Anti-inflammatory tirucallane saponins from Paramignya scandens

Article abstract of DOI:10.1248/cpb.c15-00180

Five new tirucallane saponins, paramignyosides A-E (1-5), were isolated from the water fraction of the Paramignya scandens stem and leaves. Their structures were elucidated on the basis of spectroscopic evidence including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and one dimensional (1D)- and 2D-NMR. The effects of isolated compounds on pro-inflammatory cytokines were evaluated by measuring the production of interleukin (IL)-12 p40, IL-6, and tumor necrosis factor-α (TNF-α) in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells (BMDCs). Paramignyoside C (3) exhibited selective and potent inhibitory effect (IC₅₀=5.03±0.19 μM) on the production of IL-12 p40 comparable to that of the positive control, SB203580 (IC₅₀=5.00×0.16 μM). Further studies are required to confirm efficacy in vivo and the mechanism of anti-inflammatory effects.

Full text of DOI:10.1248/cpb.c15-00180

5
58
Chem. Pharm. Bull. 63, 558–564 (2015)
Vol. 63, No. 7
Note
Anti-inflammatory Tirucallane Saponins from Paramignya scandens
a
a
b
b,c
Nguyen Huu Toan Phan, Nguyen Thi Dieu Thuan, Ninh Thi Ngoc, Nguyen Phuong Thao,
d
d
b
b
Sohyun Kim, Young Sang Koh, Nguyen Van Thanh, Nguyen Xuan Cuong,
Nguyen Hoai Nam, Phan Van Kiem, Young Ho Kim,* and Chau Van Minh*
b
b
,c
,b
a
ꢀ
TayꢀNguyenꢀInstituteꢀofꢀScientificꢀResearch,ꢀVietnamꢀAcademyꢀofꢀScienceꢀandꢀTechnologyꢀ(VAST);ꢀ116ꢀXoꢀVietꢀNgheꢀ
b
TinhꢀStreet,ꢀ7thꢀWard,ꢀDalatꢀ670000,ꢀVietnam:ꢀ ꢀInstituteꢀofꢀMarineꢀBiochemistryꢀ(IMBC),ꢀVAST;ꢀ18ꢀHoangꢀQuocꢀ
Viet,ꢀCaugiay,ꢀHanoiꢀ100000,ꢀVietnam:ꢀ ꢀCollegeꢀofꢀPharmacy,ꢀChungnamꢀNationalꢀUniversity;ꢀDaejeonꢀ305–764,ꢀ
RepublicꢀofꢀKorea:ꢀandꢀ ꢀSchoolꢀofꢀMedicine,ꢀBrainꢀKoreaꢀ21ꢀPLUSꢀProgram,ꢀandꢀInstituteꢀofꢀMedicalꢀScience,ꢀJejuꢀ
c
d
NationalꢀUniversity;ꢀJejuꢀ690–756,ꢀRepublicꢀofꢀKorea.
Received February 26, 2015; accepted March 30, 2015
Five new tirucallane saponins, paramignyosides A–E (1–5), were isolated from the water fraction of the
Paramignya scandens stem and leaves. Their structures were elucidated on the basis of spectroscopic evi-
dence including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and one dimension-
al (1D)- and 2D-NMR. The effects of isolated compounds on pro-inflammatory cytokines were evaluated by
measuring the production of interleukin (IL)-12 p40, IL-6, and tumor necrosis factor-α (TNF-α) in lipopoly-
saccharide (LPS)-stimulated bone marrow-derived dendritic cells (BMDCs). Paramignyoside C (3) exhibited
selective and potent inhibitory effect (IC ꢀ5.03ꢁ0.19µM) on the production of IL-12 p40 comparable to that
5
0
of the positive control, SB203580 (IC ꢀ5.00ꢁ0.16 µM). Further studies are required to confirm efficacy in
5
0
vivo and the mechanism of anti-inflammatory effects.
Key words Paramignya scandens; Rutaceae; tirucallane saponin; cytokine; anti-inflammatory activity
Tirucallane triterpenoids are metabolically altered triter- the molecular formula, C H O , determined by a quasi-mo-
42
66 17
+
penes and have a structure either containing or derived from a lecular ion peak at m/z 865.4233 [M+Na] in high-resolution
precursor with a (13S,14R,17S,20S)-lanostane skeleton. Recent- electrospray ionization mass spectrometry (HR-ESI-MS). The
1
ly, tirucallane-type triterpenoids have received much attention
H-NMR spectrum exhibited typical signals of six tertiary
from scientists because of their unique structures and various methyl groups [δH 0.91 (H-18), 0.74 (H-19), 1.26 (H-26), 1.24
biological activities. Published investigations demonstrated (H-27), 1.31 (H-28), and 1.07 (H-30), each 3H, s] and one ole-
that this type of compound possessed numerous interesting finic proton [δ 5.31 (1H, d, J=2.5Hz, H-7)]. In addition, two
H
1–6)
biological effects, such as cytotoxic,
anti-platelet aggrega- anomeric protons at δH 5.88 (1H, d, J=8.0Hz, H-1′) and 4.59
7,8)
9)
10) 11)
tion, anti-inflammatory, antitubercular, vasodilative ac- (1H, d, J=8.0Hz, H-1″), which correlated with the relevant
tivities, and inhibition of mouse 11β-hydroxysteroid dehydro- anomeric carbons at δC 95.0 (C-1′) and 105.1 (C-1″) on het-
genase type 1 and human immunodeficiency virus (HIV-1) eronuclear single quantum coherence (HSQC) spectrum, indi-
12)
13)
13
protease. Previously, we reported two new tirucallane triter- cated the presence of two sugar moieties. The C-NMR spec-
penes from Paramignya (P.) scandens and evaluation of their trum of 1 revealed 42 signals of a triterpene aglycon and a
14)
13
cytotoxic effects.
disaccharide moiety. Comparison of the C-NMR data of the
As a part of our ongoing investigations on chemical constit- disaccharide chain (see Table 1) with the corresponding values
uents of Vietnamese marine organisms and medicinal plants of (−)-4-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyloxy]-
2
0)
possessing inhibitory effects on production of pro-inflamma- benzyl alcohol and combination with acid hydrolysis fol-
1
5–19)
tory cytokines,
the current paper addresses the isolation, lowed by derivatization and GC analysis (see Experimental)
1
1
structural elucidation, and evaluation of the inhibitory effects as well as analysis of H– H correlation spectroscopy (COSY)
on the production of interleukin (IL)-12 p40, IL-6, and tumor and heteronuclear multiple-bond correlation (HMBC) cross-
necrosis factor (TNF)-α in lipopolysaccharide (LPS)-stimulat- peaks (see Fig. 2) indicated a β-D-glucopyranosyl-(1→3)-β-D-
13
ed bone marrow-derived dendritic cells (BMDCs) of five rare glucopyranoside structure. The C-NMR data for the aglycon
tirucallane saponins, paramignyosides A–E (1–5, see Fig. 1) showed typical signals of six methyl groups [δC 23.8 (C-18),
from the P. scandens stem and leaves.
12.8 (C-19), 26.7 (C-26), 26.4 (C-27), 24.3 (C-28), and 27.9
(
C-30)], one trisubstituted double bond [δ 119.5 (d, C-7) and
C
Results and Discussion
145.6 (s, C-8)], three oxymethine groups [δC 71.4 (C-3), 80.2
The dried stem and leaves of P. scandens were extracted (C-23), and 78.1 (C-24)], one oxygenated quaternary carbon
with methanol. The organic extract was concentrated to dry- [δC 72.8 (C-25)], and two carbonyl carbons [δC 181.3 (C-21)
ness, suspended in water, and partitioned in turn with n-hex- and 176.6 (C-29)]. The NMR data for the aglycon of 1 were
21)
ane and CH Cl . The water layer was passed through a Diaion similar to those of mesendanin M, except for the presence of
2
2
HP-20 column chromatography (CC) and further purified by a carbonyl group in 1 instead of an oxymethylene in mesend-
repeated silica gel, YMC RP-18, and Sephadex LH-20 CC to anin M. The position of the additional carbonyl group at C-29
yield metabolites 1–5.
we determined by the HMBC correlations of H-5 (δH 2.06)
Paramignyoside A (1) was obtained as a white powder with and H-28 (δ 1.31) with C-29 (δ 176.6). The anomeric proton
H
C
*
To whom correspondence should be addressed. e-mail: yhk@cnu.ac.kr; cvminh@vast.vn
©
2015 The Pharmaceutical Society of Japan

Vol. 63, No. 7 (2015)
Chem. Pharm. Bull.
559
Fig. 1. Structures of 1–5
H-1′ (δH 5.88) had a strong HMBC correlation with C-29 (δC correlation of H-2′ (δH 5.10) with the carbonyl carbon at δC
76.6) confirming the attachment of the disaccharide moiety at 171.7. Thus, the structure of paramignyoside B (2) was clearly
this carbon. Detailed analysis of other COSY and HMBC cor- elucidated.
1
relations clearly indicated the planar structure of 1 as shown
in Fig. 2.
Paramignyoside C (3) was also obtained as a white powder.
Its molecular formula was determined to be the same as that
+
1
The stereochemistry of 1 was established by comparison of 1 by HR-ESI-MS at m/z 865.4213 [M+Na] . The H- and
1
13
13
of its H- and C-NMR chemical shifts and the coupling
C-NMR data of 3 were similar to those of 1 (see Table 1) ex-
21–23)
constants with those of similar compounds,
and further cept for difference in the data of the side chain and sugar moi-
confirmed by a rotating frame Overhauser enhancement spec- eties. The oxygenated quaternary carbon C-25 was strongly
troscopy (ROESY) experiment. The configurations in the side shifted downfield at δ 80.2 suggested for the glycosylation at
C
chain of 1 were determined to be the same as those of mesen- this carbon, which was confirmed by an HMBC cross-peak of
2
1)
1
13
danin M by the essential agreement of the H- and C-NMR H-1″ (δ 4.52) with C-25 (δ 80.2). The attachment of the other
H
C
data between these two compounds. The resonance of proton glucose moiety at C-29 was determined by an HMBC correla-
H-3 at δ 4.10 (1H, d, J=2.5Hz) and carbon C-3 at δ 71.4 is tion of H-1′ (δ 5.50) with C-29 (δ 176.9).
H
C
H
C
2
1,22)
typical for a β-orientation of H-3.
methyl group C-28 was suggested by the C-NMR of C-28 fied as C H O by HR-ESI-MS at m/z 1027.4787 [M+Na] .
at δC 24.3, which was quite different from that of reference Its H- and C-NMR data were also similar to those of 1 (see
compound with β-orientation of the methyl group C-28 at δ Table 2) except for an additional presence of a glucose moi-
7.5, and further confirmed by the spatial proximities ob- ety. Similar as in case of compound 3, the downfield shifted
served of H-28 (δ 1.31) with H-5 (δ 2.06)/H -6 (δ 2.20) and signal of C-25 at δ 81.1 indicating the attachment of the ad-
The α-orientation of the
The molecular formula of paramignyoside D (4) was identi-
13
+
4
8
76 22
13
1
C
2
3)
1
H
H
α
H
C
that of H -6 (δ 2.84) with H-19 (δH 0.74) from the ROESY ditional glucose at this carbon, which was further confirmed
β
H
(
see Fig. 2).
by an HMBC correlation of H-1‴ (δ 4.55) with C-25 (δ 81.1).
H C
1
13
The H- and C-NMR data of 2 were similar to those of Detailed analysis of HSQC, HMBC, COSY, and ROESY data
(see Table 1) except for an additional presence of an acetyl clearly elucidated the structure of 4.
1
group at δ 171.7 and δ 21.2 (q)/δ 2.10 (3H, s) as also con-
Similar as in case of compounds 1 and 2, paramignyoside
C
C
H
+
firmed by HR-ESI-MS at m/z 907.4315 [M+Na] . Proton H-2′ E (5) is different from 4 only for an additional presence of
was strongly shifted downfield at δ 5.10 suggested for the an acetyl group [δ 171.6 and δ 21.2 (q)/δ 2.09 (3H, s)] as
H
C
C
H
2
4)
+
esterification at C-2′, which was confirmed by an HMBC also confirmed by HR-ESI-MS at m/z 1069.4901 [M+Na] .

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60
Chem. Pharm. Bull.
Vol. 63, No. 7 (2015)
1
13
Table 1. The H- (CD OD, 500MHz) and C-NMR (CD OD, 125MHz) Spectroscopic Data of 1–3
3
3
1
2
3
Position
δC
δH mult. (J in Hz)
δC
δH mult. (J in Hz)
δC
δH mult. (J in Hz)
1
2
3
4
5
6
7
8
9
33.0
27. 8
71.4
49.5
46.5
25.4
119.5
145.6
49.1
36.3
18.9
32.4
44.8
51.7
34.9
24.8
48.7
23.8
12.8
42.0
181.3
29.4
80.2
78.1
72.8
26.7
26.4
24.3
176.6
27.9
Glc I
95.0
73.4
88.2
69.5
78.5
62.3
1.42m/1.54m
1.67m/2.28m
4.10 d (2.5)
—
32.9
27.9
71.1
49.4
46.3
25.3
119.5
145.6
49.1
36.3
18.9
32.4
44.8
51.7
34.9
24.7
48.6
23.8
12.9
42.0
181.4
29.4
80.2
78.1
72.8
26.7
26.4
24.0
176.5
27.9
GlcOAc
93.1
72.4
85.4
69.6
78.7
62.1
1.41m/1.52m
1.62m/2.00m
4.02 brs
—
33.0
28.0
71.5
49.6
46.5
25.5
119.6
145.7
49.6
36.4
18.9
32.5
44.8
51.7
34.9
25.0
49.4
23.9
12.9
41.9
181.4
29.6
80.1
77.7
80.2
24.5
23.4
24.2
176.9
27.9
Glc I
95.5
74.1
78.5
71.2
78.0
62.8
1.42m/1.55m
1.66m/2.29m
4.10 brs
—
2.06m
2.03m
2.05m
2.20m/2.84m
5.31 d (2.5)
—
2.26m/2.78m
5.30 d (2.5)
—
2.10m/2.85m
5.30 brs
—
2.41m
2.41m
2.41m
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
—
—
—
1.56m/1.69m
1.79m
1.57m/1.68m
1.79m
1.58m/1.69m
1.80m
—
—
—
—
—
—
1.57m/1.64m
1.60m/1.82m
2.38m
1.57m/1.63m
1.60m/1.82m
2.38m
1.57m/1.64m
1.60m/1.80m
2.38m
0.91s
0.91s
0.93s
0.74s
0.70s
0.74s
2.83m
2.83m
2.81m
—
—
—
2.29m/2.31m
4.68m
2.29m/2.31m
4.68m
2.32m/2.60m
4.77m
3.63 d (2.5)
—
3.63 d (3.5)
—
3.78 d (3.0)
—
1.26s
1.25s
1.36s
1.24s
1.23s
1.33s
1.31s
1.23s
1.31s
—
—
—
1.07s
1.07s
1.07s
1
2
3
4
5
6
′
′
′
′
′
′
5.88 d (8.0)
3.58 dd (8.0, 9.0)
3.64 t (9.0)
5.69 (8.0)
5.10 dd (8.0, 9.0)
3.83 t (9.0)
3.60 t (9.0)
3.50m
5.50 (d, 8.0)
3.38*
3.44*
3.52 t (9.0)
3.40*
3.44m
3.29*
3.74 dd (4.5, 12.0)
3.77 dd (4.5, 12.0)
3.88 brd (12.0)
3.67/3.89*
3
.86 brd (12.0)
OAc
OAc
171.7
21.2
—
2.10s
Glc II
4.59 d (8.0)
Glc
Glc II
1″
2″
3″
4″
5″
6″
105.1
75.4
77.8
71.5
78.1
62.6
105.4
74.9
77.9
71.4
78.1
62.5
4.40 d (8.0)
3.21 dd (8.0, 9.0)
3.37*
98.4
75.4
78.8
71.7
78.0
62.7
4.52 d (7.5)
3.16*
3.32*
3.41 t (9.0)
3.31*
3.39*
3.31*
3.29*
3.37m
3.37m
3.39*
3.67 dd (6.0, 12.0)
3.67 dd (6.0, 12.0)
3.91 dd (1.5, 12.0)
3.70/3.85*
3
.92 dd (1.5, 12.0)
*
Overlapped signals. Assignments were confirmed by HSQC, HMBC, COSY, and ROESY experiments.
A strong HMBC cross-peak was observed between H-2′ (δH on pro-inflammatory cytokines by measuring the production
.10) and the carbonyl carbon at δC 171.6 confirming the at- of IL-12 p40, IL-6, and TNF-α in LPS-stimulated BMDCs.
tachment of the acetyl group at C-2′. Tirucallane saponins are Interestingly, all the compounds show selective effects on the
5
2
5)
relatively rare from natural sources. Noteworthy, to best of production of IL-12 p40 (see Fig. 3). Of which, paramignyo-
our knowledge, this is the first report of 25-glycosylated and/ side C (3) exhibited most potent inhibitory effect on produc-
or O-acetylglycosylated derivatives of tirucallane triterpenoids tion of IL-12 p40 with an IC50 value of 5.03± 0.19 µM. The
to date.
activity of 3 was comparable to that of the positive control,
2
6)
All the isolated compounds were evaluated for their effects SB203580
(IC =5.00± 0.16 µM). In addition, paramignyo-
5
0

Vol. 63, No. 7 (2015)
Chem. Pharm. Bull.
561
Fig. 2. Key COSY, HMBC, and ROESY Correlations of 1
sides D and E (4 and 5) showed significant inhibitory effects YMC RP-18 resins (30–50µm, Fuji Silysia Chemical, Aichi,
with IC50 values of 19.59± 0.97 and 14.09± 0.65µM, respec- Japan). Thin layer chromatography (TLC) used pre-coated
tively. Moderate inhibitory effect on IL-12 p40 production silica gel 60F254 (1.05554.0001, Merck) and RP-18F254S plates
was observed for paramignyoside B (2) with an IC50 value (1.15685.0001, Merck). Compounds were visualized by spray-
of 29.15± 1.33 µM, while weak activity (IC =48.68± 1.89 µM) ing with aqueous 10% H SO and heating for 3–5min.
5
0
2
4
was observed for paramignyoside A (1). All the isolated com-
Plant Material The stem and leaves of P. scandens
pounds did not show significant inhibition of IL-6 and TNF-α (GRIFF.) CRAIB were collected in Da Lat, Lamdong, Vietnam
in LPS-stimulated BMDCs (IC >100 µM). Among the vari- during May 2013 and identified by Dr. Nong Van Duy (Tay
5
0
ous inflammatory cytokines, IL-12 plays a central role in the Nguyen Institute of Scientific Research, VAST, Vietnam). A
initiation and regulation of cellular immunity. It is involved voucher specimen (TN3/055) was deposited at the Herbarium
in type-1 helper T-cell-mediated inflammation as a part of the of Tay Nguyen Institute of Scientific Research.
normal immune response, as well as in inflammatory diseases,
Extraction and Isolation The dried stem and leaves of
such as rheumatoid arthritis, asthma, psoriasis, and Crohn’s P. scandens (GRIFF.) CRAIB (1.5kg) were ground and extracted
2
7–29)
disease.
Although further studies are required to confirm three times with methanol (MeOH, 3×2.5L) at temperature
efficacy in vivo and mechanism of anti-inflammatory effects, 45°C in an ultrasonic bath. The resulted solution was filtered
it is the contention of the authors that compounds 3–5 could and evaporated at reduced pressure to give 85g residue. This
be used in the development of therapeutic targets for modula- residue was suspended in distilled water and partitioned
tion of IL-12 and related inflammatory diseases.
in turn with n-hexane and CH Cl2 to obtain correspond-
2
ing extracts: n-hexane (PSh, 15.2g), CH Cl2 (PSc, 37.3g),
2
Experimental
and water layer (PSw). The water layer was passed through
General Experimental High resolution mass spec- Diaion HP-20 CC eluting with increasing concentration of
tra were recorded on a MicroQ-TOF III mass spectrom- MeOH in water (0, 25, 50, 75, and 100%) to obtain four
1
eter (Bruker Daltonics, 255748 Germany). The H-NMR fractions, PSw1–PSw4, after removal of the fraction eluted
(500MHz) and 13C-NMR (125MHz) spectra were recorded with water. Fraction PSw2 (22g) was separated into eight
on a Bruker AM500 FT-NMR spectrometer (Bruker, Billerica, subfractions, PSw2A–PSw2H, by silica gel CC using gradi-
MA, U.S.A.) using tetramethylsilane (TMS) as internal stan- ent eluation of CH Cl –MeOH (20:1–1:1, v/v). Subfraction
2
2
dard. Gas chromatography (GC) analysis was carried out on PSw2G (2.5g) was futher separted on YMC RP-18 CC eluting
a Shimadzu-2010 spectrometer (Chiyoda-ku, Tokyo, Japan). with MeOH–H O (1.5:1, v/v) to give five smaller fractions,
2
CC was performed using a silica gel (Kieselgel 60, 70–230 PSw2G1–PSw2G5. Purification of fraction PSw2G3 (280mg)
mesh and 230–400 mesh, Merck, Darmstadt, Germany) or by silica gel CC using CH Cl –MeOH–H O (4:1:0.1, v/v) as
2
2
2

5
62
Chem. Pharm. Bull.
Vol. 63, No. 7 (2015)
1
13
Table 2. The H- (CD OD, 500MHz) and C-NMR (CD OD, 125MHz) Spectroscopic Data of 4 and 5
3
3
4
5
Position
δC
δH mult. (J in Hz)
δC
δH mult. (J in Hz)
1
2
3
4
5
6
7
8
9
33.0
27.8
71.3
49.5
46.6
25.4
119.6
145.6
48.8
36.3
18.9
32.4
44.8
51.7
34.9
24.7
48.5
23.9
12.8
41.6
181.5
31.6
78.5
77.7
81.1
23.6
23.3
24.3
176.6
27.9
Glc I
95.0
73.4
88.1
69.5
78.4
62.3
1.42m/1.55m
1.66m/2.28m
4.11 brs
—
32.9
27.9
71.1
49.7
46.3
25.4
119.6
145.6
49.4
36.3
18.9
32.4
44.8
51.7
34.9
24.7
49.3
23.9
12.9
41.6
181.5
31.7
78.4
77.7
81.1
23.7
23.3
24.0
176.4
27.9
GlcOAc
93.1
72.4
85.4
69.6
78.7
62.1
171.6
21.2
1.41m/1.53m
1.62m/2.00m
4.02 brs
—
2.06m
2.03m
2.20m/2.85m
5.30 d (3.0)
—
2.15m/2.80m
5.31 d (3.0)
—
2.41m
2.41m
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
—
—
1.57m/1.70m
1.79m
1.58m/1.68m
1.79m
—
—
—
—
1.58m/1.63m
1.66m/2.28m
2.40m
1.58m/1.63m
1.60m/1.82m
2.40m
0.91s
0.91s
0.75s
0.70s
2.80m
2.80m
—
—
2.27m/2.33m
4.78m
2.27m/2.33m
4.77m
3.50 d (2.0)
—
3.50 d (2.0)
—
1.38s
1.35s
1.34s
1.35s
1.31s
1.23s
—
—
1.07s
1.08s
1
2
3
4
5
6
′
′
′
′
′
′
5.58 d (8.0)
3.58 dd (8.0, 9.0)
3.64 t (9.0)
3.52 t (9.0)
3.43m
5.69 d (8.0)
5.10 dd (8.0, 9.0)
3.83 t (9.0)
3.60 t (9.0)
3.49m
3.75 dd (4.5, 12.0)/3.87 dd (2.5, 12.0)
3.77 dd (4.5, 12.0)/3.88 dd (2.5, 12.0)
OAc
OAc
—
2.09s
Glc II
Glc I
1
2
3
4
5
6
″
″
″
″
″
″
105.0
75.4
78.1
71.5
77.6
62.6
Glc III
98.5
75.1
77.8
71.7
78.1
62.7
4.58 d (8.0)
105.4
74.7
78.0
71.4
77.6
62.7
Glc II
98.5
75.2
78.1
71.7
78.1
62.5
4.39 d (8.0)
3.21 dd (8.0, 9.0)
3.37*
3.32*
3.37*
3.31*
3.31*
3.28m
3.27*
3.67*/3.85 dd (2.5, 12.0)
3.66/3.85*
1‴
2‴
3‴
4‴
5‴
6‴
4.55 d (7.5)
3.18 dd (7.5, 9.0)
3.41 t (9.0)
4.55 d (7.5)
3.17 dd (7.5, 9.0)
3.39*
3.30*
3.34*
3.37*
3.35*
3.67*/3.91 dd (2.5, 12.0)
3.67/3.90*
*
Overlapped signals. Assignments were confirmed by HSQC, HMBC, COSY, and ROESY experiments.
eluent furnished compounds 1 (5mg), 2 (4mg), and 3 (3.5mg). with MeOH–H O (1:1, v/v), followed by silica gel CC eluting
2
Fraction PSw2G4 (450mg) furnished compounds 4 (5mg) and with CH Cl –MeOH–H O (3:1:0.1, v/v).
2
2
2
2
5
5
(4.5mg) after subjecting it to Sephadex LH-20 CC eluting
Paramignyoside A (1): White powder, [α]D −5.0 (c, 0.1,

Vol. 63, No. 7 (2015)
Chem. Pharm. Bull.
563
Fig. 3. Effect of Compounds 1–5 on IL-12 p40 Production in LPS-Stimulated BMDCs
The data were presented as inhibition rate (%) compared to the value of vehicle-treated DCs.
+
MeOH); HR-ESI-MS m/z 865.4233 [M+Na] (Calcd for C57BL/6 mice (Orient Bio Inc.). All animal procedures were
+
1
C H NaO , 865.4192); H-NMR (CD OD 500MHz) and approved and performed according to the guidelines of the
42
66
17
3
13
C-NMR (CD OD, 125MHz) are given in Table 1.
Paramignyoside B (2): White powder, [α]D −12.3 (c, 0.1, University (#2010-0028). Briefly, the mouse tibia and femur
Institutional Animal Care and Use Committee of Jeju National
3
2
5
+
MeOH); HR-ESI-MS m/z 907.4315 [M+Na] (Calcd for were obtained by flushing with Dulbecco’s modified Eagle’s
+
1
C H NaO , 907.4298); H-NMR (CD OD, 500MHz) and medium to yield bone marrow cells. The cells were cultured
4
4
68
18
3
13
C-NMR (CD OD, 125MHz) are given in Table 1.
Paramignyoside C (3): White powder, [α]D −1.9 (c, 0.1, bovine serum (FBS; Gibco), 50.0µM β-ME, 2mM glutamine
in RPMI 1640 medium containing 10% heat-inactivated fetal
3
2
5
+
MeOH); HR-ESI-MS m/z 865.4213 [M+Na] (Calcd for supplemented with 3% J558L hybridoma cell culture super-
+
1
C H NaO , 865.4192); H-NMR (CD OD, 500MHz) and natant containing granulocyte-macrophage colony stimulating
42
66
17
3
13
C-NMR (CD OD, 125MHz) are given in Table 1.
Paramignyoside D (4): White powder, [α]D −6.5 (c, 0.1, every second day. At day 6 of culture, non-adherent cells and
factor. The culture medium was replaced with fresh medium
3
2
5
+
MeOH); HR-ESI-MS m/z 1027.4787 [M+Na] (Calcd for loosely adherent dendritic cell (DC) aggregates were har-
+
1
C H NaO , 1027.4720); H-NMR (CD OD, 500MHz) and vested, washed, and resuspended in RPMI 1640 supplemented
4
8
76
22
3
13
C-NMR (CD OD, 125MHz) are given in Table 2.
Paramignyoside E (5): White powder, [α]D −8.3 (c, 0.1,
with 5% FBS.
Cytokine Production Measurements
3
2
5
+
MeOH); HR-ESI-MS m/z 1069.4901 [M+Na] (Calcd for
BMDCs were incubated in 48-well plates in 0.5mL contain-
+
1
5
C H NaO , 1069.4826); H-NMR (CD OD, 500MHz) and ing 1×10 cells per well, and then treated with the isolated
5
0
78
23
3
13
C-NMR (CD OD, 125MHz) are given in Table 2.
Acid Hydrolysis of Compounds 1–5
compounds 1–5 at the indicated concentration for 1h before
Each compound stimulation with 10.0ng/mL LPS from Salmonella minnesota
3
3
0)
(
2.0mg) was dissolved in 1.0N HCl (dioxane–H O, 1:1, v/v, (ALEXIS). Supernatants were harvested 18h after stimulation.
2
1.0mL) and then heated to 80°C in a water bath for 3h. The Concentrations of murine TNF-α, IL-6, and IL-12 p40 in
acidic solution was neutralized with silver carbonate and the the culture supernatants were determined by enzyme-linked
solvent thoroughly driven out under N gas overnight. After immunosorbent assay (ELISA) (BD PharMingen) according
2
extraction with ethyl acetate, the aqueous layer was concen- to the manufacturer’s instructions. The data are presented as
trated to dryness using N gas. The residue was dissolved means± standard deviation (S.D.) of at least three independent
2
in 0.1mL of dry pyridine, and then L-cysteine methyl ester experiments performed in triplicate.
hydrochloride in pyridine (0.06M, 0.1mL) was added to the
solution. The reaction mixture was heated at 60°C for 2h, and
.1mL of trimethylsilylimidazole solution was added, followed nam national project of the Tay Nguyen 3 Program (code:
Acknowledgments This work was supported by a Viet-
0
by heating at 60°C for 1.5h. The dried product was partitioned TN3/T14) and the Priority Research Center Program through
with n-hexane and H O (0.1mL, each), and the organic layer the National Research Foundation of Korea (NRF) funded
2
was analyzed by gas liquid chromatography (GC): Column by the Ministry of Education, Science, and Technology
SPB-1 (0.25mm×30m); detector hydrogen flame ionization (2009–0093815), Republic of Korea. The authors are grateful
detecter (FID), column temp. 210°C, injector temp. 270°C, to Dr. Nong Van Duy, Tay Nguyen Institute of Scientific Re-
detector temp. 300°C, carrier gas He. The absolute configura- search, VAST for the plant identification; and the Institute of
tion of the monosaccharides in compounds 1–5 was confirmed Chemistry, VAST for measurement of the NMR spectra.
to be all D-glucose by comparison of the retention time of the
monosaccharide derivative (t 14.14min) with that of authentic
Conflict of Interest The authors declare no conflict of
R
sugar derivative samples prepared in the same manner (D-glu- interest.
cose derivative t 14.11min, L-glucose derivative t 14.26 min).
R
R
Cell Culture BMDCs were grown from wild-type
Supplementary Materials The online version of this

5
64
Chem. Pharm. Bull.
Vol. 63, No. 7 (2015)
Kim Y. H., Bioorg.ꢀMed.ꢀChem.ꢀLett., 23, 1823–1827 (2013).
7) Thao N. P., Cuong N. X., Luyen B. T., Thanh N. V., Nhiem N. X.,
article contains supplementary materials, 1D- and 2D-NMR
spectra for the new compounds 1–5.
1
Koh Y. S., Ly B. M., Nam N. H., Kiem P. V., Minh C. V., Kim Y.
H., J.ꢀNat.ꢀProd., 76, 1764–1770 (2013).
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