New ent-kauranes from the fruits of Annona glabra and their inhibitory nitric oxide production in LPS-stimulated RAW264.7 macrophages

Article abstract of DOI:10.1016/j.bmcl.2014.11.059

Three new ent-kaurane diterpenoids, 7β,16α,17-trihydroxy-ent-kauran-19-oic acid (1), 7β,17-dihydroxy-16α-ent-kauran-19-oic acid 19-O-β-d-glucopyranoside ester (2), 7β,17-dihydroxy-ent-kaur-15-en-19-oic acid 19-O-β-d-glucopyranoside ester (3) along with fi

Full text of DOI:10.1016/j.bmcl.2014.11.059

Accepted Manuscript
New ent-kauranes from the fruits of Annona glabra and their inhibitory nitric
oxide production in LPS-stimulated RAW264.7 macrophages
Nguyen Xuan Nhiem, Nguyen Thi Thu Hien, Bui Huu Tai, Hoang Le Tuan Anh,
Dan Thi Thuy Hang, Tran Hong Quang, Phan Van Kiem, Chau Van Minh,
Wonmin Ko, Seungjun Lee, Hyuncheol Oh, Seung Hyun Kim, Young Ho Kim
PII:
S0960-894X(14)01254-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.11.059
BMCL 22214
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
29 August 2014
6 November 2014
20 November 2014
Please cite this article as: Nhiem, N.X., Hien, N.T.T., Tai, B.H., Anh, H.L.T., Hang, D.T.T., Quang, T.H., Van
Kiem, P., Van Minh, C., Ko, W., Lee, S., Oh, H., Kim, S.H., Kim, Y.H., New ent-kauranes from the fruits of Annona
glabra and their inhibitory nitric oxide production in LPS-stimulated RAW264.7 macrophages, Bioorganic &
Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.11.059
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Bioorg. Med. Chem. Lett.
New ent-kauranes from the fruits of Annona glabra and their inhibitory
nitric oxide production in LPS-stimulated RAW264.7 macrophages
a
Nguyen Xuan Nhiem , Nguyen Thi Thu Hien , Bui Huu Tai , Hoang Le Tuan Anh ,
a
a,c
a
a
Dan Thi Thuy Hang , Tran Hong Quang , Phan Van Kiem , Chau Van Minh , Wonmin
a
a
a
d
d
d
b,∗
Ko , Seungjun Lee , Hyuncheol Oh , Seung Hyun Kim , and Young Ho Kim
c,∗
a
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18
Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
b
College of Pharmacy, Yonsei Institute of Pharmaceutical Science, Yonsei University,
Incheon 406-840, Korea
c
College of Pharmacy, Chungnam National University, Daejeon 305–764, Korea
d
College of Pharmacy, Wonkwang University, Iksan 570-749, Korea
∗
Corresponding author:
Tel.: +82-32-749-4514; fax: +82-32-749-4105; e-mail address: kimsh11@yonsei.ac.kr
Kim, S. H.).
(
Tel.: 82-42-821-5933; fax: 82-42-823-6566; e-mail address: yhk@cnu.ac.kr (Kim,
Y.H)
1

Abstract
Three new ent-kaurane diterpenoids, 7ꢀ,16ꢁ,17-trihydroxy-ent-kauran-19-oic acid
1), 7ꢀ,17-dihydroxy-16ꢁ-ent-kauran-19-oic acid 19-O-ꢀ-D-glucopyranoside ester (2),
(
7
ꢀ,17-dihydroxy-ent-kaur-15-en-19-oic acid 19-O-ꢀ-D-glucopyranoside ester (3) along
with five known compounds, paniculoside IV (4), 16ꢁ,17-dihydroxy-ent-kaurane (5),
1
1
6β,17-dihydroxy-ent-kaurane (6), 16β,17-dihydroxy-ent-kauran-19-al (7), and
6β,17-dihydroxy-ent-kauran-19-oic acid (8) were isolated from the fruits of Annona
glabra. Their chemical structures were elucidated by physical and chemical methods.
All compounds were evaluated for inhibitory activity against nitric oxide (NO)
production in LPS-stimulated RAW 264.7 macrophages. As the results, compound 3
showed potent inhibitory LPS-stimulated NO production in RAW 264.7 macrophages
with the IC50 value of 0.01 µM; compounds 1 and 7 showed significant inhibitory NO
production with the IC50 values of 0.39 µM and 0.32 µM, respectively.
Keywords: Annona glabra, Annonaceae, ent-kaurane diterpene, anti-inflammatory
2

Nitric oxide (NO) produced in large amounts by inducible nitric oxide synthase
(
iNOS) is known to be responsible for the vasodilation and hypotension observed
1
during septic shock and inflammation. The production of NO confers cytostatic or
2
cytotoxic activity on macrophages against microbes and tumor cells. NO mediates a
variety of biological actions ranging from vasodilatation, neurotransmission, inhibition
of platelet adherence and aggregation, as well as the macrophage and neutrophil-
3
mediated killing of pathogens. Although previous studies have demonstrated that
cytokine-induced NO production can lead to eliminate parasites or tumor cells, NO has
also been associated to damage of normal cells. Overproduction of NO causes
4
-6
excessive inflammatory response harmful to body in the inflammatory diseases.
They are secreted during the early phase of acute and chronic inflammatory diseases
such as asthma, rheumatoid arthritis, septic shock, and other allergic diseases, and
7
during the activation of T cells.
Annona glabra L., belongs to Annonaceae family is a tropical tree growing wild
in the America and Asia. It is used in traditional medicine to treat several diseases such
as inflammation, and cancer. Phytochemical investigation led to the isolation of
8
numerous acetogenins, ent-kauranes,
9-12
13
peptides, and alkaloids.
14, 15
In addition,
1
2, 16
11
anti HIV-reserve transcriptase, and anti-
compounds exhibited anticancer,
1
4
malarial activities. In the present study, three new along with five known ent-kaurane
diterpenoids were isolated from the fruits of A. glabra. All compounds were evaluated
for inhibitory against NO production in lipopolysaccharide (LPS)-stimulated RAW
2
64.7 macrophages.
The fruits of A. glabra were collected in Ho Chi Minh city, Vietnam, in May
013, and authenticated by Dr. Bui Van Thanh, Institute of Ecology and Biological
2
Resources, VAST, Vietnam. A voucher specimen (AG1605) was deposited at the
Herbarium of Institute of Marine Biochemistry, VAST, Vietnam. The dried fruits of A.
3

glabra (4.0 kg) were extracted with MeOH (3 × 5 L, 50℃ ) under sonication for 1 h to
yield 300.0 g extract after evaporation of the solvent. This extract was suspended in
H
2
O (2.0 L) and successively partitioned with n-hexane, CHCl
EtOAc) to obtain the n-hexane (AG1, 51.0 g), CHCl (AG2, 190.5 g), EtOAc (AG3,
.5 g), and H O (AG4, 54.0 g) extracts after removal of the solvents in vacuo. Using
various chromatographic resin and isolation techniques, eight ent-kaurane-type
3
, and ethyl acetate
(
3
3
2
1
7
diterpenoids were isolated.
18
Compound 1 was obtained as a white amorphous powder. The HR-ESI-MS
analysis revealed the molecular formula to be C20
H
32
O
5
, with a pseudo ion peak at m/z
+
75.2159 [M+Na] (Calcd. for C20
1
Na: 375.2142). The H-NMR spectrum of 1
3
H
32
O
5
showed the signals for protons of two tertiary methyl groups at ꢂ
.18 (3H, s), one oxymethine at ꢂ 3.63 (br s), and one oxymethylene group at ꢂ
d, J = 11.5 Hz) and 3.72 (d, J = 11.5 Hz). The C-NMR and DEPT spectra of 1
revealed signals for 20 carbons including one carboxylic, four quaternary (including an
oxygenated carbon at ꢂ 82.9), four methine, and nine methylene, and two methyl
carbons (Table 1). A comparison of the C-NMR data of 1 to those of related kaurane,
H
1.00 (3H, s) and
1
H
H
3.62
1
3
(
C
1
3
1
9
1
6ꢁ,17-dihydroxy-ent-kauran-19-oic acid suggested that compound 1 possessed the
same ent-kaurane skeleton. The HMBC correlations between H-18 (ꢂ 1.18) and C-3
39.2)/C-4 (ꢂ 44.2)/C-5 (ꢂ 48.1)/C-19 (ꢂ 182.0) confirmed the positions of both
H
(
ꢂ
C
C
C
C
methyl and carboxylic groups at C-4 (Figure 2). The configuration of the methyl group
at C-4 was determined to be ꢀ by the observation of NOESY correlation between H-18
(
ꢂ
H
1.18) and H-5 (ꢂ
.56 and 1.74) to C-16 (ꢂ
6 and C-17. In addition, the ꢁ configuration of hydroxyl group at C-16 was confirmed
H
1.77). The HMBC correlations from H-13 (ꢂ
H
2.08)/H-15 (ꢂ
H
1
1
C
82.9)/C-17 (ꢂ 66.7) confirmed two hydroxyl groups at C-
C
by comparing the chemical shifts of C-13 (ꢂ
C
46.1), C-16 (ꢂ
C
C
82.9), and C-17 (ꢂ 66.7)
4

in 1 with those of the corresponding data of the 16ꢀ,17-dihydroxy-ent-kauran-19-oic
2
0
acid [ꢂ
kauran-19-oic acid [ꢂ
hydroxyl group at C-7 was determined by the HMBC correlations from H-5 (ꢂ
C
41.7 (C-13), 79.8 (C-16), and 70.5 (C-17)] and 16ꢁ,17-dihydroxy-ent-
2
0
C
45.9 (C-13), 81.7 (C-16), and 66.5 (C-17)]. The position of the
H
1
.77)/H-6 (ꢂ
H
1.98 and 2.11)/H-15 (ꢂ
H
1.56 and 1.74) to C-7 (ꢂ
C
78.1). Moreover, the
NOESY correlations of H-7 (ꢂ
H
3.63)/H-14 (ꢂ
H
1.70) and broad singlet signal of H-7,
proved the configuration of H-7 to be ꢁ (equatorial orientation). Consequently,
compound 1 was elucidated to be 7ꢀ,16ꢁ,17-trihydroxy-ent-kauran-19-oic acid. The
structure of 1 was previously assumed in the barley grain extract by Paul Gaskin el
2
al. using GC-MS analysis. However, the isolation of pure compound and clear
1
elucidation its chemical structure have not been reported to date.
2
2
1
Compound 2 was also obtained as a white amorphous powder. The H-NMR
spectrum of 2 showed the following signals: two methyl groups at ꢂ 0.99 (3H, s) and
.22 (3H, s), assigned to the ent-kaurane aglycone; one anomeric proton at ꢂ 5.42 (1H,
H
1
H
1
3
d, J = 8.0 Hz), suggested the presence of a sugar moiety. The C-NMR and DEPT
spectra revealed the presence of 26 carbons: one carboxyl, three quaternary, ten
methine (including six oxymethine), ten methylene (including two oxymethylene), and
1 13
two methyl carbons. The H- and C-NMR data of 2 were similar to those of
23
helikauranoside A, except for the additional hydroxyl group at C-7. The position of
hydroxyl group at C-7 was confirmed by HMBC cross peaks from H-5 (ꢂ 1.78)/H-9
78.7) as well as COSY correlations of H-5
3.50). Similarly to 1, the configuration of H-7
H
(
(
ꢂ
H
H
1.43)/H-15 (ꢂ
H
1.12 and 1.71) to C-7 (ꢂ
C
ꢂ
1.78)/H-6 (ꢂ
H
1.97 and 2.18)/H-7 (ꢂ
H
in 2 was determined to be ꢁ-configuration by a broad singlet of H-7. The HMBC
correlations between H-17 (ꢂ 3.35) and C-13 (ꢂ 39.5)/C-15 (ꢂ 42.6)/C-16 (ꢂ 44.7)
H
C
C
C
suggested the hydroxyl group at C-17. In addition, the ꢁ configuration of hydrogen
5

atom at C-16 was determined by comparing the chemical shifts of C-12 (ꢂ
39.5), C-16 (ꢂ 44.7), and C-17 (ꢂ 67.7) with the corresponding data of 16ꢁ-ent-
kaurane: helikauranoside A [ꢂ 32.5 (C-12), 39.5 (C-13), 44.5 (C-16), and 67.7 (C-
7)] and 16ꢀ-ent-kaurane: 17-hydroxy-16ꢀ-ent-kauran-19-al [ꢂ 25.8 (C-12), 36.9
C-13), 43.1 (C-16), and 64.1 (C-17)]. The HMBC cross peaks from H-18 (ꢂ 1.22)
to C-3 (ꢂ 39.1), C-4 (ꢂ 44.7), C-5 (ꢂ 49.5), and C-19 (ꢂ 178.7) confirmed the
carboxyl group at C-4. Acid hydrolysis of 2 (see Experimental) provided the D-glucose
identified as TMS derivatives). In addition, the coupling constant of glc H-1ꢃ and glc
C
33.0) C-13
(
ꢂ
C
C
C
C
2
3
1
C
2
0
(
H
C
C
C
C
(
H-2ꢃ; J = 8.0 Hz indicated the configuration of hydroxyl group at anomeric carbon in
sugar to be ꢀ. The position of this sugar at C-19 of aglycone was confirmed by HMBC
correlation between glc H-1ꢃ (ꢂ
H
C
5.42) and C-19 (ꢂ 178.7). Based on the above
evidence, the structure of 2 was elucidated to be 7ꢀ,17-dihydroxy-16ꢁ-ent-kauran-19-
oic acid 19-O-ꢀ-D-glucopyranoside ester.
2
Compound 3 was obtained as a white amorphous powder. Its molecular
4
formula was determined to be C26
H
40
O
9
by the appearance of the pseudo-ion peak at
1
m/z 519.2550 in the HR-ESI-MS (Calcd. for C26
showed one olefinic proton at ꢂ
s) and 1.22 (3H, s), suggested the presence of a ent-kaurane moiety; one anomeric
H
40
O
9
Na: 519.2565). The H-NMR of
3
H
H
5.81 (1H, s) and two methyl groups at ꢂ 1.02 (3H,
1
3
proton at ꢂ
H
5.42 (1H, d, J = 7.5 Hz), assigned to a sugar moiety. The C-NMR and
DEPT spectra revealed the presence of 26 carbons, including 20 carbons of ent-
1
13
kaurane-type diterpenoid and 6 carbons of sugar moiety. The H- and C-NMR data of
were similar to those of 2, except for the additional double bond at C-15/C-16. The
HMBC correlations between H-14 (ꢂ 1.42 and 2.06)/H-17 (ꢂ 4.13) and C-15 (ꢂ
5.81) and C-7 (ꢂ 75.6)/C-8 (ꢂ 54.3)/C-9 (ꢂ
61.2) confirmed the double bond at C-
3
H
H
C
1
4
32.1) /C-16 (ꢂ
C
148.1); H-15 (ꢂ
H
C
C
C
3.6)/C-14 (ꢂ 43.5)/C-16 (ꢂ
C
C
148.1)/C-17 (ꢂ
C
6

1
5/C-16. The hydroxyl group at C-7 were determined as the same with those of 2.
Consequently, structure of 3 were elucidated to be 7ꢀ,17-dihydroxy-ent-kaur-15-en-19-
oic acid 19-O-ꢀ-D-glucopyranoside ester.
2
5
The other compounds were characterized as paniculoside IV (4), 16ꢁ,17-
9
dihydroxy-ent-kaurane (5), 16β,17-dihydroxy-ent-kaurane (6), 16β,17-dihydroxy-
9
2
ent-kauran-19-al (7), and 16β,17-dihydroxy-ent-kauran-19-oic acid (8), (see Figure
0
20
1
) on the basis of NMR spectroscopic data, which were in good agreement with those
reported in the literature.
To evaluate the inhibitory effects of the isolates on production of NO in the LPS-
stimulated RAW 264.7 macrophages, cells were seeded in the 24-well plates at a
5
density of 5×10 cells/mL. After 3 h, the cells were pretreated with various
concentrations of compounds and stimulated for 24 h with or without 1 µg/mL of LPS.
Nitrite concentration were measured in the supernatant of RAW 264.7 cells by the
2
6
Griess reaction. First, the compounds were examined their cytotoxicity on cells at
concentration of 30 ꢄM. After that, each compound was screened its effects on the LPS
induced the production of NO in RAW 264.7 cells at concentrations of 3, 10, and 30
ꢄM since none of significant cytotoxic effects on the cells was found. In absence of
both test compounds and LPS, the NO was produced at very low concentration (3.16 ±
0
.27 µM). LPS markedly induced the NO production by concentration of 14.48 ± 0.43
µM. At the concentration of 30 µM, compounds 1, 3, and 7 potently decreased NO
production with the inhibitory percent ranging from 90.0 to 97.6 %; compounds 5, 6,
and 8 significantly decreased NO production with the inhibitory percent ranging from
7
0.4 to 77.4 % (Figure 3). In order to get the IC50 values of highly potent inhibitory
compounds, dose dependent inhibitory activities were tested. Suitable concentrations of
each compound were roughly estimated from screening results and subjected for dose-
7

dependent examination. As the results, compound 3 showed potent inhibitory LPS-
stimulated NO production in RAW 264.7 macrophages with the IC50 value of 0.01
µM; compounds 1 and 7 showed significant inhibitory NO productions with the IC50
values of 0.39 µM and 0.32 µM, respectively. Dexamethasone, a well-known anti-
inflammatory and autoimmune drug, was used as a positive control throughout
experiments. It showed the inhibitory NO production with the IC50 value of 0.8 µM.
In the structure-activity relationship of ent-kaurane (1–8), the presence of the double
bond at C-15/C-16 affected on inhibitory NO production. However, the presence of
sugar moiety at C-19 did not affect to the NO production. In previous report, the
2
7
methanol extract of A. glabra leaves showed potential anti-inflammatory activity.
Moreover, several ent-kauranes have been reported to possess inhibitory LPS-induced
2
8
NO production in RAW 264.7 macrophages. In our experiment, compound 3 showed
potent inhibitory LPS-induced NO production in RAW 264.7 macrophages with the
IC50 value of 0.01 µM (Table 2). Thus, it is possible to demonstrate that isolated ent-
kauranes might be important anti-inflammatory constituent of this plant. The in vivo
anti-inflammatory effect of compound 3 need to be further studied.
Acknowledgements
This work was financially supported by Vietnam Academy of Science and Technology
(
VAST 04.04.13-14). This work was also supported by a grant from the Priority
Research Center Program through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology (2009-0093815),
Republic of Korea. The authors are grateful to Mr Dang Vu Luong, Institute of
Chemsitry, VAST for recording the NMR and mass spectra.
Supplementary data
8

Supplementary data associated with this article can be found, in the online version, at
doi:.....
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1
7. The AG2 extract was chromatographed on a silica gel column and eluted with n-
hexane−EtOAc gradient (100 : 1−1 : 1, v/v) to obtain four fractions AG2A−AG2D.
The AG2B fraction was chromatographed on a silica gel column eluting with n-
hexane−EtOAc (4 : 1, v/v) to obtain three fractions AG2B1-AG2B3. The AG2B1
fraction was further chromatographed on an YMC RP-18 column eluting with
2
acetone−H O (3 : 1, v/v) to yield 5 (51.0 mg) and 6 (321.0 mg). The AG2D
fraction was chromatographed on a silica gel column eluting with n-
hexane−acetone (2 : 1, v/v) to obtain three fractions AG2D1-AG2D3. The
AG2D1 was chromatographed on an YMC RP-18 column eluting with
acetone−water (5 : 1, v/v) to yield 7 (7.0 mg). The AG2D3 fraction was
2 2
chromatographed on a silica gel column eluting with CH Cl −EtOAc (10 : 1, v/v)
to yield 8 (10.0 mg). The water soluble fraction AG4 was chromatographed on a
Diaion HP-20P column (Mitsubishi Chem. Ind. Co., Tokyo, Japan) eluting with
water containing increasing concentrations of MeOH (0%, 25%, 50%, 75%, and
1
00% MeOH) to give four fractions, AG4A−AG4D. The AG4C fraction was
chromatographed on a silica gel column eluting with CH Cl −MeOH (6 : 1, v/v) to
give three fractions AG4C1−AG4C3. The AG4C1 was chromatographed on an
YMC RP-18 column eluting with MeOH−H O (1 : 1.5, v/v) to yield 1 (5.0 mg).
The AG4C2 was chromatographed on a silica gel column eluting with
2
2
2
1
0

2 2
CH Cl −EtOAc (10 : 1, v/v) to obtained two fractions AG4C2A and AG4C2B.
The AG4C2A fraction was further separated on an YMC RP-18 column eluting
with MeOH−H
2
O (1 : 1, v/v) to yield 2 (4.0 mg), 3 (3.0 mg), and 4 (6.0 mg).
1
: –64.9 (c 0.1, MeOH); H-NMR
1
8. Compound (1): White amorphous powder; [α]
D
1
3
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+
3
(
32 5
Calcd. for C20H O Na: 375.2142).
1
2
2
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: –40.7 (c 0.1, MeOH); H-NMR
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3
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Calcd. for C26
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3
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(
42 9
H O Na: 521.2721).
2
2
3
1
D
: –38.5 (c 0.1, MeOH); H-NMR
1
and C-NMR (CD OD): see Table 1; HR-ESI-MS found m/z 519.2550 [M+Na]
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+
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(
40 9
Calcd. for C26H O Na: 519.2565 ).
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2
2
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1

2
8. Aquila, S.; Weng, Z. Y.; Zeng, Y. Q.; Sun, H. D.; Ríos, J. L. J. Nat. Prod. 2009, 72,
269.
1
1
2

1 13
Table 1. The H and C-NMR data for compounds 1 – 3
1
2
3
C
a,b
C
a,c
H
a,b
C
a,c
H
a,b
a,c
H
δ
δ
(mult., Hz)
δ
δ
41.8 0.94 (m)
1.88 (m)
20.2 1.45 (m)
1.94 (m)
(mult., Hz)
δ
C
δ
(mult., Hz)
1
41.7 0.93 (m)
.87 (m)
20.3 1.44 (m)
.96 (m)
41.7 1.03 (dd, 3.5, 13.5)
1.87 (d, 13.5)
20.2 1.44 (dt, 5.0, 10.0)
1.96 (m)
39.1 1.12 (dd, 4.0, 13.5)
2.22 (m)
44.7 -
48.3 1.78 (m)
29.3 1.96 (m)
2.23 (m)
1
2
3
1
39.2 1.08 (dd, 4.5, 13.5) 39.1 1.13 (m)
.17 (d, 13.5) 2.22 (m)
44.7 -
2
4
5
6
44.2 -
48.1 1.77 (d, 9.0)
30.5 1.98 (m)
49.5 1.78 (d, 13.0 )
30.7 1.97 (m)
2
.11 (m)
2.18 (dd, 13.0, 14.5)
7
8
9
78.1 3.63 (br s)
49.0 -
78.7 3.50 (br s)
49.8 -
75.6 3.59 (br s)
54.3 -
51.1 1.43 (d, 7.5)
40.4 -
19.1 1.57 (m)
50.6 1.43 (m)
40.5 -
43.6 1.39 (d, 7.5)
40.9 -
10
11
19.5 1.57 (m)
1.63 (m)
19.7 1.58 (m)
1.64 (m)
26.3 1.52 (m)
1
.64 (m)
27.6 1.57 (m)
.68 (m)
46.1 2.08 (m)
12
33.0 1.43 (m)
1.63 (m)
39.5 2.11 (m)
1
1
1
3
4
42.2 2.57 (m)
37.5 1.70 (dd, 4.0, 12.0) 37.2 1.08 (m)
.83 (d, 12.0) 1.80 (d, 11.5)
50.1 1.56 (d, 13.5) 42.6 1.12 (m)
.74 (d, 13.5) 1.71 (dd, 3.5, 10.0)
44.7 1.94 (m)
43.5 1.42 (dd, 7.5, 10.5)
2.06 (d, 10.5)
132.1 5.81 (s)
1
15
1
1
1
6
7
82.9 -
66.7 3.62 (d, 11.5)
.72 (d, 11.5)
148.1 -
61.2 4.13 (d, 1.0)
67.7 3.35 (m)
3
18
19
20
29.3 1.18 (s)
182.0 -
16.1 1.00 (s)
28.8 1.22 (s)
178.7 -
16.3 0.99 (s)
28.8 1.22 (s)
178.6 -
16.1 1.02 (s)
19-O-Glc
1
2
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
95.7 5.42 (d, 8.0)
74.1 3.38 (m)
78.7 3.45 (m)
71.1 3.39 (m)
78.6 3.39 (m)
96.6 5.42 (d, 7.5)
74.1 3.38 (m)
78.7 3.42 (m)
71.1 3.40 (m)
78.7 3.39 (m)
3
4
5
6
62.4 3.71(dd, 4.0, 11.5)
3
62.4 3.71 (dd, 4.0, 12.0)
3.85 (dd, 2.0, 12.0)
.86 (d, 11.5)
a)
b)
c)
3
Measured in CD OD, 125 MHz, 500 MHz. Assignments were done by HMQC, HMBC, COSY, and
NOESY experiments
1
3

Table 2. The inhibitory effects of 1-8 on NO production in LPS-stimulated RAW
2
64.7 macrophages
Compound
IC50 (µM)
0.39±0.12
>30
0.01±0.01
>30
17.06±0.93
6.20±0.47
0.32±0.04
14.32±1.06
0.80±0.04
1
2
3
4
5
6
7
8
Dexamethasone*
*Dexamethasone was used as a positive control
1
4

Figure Legend
Figure 1. Structures of compounds 1–8 from the fruits of Annona glabra.
Figure 2. Selected HMBC and COSY correlations compounds 1–3.
Figure 3. Effect of compounds 1–8 on NO production in RAW 264.7 cells. The cells were
treated with various concentrations of compounds (3, 10, and 30 ꢄM), and then LPS
(
1 ꢄg/mL) was added and the cells were incubated for 24 h. Dexa, dexamethaxone. The
values represent the means ± SD from three independent experiments.
15

Figure 1.
16

Figure 2.
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ꢅꢆ
ꢅꢆ
ꢅꢆ
ꢅꢆ
ꢅ
ꢅ
ꢅꢆ
ꢆꢅ
ꢆꢅ
ꢆꢅ
ꢆꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢇꢅꢅꢆ
ꢆꢅ
ꢆꢅ
ꢅꢆ
ꢅꢆ
1
2
3
ꢇꢅꢈꢉ
ꢆ
ꢆ
ꢆꢊꢋꢇ
ꢆ
ꢇ
17

Figure 3.
18

Graphical abstract
New ent-kauranes from the fruits of Annona glabra and their inhibitory
nitric oxide production in LPS-stimulated RAW264.7 macrophages
Nguyen Xuan Nhiem, Nguyen Thi Thu Hien, Bui Huu Tai, Hoang Le Tuan Anh, Dan Thi Thuy
Hang, Tran Hong Quang, Phan Van Kiem, Chau Van Minh, Wonmin Ko, Seungjun Lee, Hyuncheol
Oh, Seung Hyun Kim and Young Ho Kim
19