Anti-inflammatory compounds from the aerial parts of aceriphyllum rossii

Article abstract of DOI:10.1248/cpb.c13-00664

A new megastigmane glycoside, galloyl linarionoside A (1), together with 13 known compounds (2-14) were isolated from the aerial parts of Aceriphyllum rossii ENGLER. (Saxifragaceae). The chemical structures of the isolated compounds were established mainly by using nuclear magnetic resonance spectra, mass spectrometry, and modified Mosher's method. Among the isolates, compounds 4, 5, 6 and 7 showed potent inhibitory activity against the lipopolysaccharide- induced nitric oxide production in RAW264.7 macrophage cells with IC50 values of 12.5, 9.5, 10.5 and 9.3 μM, respectively. The anti-inflammatory effect of compound 7 was accompanied by dose-dependent decreases in the production of inducible nitric oxide synthase and cyclooxygenase- 2 proteins not in the inhibitor kappa B (IκB)-dependent nuclear factor-kappa B activation.

Full text of DOI:10.1248/cpb.c13-00664

February 2014
Note
Chem. Pharm. Bull. 62(2) 185–190 (2014)
185
Anti-inflammatory Compounds from the Aerial Parts of Aceriphyllum
rossii
Tran Thi Thu Trang,a To Dao Cuong,a Tran Manh Hung,a Jeong Ah Kim,b Jeong Hyung Lee,c
,a
Mi Hee Woo,a Jae Sue Choi,d Hyeong Kyu Lee,e and Byung Sun Min*
a College of Pharmacy, Catholic University of Daegu; Gyeongbuk 712–702, Korea: b College of Pharmacy, Kyungpook
National University; Daegu 702–701, Republic of Korea: c College of Natural Science, Kangwon National University;
Kangwon 200–701, Korea: d Faculty of Food Science and Biotechnology, Pukyung National University; Busan 608–
737, Korea: and e Natural Medicine Research Center, KRIBB; Chungbuk 363–883, Korea.
Received August 21, 2013; accepted October 22, 2013
A new megastigmane glycoside, galloyl linarionoside A (1), together with 13 known compounds (2–14)
were isolated from the aerial parts of Aceriphyllum rossii ENGLER. (Saxifragaceae). The chemical structures
of the isolated compounds were established mainly by using nuclear magnetic resonance spectra, mass spec-
trometry, and modified Mosher’s method. Among the isolates, compounds 4, 5, 6 and 7 showed potent inhibi-
tory activity against the lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophage cells
with IC50 values of 12.5, 9.5, 10.5 and 9.3µM, respectively. The anti-inflammatory effect of compound 7 was
accompanied by dose-dependent decreases in the production of inducible nitric oxide synthase and cyclooxy-
genase-2 proteins not in the inhibitor kappa B (IκB)-dependent nuclear factor-kappa B activation.
Key words Aceriphyllum rossii; Saxifragaceae; megastigmane; anti-inflammatory activity
Inflammation is the normal physiological and immune to MCF-7 and LLC cancer cells,4) acyl-CoA-cholesterol O-
response to tissue injury. Increased blood supply, enhanced acyltransferase inhibition and antibacterial activity.5,6) Inhibi-
vascular permeability and migration of immune cells occur tion of protein tyrosine phosphatase 1B activity by triterpenes
at damaged sites. The inflammatory process is a protective isolated from the seeds of A. rossii has also reported.7) In
response that occurs in response to trauma, infection, tissue addition, flavonoids isolated from the aerial parts showed an-
injury or noxious stimuli.1) In this process, activated inflam- tioxidant activity.8) In our study, activity-guided fractionation
matory cells (neutrophils, eosinophils, mononuclear phago- lead to the isolation of a new megastigmane glycoside (1) to-
cytes and macrophages) secrete increased amounts of nitric gether with 13 known compounds (2–14) (Fig. 1). The present
oxide (NO), prostaglandin E2 (PGE2) and cytokines, such as paper reports the isolation and structural elucidation of these
interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. compounds as well as their anti-inflammatory activities.
These substances not only induce cell and tissue damage,
but also activate macrophages in rheumatoid arthritis and
Results and Discussion
Compound 1 was isolated as an amorphous powder, and
chronic hepatitis.2) NO is a major product and its production
is controlled by nitric oxide synthases (NOSs), which include had a molecular formula of C26H38O11 based on the ion peak
inducible nitric oxide synthase (iNOS), endothelial nitric oxide at m/z 549.2316 ([M+Na]+) in the high resolution-fast atom
synthase (eNOS) and neuronal nitric oxide synthase (nNOS). bombardment mass spectrometry (HR-FAB-MS) spectrum.
Most importantly, iNOS is highly expressed in macrophages; The infrared (IR) spectrum of 1 suggested the presence of
its activation leads to organ destruction in some inflamma- hydroxyl group (3328cm−1), carbonyl group (1748cm−1) and
tory and autoimmune diseases. PGE2 is another important aromatic absorption (1418cm−1). The 1H-NMR spectrum of
inflammatory mediator and is produced from arachidonic acid 1 displayed signals for four methyl groups at δH 0.88 (H-11),
metabolites by the catalysis of cyclooxygenase-2 (COX-2).3) 0.93 (H-12), 1.15 (H-10) and 1.59 (H-13), eight methylene pro-
Inflammatory stimuli including lipopolysaccharide (LPS) and ton signals at δH 1.41 (Hax-2), 1.77 (Heq-2), 1.99 (Hax −4), 2.26
pro-inflammatory cytokines activate immune cells to up-regu- (Heq-4), 1.86 (H-7a), 2.14 (H-7b), 1.43 (H-8a), and 1.45 (H-8b),
late inflammatory states. Therefore, NO and PGE2 production and two oxymethine proton signals at δH 3.93 (H-3) and 3.68
induced by LPS through iNOS and COX-2, respectively, can (H-9) as well as aromatic proton signal at δH 7.10 (H-2″, H-6″)
reflect the degree of inflammation, and the change in NO and (Table 1). The 13C-NMR and distortionless enhancement by
PGE2 level through the inhibition of iNOS and COX-2 activ- polarization transfer (DEPT) spectra showed the presence of
ity provides a means of assessing the effect of agents on the a sugar moiety, four methyl groups at δC 23.3 (C-10), 28.8
inflammatory process.
(C-11), 30.2 (C-12), and 20.1 (C-13), four methylene groups at
In a preliminary study, a methanol extract of the aerial δC 47.7 (C-2), 39.9 (C-4), 25.6 (C-7), and 40.7 (C-8), two me-
parts of Aceriphyllum rossii ENGLER. (Saxifragaceae) showed thines with oxygen function at δC 74.5 (C-3) and 69.3 (C-9),
inhibitory effect against the LPS-induced NO production in one quaternary carbon at δC 38.9 (C-1) and a tetrasubstituted
RAW264.7 macrophage cells. A. rossii, an endemic species double bond at δC 125.0 (C-5) and 138.7 (C-6) and remain-
in Korea, is a perennial herb that grows on damp rocks along ing five signals of a galloyl unit which was characterized by
valleys in the central northern part of Korea. The roots of A. δC 168.4 (C-7″), 110.4 (C-2″, C-6″), 146.7 (C-3″, C-5″), 139.9
rossii contain oleanane-type triterpenoids, which are cytotoxic (C-4″) and 121.5 (C-1″).9) These results suggested that 1 was
a derivative of ionol (megastigmane) glycoside.10) The place
of tetrasubstituted double bond in the megastigmane skeleton
The authors declare no conflict of interest.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: bsmin@cu.ac.kr

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Vol. 62, No. 2
Fig. 1. Chemical Structure of Isolated Compounds (1–14) from Aceriphyllum rossii
1
was between the 5- and 6-positions. This fact was confirmed 2B). In addition, the H-NMR spectrum of (S)-MTPA ester
by heteronuclear multiple bond connectivity (HMBC) between showed the downfield chemical shift for H-10 and upfield
H-11, H-12 and C-6; H-13 and C-5, C-6 (Fig. 2A). The signals chemical shift for H2-8, H2-7 compared with the same groups
from the sugar moiety appeared at δC 103.1 (C-1′), 78.1 (C-3′), in other diastereomer obtained from (S)-(+)-MTPA-Cl, which
75.5 (C-2′), 75.3 (C-5′), 72.2 (C-4′), 65.1 (C-6′) suggested also lead to determine the absolute configuration at C-9 was
the presence of D-glucopyranose.10) The coupling constant R11) (Fig. 2B). From the above, 1 was identified as (3R,9R)-5-
J=7.6Hz of the anomeric proton of D-glucopyranose indicated megastigmen-3,9-diol-3-O-(6′-galloyl)-β-D-glucopyranoside
it to be the β-form.10) The glycosidic and galloyl unit sites named galloyl linarionoside A.
were determined base on the long-range correlation between
the H-1′ (δH 4.42, d) and C-3 (δC 74.5) as well as H-6′ (δH
The known compounds were identified as linarionoside
A
(2),12) 2,3-trans-3,4-trans-4,5-trans-2,5-bis-(4-hydroxy-
4.48, dd; 4.42, m) and C-7″ (δC 168.4) in the HMBC spectrum. phenyl)-3,4-dimethyltetrahydrofuran (3),13) 3α,24-dihydroxy-
Furthermore, the long-range correlations between the follow- olean-12-en-27-oic acid (4),14) 3α-hydroxyolean-12-en-27-oic
ing protons and carbons (H-2, H-4, H-7, H-8, H-11, H-12, acid (5),15) β-amyrin (6),16) 3-oxolean-12-en-27-oic acid (7),15)
H-13 and C-6; H-2 and C-3; H-4, H-13 and C-5; H-2″ and C-1″, isoquercitrin (8), astragalin (9), kaempferol-3-O-α-L-rhamno-
C-3″, C-4″, C-7″) were also observed in the HMBC spectrum pyranosyl-(1→6)-β-D-glucopyranoside (10), rutin (11),8) quer-
(Fig. 2A). The glycosylated proton of H-3 at δH 3.93 showed cetin-3-O-(6′-galloyl)-β-D-glucopyranoside (12),9) 1,2,3,4,6-
couplings in the correlation spectroscopy (COSY) with Hax-2, penta-O-galloyl-β-D-glucopyranoside (13)17) and dihydromyric-
Heq-2, Hax-4 and Heq-4. Thus, compound 1 was determined as etin (14)18) on basis of spectroscopic analysis, chemical evi-
5-megastigmen-3,9-diol-3-O-(6′-galloyl)-β-D-glucopyranoside. dence and the literature data.
To determine the absolute configuration of 3- and 9-posi-
The cytotoxic effects of isolated compounds (1–14) were
tions, enzymatic hydrolysis of 1 with β-glucosidase yielded evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
an aglycone as 5-megastigmen-3,9-diol, and then the modi- lium bromide (MTT) assay.19) These compounds did not affect
fied Mosher’s method was applied.11) The (R)-α-methoxy-α- the cell viabilities of RAW264.7 cells in either the present or
trifluoromethylphenyl acetyl (MTPA) and (S)-MTPA esters absence of LPS, even at a dose of 30µM after a period of 24h
were prepared. The 1H-NMR spectrum of (S)-MTPA ester (data not shown). In murine RAW264.7 macrophages, LPS
showed the upfield chemical shift for H2-2, H3-11, and H3-12 stimulation alone can induce iNOS transcription and protein
and downfield chemical shift for the H2-4 relative to the same synthesis and subsequent NO production.20) Therefore, this
groups in the alternate diastereomer prepared from (S)-(+)- cell system is an excellent model for evaluating topical agents
MTPA-Cl, so that the C-3 position has R configuration (Fig. and screening potential inhibitors of the pathways that induce

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Table 1. 1H- and 13C-NMR Spectroscopic Data for Compound 1
δH (J in Hz)a)
δC
b)
Position
1
38.9
47.7
2ax
2eq
3
1.41 (m)
1.77 (brd, 12.5)
3.93 (m)
74.5
39.9
4ax
4eq
5
1.99 (dd, 9.6,16.0)
2.26 (brdd, 4.8, 16.0)
125.0
138.7
25.6
6
7a
7b
8a
8b
9
1.86 (td, 6.4, 11.2)
2.14 (td, 6.4, 11.2)
1.43 (m)
40.7
1.45 (m)
3.68 (sext, 6.4)
1.15 (d, 6.4)
0.88 (s)
69.3
23.3
28.8
30.2
20.1
10
11
12
13
Glc
1′
2′
3′
4′
5′
0.93 (s)
1.59 (s)
4.42 (d, 7.6)
3.19 (t, 7.6)
103.1
75.5
78.1
72.2
75.3
65.1
3.38 (m)
Fig. 2. Selected 1H–1H COSY and Key HMBC Correlations of Com-
pound 1 (A) and Chemical Shift Difference for the (S)-MTPA Ester (1c)
and (R)-MTPA Ester (1b) in ppm (δS–δR) (B)
3.34 (m)
3.58 (m)
6′
4.48 (dd, 4.8, 11.2), 4.42 (m)
Galloyl
Table 2. Inhibition of NO Production in Macrophage RAW264.7 Cells
1″
2″
3″
4″
5″
6″
7″
121.5
110.4
146.7
139.9
146.7
110.4
168.4
by Compounds 1–14
7.10 (s)
7.10 (s)
Compound
IC50 (µM)a)
1
>30
>30
>30
2
3
4
12.5 3.0
9.5 2.0
10.5 1.8
9.3 2.4
>30
a) 1H-NMR (CD3OD, 400MHz, δ value) spectroscopic data. b) 13C-NMR (CD3OD,
100MHz) spectroscopic data.
5
6
7
8
9
>30
>30
>30
>30
iNOS and NO production. Due to the rapid half-life of NO in
vivo, we used nitrite production as a biomarker of NO produc-
tion in LPS-stimulated RAW264.7 cells. To investigate the
effect of compounds 1–14 on NO production, the Griess assay
was applied.19) RAW264.7 cells were stimulated with LPS
in the presence or absence of 1–14 for 24h. Nitrite levels in
LPS-stimulated cells increased significantly compared to con-
trol. As shown in Table 2, compounds 4, 5, 6 and 7 showed
potent inhibitory activity with IC50 values of 12.5, 9.5, 10.5
and 9.3µM, respectively. Compound 13 displayed moderate in-
hibitory effect with IC50 value of 17.2µM, but the others were
10
11
12
13
17.2 2.8
14
>30
1.0 0.1
Celastrolb)
a) The inhibitory effects are represented as the molar concentration (µM) giving
50% inhibition (IC50) relative to the vehicle control. These data represent the aver-
age values of three repeated experiments (mean S.D.). b) Positive control for NO
production.
inactive. In this experiment, celastrol, a positive inhibitor, edly upregulated their protein levels, and pre-treatment with
significantly inhibited LPS-induced NO production with IC50 7 inhibited these up-regulations. Compound 7 (1–10 µg/mL)
value of 1.0µM.21–23)
dose-dependently reduced the LPS-induced iNOS and COX-2
In a variety of inflammatory cells, including macrophages, expressions, but did not change the α-tubulin expression. Par-
COX-2 is induced by cytokines and other activators, such as ticularly, the LPS-induced COX-2 was changed clearly as the
LPS, resulting in the release of large amounts of PGE2 at in- concentration of compound 7 increased from 3 to 10µg/mL
flammatory sites.3) Among the tested compounds, 7 was most (Fig. 3).
effective on the LPS-induced production of NO in RAW264.7
The translocation of nuclear factor-kappa B (NF-κB) to the
cells. Therefore, Western blot was performed to determine the nucleus is preceded by the phosphorylation, ubiquitination,
inhibitory effects of compound 7 on the modulation of iNOS and proteolytic degradation of inhibitor kappa B-alpha (IκB-
and COX-2 expression. In unstimulated RAW264.7 cells, α).24) We determined whether the inhibition of LPS-induced
iNOS and COX-2 proteins were not detected, but LPS mark- NF-κB activation by compound 7 was caused by inhibition

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Vol. 62, No. 2
of IκB-α degradation. Cells were exposed to LPS for various Merck), eluted with CHCl3–EtOAc (15:1→2:1, each 0.5L)
times. Cytoplasmic extracts were analyzed for the presence of to obtain compounds 3 (3.4mg) and 7 (50.0mg). Subfraction
IκB-α using Western blots. IκB-α was almost completely de- F6 (280mg) was applied to a silica gel column (3×40cm,
graded in 15min after stimulation with LPS and resynthesized 40–63µm particle size, Merck), eluted with CHCl3–EtOAc
in 30min (Fig. 4A). We pretreated cells with difference con- (25:1→5:1, each 0.5L) to afford six subfractions (F6.1–
centrations of 7 (3–30 µg/mL) and then exposed them to LPS F6.6). The subfraction F6.2 (55.6mg) was chromatograped
for 10min. However, compound 7 could not significantly pre- over a silica gel column (2×40cm, 40–63µm particle size)
vent LPS-induced degradation and resynthesis of IκB-α (Fig. and eluted with CHCl3–MeOH (30:1→5:1, each 0.4L) to
4B). Thus, this study could be combined suggesting that com- obtain compounds 4 (7.0mg) and 5 (7.2mg). Compound 6
pound 7 produced anti-inflammatory activity through reducing (7.1mg) was crystallized from subfraction F6.4 (18mg) with
inducible nitric oxide synthase and cyclooxygenase-2 proteins, CHCl3–MeOH (10:1). The EtOAc-soluble fraction (53.8g) was
not through IκB-α dependent nuclear factor-kappa B pathway.
also subjected to a silica gel column (12×60cm, 63–200µm
particle size), eluted with CHCl3–MeOH–H2O (9:1:0.1→
1:1:0.02, each 2L) to obtain nine subfractions (E1–E9). Sub-
Experimental
Materials and Methods The aerial parts of A. rossii fraction E3 (3.5g) was applied to RP-C18 silica gel column
were collected at Jeongbong-san, Kangwondo, Korea, in June (6×60cm, 40–63µm particle size), eluted with MeOH–H2O
2007 and identified by Dr. Hyeong Kyu Lee. A voucher speci- (1:4→1:1) to yield ten subfractions (E3.1–E3.10). Subfrac-
men (PB-1636) was deposited at the herbarium of the Korea tion E3.4 (72.0mg) was applied to one more RP-C18 silica
Research Institute of Bioscience and Biotechnology, Korea. gel column (2×60cm, 40–63µm particle size) and eluted
The aerial parts (3.0kg) were extracted reflux with MeOH with ACN–H2O (1:8→2:3, each 1L) to afford compounds 1
(5 L×3 times). After evaporation of the solvent under reduced (15.0mg) and 8 (25.0mg). Subfraction E3.6 (85.0mg) was puri-
pressure, the crude MeOH extract (290g) was obtained and fied by HPLC [eluting with MeOH–H2O (75:25→55:45) over
suspended in hot water and partitioned with n-hexane, CHCl3, 90min; flow rate: 5mL/min; UV detection at 210nm; column
EtOAc and BuOH, successively, to afford n-hexane- (39.5g), COSMOSIL (20×250mm, 5µm)] to obtain compounds 9
CHCl3- (3.2g), EtOAc- (53.8g), BuOH- (67.2g) soluble frac- (8.0mg, tR=29.8min), 10 (3.4mg, tR=42.6min) and 11 (7.1mg,
tions, respectively. By the activity-guided fractionation, the tR=51.3min). Subfraction E8 (2.2g) was subjected to RP-C18
CHCl3 soluble fraction (3.2g) was chromatographed over a silica gel column (6×60cm, 40–63µm particle size), eluted
silica gel column (6×60cm, 63–200µm particle size, Merck) with ACN–H2O (1:5→2:3, each 1.2L) to afford 8 subfractions
and eluted with n-hexane–EtOAc (20:1→1:1, each 1L) yield- (E8.1–E8.8). Subfraction E8.4 (65.0mg) was applied to RP-C18
ed eight fractions (F1–F8). Subfraction F4 (430.0mg) was sub- silica gel column (2×40cm, 40–63µm particle size) using
jected to a silica gel column (4×50cm, 40–63µm particle size, MeOH–H2O (1:4→2:3, each 0.6L) to obtain compounds 12
(20.0mg) and 14 (10.8mg). Compound 2 (30.0mg) was crys-
tallized from subfraction E8.6 (45.0mg). Subfraction E8.5
(48.0mg) was chromatogaphed over RP-C18 silica gel column
(2×40cm, 40–63µm particle size), eluted with ACN–H2O
(1:5→1:2, each 0.5L) to afford compound 13 (32.0 mg).
Galloyl Linarionoside A (1): Amorphous powder; [α]D25
0 (c=0.1, MeOH); UV (MeOH) λmax (logε) 277 (3.98), 216
(4.36); IR νmax (KBr) 3328, 2943, 2831, 2527, 2356, 1748, 1449,
1418, 1116, 1024cm−1; HR-FAB-MS m/z 549.2316 [M+Na]+
1
(Calcd for C26H38O11Na, 549.2312). H- and 13C-NMR spectro-
scopic data, see Table 1.
Enzymatic Hydrolysis of
Fig. 3. Inhibition of LPS-Induced iNOS and COX-2 Expression in
RAW264.7 Cells by Compound 7 (C7)
1
Compound 1 (10.0mg)
was dissolved in AcOH–NaOAc buffer (pH 5.0, 5mL) with
naringinase (Sigma, EC 232-962-4, β-glucosidase activity:
69units/g) (25.0mg) and incubated at room temperature for
14h. The crude reaction mixture was extracted with EtOAc
RAW264.7 cells were pretreated for 30min indicated concentrations of 7, fol-
lowed by stimulation with LPS (1µg/mL) for 18h. Total lysates were prepared, the
expression levels of iNOS and COX-2 were determined by western blot analysis.
Histograms show densitometry analyses of relative iNOS, COX-2 expression levels
normalized against tubulin.
Fig. 4. Effect of Compound 7 on the LPS-Induced IκB-α Degradation
RAW264.7 cells were exposed with 1µg/mL LPS for the various time periods (A). Cytoplasmic extracts were analyzed for the presence of IKB-α with Western blot
analysis. Preincubation with 7 and then exposed them to 1µg/mL LPS for the indicated time (B).

February 2014
189
(5 mL×3 times). The organic layer was dried under reduced phenylmethyl sulfonyl fluoride, 1µg/mL leupeptin, 1mM
pressure. The residue (5.1mg) was applied to a RP-C18 silica sodium vanadate, 150mM NaCl). Fifty micrograms of protein
gel column (2×20cm, 40–63µm particle size, Merck), eluted (for iNOS) per lane was separated by sodium dodecyl sulfate
with acetone–H2O (2:3, 0.5L) to yield 1a (3.2mg).25)
(SDS)-polyacrylamide gel electrophoresis (PAGE), and trans-
Preparation of (R)- and (S)-MTPA Esters (1b,c) from 1a ferred to a polyvinylidene difluoride membrane (Millipore,
A solution of 1a (1.0mg) in dry pyridine (50µL) was reacted Bedford, MA, U.S.A.). The membrane was blocked with 5%
with 5µL of (S)-(−)-MTPA-Cl and 2mg of 4-DMAP at room skim milk, and then incubated with the corresponding anti-
temperature for 30min. The reaction mixture was dried by body. The antibody for iNOS was obtained from Santa Cruz
N2 gas. The dried product was partitioned with CH2Cl2 and Biotechnology (Santa Cruz, CA, U.S.A.). The antibody for
H2O. The organic layer was dried using Na2SO4 and con- α-tubulin was obtained from Sigma. Antibody for IκB-α was
centrated under reduced pressure. The residue was purified obtained from Cell Signaling Technology (Danvers, MA,
by preparative TLC silica gel (0.25mm thickness) developed U.S.A.). After binding of an appropriate secondary antibody
with CH2Cl2, and the product was eluted with CH2Cl2–MeOH coupled to horseradish peroxidase, proteins were visualized by
(10:1, 0.2L) to furnish (R)-MTPA ester, 1b (1.3mg). In a simi- enhanced chemiluminescence according to the instructions of
lar manner, 1a (1.7mg) yielded (S)-MTPA ester (1c, 1.1mg).26) the manufacturer (Amersham Pharmacia Biotec, Buckingham-
5-Megastigmen-3,9-di-(R)-MTPA Ester (1b): Colorless oil; shire, U.K.).23,27)
1H-NMR (CDCl3, 400MHz) δH 1.571 (1H, m, Hax-2), 1.796
(1H, ddd, J=2, 3.6, 12Hz, Heq-2), 5.228 (1H, m, H-3), 2.283
Acknowledgments This research was supported by Basic
(1H, dd, J=6.4, 16.8Hz, Heq-4), 2.114 (1H, m, Hax-4), 1.899 Science Research Program through the National Research
(1H, td, J=4.4, 12.4Hz, H-7a), 2.051 (1H, m, H-7b), 1.617 (2H, Foundation of Korea (NRF) funded by the Ministry of Educa-
m, H-8a, H-8b), 5.090 (1H, sext, J=5.6Hz, H-9), 1.271 (3H, tion, Science and Technology (KRF-2012R1A2A2A06046921)
d, J=6.4Hz, H-10), 1.045 (3H, s, H-11), 0.990 (3H, s, H-12), and BK21 Plus. The authors are grateful at the Korea Basic
1.520 (3H, s, H-13).
Science Institute for measuring the MASS spectrometry.
5-Megastigmen-3,9-di-(S)-MTPA Ester (1c): Colorless oil;
1H-NMR (CDCl3, 400MHz) δH 1.521 (1H, m, Hax-2), 1.708
(1H, ddd, J=1.2, 3.2, 12Hz, Heq-2), 5.207 (1H, m, H-3), 2.338
(1H, dd, J=5.6, 16.4Hz, Heq-4), 2.128 (1H, m, Hax-4), 1.826
(1H, td, J=5.2, 16.1Hz, H-7a), 1.982 (1H, m, H-7b), 1.582 (2H,
m, H-8a, H-8b), 5.117 (1H, sext, J=6.0Hz, H-9), 1.340 (3H, d,
J=6.4Hz, H-10), 1.000 (3H, s, H-11), 0.910 (3H, s, H-12), 1.470
(3H, s, H-13).
Cell Culture The murine macrophage cell line
(RAW264.7) was purchased from the Korean Cell Line Bank
(KCLB, Seoul, Korea). These cells were cultured in Dul-
becco’s modified Eagle’s medium (DMEM; GIBCO, Inc., NY,
U.S.A.) supplemented with 100U/mL of penicillin, 100U/mL
of streptomycin and 10% fetal bovine serum (FBS, GIBCO,
Inc., NY, U.S.A.). The cells were incubated in an atmosphere
of 5% CO2 at 37°C and were subcultured every 3d.
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