Chemical constituents of Miliusa balansae leaves and inhibition of nitric oxide production in lipopolysaccharide-induced RAW 264.7 cells

Article abstract of DOI:10.1016/j.bmcl.2015.07.056

Abstract Methanolic extract of Miliusa balansae Finet et Gagnep exerts an anti-inflammatory effect via inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophage cells. Three new megastigmane glycosides, milbasides A-C (1-3), together with fifteen known compounds (4-18), were isolated from the active fraction. Their chemical structures were elucidated using extensive spectroscopic analyses, including 1D and 2D NMR, HR ESI MS, and CD analysis, as well as comparison with previously reported data. Compounds 1-3, 11 and 14 (20.0 μM) showed potent inhibitory activities with inhibition values of 98.5 ± 1.6%, 90.9 ± 7.8%, 84.8 ± 3.5%, 91.5 ± 8.7%, and 91.8 ± 2.7%, respectively. Our results suggest that megastigmane glycosides from M. balansae leaves may be used to treat inflammatory diseases.

Full text of DOI:10.1016/j.bmcl.2015.07.056

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chemical constituents of Miliusa balansae leaves and inhibition of
nitric oxide production in lipopolysaccharide-induced RAW 264.7
cells
Nguyen Phuong Thao a,b, , Bui Thi Thuy Luyen a, , Bui Huu Tai b, Nguyen Manh Cuong c, Youn Chul Kim d,
Chau Van Minh b, Young Ho Kim a,
⇑
a College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
b Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam
c Institute of Natural Products Chemistry (INPC), VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam
d College of Pharmacy, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Methanolic extract of Miliusa balansae Finet et Gagnep exerts an anti-inflammatory effect via inhibition of
nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophage
cells. Three new megastigmane glycosides, milbasides A–C (1–3), together with fifteen known
compounds (4–18), were isolated from the active fraction. Their chemical structures were elucidated
using extensive spectroscopic analyses, including 1D and 2D NMR, HR ESI MS, and CD analysis, as well
Received 7 June 2015
Revised 9 July 2015
Accepted 18 July 2015
Available online xxxx
as comparison with previously reported data. Compounds 1–3, 11 and 14 (20.0 lM) showed potent inhi-
Keywords:
Miliusa balansae
Annonaceae
Milbasides A–C
Anti-inflammatory
bitory activities with inhibition values of 98.5 1.6%, 90.9 7.8%, 84.8 3.5%, 91.5 8.7%, and 91.8 2.7%,
respectively. Our results suggest that megastigmane glycosides from M. balansae leaves may be used to
treat inflammatory diseases.
Ó 2015 Elsevier Ltd. All rights reserved.
Inflammation is a complex process initiated by several factors,
ranging from bacterial infection and chemical agents to environ-
mental pollution resulting in cellular injury or tissue destruction.
This trauma causes the release of inflammatory mediators, includ-
Lipopolysaccharide is the major component which derived from
the outer membrane of the Gram-negative bacteria, such as
Escherichia coli. The exposure of macrophages to bacterial LPS
increase iNOS gene expression and excessive production of NO
over prolonged periods, leading to the initiation of an inflamma-
tory response. This excessive NO production also leads to attack
of normal tissues surrounding the infected area by binding with
other superoxide radicals and acting as a reactive radical that dam-
ages normal cell function.5,6 Therefore, inhibition of NO production
may be beneficial in the therapeutic consideration of several
inflammatory disorders.
Annonaceae, the custard-apple (or Annona), represents the
largest family of the magnolia order (Magnoliales) and vines com-
prising approximately 130 genera and 2300 species present in
Southeast Asia, New Guinea, Pacific Islands, Australia, India,
Africa, Madagascar, Neotropics, and North America. Miliusa
balansae Finet et Gagnep is an evergreen shrub, belonging to the
family Annonaceae.7 In Chinese traditional medicine, this plant is
used to treat various diseases, such as gastropathy and glomeru-
lonephropathy.8 In previous studies, the main constituents of M.
balansae have resulted in the isolation of a variety of secondary
metabolites, including flavonoids,9 dihydrochalcones,10 styryl
derivatives,11 homogentistic acid derivatives,12 norditerpenes,13
ing cytokines such as tumor necrosis factor (TNF)-a, and reactive
nitrogen intermediates, such as NO, which are produced by
immune effector cells (i.e., macrophages).1
Nitric oxide is synthesized by inducible nitric oxide synthase
(NOS) and is considered as a mediator and regulator in the pathol-
ogy of inflammatory diseases including atherosclerosis, rheuma-
toid arthritis, diabetes, septic shock, transplant rejection, and
multiple sclerosis, all of which lead to cell death.2 The constitutive
NOS isoforms produce small quantities of NO that are required for
normal physiological functions, while iNOS is expressed under
pathological conditions, including those in response to bacterial
LPS or pro-inflammatory cytokines, such as TNF-
a
, interleukin
(IL)-1b, and interferon (IFN)-
a, and it could be involved in the
multistage process of carcinogenesis.3,4
⇑
Corresponding author. Tel.: +82 42 821 5933; fax: +82 42 823 6566.
E-mail address: yhk@cnu.ac.kr (Y.H. Kim).
These authors attributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2015.07.056
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Thao, N. P.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.056

2
N. P. Thao et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
and alkaloids.14 Published investigations have previously demon-
strated that this type of compound possesses several interesting
cytotoxic effects.9
As a part of our ongoing investigations on the chemical con-
stituents of Vietnamese medicinal plants with anti-inflammatory
activities, we found that the methanolic extract of M. balansae
showed significant anti-inflammatory effects in vitro with an inhi-
to a megastigmane aglycone moiety and the remaining six to the
one monosaccharide moiety. The 13C NMR spectra indicated the
presence of four methyls [dC 23.9 (C-12), 26.9 (C-13), 25.4 (C-11),
and 27.4 (C-10)], one methylene [dC 43.7 (C-4)], one 3,6-epoxy
functionality [dC 83.9 (CH, C-3) and 93.1 (C, C-6)], one oxygenated
carbon [dC 85.1 (CH, C-2)], and four none-protonated carbons [dC
51.1 (C-1), 88.5 (C-5), 93.1 (C-6), and 201.1 (C-9)], together with
a pair of trans-olefinic methine carbons [dC 144.3 (CH, C-7) and
131.6 (CH, C-8)]. Signals from one sugar moiety were also observed
[dC 100.5 (CH, C-10), 74.9 (CH, C-20), 78.6 (CH, C-30), 71.3 (CH, C-40),
77.7 (CH, C-50), and 62.5 (CH2, C-60)] (see Table 1).
bition value of 93.9% (20 lg/mL). The current Letter addresses the
isolation and structural elucidation of three new megastigmane
glycosides (1–3), along with 15 known compounds, from M. bal-
ansae (1–18, see Fig. 1) and evaluates their inhibitory activity
against NO production in LPS-stimulated murine RAW 264.7
macrophage cells.
NMR data of 1 were similar to those of (3S,5R,6R,7E,9R)-3,
6-epoxy-7-megastigmene-5,9-diol-9-O-b-D
-glucopyranoside,16
M. balansae samples were collected in May 2013 at Hoabinh
province, Vietnam, and were taxonomically identified by Prof.
Ngo Van Trai (National Institute of Medicinal Materials). A voucher
specimen (VHKC-0298) was deposited at the Herbarium of
National Institute of Medicinal Materials and College of
Pharmacy, Chungnam National University, Korea.
Air-dried leaves of M. balansae were extracted using MeOH, and
the concentrated extract was partitioned using solvents of increas-
ing polarity (CH2Cl2, EtOAc, and water). From these fractions, three
new megastigmane glycosides, named milbasides A–C (1–3), along
with 15 known compounds (4–18), were separated and purified
using combined means of various chromatographic procedures.
Their structures were elucidated using physicochemical and spec-
troscopic methods.
except for the replacement of a carbonyl at C-9, hydroxyl group at
C-2, and a glucose moiety attached to C-5. The structure of 1 was
further confirmed by HMQC and HMBC experiments. The methyl
proton H-11 (dH 1.49) observed HMBC correlations with C-1 (dC
51.1)/C-2 (dC 85.1)/C-6 (dC 93.1)/C-12 (dC 23.9) and cross-peaks
with methine H-3 (dH 4.16) with C-1 (dC 51.1)/C-2 (dC 85.1)/C-4
(dC 43.7)/C-6 (dC 93.1), confirming the location of a hydroxy group
at C-2 and authenticating the presence of a 3,6-epoxy bridge.16 The
presence of a carbonyl group at C-9 was confirmed using HMBC
correlations between H-10 (dH 2.33) and C-9 (dC 201.1)/C-8 (dC
131.6); H-7 (dH 7.02) and C-6 (dC 93.1)/C-8 (dC 131.6)/C-9 (dC
201.1). Moreover, a sugar moiety was located at C-5, which was
also supported by HMBC correlations between the anomeric pro-
ton H-10 (dH 4.44) and C-5 (dC 88.5). A detailed comparison of
NMR data for the glycosyl chain of 1 with those of previous
megastigmane glycosides16 and a combination of HMQC, HMBC,
1H–1H COSY, and NOESY data indicated the same kind of sugar
and its attachment position in 1. Configurations of a glucose moi-
ety were further confirmed by acid hydrolysis of 1 followed by
derivatization, and HPLC analysis (see Supporting information)
Milbaside A (1)15 was obtained as a white amorphous powder
with the molecular formula, C19H30O9 as determined by HR ESI
MS at m/z 425.1782 [M+Na]+. The 1H NMR spectrum indicated
the presence of four methyl groups [dH 0.84 (H-12), 1.25 (H-13),
1.49 (H-11), and 2.33 (H-10); each 3H, s] and trans-olefinic protons
[dH 6.28 and 7.02 (each 1H, dd, J = 16.0 Hz)]. The anomeric proton
[dH 4.44 (1H, d, J = 7.5 Hz)] suggested a sugar moiety binding to
aglycone via b-glycosidic linkage. The 13C NMR and DEPT-135
spectra of 1 revealed 19 carbon signals, 13 of which were assigned
confirmed the presence of
D-glucose as the sugar component.
The absolute structure of 1 was determined using 1H NMR,
NOESY and CD experiments. A negative Cotton effect (De240
nm
11
12
OH
O
O
9
OR2
7
H3CO
HO
6
1
OH
10
2
8
13
O
O
R1
5
OH
4
HO
OR
OR
R3
OCH3
1
2
R = Glc
R = Glc(6
4
5
6
R1 = H, R2 = Glc, R3 = αOH
R1 = OH, R2 = Glc, R3 = O, Δ7
R1 = H, R2 = Glc, R3 = O
3
R = Glc
1)Api
OH
7
8
threo-
erythro-
OH
OH
R
H
OH
OH
O
OR
O
OR
HO
HO
OH
OH
OCH3
OCH3
12 R= αOH
13 R= βOH
9
10
threo-
14
R = Glc
OR3
11
R = Glc
erythro-
OH
OH
OH
HO
OH
O
R1O
R2
O
HO
O
OR4
OH
OGlc
OH
R1 = R3 = R4 = CH3, R2 = OH,
18 R1 = R2 = R3 = H, R4 = Glc(6
O
OH
15
16
17
1)Rha
O
OH
HO
O
HO
HO
H3C
HO
O
HO
-glucopyranosyl
OH
OH
Api: -D-apiofuranosyl
β
HO
Rha: α-L-rhamnopyranosyl
OH
Glc
: β-
D
Figure 1. Chemical structures of isolated compounds (1–18) from M. balansae.
Please cite this article in press as: Thao, N. P.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.056

N. P. Thao et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
NMR spectroscopic data (CD3OD) of milbasides A–C (1–3)
Pos.
1
2
3
a
b
a
b
a
b
dC
dH mult. (J in Hz)
dC
dH mult. (J in Hz)
dC
dH mult. (J in Hz)
1
2
51.1, C
85.1, CH
—
51.2, C
85.2, CH
—
45.4, C
49.0, CH2
—
3.80 br s
3.75 br s
1.70 d (9.9)
1.81 dd (4.7, 9.9)
4.38 m
1.90 d (4.7, 12.0)
1.99 d (12.0)
—
3
4
83.9, CH
43.7, CH2
4.16 d (6.6)
1.96 dd (6.6, 12.0)
2.06 d (12.0)
—
84.0. CH
43.7, CH2
4.10 d (6.5)
1.92 dd (6.5, 12.6)
2.01 d (12.6)
—
75.6, CH
47.2, CH2
5
88.5, C
88.6, C
89.1, C
6
93.1, C
—
93.2, C
—
92.9, C
—
7
8
9
10
11
12
13
144.3, CH
131.6, CH
201.1, C
27.4, CH3
25.4, CH3
23.9, CH3
26.9, CH3
7.02 d (16.0)
6.28 d (16.0)
—
2.33 s
1.49 s
144.5, CH
132.0, CH
200.9, C
27.3, CH3
25.4, CH3
23.9, CH3
27.0, CH3
6.98 d (16.2)
6.21 d (16.2)
—
2.27 s
1.46 s
144.9, CH
131.6, CH
200.9, C
27.3, CH3
25.8, CH3
23.7, CH3
27.2, CH3
7.08 d (16.2)
6.28 d (16.2)
—
2.32 s
1.51 s
0.84 s
1.25 s
0.81 s
1.24 s
0.87 s
1.25 s
Glc
10
100.5, CH
74.9, CH
78.6, CH
71.3, CH
77.7, CH
62.5, CH2
4.44 d (7.5)
3.22 dd (7.5, 9.0)
3.37c
100.6, CH
74.9, CH
78.0, CH
71.4, CH
76.6, CH
68.6, CH2
4.42 d (7.5)
3.15 d (7.5, 9.0)
3.35c
100.7, CH
75.1, CH
78.8, CH
71.5, CH
77.8, CH
62.7, CH2
4.45 d (7.5)
3.21 dd (7.5, 9.0)
3.36c
20
30
40
3.32c
3.27c
3.29c
50
3.27 m
3.86 dd (2.0, 12.0)
3.70 dd (5.5, 12.0)
3.33 m
3.95 dd (2.5, 12.0)
3.57 dd (5.0, 12.0)
3.26 m
3.82 dd (2.0, 12.0)
3.62 dd (5.5, 12.0)
60
Api
100
111.0, CH
78.7, CH
80.5, C
4.94 d (2.0)
3.82 d (2.4)
—
3.71 d (9.0)
3.92 d (9.0)
3.54 d (10.8)
3.58 d (10.8)
200
300
400
75.0, CH2
500
65.5, CH2
Assignments were confirmed by DEPT, HMQC, HMBC, and COSY spectra.
a
150 MHz.
600 MHz.
Overlapped signals.
b
c
ꢀ3.27) in its CD spectrum (see Supporting information) indicated
an S configuration of C-6 ( -substituted configuration), and hence,
configuration of C-3 was also in the
-orientation.17,18 In addition,
NOESY correlations including H3-11 (dH 1.49)/H-2 (dH 3.80)/H-4b
(dH 2.06) suggested spatial proximities of those protons and
ESI MS spectrum with a quasi-molecular ion at m/z 557.2205
[M+Na]+. The 1H and 13C NMR data of 2 were similar to those of
1, except for the presence of an additional apiofuranose moiety
(see Table 1). The downfield shift of oxygenated methylene carbon
C-60 (dC 68.6) suggested glycosylation at this carbon, which was
confirmed by an HMBC cross-peak of H-100 (dH 4.94, d, J = 2.0 Hz)
with C-60 (dC 68.6). Attachment of the sugar moiety at C-5 was also
determined by an HMBC correlation of H-10 (dH 4.42, d, J = 7.5 Hz)
with C-5 (dC 88.6) (see Fig. 2). Furthermore, acid hydrolysis of 2
a
a
confirmed
a-configuration at C-2. The a-configurations of C-2
and C-3 were in agreement with a broad singlet signal of H-2 in
the 1H NMR spectrum. On the other hand, a NOESY correlation
between H-3 (dH 4.16)/H-4a (dH 1.96) and H-4a/H3-13 (dH 1.25)
indicated a b-configuration of C-5 (see Fig. 2). Consequently, the
revealed D-glucose and D-apiose as sugar components by derivati-
structure of 1 was elucidated to be that of (2R,3S,5S,6S,7E)-3,
zation and HPLC analysis (see Supporting information).
6-epoxy-7-megastigmen-9-one-2,5-diol 5-O-b-
D
-glucopyranoside.
The similarity between CD spectra [MeOH, nm (De)]: 239
Milbaside B (2)15 was obtained as a white amorphous powder
(ꢀ1.73), see Supporting information], NOESY correlations (see
and its molecular formula was determined to be C24H38O13 by HR
Fig. 2) and agreement with the 13C NMR chemical shifts at C-1
O
HO
12
11
O
2
O
3
HO
O
O
O
4
OH
OH
13
OH
NOESY
OH
Sugar moiety
OH
COSY
HMBC
2
1 2
−
Figure 2. Key HMBC, 1H–1H COSY, and NOESY correlations of 1 and 2.
Please cite this article in press as: Thao, N. P.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.056

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N. P. Thao et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
(dC 51.2), C-2 (dC 85.2), C-3 (dC 84.0), C-5 (dC 88.6), and C-6 (dC 93.2)
of 2 with those of milbaside A (1) at dC 51.1 (C-1), 85.1 (C-2), 83.9
(C-3), 88.5 (C-5), and 93.1 (C-6), respectively, suggested that
absolute configurations of 2 were the same as those in 1. Thus,
compound 2 was determined to be (2R,3S,5S,6S,7E)-3,6-epoxy-7-
The stereochemistry of 3 was also determined on the basis of
NOESY and CD experiments. The positive Cotton effects of 3
(De245 nm +1.52) in its CD spectrum (see Supporting information),
which indicated R and S configurations of C-6 and C-3, respec-
tively.16–18 Moreover, NOESY correlations at H-3 (dH 4.38)/H-4b
(dH 1.90), H-4b/H3-13 (dH 1.25) suggested a b-orientation of C-13,
confirming the R configuration of C-5 (see Fig. 3). Therefore, the
structure of 3 was elucidated to be (3S,5R,6R,7E)-3,6-epoxy-7-
megastigmen-9-one-2,5-diol
glucopyranoside.
5-O-b-D D-apiofuranosyl)
-(60-O-b-
Milbaside C (3)15 was isolated as a white amorphous powder.
The molecular formula was determined to be C19H30O8, based on
a pseudo-molecular ion peak at m/z 409.1833 [M+Na]+ in HR ESI
MS. The 13C NMR and DEPT-135 spectra of 3 revealed 19 carbons,
including four methyls, three methylenes, eight methines, and four
non-protonated carbons. The NMR spectroscopic data for 3 was the
same as that of milbaside A (1), except for the presence of one
additional hydrogen atom in 3 instead of one hydroxy group in 1.
The 1H NMR spectra of 3 revealed a strong upfield shift of H2-2
[dH 1.70 (1H, d, J = 9.9 Hz) and 1.81 (1H, dd, J = 4.7, 9.9 Hz)], sug-
gested a methylene at C-2, which was confirmed by HMBC correla-
tions between the proton signal of the methyl group at dH 1.51 (3H,
s, H-11) and C-1 (dC 45.4)/C-2 (dC 49.0); H-3 (dH 4.38) and C-2 (dC
49.0)/C-4 (dC 47.2)/C-6 (dC 92.9) (see Fig. 3). The latter anomeric
proton was assigned to a glucose moiety (dH 4.45, d, J = 7.5 Hz),
and acid hydrolysis of 3 (see Supporting information) confirmed
megastigmen-9-one-5-ol 5-O-b-D-glucopyranoside.
On the basis of spectral data and chemical evidence in agree-
ment with those reported in the literature, the other compounds
were identified as myrsinionoside D (4),19 ampelopsisionoside
(5),20 myrsinionoside A (6),19 threo- and erythro-1-C-syringylglyc-
erol (7 and 8),21 threo- and erythro-guaiacylglycerol (9 and 10),22
(
L
)-guaiacyl glycerol 20-O-b-
D
-glucopyranoside (11),23 curcolide
-glucopyranosyl (Z)-3-hexenol (14),25
(12), serralactone (13),24 b-
D
1-(3-methylbutyryl)phloroglucinol-glucopyranoside (15),26 (ꢀ)
epicatechin (16),27 chrysosplenol C (17),12 and rutin (18)28 (see
Fig. 1).
Recent studies on the chemical constituents of M. balansae
demonstrated interesting cytotoxic activity against three human
cell lines (KB, HepG2, and RD), with IC50 values <5.0 mg/mL.9
However, there are no reports concerning the anti-inflammatory
effects of the extracts and/or pure compounds from this plant in
the literature. The anti-inflammatory effects of the isolated com-
pounds were analyzed to evaluate their mode of action, such as
the presence of
D-glucose as the sugar component. Detailed
analyses of other HMBC and COSY correlations clearly indicated
the planar structure of 3, as shown in Figure 1.
O
11
O
2
12
O
HO
3
O
4
OH
13
Sugarmoiety
HMBC
OH
OH
NOESY
COSY
Figure 3. Key HMBC, 1H–1H COSY, and NOESY correlations of 3.
10.0 μM
20.0 μM
40.0 μM
12
10
8
6
4
2
0
e
l
n
1*
2
3
4
5
6
7+8 9+10 11
12
13
14
15
16
17
18
S
c
i
ti
e
h
LP
r
Ve
fu
l
u
S
Compounds
Figure 4. Effects of isolated compounds (1–18) on NO production in LPS-stimulated RAW 264.7 cells. Lipopolysaccharide (LPS) stimulation (1.0
mean SD of at least three independent experiments performed in triplicate. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple
comparison test (P <0.05). Sulfuretin (20.0 M.
M) was used as a positive control. ⁄ At the concentrations of 5.0, 10.0, and 20.0
lg/mL). Data represent the
l
l
Please cite this article in press as: Thao, N. P.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.056

N. P. Thao et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
NO. NO plays an important role in the regulation of various patho-
Supplementary data
physiological processes such as neuronal communication, vasodi-
latation, and neurotoxicity. However, overproduction of NO
induces tissue damage associated with acute and chronic inflam-
mations.3–5
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.07.
056.
The effects of all isolated compounds (1–18) on LPS-induced
production of the inflammatory mediator NO by RAW 264.7
macrophage cells were evaluated (see Supporting information).
Cell viability was assessed by determining mitochondrial reductase
function using an assay based on the reduction of the tetrazolium
salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide (MTT) into formazan crystals. The formation of formazan
from MTT is proportional to the number of functional mitochon-
dria in living cells. The results showed that compounds 1–18 did
not exhibit significant cytotoxicity in RAW 264.7 cells at the
concentrations evaluated (data not shown). Thus, the effects of
compounds 1–18 on LPS-induced production of the inflammatory
mediator NO in RAW 264.7 cells were evaluated at concentrations
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less than 50.0 lM. Bacterial LPS, either alone or in combination
with interferon, is the best-characterized stimulus for the
induction of inflammatory mediators, cytokines, and oxygen and
nitrogen species.29 NO production was measured using Griess reac-
tion assays.30
Each compound was screened at concentrations of 10.0, 20.0
and 40.0 lM. At the concentration of 20.0 lM, the results showed
that compounds 1–3, 11 and 14 showed potent inhibitory activi-
ties, with inhibition values of 98.5 1.6%, 90.9 7.8%, 84.8 3.5%,
91.5 8.7%, and 91.8 2.7%, respectively (see Fig. 4), relative com-
15. Physical and spectroscopic data of new compounds: Milbaside A (1): Amorphous
powder; ½a 2D4
ꢁ
: ꢀ52.8 (c 0.15, MeOH); IR mmax (KBr): 3237, 2926, 2911, 2809,
1632, 1427, 1347, and 1050 cmꢀ1
;
1H NMR (CD3OD, 600 MHz) and 13C NMR
(CD3OD, 150 MHz) spectroscopic data, see Table 1; HR ESI MS (positive-
ionmode) m/z: 425.1782 [M+Na]+ (Calcd for C19H30NaO9, 425.1788); CD
[MeOH, nm (
D
e)]: 240 (ꢀ2.27). Milbaside B (2): ½a D24
ꢁ : ꢀ23.0 (c 0.15, MeOH);
IR mmax (KBr): 3357, 2955, 2920, 2870, 1618, 1436, 1358, and 1150 cmꢀ1 1H
;
NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) spectroscopic data,
pared to the positive control, sulfuretin (81.3 4.9% at 20.0
Interestingly, myrsinionoside D (4) and (ꢀ) epicatechin (16) which
showed moderate or weak activity at 10.0 and 20.0 M, but strong
inhibitory effects at 40.0 M (with inhibition values of 82.0 5.9%
lM).
see Table 1; HR ESI MS (positive-ionmode) m/z: 557.2205 [M+Na]+ (Calcd for
C
24H38NaO13, 557.2210); CD [MeOH, nm (
D
e)]: 239 (ꢀ1.73). Milbaside C (3):
Amorphous powder; ½a D24
ꢁ : ꢀ68.3 (c 1.0, MeOH); IR mmax (KBr): 3351, 2940,
l
2907, 2868, 1621, 1467, 1360, and 1055 cmꢀ1 1H NMR (CD3OD, 600 MHz) and
;
l
13C NMR (CD3OD, 150 MHz) spectroscopic data, see Table 1; HR ESI MS
(positive-ionmode) m/z: 409.1833 [M+Na]+ (Calcd for C19H30NaO8, 425.1838);
and 91.8 5.6%, respectively). The remaining compounds were
CD [MeOH, nm (De)]: 245 (+1.52).
16. Gao, P. Y.; Li, L. Z.; Peng, Y.; Li, F. F.; Niu, C.; Huang, X. X.; Ming, M.; Song, S. J.
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2001, 49, 1093.
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1989, 28, 883.
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weak or inactive at the concentrations tested.
Structure–activity relationship (SAR) assays of compounds 1–18
revealed three new megastigmane glycoside milbasides A–C (1–3)
exerting strong inhibitory activities against NO production with
similar structures. When an epoxy group was linked from C-3 to
C-6 of the aglycone, its inhibitory activity increased significantly
and was comparable to those of other megastigmane glycosides
(4–6). Therefore, the epoxy group at C-3/C-6 appears to be a key
functional element. In addition, compound 11 (inhibition value
98.0 5.9%), which contains a sugar unit at C-8 of the aglycone,
showed stronger inhibitory activity against NO production than
its aglycones 9 and 10 (inhibition value 48.4 3.9%). These results
indicate that sugar unit play an important role in the inhibition of
NO production.
24. Lou, Y.; Zhao, F.; Hea, H.; Penga, K. F.; Chen, L. X.; Qiu, F. Chem. Biodivers. 2010,
7, 1245.
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Acknowledgments
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This study was supported by the Priority Research Center
Program (2009-0093815) through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education,
Science and Technology, Republic of Korea.
Please cite this article in press as: Thao, N. P.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.056