Triterpenoid saponins of pulsatilla koreana root have inhibition effects of tumor necrosis factor-α secretion in lipopolysaccharide-induced RAW264.7 cells

Article abstract of DOI:10.1248/cpb.c12-01034

In the present study, a new oleanane-type triterpenoid saponin, pulsatilloside F (1), along with 21 known compounds (2-22), were isolated from the root of Pulsatilla koreana. Their chemical structures were elucidated by mass, ¹H-, ¹³C-NMR, correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond connectivity (HMBC) spectroscopy. Anti-inflammatory effects of the compounds were evaluated in terms of inhibitory of tumor necrosis factor a (TNF-α) secretion in the lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophage cell line. Compounds 19 and 20 exhibited particularly inhibitory effects with respective IC₅₀ values of 0.32 and 0.65 μm. Compounds 1-4, 7 and 10-13 exhibited inhibitory effects with inhibition rates up to 41.55-73.76% at a concentration of 5 μm, respectively.

Full text of DOI:10.1248/cpb.c12-01034

April 2013
Note
Chem. Pharm. Bull. 61(4) 471–476 (2013)
471
Triterpenoid Saponins of Pulsatilla koreana Root Have Inhibition Effects
of Tumor Necrosis Factor-α Secretion in Lipopolysaccharide-Induced
RAW264.7 Cells
Wei Li,a Yan Ding,a,b Ya Nan Sun,a Xi Tao Yan,a Seo Young Yang,a Chun Whan Choi,a
,a
Ji Yun Cha,c Young Mi Lee,c and Young Ho Kim*
b
a College of Pharmacy, Chungnam National University; Daejeon 305–764, Republic of Korea: School of Food
c
Science and Technology, Dalian Polytechnic University; Dalian, Liaoning 116034, P. R. China: and Department of
Oriental Pharmacy, College of Pharmacy, Wonkwang University; Iksan, Jeonbuk 570–749, Republic of Korea.
Received November 30, 2012; accepted January 7, 2013; advance publication released online January 25, 2013
In the present study, a new oleanane-type triterpenoid saponin, pulsatilloside F (1), along with 21 known
compounds (2–22), were isolated from the root of Pulsatilla koreana. Their chemical structures were eluci-
1
dated by mass, H-, 13C-NMR, correlation spectroscopy (COSY), heteronuclear multiple quantum coherence
(HMQC) and heteronuclear multiple bond connectivity (HMBC) spectroscopy. Anti-inflammatory effects of
the compounds were evaluated in terms of inhibitory of tumor necrosis factor α (TNF-α) secretion in the li-
popolysaccharide (LPS)-stimulated murine RAW264.7 macrophage cell line. Compounds 19 and 20 exhibited
particularly inhibitory effects with respective IC50 values of 0.32 and 0.65µm. Compounds 1–4, 7 and 10–13
exhibited inhibitory effects with inhibition rates up to 41.55–73.76% at a concentration of 5µm, respectively.
Key words Pulsatilla koreana; Ranuculaceae; triterpenoid saponin; anti-inflammatory; murine RAW264.7
macrophage cell
The Pulsatilla genus, Pulsatilla koreana Nakai (Ranucula- oleanane-type triterpenoid saponin (1), along with 21 known
ceae), from Korea, is an important herb in traditional medicine compounds (2–22) were isolated.
used to treat amoebic dysentery and malaria.1) Phytochemical
Pulsatilloside F (1) was obtained as a white crystal. The
studies on P. koreana root have demonstrated the presence of molecular formula, C77H126O41, was determined from the
protoanemonin,2) deoxypodophyllotoxin,3) oleanane and lu- pseudomolecular ion peak [M−H]− at m/z 1705.7692 (Calcd for
pane-type triterpenoid saponins.4–6) Root extract of P. koreana C77H125O41, 1705.7696) in the high-resolusion electrospray ion-
has been reported to possess antitumor,7) antibiotic,2) and anti- ization quadrupole time-of-flight mass spectrometry (HR-ESI-
inflammatory activities.8) In this report, a new compound (1), QTOF-MS). Its Fourier transform infrared (FT-IR) spectrum
and 21 known compounds (2–22), were isolated from a metha- showed absorption bands at 3367 and 1731cm−1, suggesting
nol extract of P. koreana. The structures were elucidated by the presence of hydroxyl and carbonyl groups, respectively.
one-dimensional (1D)-, and two-dimensional (2D)-nuclear Acid hydrolysis of compound 1 with 10% HCl resulted in
magnetic resonance (NMR) and mass spectrometry.
hederagenin, l-arabinose, d-glucose, and l-rhamnose. The
Inflammatory cytokines are produced by innate immune 13C-NMR and distortionless enhancement by polarization
cells, such as macrophages and dendritic cells, during in- transfer (DEPT) spectra exhibited 30 aglycone carbon sig-
fection. Lipopolysaccharide (LPS) is an endotoxin that in- nals, which were assignable to six methyl groups at δC 14.0
duces septic shock syndrome and stimulates the production (C-24), 16.0 (C-25), 17.3 (C-26), 23.4 (C-30), 25.8 (C-27), and
of inflammatory mediators such as tumor necrosis factor 32.9 (C-29); eleven methylene groups at δC 17.9 (C-6), 23.1
(TNF)-α.9) TNF-α is a well-characterized, pro-inflammatory (C-16), 23.6 (C-11), 26.2 (C-2), 28.1 (C-15), 32.3 (C-22), 32.5
cytokine released primarily from monocytes and macrophages (C-7), 33.7 (C-21), 38.8 (C-1), 45.9 (C-19), and 63.7 (C-23);
upon invasion of the host by a wide variety of pathogens. It four methine groups at δC 41.4 (C-18), 47.3 (C-5), 47.9 (C-9),
plays a crucial role in host defense and the inflammatory re- and 80.6 (C-3); six quaternary carbons at δC 30.5 (C-20), 36.6
sponse. Although it has numerous beneficial roles in immune (C-10), 39.6 (C-8), 41.9 (C-14), 43.3 (C-4), and 46.8 (C-17); two
regulation, it has also been implicated in the pathogenesis of olefinic carbons at δC 122.7 (C-12) and 143.9 (C-13); and one
both acute and chronic inflammatory disease.10) A reduction in carbonyl group at δC 176.3 (C-28). Another 47 carbon signals
1
the secretion of TNF-α would greatly aid in the treatment or were assigned to eight sugar moieties. The H-NMR spectrum
mitigation of chronic inflammatory diseases.11) Here, we report revealed six quaternary methyl groups at δH 0.87 (Me-29),
the effects of compounds isolated from P. koreana on the in- 0.88 (Me-30), 0.98 (Me-25), 1.12 (Me-26), 1.16 (Me-24), and
hibitory activity of TNF-α secretion in the presence of LPS in 1.18 (Me-27), and a broad singlet for an olefinic proton at
a murine RAW264.7 macrophage cell line.
δH 5.31 (brs) characteristic of the Δ12 proton in pentacyclic
triterpenes.12) Oxymethylene groups at δH 3.80 (d, J=11.2Hz,
H-23a) and 4.23 (d, J=11.2Hz, H-23b) were assigned to H-23
Results and Discussion
A MeOH extract (150g) of the roots of P. koreana was by heteronuclear multiple quantum coherence (HMQC), and
suspended in H2O and successively extracted with EtOAc and heteronuclear multiple bond correlation (HMBC) spectra
n-BuOH. Using combined chromatographic separations, a new (Table 1). All of the above signals showed a characteristic
hederagenin skeleton.13) In contrast, eight anomeric proton
signals at δH 4.80 (d, J=6.9Hz, ara-H1), 4.91 (d, J=7.0Hz,
The authors declare no conflict of interest.
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: yhk@cnu.ac.kr

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Vol. 61, No. 4
Table 1. NMR Spectral Data for Aglycone of Compound 1
a,b)
a,c)
a,b)
a,c)
Position
1
δC
δH [mult. (J in Hz)]
Position
16
δC
δH [mult. (J in Hz)]
38.8
26.2
1.07 (m)
1.54 (m)
2.00 (m)
2.25 (m)
23.1
1.91 (m)
2.05 (m)
2
17
18
19
46.8
41.4
45.9
3.17 (d, 11.7)
1.20 (m)
1.69 (m)
3
4
5
6
7
80.6
43.3
47.3
17.9
32.5
4.30 (dd, 3.5, 10.5)
1.75 (d, 11.8)
1.55 (m)
1.56 (m)
20
21
22
30.5
33.7
32.3
1.08 (m)
1.73 (m)
1.87 (m)
3.80 (d, 11.2)
4.23 (d, 11.2)
1.16 (s)
0.98 (s)
1.12 (s)
1.18 (s)
1.56 (m)
8
9
39.6
47.9
36.6
23
63.7
1.77 (m)
10
11
12
13
14
15
24
25
26
27
28
29
30
14.0
16.0
17.3
25.8
176.3
32.9
23.4
23.6
1.96 (m)
5.31 (br s)
122.7
143.9
41.9
28.1
1.09 (m)
2.25 (m)
0.87 (s)
0.88 (s)
Assignments were done by HMQC, HMBC and 1H–1H COSY experiments; a) measured in pyridine-d5; b) 150MHz; c) 600MHz.
Table 2. NMR Spectral Data for Suger Moieties of Compound 1
a,b)
a,c)
a,b)
a,c)
Position
δC
δH [mult. (J in Hz)]
Position
δC
δH [mult. (J in Hz)]
C-3
ara-1
glc″-1
104.8
75.1
77.9
71.2
78.2
62.2
5.07 (d, 7.0)
3.92 (m)
4.15 (m)
4.87 (m)
4.10 (m)
4.24 (m)
4.50 (m)
104.6
75.5
73.8
80.5
65.7
4.80 (d, 6.9)
4.13 (m)
4.35 (m)
4.76 (m)
3.70 (d, 10.8)
4.40 (m)
2
3
4
5
6
2
3
4
5
glc-1
106.6
75.3
78.2
71.0
78.6
62.3
5.01 (d, 7.7)
4.13 (m)
4.18 (m)
4.88 (m)
3.92 (m)
C-28
glc⁗-1
2
3
4
5
6
95.4
74.5
78.5
70.5
77.8
68.9
6.16 (d, 8.0)
4.10 (m)
4.33 (m)
4.15 (m)
3.87 (m)
4.30 (m)
4.59 (m)
4.91 (d, 7.0)
3.93 (m)
4.12 (m)
4.07 (m)
3.60 (d, 8.9)
4.05 (m)
4.15 (m)
5.77 (brs)
4.65 (m)
4.53 (m)
4.33 (m)
4.29 (m)
1.60 (d, 6.0)
2
3
4
5
6
4.37 (m)
4.54 (m)
rha-1
101.1
71.4
82.9
72.7
69.5
18.3
106.4
75.2
76.5
80.9
76.3
61.5
6.17 (brs)
4.92 (m)
4.75 (dd, 2.5, 9.3)
4.50 (m)
2
3
4
5
glc⁗-1
104.7
75.0
76.5
77.9
77.0
61.0
2
3
4
5
6
4.98 (m)
6
1.47 (d, 6.0)
5.37 (d, 7.7)
3.92 (m)
4.28 (m)
4.31 (m)
glc′-1
2
3
4
5
6
rha′-1
102.5
72.3
72.5
73.6
70.1
18.3
2
3
4
5
6
4.08 (m)
3.83 (m)
4.44 (m)
Assignments were done by HMQC, HMBC and 1H–1H COSY experiments; glc: d-glucopyranosyl; rha: l-rhamnopyranosyl; ara: l-arabinopyranosyl; a) Measured in pyri-
dine-d5; b) 150MHz; c) 600MHz.
glc⁗-H1), 5.01 (d, J=7.7Hz, glc-H1), 5.07 (d, J=7.0Hz, (ara-C1), 104.7 (glc⁗-C1), 106.6 (glc-C1), 104.8 (glc″-C1),
glc″-H1), 5.37 (d, J=7.7Hz, glc′-H1), 5.77 (brs, rha′-H1), 106.4 (glc′-C1), 102.5 (rha′-C1), 95.4 (glc‴-C1) and 101.1
6.16 (d, J=8.0Hz, glc‴-H1), and 6.17 (brs, rha-H1) showed (rha-C1), indicating that compound 1 possessed eight sugar
HMQC correlations with anomeric carbon signals at δC 104.6 units (Table 2). The downfield chemical shift of C-3 (δC 80.6)

April 2013
473
Table 3. IC50 Values of Compounds 1–22 for TNF-α Production in LPS-
Stimulated RAW264.7 Cells
Compounds
IC50 (µm)
1
>5
2
3
4
5
2.38 0.4
2.01 0.01
2.89 0.2
>5
6
>5
7
3.78 0.4
8
>5
9
>5
10
11
12
13
2.76 0.2
3.89 0.4
3.64 0.3
2.81 0.1
>5
Fig. 1. Key HMBC Correlations of Compound 1
14
15
16
17
18
19
20
21
22
>2
>2
>2
>2
0.32 0.1
0.65 0.1
>2
and the upfield chemical shift of C-28 (δC 176.3) indicated that
compound 1 was a bisdesmosidic saponin. The sequence of
sugar residues of compound 1 was assigned unambiguously on
the basis of HMBC spectra. The location of the sugar chain
at C-3 was identified as O-β-d-glucopyranosyl-(1→4)-[O-β-
d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→3)-O-α-l-
rhamnopyranosyl-(1→2)]-O-α-l-arabinopyranosyl based on
the HMBC correlations: H-1 of glucose″ (δH 5.07) with C-4
of glucose′ (δC 80.9), H-1 of glucose′ (δH 5.37) with C-3 of
rhamnose (δC 82.9), H-1 of rhamnose (δH 6.17) with C-2 of
arabinose (δC 75.5), H-1 of glucose (δH 5.01) with C-4 of
>2
Dexsamethasonea)
0.01 0.0001
a) Dexsamethasone was used as positive control. Data are presented as the
mean S.D. and all experiments measured in triplicate.
arabinose (δC 80.5), and a cross-peak between H-1 of arabi- (13),6) 3-O-β-d-glucopyranosyl(1→4)-β-d-glucopyranosyl(1→
nose (δH 4.80) and C-3 of the aglycone (δC 80.6). The sugar 3)-α-l-rhamnopyranosyl(1→2)-α-l-arabinopyranosyl oleano-
chain at C-28 was established as O-α-l-rhamnopyranosyl-(1→ lic acid (14),6) 23-hydroxy-3β-[(O-α-l-rhamnopyranosyl-(1→
4)-O-β-d-glucopyranosyl-(1→6)-O-β-d-glucopyranosyl from 2)-O-[O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl(1→
the following HMBC correlations: H-1 of rhamnose′ (δH 4)]-O-α-l-arabinopyranosyl)oxy]lup-20(29)-en-28-oic acid 28-
5.77) with C-4 of glucose⁗ (δC 77.9), H-1 of glucose⁗ (δH O-α-l-rhamnopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→6)-
4.91) with C-6 of glucose‴ (δC 68.9), and H-1 of glucose‴ O-β-d-glucopyranosyl ester (15),22) 3β-[O-β-d-glucopy-
(δH 6.16) with C-28 of the aglycone (δC 176.3) (Fig. 1). On ranosyl-(1→3)-O-α-l-rhamnopyranosyl-(1→2)-O-α-l-arabino-
the basis of the above analyses, the structure of compound pyranosyl)oxy]lup-20(29)-en-28-oic acid (16),6) pulsatilloside
1 was established as 3-O-β-d-glucopyranosyl-(1→4)-[O-β- E (17),8) 3β-[(O-α-l-rhamnopyranosyl-(1→2)-O-α-l-arabinopy-
d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→3)-α-O-l- ranosyl)oxy]lup-20(29)-en-28-oic acid 28-O-β-d-glucopyrano-
rhamnopyranosyl(1→2)]-O-α-l-arabinopyranosyl hederagenin syl-(1→6)-O-β-d-glucopyranosyl ester (18),8) pulchinenoside
28-O-α-l-rhamnopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→ C (19),22) cussosaponin C (20),8) hederagenin 3-O-α-l-rham-
6)-O-β-d-glucopyranosyl ester, and was termed pulsatilloside nopyranosyl-(1→2)-O-α-l-arabinopyranoside (21),23) and 23-
F.
hydroxy-3β-[O-α-l-rhamnopyranosyl-(1→2)-[O-β-d-glucopy-
Comparison of the 1D- and 2D-NMR and mass spec- ranosyl-(1→4)]-O-α-l-arabinopyranosyl)oxy]lup-20(29)-en-28-
tral data with reported values led to the identifica- oic acid (22)8) (Fig. 2).
tion of structures of the 21 known compounds (2–22)
When RAW264.7 cells were cultured with the compounds
as hederacolchiside F (2),14) pulsatilla saponin A (3),14) isolated from P. koreana, the secretion of TNF-α was se-
cernuoside
A E
(4),15) hederacholchiside (5),16) beesio- verely decreased by treatment with compounds 1–4, 7, and
side Q (6),17) oleanolic acid 3-O-β-d-glucopyranosyl(1→ 10–13, and exhibited inhibitory effects with inhibition rates
4)-β-d-glucopyranosyl(1→3)-α-l-rhamnopyranosyl(1→2) up to 41.55–73.76% at a concentration of 5µm, respectively.
[β-d-glucopyranosyl(1→4)]-α-l-arabinopyranosyl-28-O-α- Compounds 19 and 20 showed the strongest inhibitory effect
l-rhamnopyranosyl(1→4)-β-d-glucopyranosyl(1→6)-β-d- with IC50 values of 0.32 0 .1 µm and 0.65 0 .1 µm, respec-
glucopyranosyl ester (7),18) hederacoside
B
(8),19) 3-O- tively (Table 3). Other compounds had negligible effects at
β-d-glucopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→ the same concentration. Therefore, compounds 1–4, 7, 10–13,
2)-[O-β-d-glucopyranosyl-(1→4)]-O-α-l-arabinopyranosyl 19, and 20 may be excellent therapeutics for the treatment of
oleanolic acid (9),20) raddeanoside R17 (10),21) 3-O- abnormally-increased-TNF-α levels, such as rheumatoid ar-
β-d-glucopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→ thritis,24) atherosclerosis,25) and graft rejection.26)
2)-O-α-l-arabinopyranosyl oleanolic acid (11),6) radd-
Upon examination of the structure–activity relationship of
eanoside R13 (12),6) 3-O-β-d-glucopyranosyl(1→4)-β-d- the lupane-type triterpenoid saponins (15–22), compounds 19
glucopyranosyl(1→3)-α-l-rhamnopyranosyl(1→2)[β-d- and 20 showed the strongest inhibitory effects on TNF-α se-
g l u c o p y r a n o s y l ( 1 → 4 ) ] - α-l-arabinopyranosyl oleanolic acid cretion, and their structures were similar. When the sugar unit

474
Vol. 61, No. 4
Fig. 2. Structures of Compounds 1–22 from the Roots of P. koreana
at C-3 of the aglycone was linked to a rhamnose-arabinose at C-3 and the rhamnose-glucose-glucose chain at C-28 of
chain, the anti-inflammatory activity increased significantly the bisdesmosidic lupane-type saponin appear to be the key
when compared to a rhamnose-(glucose-glucose-)arabinose functional elements. These data may be useful in evaluating
chain (15), and a rhamnose-(glucose-)arabinose chain (17). the structure–activity relationships of other lupane-type triter-
Additionally, the sugar moieties of compounds 19 and 20 penoid saponins and in developing anti-inflammatory agents
at C-28 linked to a rhamnose-glucose-glucose chain of the for medical uses.
aglycones compared to a carboxyl group (21) and a glucose-
glucose chain (18) indicate that the anti-inflammatory activity
could be increased significantly. Compounds 15 and 17 were
Experimental
General Experimental Procedures Melting points were
also linked to a rhamnose-glucose-glucose chain; however, determined using an Electrothermal IA-9200 system. Opti-
they did not display significant anti-inflammatory effects. As cal rotations were determined using a Jasco DIP-370 auto-
previous reported, compounds 19 and 20 showed significant matic polarimeter. The FT-IR spectra were measured using
inhibitory activities against nitric oxide (NO) production on a Nicolet 380 FT-IR spectrometer. GC was carried out on a
LPS-stimulated RAW264.7 cells, with relative NO production Shimadzu-2010 spectrometer: detector, flame ionization de-
values of 12.7 3.1 and 13.0 2.5% and at 100µm, respec- tector (FID); detection temperature, 300°C; column, SPB-1
tively.8) Moreover, bisdesmosidic lupane-type saponin also (0.25mm i.d.×30m); column temperature, 230°C; carrier
showed significant effect against carrageenan, tetradecanoyl- gas, He (2mL/min) injection temperature, 250°C; injection
phorbol acetate, arachidonic acid and ethyl phenylpropiolate volume, 0.5µL; The NMR spectra were recorded using a
acute edemas.27) Therefore, the rhamnose-arabinose chain JEOL ECA 600 spectrometer (1H, 600MHz; 13C, 150MHz),

April 2013
475
and high-resolution electrospray ionization mass spectra (HR- 4L for each step) to give 5 fractions (Frs. 3B1–B5). Fraction
ESI-MS) were obtained using an Agilent 6530 Accurate-Mass 3B2 using an YMC column (2×80cm) with a MeOH–ace-
Q-TOF LC/MS system. Column chromatography was per- tone–H2O (1:0.3:1, 650mL) elution solvent to give compound
formed using a silica gel (Kieselgel 60, 70–230, and 230–400 2 (12.0mg). Fraction 3B4 was separated using a silica gel
mesh, Merck, Darmstadt, Germany), YMC RP-18 resins, and column (1.5×80cm) with CHCl3–MeOH–H2O (1.2:1:0.1,
thin layer chromatography (TLC) was performed using pre- 1.5L) to give compound 15 (4.0mg) and 1 (130.0mg). Frac-
coated silica-gel 60 F254 and RP-18 F254S plates (both 0.25mm, tion 3B4 was further chromatographed on RP chromatography
Merck, Darmstadt, Germany).
column with acetone–MeOH–H2O (0.3:1:1–0.4:1:1–0.45:
Plant Material Dried roots of P. koreana were purchased 1:1.4) to yield compounds 5 (50.0mg), 6 (44.0mg). Compound
from herbal market, Kumsan, Chungnam, Korea in March 4 (44.0mg) was isolated from 3B6 using RP chromatography
2009 and identified by one of the authors (Prof. Young Ho column with MeOH–H2O (2.5:1). Fraction 3B5 was fur-
Kim). A voucher specimen (CNU 09106) was deposited at ther chromatographed on RP chromatography column with
the Herbarium of College of Pharmacy, Chungnam National acetone–MeOH–H2O (0.3:1:1–0.6:1:1–0.7:1:1.5) to yield
University, Korea.
compounds compounds 7 (78.0mg), 8 (300.0mg), 9 (12.0mg),
Extraction and Isolation Dried roots of P. koreana and 10 (36.0mg).
(2.0kg) were extracted with MeOH under reflux for 10h
Pulsatilloside F (1): White crystals (MeOH); mp 270–275°C;
(7L×3 times) to yield 500.0g of extract. This extract was [α]D28−68 (c=0.25, MeOH); FT-IR (CH3CN) νmax 3373, 2933,
suspended in water and partitioned with ethyl acetate to 1731, 1621, 1377, 1257, 1057, 1031cm−1; HR-ESI-QTOF-MS:
1
yield 37.0g ethyl acetate extract and 463.0g water extract. m/z 1705.7692 [M−H]−, (Calcd for C77H125O41: 1705.7696); H
The water extract was partitioned with n-BuOH to yield (pyridine-d5, 600MHz) and 13C-NMR (pyridine-d5, 150MHz)
130.0g BuOH extract. The ethyl acetate extract was sub- data: see Tables 1 and 2.
jected to silica gel (5×30cm) column chromatography with
Acid Hydrolysis Compound 1 (5mg) was heated in 3mL
a gradient of CHCl3–MeOH (1:0, 50:1, 20:1,10: 1 and 1:1; 10% HCl (dioxane–H2O, 1:1) at 90°C for 3h. The residue
2L for each step) to give 3 fractions (Frs. 1A–C). Fraction was partitioned between EtOAc and H2O to give the aglycone
1B was separated using an YMC column (3×80cm) with a and sugar, respectively. The aqueous layer was evaporated
MeOH–acetone–H2O (1.3:1.3:1, 1.2L) elution solvent to give until dry to yield a residue; this was dissolved in anhydrous
compound 21 (62.0mg). Fraction 1C was separated using a pyridine (200µL) and then mixed with a pyridine solution of
YMC column (1×60cm) with a MeOH–H2O (2.7:1–3.5:1, 0.1 m l-cysteine methyl ester hydrochloride (200µL). After
800mL) elution solvent to give compounds 22 (28.0mg) warming to 60°C for 1h, trimethylsilylimidazole solution was
and 14 (19.0mg). The BuOH extract was subjected to silica added, and the reaction solution was warmed at 60°C for 1h.
gel (5×30cm) column chromatography with a gradient of The mixture was evaporated in vacuo to yield a dried product,
CHCl3–MeOH–H2O (7:1:0.1, 5:1:0.1, 2:1:0.1 and 0:1:0; which was partitioned between n-hexane and H2O. The n-hex-
3L for each step) to give 4 fractions (Frs. 2A–D). Fraction ane layer was filtered and analyzed by gas chromatography.
2B was separated using a silica gel column (3×80cm) with Retention times of the persilylated monosaccharide deriva-
CHCl3–MeOH–H2O (5:1:0.1, 4:1:0.1 and 3:1:0.1, 1L for tives were as follows: l-arabinose (tR, 4.72min), l-rhamnose
each step) to give 4 sub-fractions (Frs. 2B1–B4). Fraction (tR, 5.31min) and d-glucose (tR, 14.11min) were confirmed by
2B1 was separated using an YMC column (1×80cm) with a comparison with those of authentic standards.
MeOH–H2O (4.5:1, 1.1L) elution solvent to give compound
Cell Culture RAW264.7 cells were obtained from Ameri-
3 (37.0mg). Fraction 2B2 was separated using an YMC col- can Type Culture collection (ATCC, Manassas, VA, U.S.A.)
umn (1.5×80cm) with a MeOH–acetone–H2O (2.5:0.7:1, and maintained in Dulbecco’s modified Eagle’s medium
650mL) elution solvent to give compound 16 (12.0mg). Frac- (DMEM), DMEM containing 2mm l-glutamine, 10% heat-
tion 2B4 was separated using an YMC column (1×80cm) inactivated fetal bovine serum (FBS), 100U/mL penicillin,
with a MeOH–acetone–H2O (2:0.5:1, 700mL) elution solvent and 100µg/mL streptomycin at 37°C in a 5% CO2 humidified
to give compound 17 (12.0mg). Fraction 2C was separated incubator.
using a silica gel column (3×80cm) with CHCl3–MeOH–H2O
Inflammatory Cytokine Assay RAW264.7 cells were
(3.5:1:0.1, 2:1:0.1 and 1:1:0.2, 1L for each step) to give 3 cultured at 2×105 cells/mL in DMEM containing 10% fetal
sub-fractions (Frs. 2C1–C3). Fraction 2C1 was further chro- bovine serum in 24-well tissue culture plates. The cells were
matographed on RP chromatography column with acetone– pretreated with 0.5, 1 and 2µm concentrations of compound
MeOH–H2O (0.7:2.3:1–0.85:2.3:1) to yield compounds 1h before LPS stimulation. Twenty four hours after LPS
11 (78.0mg), 12 (110.0mg), and 13 (25.0mg). Fraction 2C2 stimulation (1µg/mL), TNF-α level in the supernatant were
was separated using a silica gel column (1.5×80cm) with measured by enzyme-linked immunosorbent assay (ELISA)
CHCl3–MeOH–H2O (2.9:1:0.1, 1.5L) to give compound 18 according to the commercial instruction (BD Biosciences, San
(130.0mg). Fraction 2C3 was separated using an YMC column Diego, CA, U.S.A.). For the cytokine assay using the sandwich
(1.5×80cm) with a MeOH–acetone–H2O (1:0.5:1, 1.5:0.5:1, method, a capture antibody (1:250 dilution) solution was in-
each 550mL) elution solvent to give compounds 19 (7.0mg) cubated overnight at 4°C in 96-well plates. The plates were
and 20 (20.0mg). Water extract was chromatographed on a washed three times with washing buffer (0.05% Tween-20 in
column of highly porous polymer (Diaion HP-20) and eluted phosphate buffered saline (PBS)) and blocked with assay dilu-
with H2O and MeOH, successively, to give three fractions ent (10% fetal bovine serum in PBS) for 1h at room tempera-
(Frs. 3A–C). Fraction 3B was subjected to silica gel (8×30cm, ture, and then 100µL of the culture supernatants was added to
70–230µm) column chromatography with a gradient of the wells and was incubated at room temperature. After 2h,
CHCl3–MeOH–H2O (6:1:0.1, 4:1:0.1, 2:1:0.1 and 0:1:0; the plates were washed and incubated for 1h with a detection

476
Vol. 61, No. 4
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Statistical Analysis All data represent the mean S .D .
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one-way ANOVA followed by Dunnett’s multiple comparison
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Supporting Information FT-IR, 1H- and 13C-NMR,
HMQC, HMBC, COSY and HR-ESI-QTOF-MS spectrum of
compound 1 are available as Supporting Information.
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Acknowledgments This study was supported by the Pri-
ority Research Center Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Edu-
cation, Science and Technology (2009-0093815), and by the
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