 5.74 (1H, m, H-5).
(1:1) at room temperature. The extract was concentrated in
4-(S)-MTPA Ester Derivative (9a): 1H-NMR (CDCl3,
vacuo to obtain a residue of 122g (6.77%). The residue was 600MHz) δ: 1.10 (3H, d, J=6.9Hz, H3-14), 1.29 (3H, d,
fractionated on a silica gel column (6×120cm) eluted with n- J=6.2Hz, H3-15), 1.84, 2.15 (1H each, both m, H2-3), 2.03
hexane (3000mL) followed by a gradient of n-hexane–CHCl3 (3H, s, CH3CO–), 4.62 (1H, m, H-8), 5.05 (1H, dd, J=5.5,
up to 100% CHCl3 and CHCl3–MeOH up to 50% MeOH 8.9Hz, H-2), 5.11 (1H, m, H-4), 5.54, 6.28 (1H each, both d,
(3000mL each of the solvent mixture). The n-hexane–CHCl3 J=2.8Hz, H2-13), 5.78 (1H, dd, J=6.2, 9.6Hz, H-5).
(1:3) fraction (22g) was chromatographed on a Sephadex
4-(R)-MTPA Ester Derivative (9b): 1H-NMR (CDCl3,
LH-20 column (3×90cm) eluted with n-hexane–CH2Cl2– 600MHz) δ: 1.08 (3H, d, J=6.9Hz, H3-14), 1.36 (3H, d,
methanol (7:4:0.25). Fractions were obtained and combined J=6.2Hz, H3-15), 1.77, 2.08 (1H each, both m, H2-3), 2.03 (3H,
into three main subfractions A, B, and C. Sub-fraction A (7g) s, CH3CO–), 4.60 (1H, m, H-8), 4.99 (1H, dd, J=4.8, 8.2Hz,
was re-purified by reversed-phase HPLC using MeOH/H2O H-2), 5.10 (1H, m, H-4), 5.54, 6.28 (1H each, both d, J=2.8Hz,
—
(55 45%) to afford compounds 4 (23mg, 0.0013%), 5 (40mg, H2-13), 5.74 (1H, m, H-5).
0.0022%), 6 (16mg, 0.0009%), and 7 (65mg, 0.0036%). Sub-
Preparation of the (S)- and (R)-MTPA Esters (8a,b,
fraction B (13g) was re-purified by using silica gel column 9a,b) from 5 The mixture of (S)-MTPA ester derivatives,
(6×90cm) eluted with n-hexane–EtOAc (4:1) followed by 8a and 9a (4.5mg, 5:4), and the mixture of (R)-MTPA ester
—
reversed-phase HPLC using MeOH/H2O (55 45%) to afford derivatives, 8b and 9b (4.3mg, 5:4), was each obtained from
compounds 2 (22mg, 0.0012%) and 3 (300mg, 0.017%). Sub- 5 (each 3.0mg, 0.01mmol) using a similar procedure to when
fraction C (2g) was re-purified by reversed-phase HPLC using MTPA ester derivatives were obtained from 4.
—
MeOH/H2O (60 40%) to afford 1 (7mg, 0.0004%).
Preparation of the Diacetate (10) from 4 and 5 A solu-
tion of 4 (3.0mg, 0.01mmol) in pyridine (0.5mL) was treated
with Ac2O (0.5mL), and the mixture was stirred at rt for 12h.
—
°
5α-Hydroperoxyivalin (1): Colorless needles; mp. 218.0
25
°
220.0 C (recrystallized from CHCl3–MeOH); [α]D +22.0
(c=0.5, MeOH); IR (KBr) νmax 3450, 2926, 1735, 1650cm−1; The reaction mixture was poured into H2O and extracted with
1H-NMR data see Table 1; 13C-NMR data see Table 1; FAB- EtOAc. The EtOAc extract was dried over Na2SO4 powder and
MS m/z 303 [M+Na]+; HR-FAB-MS: m/z 303.1204 (Calcd for filtered. Removal of the solvent from the filtrate under reduced
C15H20O5Na [M+Na]+, 303.1208).
pressure furnished a residue, which was purified by normal-
8-epi-Xanthanol (4): Colorless oil; [α]D25 −15.0 (c=1.0, phase silica gel column chromatography [n-hexane–EtOAc
MeOH); IR (KBr) νmax 3450, 1738, 1732, and 1652cm−1; (2:1, v/v)] to give 10 (3.2mg). Through the same procedure,
1H-NMR data see Table 1; 13C-NMR data see Table 1; CI- diacetate (10, 3.0mg) was obtained from 5 (3.0mg, 0.01mmol).
°
MS m/z 309 [M+H]+; HR-CI-MS: m/z 309.1705 (Calcd for
X-Ray Crystallography Data for 1 Single crystal X-ray
analysis established the complete structure and relative
C17H25O5 [M+H]+, 309.1702).
8-epi-Isoxanthanol (5): Colorless oil; [α]D25 −22.0 (c=1.0, configuration of 1. The crystal data can be summarized as
MeOH); IR (KBr) νmax 3450, 1738, 1732, and 1652cm−1; follows: C15H20O5, formula wt 280.32, Orthorhombic, space
1H-NMR data see Table 1; 13C-NMR data see Table 1; CI- group P212121, a=9.6395 (2)Å, b=11.0925 (2)Å, c=13.0510
MS m/z 309 [M+H]+; HR-CI-MS: m/z 309.1705 (Calcd for (2)Å, V=1395.49 (5)Å3, Z=4, Dcacld=1.334g/cm3, crystal size
°
C17H25O5 [M+H]+, 309.1702).
0.369×0.344×0.267mm3. All diagrams and calculations were
Preparation of the (S)- and (R)-MTPA Esters (8a,b, performed using Rigaku R-AXIS RAPID diffractometer, with
9a,b) from 4 A solution of 4 (3mg, 0.01mmol) in pyri- graphite monochromated CuKα radiation (λ=1.54187Å). The
dine (0.5mL) was treated with (−)-MTPA-Cl (0.01mL, structures were refined by full matrix least squares on F2
0.066mmol), and the mixture was stirred at r.t. for 12h. The using Brucker SHELX-97.22) The final R and Rw were 0.0409
reaction mixture was poured into H2O and extracted with and 0.0888, respectively.
EtOAc. The EtOAc extract was dried over Na2SO4 powder
Bioassay Method. Cell Culture The murine macro-
and filtered. Removal of the solvent from the filtrate under phage cells (RAW264.7, ATCC No. TIB-71) were obtained
reduced pressure furnished a residue, which was purified by from Dainippon Pharmaceutical, Osaka, Japan and cultured
’
’
normal-phase silica gel column chromatography [n-hexane– in Dulbecco s modified Eagle s medium (DMEM, 4500mg/L
EtOAc (2:1, v/v)] to give the mixture of (S)-MTPA ester glucose) supplemented with 10% fetal calf serum, penicillin
derivatives, 8a and 9a (4.6mg, 4:3). Through a similar pro- (100U/mL), and streptomycin (100μg/mL) (Sigma Chemical
°
cedure, the mixture of (R)-MTPA ester derivatives, 8b and 9b Co., St. Louis, MO, U.S.A.). The cells were incubated at 37 C
(4.5mg, 4:3), was obtained from 4 (3.0mg, 0.01mmol) using in 5% CO2/air.
(+)-MTPA-Cl.
Effects on Production of NO in LPS-Stimulated
2-(S)-MTPA Ester Derivative (8a): 1H-NMR (CDCl3, RAW264.7 Macrophages The total amount of nitrite in a

March 2012
369
medium is used as an indicator of NO synthesis. The screen- onto 10% SDS-polyacrylamide gels (BIO-RAD ready gel J).
ing test for NO production using RAW264.7 cells was de- For the Western blot analysis, β-actin was used as an internal
scribed previously.23) Briefly, RAW264.7 cells were cultured in standard. After electrophoresis, the proteins from each experi-
DMEM, and the suspension seeded into a 96-well microplate ment were transferred onto a polyvinylidene fluoride (PVDF)
at 2.5×105 cells/100μL/well. After 6h, nonadherent cells were membrane (BIO-RAD, HC, U.S.A.). The membrane was then
removed by washing with phosphate buffered saline (PBS), soaked in Tris-buffered saline containing 0.1% Tween 20
the adherent cells were cultured in 100μL of fresh medium (T-TBS) with gentle shaking at 75rpm for 10min, three times.
containing the test compounds for 10min, and then 100μL For the blocking of the nonspecific sites, the membrane was
of the medium containing LPS (from E. coli, 055: B5, Sigma) soaked in Blocking One (Nacalai Tesque, Japan) by shaking
was added to stimulate the cells for 18h (final concentration of for 0.5h. The membrane was rinsed with T-TBS and incubated
LPS, 10μg/mL). The nitrite concentration was measured from with specific primary antibodies: iNOS and β-actin (1:1000,
the supernatant by a Griess reaction. NO production in each Cell Signaling Technology). After incubation 1h at r.t., the
well was assessed by measuring the accumulation of nitrite in membrane was rinsed in T-TBS, and incubated in secondary
the culture medium using Griess reagent. Inhibition (%) was antibodies (horse-radish peroxidase (HRP)-conjugated goat
calculated using the following formula and the IC50 was deter- anti-mouse and anti-rabbit immunoglobulin G (IgG), 1:5000)
mined graphically (n=4).
in an immunoreactions enhancer solution (Can Get Signal,
Toyobo, Japan) for 1h. Next, the membrane was shaken in
T-TBS for 10min, three times. The proteins were detected
inhibition (%)=[(A− B) / (A−C)]×100
A−C: nitrite concentration (μM). A: LPS (+), sample (–); B: using an enhanced chemiluminescence (ECL) plus Western
LPS (+), sample (+); C: LPS (−), sample (−)
blotting detection system (AmershamTM GE Healthcare,
Determination of Cytotoxic Effects Cytotoxicity was Biosciences). The images of membranes were recorded using
evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- a luminescent image analyzer, LAS-4000 mini (FUJIFILM,
tetrazolium bromide (MTT) colorimetric assay according Japan).
to a previous report with a slight modification.23) Briefly,
Statistical Analysis All data are expressed as the
RAW264.7 cells (1.0×105 cells/200μL/well) were incubated mean S.E.M. The data analysis was performed with a one-
’
with test compounds without LPS for 18h. An aliquot of the way analysis of variance (1-ANOVA), followed by Dunnett s
medium (100μL) was removed and MTT solution (10μL, 5mg/ test. A p value of less than 0.05 was considered to be signifi-
°
mL in PBS) was then added. After a 2-h incubation at 37 C, cant.
the medium was removed, and isopropanol containing 0.04M
HCl was added to dissolve the formazan produced in the cells.
Acknowledgements The research described in this paper
The optical density (OD) of the formazan solution was mea- was supported by a JSPS (Japan Society for the Promotion of
sured with a microplate reader at 570nm (reference: 655nm). Science) postdoctoral fellowship (ID No. P10117).
If the OD of the sample-treated group dropped below 80% of
that in the vehicle-treated group, the test compound was con-
sidered cytotoxic.
SDS-PAGE and Western Blot Analysis The detection
of iNOS was determined according to methods described
previously.23) RAW 264.7 cells (5.0×106 cells/2mL/well)
were seeded into a 6-well multiplate and allowed to adhere
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