Structure-activity relationship of lupane-triterpene glycosides from Acanthopanax koreanum on spleen lymphocyte IL-2 and IFN-γ

Article abstract of DOI:10.1016/j.bmcl.2010.06.044

Phytochemical investigation resulted in the isolation of three new lupane-triterpene glycosides acankoreosides M-O (1, 2 and 8) together with eight known lupane-triterpene glycosides (3-7, 9-11) from the leaves of Acanthopanax koreanum (Araliaceae). Their chemical structures were elucidated by mass, 1D and 2D NMR spectroscopy. The effect of eleven lupane-triterpene glycosides on Con A-induced splenolytic production of IL-2 and IFN-γ were measured as markers of acquired immune responses. Compounds 4, 5, 7, and 11 (5, 25, and 100 μM) significantly increased IFN-γ and IL-2 release in spleen cells.

Full text of DOI:10.1016/j.bmcl.2010.06.044

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4927–4931
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship of lupane-triterpene glycosides from
Acanthopanax koreanum on spleen lymphocyte IL-2 and IFN-
c
Nguyen Xuan Nhiem a,b, Phan Van Kiem b, Chau Van Minh b, Bui Huu Tai a,b, Nguyen Huu Tung a,b
,
Do Thi Ha a, Kwang Su Soung c, Jun Ho Kim d, Ji Young Ahn d, Young-Mi Lee d, Young Ho Kim a,
*
a College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
b Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caug Caugiay, Hanoi, Viet Nam
c 222, Haejeong-ri, Susin-myeon, Cheonan-si, Chungcheongnam-do 330-881, Republic of Korea
d Department of Oriental pharmacy, College of Pharmacy, Wonkwang University, Iksan, Jonbuk 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:
Phytochemical investigation resulted in the isolation of three new lupane-triterpene glycosides acanko-
reosides M–O (1, 2 and 8) together with eight known lupane-triterpene glycosides (3–7, 9–11) from the
leaves of Acanthopanax koreanum (Araliaceae). Their chemical structures were elucidated by mass, 1D and
2D NMR spectroscopy. The effect of eleven lupane-triterpene glycosides on Con A-induced splenolytic
Received 11 April 2010
Revised 4 June 2010
Accepted 8 June 2010
Available online 15 June 2010
production of IL-2 and IFN-
5, 7, and 11 (5, 25, and 100
c
l
were measured as markers of acquired immune responses. Compounds 4,
M) significantly increased IFN- and IL-2 release in spleen cells.
c
Keywords:
Acanthopanax koreanum
Araliaceae
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Lupane-triterpene glycoside
Acankoreosides M–O
IL-2
IFN-c
Both IL-2 and IFN-
c
play an important role in the immune-reg-
biological investigations of A. koreanum have yielded a number of
triterpenes.10–14
In the preliminary data, the methanol extracts of leaves and
stems of A. koreanum showed the immune enhancement activity
ulation in the body. IL-2 is normally produced by the body during
an immune response.1,2 IL-2 is indispensable for the development
of T-cell immunologic memory, one of the unique characteristics
of the immune system, and also vital during T cell development
in the thymus for the maturation of a unique subset of T cells that
is termed regulatory T cells.3 Thus, IL-2 is required to discriminate
between self and none-self, another one of the unique characteris-
tics of the immune system.1,2 In addition, IL-2 is able to promote
production of immunoglobulins made by B cells and induce the
on Con A-induced splenolytic production of IL-2 and IFN-c. In or-
der to clarify the active compounds for immune responses, three
new lupane-type glycosides (1, 2, and 8) and eight compounds
[acankoreoside I (3), acankoreoside A (4), acankoreoside D (5),
acankoreoside F (6), and acantrifoside A (7), acankoreosides J (9),
acankoreoside K (10), and acankoreoside L (11)] (see Fig. 1) previ-
ously isolated from the leaves of A. koreanum,15,16 were tested
their immune enhancement activity.
By using combined chromatographic separations, three new lu-
pane-triterpene glycosides were isolated from the methanol ex-
tract of A. koreanum leaves.17 Compound 1 was obtained as a
white amorphous powder. Its basic ion peak at m/z 989.5 [M+H]+
was observed in the positive-ion ESI-MS, and HRTOFMS analysis
revealed the molecular formula to be C48H76O21, with a cluster
differentiation and proliferation of natural killer cells.4 IFN-
mediates a number of modulator signals at several points in the
pathway of the progression of B-lymphocyte responses.5,6 Conca-
navalin A (Con A), a lymphocyte mitogen, induced splenolytic pro-
c
duction of IL-2 and IFN-
c were measured as markers of acquired
immune responses.7
Acanthopanax koreanum Nakai is deciduous scrub of the Aralia-
ceae family and an endemic species in Korea. Its root and stem
barks have been traditionally used by practitioners of oriental
medicine as a tonic, as a prophylactic, and to treat rheumatism,
paralysis, hepatitis, and diabetes.8,9 Previous phytochemical and
ion peak at m/z 989.4989 [M+H]+ (calcd for C48H77O21
:
989.4957). The 1H NMR spectrum of 1 (in methanol-d4) showed
the following signals: four tertiary methyl groups at dH 0.96,
1.08, 1.10, and 1.18 (each 3H, s); one secondary methyl group at
dH 1.27, assigned to H-600 of the rhamnose; and three anomeric pro-
tons were at dH 4.40, 4.85, and 5.46, suggesting the appearance of
* Corresponding author. Tel.: +82 42 821 5933; fax: +82 42 823 6566.
E-mail address: yhk@cnu.ac.kr (Y.H. Kim).
0960-894X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.044

4928
N. X. Nhiem et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4927–4931
R3
H
R2
R4
R1
20
19
H
R3
O
O
28
O
O
3
R1O
OH
OH
HO
O
O
R2
23
COOH
glc
24
O
O
O
O
HO
O
HO
O
HO
OH
HO
OH
OH
OH
HO
glc'
HO
O
O
Me
HO
Me
HO
HO
HO
OH
OH
rha
R1
OH
H
H
H
R2
H
=O
OH
H
R3
CH3
-
CH2OH
CH(OCH3)2
R1
H
Glc
H
H
H
R2
R3
R4
8
9
10
11
1
2
3
4
5
6
7
COOH OH
CH3 OH
CHO OH
COOH H
CHO OH
COOH H
OH
OH
OH
H
H
OH
H
H
H
CH3
OH
Figure 1. Structure of triterpenes (1–11) isolated from leaves of A. koreanum.
three sugar units. The 13C NMR and DEPT spectra revealed 48 car-
bon signals, of which, 30 signals were assigned to a triterpenoid
sapogenol moiety and 18 signals belong to three monosaccharide
moieties. The aglycone of 1 was recognized to be a lupane-type tri-
terpene by 1H and 13C NMR analysis (see Table 1), with the typical
olefinic carbons at dC 155.9 and 107.7, four quaternary methyl car-
bons at dC 15.1, 17.5, 18.1, and 18.2, two oxymethine carbons at dC
71.1 and 73.8, one oxymethylene carbon at dC 65.5, and two car-
was determined as
dioic acid 28-O-[ -rhamnopyranosyl-(1?4)-b-
(1?6)-b- -glucopyranosyl] ester (1), named acankoreoside M.
3
a
,11
a
,30-trihydroxylup-20(29)-en-23,28-
a-L
D-glucopyranosyl-
D
Compound 2 was also obtained as a white amorphous powder.
Its basic ion peak at m/z 1121.6 [M+H]+ was observed in the posi-
tive-ion ESI-MS, and HRTOFMS analysis revealed the molecular for-
mula to be C54H88O24, with a cluster ion peak at m/z 1121.5824
[M+H]+ (calcd for C54H89O24: 1121.5744). The 1H NMR spectrum
of 2 (in methanol-d4) showed the following signals: five tertiary
methyl groups at dH 0.88, 0.95, 0.96, 1.07, and 1.08 (each 3H, s);
one secondary methyl group at dH 1.27, assigned to H-600 of the
rhamnose; and four anomeric protons were at dH 4.27, 4.40, 4.84,
and 5.45, suggesting the appearance of four sugar units. The 13C
NMR and DEPT spectra revealed 54 carbon signals, of which, 30
signals were assigned to a triterpenoid sapogenol moiety and 24
signals belong to four sugar moieties. The 1H and 13C NMR data
of 2 (Table 1) were similar to those of acankoreoside D,11 except
difference of isopropenyl group in E ring. On the other hand, the
HMBC correlations were observed between methylene protons
H-29 (dH 4.94 and 5.00) and C-19 (dC 43.8), C-20 (dC 156.0), and
C-30 (dC 65.6), as well as between H-30 (dH 4.08) and C-19
(dC 43.8), C-20 (dC 156.0), and C-29 (dC 107.6), confirming the dou-
ble bond was at C-20/C-29 and the hydroxyl group was at C-30.
The sugar linked to C-3 confirmed by HMBC correlations between
H-3 (dH 3.41) and C-1000 (dC 101.7) as well as between anomeric pro-
boxyl signals at dC 176.3 and d 180.6. Assignment of the a-hydroxyl
group at C-3 was performed by comparing its spectral data with
literature values.10–14 The chemical shifts of C-1 (35.5), C-3 (dC
73.8), and C-4 (dC 53.0) in the aglycone further confirmed the axial
configuration of the 3-hydroxyl group by comparing with the cor-
responding data of the 3b-hydroxy-lup-20(29)-ene-23,28-dioic
acid18 [dC values for C-1 (39.0), C-3 (dC 84.4), and C-4 (dC 43.0)].
Moreover, the chemical shifts of C-11 (dC 71.1) and C-23 (dC
180.6) were also similar to those of
3
a,11a
-dihydroxy-lup-
-hydroxyl
20(29)-ene-23,28-dioic acid19 suggesting that the
a
group was at C-11 and the carboxylic group was at C-23. In the
HMBC spectrum, the methyl group H-24 (dH 1.18) correlated with
carbons C-3 (dC 73.8), C-4 (dC 53.0), and C-23 (dC 180.6). In the
ROESY spectrum, cross peaks between H-24 (dH 1.18) and H-25
(dH 1.10) as well as H-3 (dH 3.67) indicated that the methyl group
at C-4 was axial, which in turn suggested that the carboxylic group
at C-4 was a-positioned. Furthermore, the HMBC cross peaks from
H-29 (dH 4.95 and 5.01) to C-19 (dC 43.7), C-20 (dC 155.9), and C-30
(dC 65.5) confirmed that the double bond was at C-20/C-29 and the
other hydroxyl group was at C-30. Acid hydrolysis of 1 provided
ton H-1000 (dH 4.27) and C-3 (dC 82.4). Assignment of the O-b-
copyranosyl moiety group at C-3 was performed as configuration
by comparing its spectral data [C-1 (dC 36.7), C-3 (dC 82.4), and C-5
(dC 51.0)] in the aglycone and those of the 3-O-b- -glucopyranosyl
betulinic acid 28-O- -arabinopyranosyl(1?6)-b- -glucopyrano-
D-glu-
a
the monosaccharide components of
L-rhamnose and
D-glucose
D
(identified as TMS derivatives by GC method).20 Moreover, the
HMBC correlations between the inner glc H-1 (d 5.46) and
C-28 of the aglycone (d 176.3), between outer glc H-10 (d 4.40)
and inner glc C-6 (d 69.6), between rha H-100 (d 4.85) and glc
C-40 (d 79.6) were observed. This evidence suggested the se-
quence of sugar linkages of 1. Consequently, the structure of 1
a
-L
D
syl ester (cussosaponin A)21 [dC values for C-1 (39.0), C-3 (dC 88.7),
and C-5 (dC 55.9)]. The HMBC correlations between the inner glc
H-1 (d 5.45) and C-28 of the aglycone (d 176.4), between outer glc
H-10 (d 4.40) and inner glc C-6 (d 69.5), between rha H-100 (d 4.84)
and glc C-40 (d 79.7) were observed. Moreover, the NMR data of

N. X. Nhiem et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4927–4931
4929
Table 1
The 1H and 13C NMR data of compounds 1, 2, and 8
Pos.
1
2
8
a,b
a,c
a,b
a,c
a,b
a,c
dC
dH mult. (J in Hz)
dC
dH mult. (J in Hz)
dC
dH mult. (J in Hz)
Aglycone
1
2
3
4
5
6
7
8
35.5
26.4
73.8
53.0
45.4
22.6
36.1
43.5
56.7
40.0
71.1
39.4
38.2
44.3
30.8
32.9
57.9
50.9
43.7
155.9
33.6
37.4
1.40
1.92
3.67
—
1.91
1.47
2.34
—
1.60 (d, 10.5)
—
3.42
1.31
2.42
—
2.21
2.37 (t, 9.5)
36.7
19.2
82.4
38.5
51.0
22.2
36.4
43.5
56.3
40.4
71.2
39.5
38.3
44.0
30.9
32.9
58.0
51.0
43.8
156.0
33.6
37.4
2.24 (dd, 3.5, 13.5)
1.36
3.41
—
1.30
1.62, 1.77
2.30
34.5
26.1
73.3
52.1
47.4
22.2
35.5
43.1
52.0
38.2
22.2
26.1
39.1
44.3
30.9
34.5
59.4
50.4
85.4
36.8
34.5
37.2
1.43
1.39
3.58
—
1.86
1.46
1.36
—
1.51 (d, 10.5)
—
1.51
1.29
2.42
—
1.29
1.39 (t, 9.5)
—
1.65
—
1.48
1.39
1.83
—
9
1.55 (d, 10.5)
—
3.42
1.29
2.42
—
1.50
2.37 (t, 9.5)
—
1.83
2.88 (dt, 4.5, 11.0)
—
2.07
1.51
1.97
10
11
12
13
14
15
16
17
18
19
20
21
22
—
1.82
2.90 (dt, 4.5, 11.0)
—
2.26
1.55
1.98
23
24
25
26
27
28
29
180.6
18.2
17.5
18.1
15.1
29.8
23.3
17.3
18.0
15.2
0.96 (s)
0.88 (s)
1.07 (s)
0.95 (s)
1.08 (s)
—
4.94 (br s)
5.00 (br s)
4.08 s
185.2
18.6
17.4
17.1
15.5
176.7
21.9
1.18 (s)
1.10 (s)
0.96 (s)
1.08 (s)
—
1.08 (s)
0.90 (s)
0.95 (s)
1.03 (s)
—
176.3
107.7
176.4
107.6
4.95 (br s)
5.01 (br s)
1.16 (d, 6.0)
*
30
65.5
4.09
65.6
27.0
1.31 (d, 6.0)
C-28 O-Glc
1
2
3
4
5
6
95.3
74.0
78.2
71.0
78.3
69.6
5.46 (d, 8.0)
3.33
3.43
3.41
3.43
95.4
74.1
78.3
71.1
78.4
69.5
5.45 (d, 8.0)
3.31
3.43
3.41
3.43
95.6
74.1
78.2
71.2
78.5
69.8
5.45 (d, 8.0)
3.33
3.43
3.42
3.43
3.80
3.80
3.80
4.10 (d, 10.5)
4.11 (d, 10.5)
4.11 (d, 10.5)
Glc0 (1?6) Glc
10
20
30
40
50
60
104.5
75.4
76.7
79.6
77.0
62.0
4.40 (d, 8.0)
3.25
3.46
3.54
3.30
104.5
75.4
76.9
79.7
77.1
62.1
4.40 (d, 8.0)
3.20
3.46
3.54
3.44
104.8
75.5
76.9
79.8
77.1
62.1
4.40 (d, 8.0)
3.25
3.46
3.53
3.44
3.66
3.66
3.66
3.81 (d, 6.5)
3.81 (d, 6.5)
3.81 (d, 6.5)
Rha (1?4) Glc0
100
200
300
400
500
103.0
72.5
72.3
73.6
70.7
18.0
4.85 (br s)
3.64 (dd, 3.5, 9.5)
3.84
3.41 (t, 9.5)
3.95 (dd, 6.0, 9.5)
1.27 (d, 6.0)
103.1
72.3
72.6
73.9
70.8
18.0
4.84 (br s)
3.62 (dd, 3.5, 9.5)
3.82
3.40 (t, 9.5)
3.97 (dd, 6.0, 9.5)
1.27 (d, 6.0)
103.1
72.6
72.4
73.3
70.9
18.0
4.84 (br s)
3.62 (dd, 3.5, 9.5)
3.82
3.40 (t, 9.5)
3.96 (dd, 6.0, 9.5)
1.27 (d, 6.0)
C-3 O-Glc00
1000
2000
3000
4000
5000
6000
101.7
75.5
78.6
72.1
77.9
63.2
4.27 (d, 8.0)
3.26
3.36
3.28 (t, 9.5)
3.21
3.66
3.85 (d, 6.5)
a
b
c
Measured in CD3OD.
125 MHz.
500 MHz, Assignments were done by HMQC, HMBC, 1H–1H COSY and ROESY experiments, Glc:
D-glucopyranosyl, Rha:
L-rhamnopyranosyl.
D
-glucose (identified as TMS derivatives by GC method).20 This
evidence suggested the sequence of sugar linkages of 2 was
the sugar moiety were superimposable on those of compound 1
and sugar moieties were also determined as L-rhamnose and

4930
N. X. Nhiem et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4927–4931
Table 2
28-O-[
a
-
L
-rhamnopyranosyl-(1?4)-b-
D
-glucopyranosyl-(1?6)-b-
The effect of compounds isolated from leaves of A. koreanum on INF-c release
D
-glucopyranosyl. Consequently, the structure of 2 was determined
as 3-O-b- -glucopyranosyl ,11 ,30-trihydroxylup-20(29)-en-
23,28-dioic acid 28-O-[ -rhamnopyranosyl-(1?4)-b- -glucopyran-
osyl-(1?6)-b- -glucopyranosyl] ester (2), named acankoreoside N.
D
3a
a
Compound
IFN-
c release (pg/mL)
a-L
D
5
lM
25
l
M
100 lM
D
Blank
1.94 0.09
Compound 8 was also obtained as a white amorphous powder.
Its basic ion peak at m/z 975.5 [M+H]+ was observed in the posi-
tive-ion ESI-MS, and HRTOFMS analysis revealed the molecular for-
mula to be C48H78O20, with a cluster ion peak at m/z 975.5162
[M+H]+ (calcd for C48H79O20: 975.5165). The 1H NMR spectrum of
8 (in methanol-d4) showed the following signals: four tertiary
methyl groups at dH 0.90, 0.95, 1.03, and 1.08 (each 3H, s); three
secondary methyl groups at dH 1.16, 1.27, and 1.31; and three ano-
meric protons were at dH 4.40, 4.84, and 5.45, suggesting the
appearance of three sugar units. The 13C NMR and DEPT spectra
of 8 revealed 48 carbon signals, of which, 30 signals were assigned
to a triterpenoid sapogenol moiety and 18 signals belong to three
sugar moieties. The 1H and 13C NMR data of 8 (Table 1) were sim-
ilar to those of acankoreoside A,10 except difference of branch chain
in E ring. The HMBC correlations were observed between two sec-
ondary methyl protons H-29 (dH 1.16), H-30 (dH 1.31) and C-19 (dC
85.4) and C-20 (dC 36.8), confirming two methyl groups was at
C-20; and one hydroxyl group was at C-19. On the other hand, the
Con A (1
l
g/mL)
15.67 3.78*
1
2
3
4
5
6
7
8
1.57 0.61
1.50 0.35
1.25 0.26
2.89 0.38*
3.69 0.22**
3.30 1.06**
4.55 0.49**
1.81 0.27
2.75 0.35*
2.78 0.92
3.53 2.34*
2.45 0.11
1.55 0.40
4.18 0.47*
4.62 0.09**
4.62 0.09*
4.00 0.57**
5.23 0.30**
1.68 0.14
3.78 0.57**
3.2 0.27
2.48 0.54
1.92 0.32
2.59 0.08
4.57 0.14**
5.29 0.08**
4.20 0.17**
5.41 0.20**
2.44 0.16
4.36 0.08**
3.66 0.33
6.07 0.17**
9
10
11
3.71 2.22*
Data are expressed as mean S.D. Statistically significant compared with blank.
p <0.05.
p <0.01.
*
**
Table 3
The effect of compounds isolated from leaves of A. koreanum on IL-2 release
ROE correlation between H-18a (dH 1.65) and H-29 (dH 1.16) con-
Compound
IL-2 release (pg/mL)
25
firmed hydroxyl group at C-19 was b. Moreover, the HMBC cross
peaks from H-3 (dH 3.58) to C-1 (dC 34.5) and C-5 (dC 47.4), from
H-5 (dH 1.86) to C-4 (dC 52.1), C-23 (dC 185.2), and C-24 (dC 18.6),
from H-24 (dH 1.08) to C-3 (dC 73.3), C-4 (dC 52.1), C-5 (dC 47.4),
and C-23 (dC 185.2) confirmed location of the hydroxyl and car-
boxyl groups at C-3 and C-4, respectively. Similar to 1, 3-hydroxyl
5
lM
l
M
100 lM
Blank
13.23 0.42
158.53 27.36*
Con A (1
l
g/mL)
1
2
3
4
5
6
7
8
15.27 0.53
17.11 0.83
10.07 2.21
17.04 5.67*
19.26 10.45*
16.09 3.11*
20.07 1.66*
13.89 2.50
15.78 6.95
16.69 0.34
20.68 0.85**
16.92 5.55
13.66 0.65
12.91 3.47
24.87 3.22*
25.32 5.57*
23.47 1.47**
25.57 4.76*
11.82 5.18
16.14 4.45
20.24 2.13
45.06 0.65**
17.26 5.3
19.06 0.08
14.76 6.55
29.78 0.25**
41.34 1.40**
27.57 1.75**
50.66 4.54**
16.30 7.21
28.27 2.84*
21.57 2.56
74.47 4.13**
and 4-carboxyl groups were assigned as the a-positions. The HMBC
correlations between the inner glc H-1 (d 5.45) and C-28 of the
aglycone (d 176.7), between outer glc H-10 (d 4.40) and inner glc
C-6 (d 69.8), between rha H-100 (d 4.84) and glc C-40 (d 79.8) were
observed. Moreover, the NMR data of the sugar moiety were
superimposable on those of compound 1, and sugar moieties were
9
10
11
also determined as L-rhamnose and D-glucose (identified as TMS
derivatives by GC method).20 This evidence suggested the se-
quence of sugar linkages of 8. Consequently, the structure of
Data are expressed as mean S.D. Statistically significant compared with blank.
*
p <0.05.
8 was determined as 3
28-O-[ -rhamnopyranosyl-(1?4)-b-
-glucopyranosyl] ester (8), named acankoreoside O. The lympho-
a,19b-dihydroxylupane-23,28-dioic acid
**
p <0.01.
a
-
L
D
-glucopyranosyl-(1?6)-b-
D
proliferation test was conducted as previously described with
minor modifications.22 Spleen cells (1 ꢀ 106/ml) were cultured
and incubated with three different concentrations of isolated com-
4, 5, 7, and 11 (5, 25, and 100
ductions ranging from 4.20 0.17 pg/mL to 6.07 0.17 pg/mL in
spleen cells. Compounds 4, 5, 7, and 11 (5, 25, and 100 M) also
lM) significant increased IFN-c pro-
l
pounds (5, 25, and 100
mL). The results were expressed as mean values of picograms per
milliliter of IL-2 and IFN- produced with standard error in tripli-
cate cultures comparing with IL-2 and IFN- standard.
The water extracts of A. koreanum stems increased to 6.6 times
for IL-2 release, 3.1 times for IFN- release compare to negative
control. Moreover, the water extracts of A. koreanum leaves in-
creased to 3.2 times for IL-2 release, 1.5 times for IFN- release
l
M) or the positive control, Con A (1
l
g/
exhibited potent effect on IL-2 release within 27.57 1.75 to
74.47 4.13 pg/mL. In the structure–activity relationship of these
lupane-triterpene glycosides, the functional groups as methyl,
aldehydic, carboxyl at C-4, hydroxyl group at C-11, and double
c
c
bond at C-20/C-29 did not affect on IL-2 and IFN-c increasing activ-
c
ity. However, the presence of hydroxyl group at C-30 or absence of
C-30 affected the immune stimulatory activity. The compounds 4,
5, 7, and 11 having no hydroxyl group at C-30 showed significant
c
compare to negative control. The effect of methanol extract of A.
koreanum leaves on both cytokine releases was greater than those
of water extract of A. koreanum leaves. These results revealed that
the immunomodulatory agents are present in both leaves and
stems of A. koreanum. Furthermore, the genuine immune stimulat-
increasing activity for both IL-2 and IFN-c. These results revealed
that the immunomodulatory effects of A. koreanum were due to
the lupane-triterpene glycosides in the leaves and stems of this
plant.
ing effects were assessed by IL-2 and IFN-
c
release in splenocytes
Acknowledgments
treated with lupane-triterpene glycosides (1–11) isolated from
the leaves of A. koreanum (see Tables 2 and 3). In absence of both
test compounds and Con A, IL-2 and IFN-c were secreted at very
low concentration (1.94 0.09 and 13.23 0.42 pg/mL, respec-
tively). In contrast, with the presence of Con A, the IL-2 and
This study was supported by Priority Research Centers 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 would like to thank Kor-
ean Basic Science Institute (KBSI) for taking NMR and MS
experiments.
IFN-c were released 15.67 3.78 and 158.53 27.36 pg/mL,
respectively. As compared with the blank and Con A, compounds

N. X. Nhiem et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4927–4931
4931
of MeOH (100% H2O, 25% MeOH, 50% MeOH, and 100% MeOH, 0.5 L each) to
give five corresponding fractions, AK2A–AK2D. Fraction AK2D (10 g) was
chromatographed on an YMC RP-18 column (4 ꢀ 30 cm) eluting with acetone/
water (1:1, v/v, 1.5 L) to yield three sub-fractions, AK2D1–AK2D3. Sub-fraction
AK2D1 (1.5 g) was further chromatographed on a silica gel column (2 ꢀ 40 cm)
eluting with chloroform/methanol/water (3:1:0.1, v/v/v, 1.5 L) to obtain
compound 2 (white amorphous powder, 5 mg). Sub-fraction AK2D2 (1.3 g)
was further chromatographed on a silica gel column (1.5 ꢀ 30 cm) eluting with
dichloromethane/methanol/water (35:10:1, v/v/v, 1.5 L) to obtain compounds
1 (white amorphous powder, 8 mg) and 8 (white amorphous powder, 7 mg).
18. Jahan, N.; Ahmed, W.; Malik, A. Phytochemistry 1995, 39, 255.
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20. Each compound (2.0 mg) was dissolved in 1.0 N HCl (dioxane/H2O, 1:1, v/v,
1.0 mL) and then heated to 80 °C in a water bath for 3 h. The acidic solution
was neutralized with silver carbonate and the solvent thoroughly driven out
under N2 gas overnight. After extraction with CHCl3, the aqueous layer was
concentrated to dryness using N2 gas. The residue was dissolved in 0.1 mL of
dry pyridine, and then L-cysteine methyl ester hydrochloride in pyridine
(0.06 M, 0.1 mL) was added to the solution. The reaction mixture was heated at
60 °C for 2 h, and 0.1 mL of trimethylsilylimidazole solution was added,
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n-hexane and H2O (0.1 mL, each), and the organic layer was analyzed by gas
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detector FID, column temp 210 °C, injector temperature 270 °C, detector
temperature 300 °C, carrier gas He (2.0 mL/min). The retention times of
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times, 50 °C, 5 L each) to yield the methanol extract (80 g), which was then
suspended in water (2 L) and extracted in turn with ethyl acetate (3 ꢀ 2 L) to
give the ethyl acetate (AK1, 30 g), and water (AK2, 50 g) extracts. The AK2
extract (50 g) was chromatographed on a Diaion HP-20P column (Mitshubishi
Chem. Ind. Co., Japan) eluting with water containing increasing concentrations
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D-glucose, L-glucose, and L-rhamnose were 14.11, 14.26, and
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