A new ursane-type triterpenoid glycoside from Centella asiatica leaves modulates the production of nitric oxide and secretion of TNF-α in activated RAW 264.7 cells

Article abstract of DOI:10.1016/j.bmcl.2011.01.066

One new ursane-type triterpenoid glycoside, asiaticoside G (1), five triterpenoids, asiaticoside (2), asiaticoside F (3), asiatic acid (4), quadranoside IV (5), and 2α,3β,6β-trihydroxyolean-12-en-28-oic acid 28-O-[α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl- (1→6)-β-d-glucopyranosyl] ester (6), and four flavonoids, kaempferol (7), quercetin (8), astragalin (9), and isoquercetin (10) were isolated from the leaves of Centella asiatica. Their chemical structures were elucidated by mass, 1D- and 2D-nuclear magnetic resonance (NMR) spectroscopy. The structure of new compound 1 was determined to be 2α,3β,23,30-tetrahydroxyurs-12-en-28- oic acid 28-O-[α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl- (1→6)-β-d-glucopyranosyl] ester. The anti-inflammatory activities of the isolated compounds were investigated on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Asiaticoside G (1) potently inhibited the production of nitric oxide and tumor necrosis factor-α with inhibition rates of 77.3% and 69.0%, respectively, at the concentration of 100 μM.

Full text of DOI:10.1016/j.bmcl.2011.01.066

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1777–1781
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Bioorganic & Medicinal Chemistry Letters
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A new ursane-type triterpenoid glycoside from Centella asiatica leaves
modulates the production of nitric oxide and secretion of TNF-
activated RAW 264.7 cells
a in
Nguyen Xuan Nhiem a,b, Bui Huu Tai a,b, Tran Hong Quang a,b, Phan Van Kiem b, Chau Van Minh b,
Nguyen Hoai Nam b, Jun-Ho Kim c, Lee-Rang Im c, Young-Mi Lee c, Young Ho Kim a,
⇑
a College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
b Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Viet Nam
c 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:
One new ursane-type triterpenoid glycoside, asiaticoside G (1), five triterpenoids, asiaticoside (2), asiati-
coside F (3), asiatic acid (4), quadranoside IV (5), and 2 ,3b,6b-trihydroxyolean-12-en-28-oic acid
28-O-[ -rhamnopyranosyl-(1?4)-b- -glucopyranosyl-(1?6)-b- -glucopyranosyl] ester (6), and four
flavonoids, kaempferol (7), quercetin (8), astragalin (9), and isoquercetin (10) were isolated from the
leaves of Centella asiatica. Their chemical structures were elucidated by mass, 1D- and 2D-nuclear
magnetic resonance (NMR) spectroscopy. The structure of new compound 1 was determined to be
Received 5 September 2010
Revised 31 December 2010
Accepted 18 January 2011
Available online 22 January 2011
a
a
-
L
D
D
Keywords:
Centella asiatica
Apiaceae
Ursane-type triterpene glycoside
Asiaticoside G
IL-2
2
a
,3b,23,30-tetrahydroxyurs-12-en-28-oic acid 28-O-[
syl-(1?6)-b- -glucopyranosyl] ester. The anti-inflammatory activities of the isolated compounds were
investigated on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Asiaticoside G (1) potently inhib-
ited the production of nitric oxide and tumor necrosis factor- with inhibition rates of 77.3% and
69.0%, respectively, at the concentration of 100 M.
a-L-rhamnopyranosyl-(1?4)-b-D-glucopyrano-
D
a
l
Anti-inflammatory
Nitric oxide
Ó 2011 Elsevier Ltd. All rights reserved.
Tumor necrosis factor-
a
Nitric oxide (NO) and tumor necrosis factor (TNF) are essential
mediators of several biological processes, including the immune
response. TNF stimulates NO production via the expression of
terpenes,6–8 polyacetylenes,9 and flavonoids.10 Studies have cited
the anti-inflammatory activity of the ursane triterpenoids and a
number of derivatives from this plant with the ursane-type skele-
ton been found to possess anti-inflammatory activity and the
inducible NO synthase (iNOS), with
strate.1 The production of NO confers cytostatic or cytotoxic activ-
ity on macrophages against microbes and tumor cells.2 TNF-
is
one of the most important pro-inflammatory cytokines and is pro-
duced by activated monocytes and macrophages. NO and TNF-
L-arginine being the only sub-
inhibitory production of NO and the secretion of TNF-a in lipopoly-
a
saccharide (LPS)-stimulated RAW 264.7 cells; these compounds in-
clude madecassic acid,11 madecassoside,12 asiatic acid,13,14 and
asiaticoside.14,15 Interestingly, our extensive phytochemical study
led to the isolation of one new ursane-type triterpenoid glycoside
as well as nine known compounds. In this study, we examined the
effect of the new ursane-type triterpenoid glycoside and the nine
known compounds on the production of NO and the secretion of
a
generally act as defense factors against invading bacterial patho-
gens and are essential elements of antimicrobial immunity during
inflammation.3 They are secreted during the early phase of acute
and chronic inflammatory diseases such as asthma, rheumatoid
arthritis, septic shock and other allergic diseases, as well as during
the activation of T cells.4
Centella asiatica (L.) Urban (Umbelliferae) is widely cultivated as
a vegetable or spice in Asia, Africa, and Oceania. It is also used to
treat skin diseases, rheumatism, inflammation, syphilis, mental ill-
ness, epilepsy, diarrhea, and wounds.5 Previous phytochemical and
biological investigations of C. asiatica have yielded a number of tri-
TNF-
In a preliminary study, the methanol extract of the leaves of C.
asiatica inhibited the production of NO and secretion of TNF- in
a in LPS-stimulated RAW 264.7 cells.
a
LPS-stimulated RAW 264.7 cells. To clarify the active compounds
from the leaves of C. asiatica caused this inhibition, one new ur-
sane-type triterpenoid glycoside (1), five triterpenoids, asiaticoside
(2),10 asiaticoside F (3),6 asiatic acid (4),16 quadranoside IV (5),17
and
rhamnopyranosyl-(1?4)-b-
pyranosyl] ester (6),7 and four flavonoids, kaempferol (7),10
2
a
,3b,6b-trihydroxyolean-12-en-28-oic acid 28-O-[
a-L-
D
-glucopyranosyl-(1?6)-b- -gluco-
D
⇑
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 Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.066

1778
N. X. Nhiem et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1777–1781
quercetin (8),10 astragalin (9),18 and isoquercetin (10)18 (see Fig. 1)
isolated from the leaves of C. asiatica,19 were tested their inhibitory
methyl carbon at dC 17.4, two oxymethine carbons at dC 69.6 and
77.9, two oxymethylene carbons at dC 65.8 and 66.3, and one car-
boxyl signal at dC 177.9. The 1H and 13C NMR data of 1 were similar
to those of asiaticoside (2),10 except for a difference of methyl
group at C-20. In the heteronuclear multiple bond correlation
(HMBC) spectrum (see Fig. 2), the methyl group H-24 (dH 0.67) cor-
related with carbons C-3 (dC 77.9), C-4 (dC 44.1), and C-23 (dC 66.3).
The large coupling constant J23 = 10.8 Hz of the two protons at C-2
and C-3 and the chemical shifts of C-2 (dC 69.6) and C-3 (dC 77.9) in
the aglycone confirmed the equatorial position of the 2- and 3-hy-
droxyl groups in comparison with the corresponding data for
effects on NO and TNF-
cells.
a production in LPS-stimulated RAW 264.7
By using combined chromatographic separations, one new
ursane-type triterpenoid glycoside together with nine known com-
pounds were isolated from the methanol extract of C. asiatica
leaves.20 Compound 1 was obtained as a white amorphous powder.
Its basic ion peak at m/z 975.5 [M+H]+ was observed on positive-
ion electrospray ionization mass spectrometry (ESI-MS), and
high-resolution electronspray ionization mass spectroscopy
(HR-ESI-MS) analysis revealed the molecular formula to be
2b,3
tween H-2 and H-3, J23 = 3.0 Hz, and dC values for C-2 (66.6) and
C-3 (78.6)] and methyl 2 ,3
,24-trihydroxyurs-12-en-28-oate22
a
,23-trihydroxyurs-12-en-28-oic acid21 [coupling constant be-
C
48H78O20, with a cluster ion peak at m/z 975.5160 [M+H]+ (calcd
for C48H79O20: 975.5165). The 1H NMR spectrum of 1 (in metha-
nol-d4) showed the following signals: four tertiary methyl groups
at dH 0.67, 0.81, 1.03, and 1.10 (each 3H, s); two secondary methyl
groups at dH 0.90 (d, 6.0 Hz) and 1.24 (d, 6.6 Hz); and three ano-
meric protons were at dH 4.35, 4.82, and 5.26, suggesting the pres-
ence of three sugar units (see Table 1). The 13C NMR and
distortionless enhancement by polarization transfer (DEPT) spectra
revealed 48 carbon signals, of which, 30 were assigned to a
triterpenoid sapogenol moiety and 18 belonged to three monosac-
charide moieties. The aglycone of 1 was recognized to be an
ursane-type triterpenoid on 1H and 13C NMR analysis (see Table 1),
with the typical olefinic carbons at dC 126.9 and 139.4, four quater-
nary methyl carbons at dC 13.9, 17.8, 18.1, and 24.0, one secondary
a a
[coupling constant between H-2 and H-3, J23 = 3.0 Hz, and dC values
for C-2 (66.2) and C-3 (73.3)]. This was further confirmed by corre-
lation between proton signal at dH 3.67 (H-2) and proton signal at
dH 1.03 (H-25). Moreover, the chemical shifts of C-3 (dC 77.9) and
C-23 (dC 66.3) were quite difference from those reported for
2a
,3b,24-trihydroxyurs-12-en-28-oic acid 28-O-[
a-
L-rhamnopyr-
anosyl-(1?4)-b- -glucopyranosyl-(1?6)-b-
D
D-glucopyranosyl] es-
ter (scheffursoside F)23 [dC values for C-3 (85.7) and C-24 (65.7)].
These results suggested that there was a hydroxyl group at C-23,
which was confirmed by a cross peak between H-24 (dH 0.67)
and H-25 (dH 1.03) in the rotating overhouser effect spectroscopy
(ROESY) spectrum. Furthermore, in the HMBC spectrum, the
R2
30
29
18
28
O
O
H
H
R1
HO
9
OR3
OSugar
3
HO
HO
OH
OH
24
23
R1
OH
OH
H
R2
OH
H
R3
6
1
2
3
4
5
Sugar
Sugar
Sugar
H
Glc
R2
OH
O
H
OH
OH
H
H
HO
OH
O
Sugar:
O
OH
OH
HO
O
HO
O
O
HO
O
Me
HO
HO
OR1
OH
OH
R1
R2
7
8
9
H
H
H
OH
H
O
HO
HO
Glc:
Glc
Glc
HO
OH
10
OH
Figure 1. Structure of compounds (1–10) isolated from leaves of C. asiatica.

N. X. Nhiem et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1777–1781
1779
Table 1
OH
The NMR spectral data of compound 1
Position
1
a,b
a,c
dC
dH (mult., Hz)
Aglycone
1
48.1
1.25 (m)
1.94 (m)
3.67 (dt, 3.6, 10.8)
O
H
2
3
4
5
6
7
69.6
77.9
44.1
48.3
19.1
33.6
HO
3.49 (d, 10.8)
O
—
0.87*
HO
OH
O
1.42 (m)
1.27 (m)
1.60*
—
OH
glc
8
9
41.0
48.9
39.0
24.5
126.9
139.4
43.4
29.3
25.2
49.4
54.1
34.3
47.7
25.4
37.6
O
O
1.61*
HO
O
HO
OH
10
11
12
13
14
15
16
17
18
19
20
21
22
OH
1.95 (m)
5.22 (t, 3.0)
—
HO
glc'
O
Me
HO
1.08*, 1.89*
1.73 (m)
—
HO
rha
OH
2.21 (d, 11.5)
1.61 (m)
1.01 (m)
1.50*
Figure 2. Selected HMBC spectrum of compound 1.
1.58*
the absence of a correlation between H-30 (dH 3.52 and 3.62) and
H-18 (dH 2.21) indicated that the methyl and hydroxyl methyl
1.72*
23
66.3
3.23 (d, 12.6)
3.47 (d, 12.6)
0.67 (s)
1.03 (s)
0.81 (s)
groups at C-19 and C-20 had the b and
tively. Acid hydrolysis of 1 provided the monosaccharide compo-
nents of -rhamnose and -glucose (identified as TMS derivatives
a configurations, respec-
24
25
26
27
28
29
30
13.9
17.8
18.1
24.0
177.9
17.4
65.8
L
D
by gas chromatography (GC) method).24 Moreover, HMBC correla-
tions between the inner glc H-1 (dH 5.26) and C-28 of the aglycone
(dC 177.9), between the outer glc H-10 (dH 4.35) and the inner glc
C-6 (dC 69.7), and between the rha H-100 (dH 4.82) and the glc
C-40 (dC 79.6) were observed. These evidences suggested the
sequence of the sugar linkages of 1. The carbon signals of the
sugar moieties were superimposable on those of characteristic
triterpenoid glycosides isolated from Centella species.6–8 Conse-
1.10 (s)
—
0.90 (d, 6.0)
3.52*
3.62 (dd, 3.0, 10.8)
C-28 O-glc
1
2
3
4
5
6
95.8
73.8
78.3
71.0
78.2
69.7
5.26 (d, 7.8)
3.38 (m)
3.32 (m)
3.37 (m)
3.38 (m)
quently, the structure of 1 was determined to be 2
hydroxyurs-12-en-28-oic acid 28-O-[ -rhamnopyranosyl-(1?4)-
b- -glucopyranosyl-(1?6)-b- -glucopyranosyl] ester, named asia-
ticoside G.25
a,3b,23,30-tetra-
a
-L
D
D
3.76 (dd, 4.2, 12.0)
4.05 (d, 12.0)
To evaluate the inhibitory effects of the isolates on the pro-
duction of NO in the LPS-stimulated RAW 264.7 cells, cells were
seeded in 96-well plates at a density of 1 ꢀ 105 cells/mL. After
3 h, the cells were treated with various concentrations of the
glc0(10?6)glc
10
20
30
40
50
60
104.5
75.3
76.8
79.6
76.7
61.9
4.35 (d, 7.8)
3.21 (t, 7.8)
3.27*
3.51 (t, 7.8)
3.42 (m)
compounds (1, 10, and 100
lM) and stimulated for 24 h with
or without 1 g/mL of LPS (Sigma Chemical Co., St. Louis, MO,
l
3.62 (dd, 4.2, 11.4)
3.79 (d, 11.4)
USA). The nitrite concentration was measured in the supernatant
of RAW 264.7 cells by the Griess reation.26 In absence of both
test the compounds and LPS, NO was produced at a very low
rha(100?40)glc0
100
200
300
400
500
600
102.8
72.4
72.2
73.7
70.7
17.9
4.82 (d, 1.8)
3.81 (m)
3.60 (dd, 2.7, 9.0)
3.30 (m)
3.93 (dd, 6.6, 9.6)
1.24 (d, 6.6)
concentration (0.87 0.11
production by concentration of 6.09 0.50
dexamethasone (10 M), positive control, resulted in a level of
1.54 0.07 M and compounds 1, 2, 4, and 7–10 (1, 10, and
100 M) significantly decreased the NO production to levels be-
tween 1.38 0.26 to 5.16 0.52 M (see Fig. 3). One oleane-type
lM). LPS markedly induced the NO
lM. In contrast, the
l
l
l
a
b
c
Measured in CD3OD.
150 MHz.
600 MHz.
l
triterpenoid glycoside (6) and two ursane-type triterpenoid gly-
*
Overlapped signals, assignments were done by HMQC, HMBC, COSY and ROESY
experiments; glc, b- -glucopyranosyl; rha, -rhamnopyranosyl.
cosides (3, 5) showed no activity at the concentration of
D
L
100
cantly inhibited production of NO, we investigated the effects
of these compounds on the secretion of TNF- in LPS-stimulated
RAW 264.7 cells.27 LPS markedly induced the TNF-
production,
to a concentration of 80.07 1.00 ng/mL. In contrast, the dexa-
methasone (10 M), positive control, resulted in level of
27.01 0.73 ng/mL and compounds 1, 2, 4, 5, and 7–10 (1, 10,
lM (IC50 >100 lM). Since isolates from C. asiatica signifi-
a
methyl group H-29 (dH 0.90) correlated with carbons C-18 (dC
54.1), C-19 (dC 34.3), and C-20 (dC 47.7), and the hydroxymethyl
group H-30 (dH 3.52 and 3.62) correlated with carbons C-19 (dC
34.3), C-20 (dC 47.7), and C-21 (dC 25.4). In the ROESY spectrum,
the cross peak between H-18 (dH 2.21) and H-29 (dH 0.90) and
a
l
a

1780
N. X. Nhiem et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1777–1781
Table 2
10
8
1 μM
10 μM
IC50 values of the compounds for NO and TNF-
264.7 cells
a production in LPS-stimulated RAW
100 μM
Compounds
IC50 (lM)
NO
8.4 1.1
67.9 18.4
>100
TNF-a
1
2
3
35.3 2.1
85.7 1.3
>100
6
4
5
6
8.6 1.8
70.2 10.3
>100
74.8 2.7
86.4 4.9
>100
4
7
8
9
10
60.2 36.3
63.0 27.9
64.0 19.4
61.2 17.2
0.8 0.07
80.3 8.5
66.6 3.0
67.1 8.9
76.3 2.6
0.01 0.0001
2
Dexsamethasone*
*
Dexsamethasone was used as positive control. Data presented is the mean S.D.
of samples run in triplicate.
0
Compounds
100 lM in the presence or absence of LPS using MTT assays. It
Figure 3. Effect of isolated compounds on NO production in RAW 264.7 cells. The
cells were seeded in 96 well plates at a density of 1 ꢀ 105 cells/well. After 3 h, the
was found that neither compound affected the viability of RAW
264.7 cells after 24 h (data not shown), indicating that their inhib-
itory effects were not due to any cytotoxic effects. Our results
showed that ursane-type triterpenoids (1–4) from C. asiatica, ex-
cept compound 3 with absence hydroxyl group at C-2, suppressed
cells were treated with various concentrations of compounds (1, 10, and 100 lM),
and then LPS (1 lg/mL) was added and the cells were incubated for 24 h.
Dexamethasone was used as
a positive control. The values represent the
means S.E.M. from three independent experiments. ⁄p <0.05, ⁄⁄p <0.001 versus
LPS-treated group.
the production of NO and secretion of TNF-a in LPS-stimulated
RAW 267.4 cells. Thus, the isolated ursane-type triterpenoid glyco-
sides might be important anti-inflammatory constituents of this
plant. Among the tested compounds, asiaticoside G (1), with a hy-
droxyl group at C-30, had the most potent the anti-inflammatory
100
1 μM
10 μM
100 μM
effect, with IC50 of 8.4 and 35.3
lM for the production of NO and
80
secretion of TNF- , respectively (see Table 2). The in vivo anti-
a
**
inflammatory effects of asiaticoside G (1) need to be further
studied.
**
*
**
**
**
*
**
60
40
20
0
**
**
**
**
**
**
**
Acknowledgements
**
**
**
**
**
This study was supported by the Priority Research Center Pro-
gram through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(2009-0093815), Republic of Korea. The authors thank the Korean
Basic Science Institute (KBSI) for taking NMR and MS experiments.
**
**
**
**
**
References and notes
Compounds
1. Sinha, B.; Eigler, A.; Baumann, K. H.; Greten, T. F.; Moeller, J.; Endres, S. Res.
Immunol. 1998, 149, 139.
2. Wang, C.; Levis, G. B. S.; Lee, E. B.; Levis, W. R.; Lee, D. W.; Kim, B. S.; Park, S. Y.;
Park, E. Int. Immunopharmacol. 2004, 4, 1039.
3. Ishibe, K.; Yamanishi, T.; Wang, Y.; Osatomi, K.; Hara, K.; Kanai, K.; Yamaguchi,
K.; Oda, T. Fish Shellfish Immunol. 2009, 27, 386.
4. Palladino, M. A.; Bahjat, F. R.; Theodorakis, E. A.; Moldawer, L. L. Nat. Rev. Drug
Discovery 2003, 2, 736.
5. Bich, D. H.; Chung, D. Q.; Chuong, B. X.; Dong, N. T.; Dam, D. T.; Hien, P. V.; Lo, V.
N.; Mai, P. D.; Man, P. K.; Nhu, D. T.; Tap, N.; Toan, T. The Medicinal Plants and
Animals in Vietnam; Hanoi Science and Technology Publishing House: Hanoi,
2004.
Figure 4. Effect of isolated compounds on TNF-
a production in RAW 264.7 cells.
The cells were seeded in 96 well plates at a density of 1 ꢀ 104 cells/well. After 1 h,
the cells were treated with various concentrations of compounds (1, 10, and
100 lM), and then LPS (1 lg/mL) was added and the cells were incubated for 18 h.
Dexamethasone was used as
a positive control. The values represent the
means S.E.M. from three independent experiments. ⁄p <0.05, ⁄⁄p <0.001 versus
LPS-treated group.
6. Jiang, Z. Y.; Zhang, X. M.; Zhou, J.; Chen, J. Helv. Chim. Acta 2005, 88, 297.
7. Kuroda, M.; Mimaki, Y.; Harada, H.; Sakagami, H.; Sashida, Y. Nat. Med. 2001,
55, 134.
8. James, J. T.; Dubery, I. A. Molecules 2009, 14, 3922.
9. Govindan, G.; Sambandan, T. G.; Govindan, M.; Sinskey, A.; Vanessendelft, J.;
Adenan, I.; Rha, C. K. Planta Med. 2007, 73, 597.
10. Satake, T.; Kamiya, K.; An, Y.; Oishi, T.; Yamamoto, J. Biol. Pharm. Bull. 2007, 30,
935.
11. Won, J. H.; Shin, J. S.; Park, H. J.; Jung, H. J.; Koh, D. J.; Jo, B. G.; Lee, J. Y.; Yun, K.;
Lee, K. T. Planta Med. 2010, 76, 251.
12. Li, H.; Gong, X.; Zhang, L.; Zhang, Z.; Luo, F.; Zhou, Q.; Chen, J.; Wan, J.
Phytomedicine 2009, 16, 538.
13. Krishnamurthy, R. G.; Senut, M. C.; Zemke, D.; Min, J.; Frenkel, M. B.;
Greenberg, E. J.; Yu, S. W.; Ahn, N.; Goudreau, J.; Kassab, M.; Panickar, K. S.;
Majid, A. J. Neurosci. Res. 2009, 87, 2541.
and 100 lM) significantly decreased TNF-a secretion to levels
from 78.77 1.14 to 24.78 1.28 ng/mL (see Fig. 4).
In the structure–activity relationship of ursane triterpenoids
(1–5), the presence of the hydroxyl group at C-30 affected NO pro-
duction and increase TNF-a activity. In contrast, the presence of
sugar moieties at C-28 decreased activity; as previous reported,
the aglycones were more active than their glycosides.11,14 Specifi-
cally, the absence a hydroxyl group at C-2 (compound 3) caused
inactivity. When compared activity of flavonoids (7–10), the pres-
ence of the hydroxyl group at C-30 or glucose moiety at C-3 affected
on NO and TNF-
a production decrease activity. We also examined
the cytotoxic effects of isolated compounds at concentration of

N. X. Nhiem et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1777–1781
1781
14. Yun, K. J.; Kim, J. Y.; Kim, J. B.; Lee, K. W.; Jeong, S. Y.; Park, H. J.; Jung, H. J.; Cho,
Y. W.; Yun, K.; Lee, K. T. Int. Immunopharmacol. 2008, 8, 431.
CA2E1ꢁCA2E3. Fraction CA2E1 was chromatographed on a silica gel column
eluting with CHCl3/MeOH/H2O (55:10:1, v/v/v) to yield compound 9 (5.0 mg).
Fraction CA2E2 was chromatographed on a silica gel column eluting with
CHCl3/MeOH/H2O (35:10:1, v/v/v), yielding compound 10 (7.0 mg).
15. Guo, J. S.; Cheng, C. L.; Koo, M. W. L. Planta Med. 2004, 70, 1150.
16. Aguirre, M. C.; Delporte, C.; Backhouse, N.; Erazo, S.; Letelier, M. E.; Cassels, B.
K.; Silva, X.; Alegria, S.; Negrete, R. Bioorg. Med. Chem. 2006, 14, 5673.
17. Adnyana, I. K.; Tezuka, Y.; Banskota, A. H.; Xiong, Q.; Tran, K. Q.; Kadota, S. J.
Nat. Prod. 2000, 63, 496.
21. Ahmad, V. U.; Bano, S.; Bano, N. Phytochemistry 1986, 25, 1487.
22. Kojima, H.; Tominaga, H.; Sato, S.; Ogura, H. Phytochemistry 1987, 26, 1107.
23. Maeda, C.; Ohtani, K.; Kasai, R.; Yamasaki, K.; Duc, N. M.; Nham, N. T.; Cu, N. K.
Q. Phytochemistry 1994, 37, 1131.
24. 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
18. Han, J. T.; Bang, M. H.; Chun, O. K.; Kim, D. O.; Lee, C. Y.; Baek, N. I. Arch.
Pharmacol. Res. 2004, 27, 390.
19. The sample of Centella asiatica (L.) was collected at Tam Dao National Park,
Vinh Phuc, Vietnam, during June 2009 and identified by Dr. Ninh Khac Ban
(Institute of Ecology and Biological Resources, VAST). A voucher specimen
(IMBC CA-0609) was deposited at the Herbarium of Institute of Marine
Biochemistry, VAST.
dry pyridine, and then L-cysteine methyl ester hydrochloride in pyridine
20. The dried leaves of C. asiatica (3.0 kg) were extracted with MeOH three times
under reflux for 15 h to yield 250 g of a dark solid extract. This extract was
suspended in water and partitioned with ethyl acetate to yield ethyl acetate
extract (CA1, 100 g) and water extract (CA2, 150 g). The ethyl acetate extract
(CA1) was then subjected to chromatography on a silica gel column eluting
with a gradient of CHCl3/MeOH (from 50:1 to 5:1 v/v) yielded five fractions
CA1AꢁCA1E. Fraction CA1A was chromatographed on an LH-20 column eluting
with CHCl3/MeOH (1:1 v/v) to yield 7 (30.5 mg) and 8 (84.7 mg). Fraction CA1C
was chromatographed on a silica gel column using CHCl3/MeOH (10:1, v/v) as
an eluent to give fractions CA1C1ꢁCA1C4. The CA1C3 fraction was
chromatographed on an YMC RP-18 column eluting with MeOH/H2O (5:1 v/
v) to give 4 (60.0 mg). Fraction CA1C4 was chromatographed on an YMC RP-18
column eluting with MeOH/H2O (4:1 v/v) to yield 5 (10.5 mg). The H2O soluble
fraction (CA2) was chromatographed on a Diaion HP-20P column (Mitshubishi
Chemical Co., Japan) eluted with a step gradient of MeOH in water (0%, 25%,
50%, 75%, and 100% MeOH) yielding the five fractions, CA2AꢁCA2E. The
fraction CA2B was chromatographed on a silica gel column eluting with CHCl3/
MeOH/H2O (75:20:3, v/v/v) yielding the four fractions, CA2B1ꢁCA2B4. Fraction
CA2B2 was chromatographed on a silica gel column eluting with CHCl3/MeOH/
(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,
followed by heating at 60 °C for 1.5 h. The dried product was partitioned with
n-hexane and H2O (0.1 mL, each), and the organic layer was analyzed by gas
liquid chromatography (GC): Column: column SPB-1 (0.25 mm ꢀ 30 m);
detector FID, column temp 210 °C, injector temp 270 °C, detector temp
300 °C, carrier gas He (2.0 mL/min). The retention times of persilylated
glucose and rhamnose were founded to be 14.11 and 4.50 min, respectively,
when compared with the standard solutions prepared by the same reaction
from the standard monosaccharides. (The retention times of persilylated
D-
glucose, -glucose, and -rhamnose were 14.11, 14.26, and 4.50 min,
L
L
respectively).
25. White amorphous powder; ½a D25
ꢂ
ꢁ18° (c 0.1, MeOH); UV kmax (log
e
, MeOH)
;
211 (1.75) nm; IR (KBr) nmax 3407, 2954, 1728, 1231, and 1064 cmꢁ1
1H and
13C NMR are given in Table 1; ESI-MS m/z 975 [M+H]+, 997 [M+Na]+;
HR-ESI-MS m/z 975.5160 [M+H]+ (calcd for C48H79O20: 975.5165).
26. The nitrite, which accumulated in the culture medium, was measured as an
indicator of NO production by means of the Griess reaction. Briefly, 100 mL of
cell culture medium (without phenol red) was mixed with an equal volume of
Griess reagent (equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric
acid and 0.1% (w/v) naphthylethylenediamine-HCl), incubated at room
temperature for 10 min, and then the absorbance was measured at 550 nm
using a microplate reader. Fresh culture medium was used as the blank in all
experiments. The amount of nitrite in the samples was obtained by means of
the NaNO2 serial dilution standard curve and the nitrite production was
measured.
H2O (75:20:3, v/v/v) yielded compound
6 (120.9 mg). Fraction CA2C was
chromatographed on an YMC RP-18 column eluting with acetone/H2O (2:1, v/
v) to yield four sub-fractions, CA2C1ꢁCA2C4. Fraction CA2C1 was
chromatographed on
a
silica gel column (50 g, 2 ꢀ 50 cm) eluting with
CHCl3/MeOH/H2O (30:10:1, v/v/v), yielding compound 1 (7.1 mg). Fraction
CA2C2 was further chromatographed on a silica gel column (50 g, 2 ꢀ 50 cm)
eluting with CH2Cl2/MeOH/H2O (35:10:1, v/v/v), yielding compounds
2
(10.2 mg) and 3 (8.0 mg). Fraction CA2E was chromatographed on an YMC
27. TNF-a production in the supernatant of RAW 264.7 cells was quantified using
RP-18 column eluting with acetone/H2O (1:1, v:v) to yield three fractions,
an OptEIA™ assay kits according to the manufacturer’s instructions.