Triterpene glucosides from the leaves of aralia elata and their cytotoxic activities

Article abstract of DOI:10.1002/cbdv.201200087

Three new triterpene glucosides, named congmuyenosides C-E (1-3, resp.), along with four known ones, were isolated from an EtOH extract of Aralia elata (Miq.) Seem. leaves. The structures of the new compounds were identified as 3-O-{β-D-glucopyranosyl-(1→

Full text of DOI:10.1002/cbdv.201200087

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
703
Triterpene Glucosides from the Leaves of Aralia elata and Their Cytotoxic
Activities
a
a
a
a
a
by Hai-Xue Kuang* ), Zhi-Bin Wang ), Qiu-Hong Wang ), Bing-You Yang ), Hong-Bin Xiao ),
b
b
Yoshihito Okada ), and Tohru Okuyama )
a) Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of
Chinese Medicine, No. 24 Heping Road, Xiangfang District, Harbin 150040, P. R. China
(
phone: þ86-451-82193001; fax: þ86-451-82110803; e-mail: hxkuang@hotmail.com)
b
)
Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan
Three new triterpene glucosides, named congmuyenosides C–E (1–3, resp.), along with four known
ones, were isolated from an EtOH extract of Aralia elata (Miq.) Seem. leaves. The structures of the new
compounds were identified as 3-O-{b-d-glucopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-[b-d-gluco-
pyranosyl-(1!2)]-b-d-glucopyranosyl}caulophyllogenin (1), 3-O-{b-d-glucopyranosyl-(1!3)-b-d-glu-
copyranosyl-(1!3)-[b-d-glucopyranosyl-(1!2)]-b-d-glucopyranosyl}hederagenin 28-O-b-d-glucopyr-
anosyl ester (2), 3-O-{b-d-glucopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-[b-d-glucopyranosyl-(1!
2
)]-b-d-glucopyranosyl}echinocystic acid 28-O-b-d-glucopyranosyl ester (3) on the basis of spectral
1
13
analyses, including MS, H-NMR, C-NMR, DEPT, HSQC, HMBC, NOESY, and HSQC-TOCSY
experiments. All isolates obtained were evaluated for their cytotoxic activities against three human
tumor cell lines (HepG2, SKOV3, and A549). Compound 3 showed significant cytotoxicity against A549
cell line (IC50 9.9ꢀ1.5 mm).
Introduction. – The young leaves of Aralia elata (Miq.) Seem. (Araliaceae,
CiLaoYa in Chinese) are cultivated as a vegetable crop in Northeast China.
Additionally, the bark of its root cortex is widely used in folk medicine due to its
tonic, antiarthritic, and antidiabetic effects [1]. Regarding the biologically active
constituents of A. elata, many triterpenes were reported to have antidiabetic activities
[
2], cytoprotective effects on CCl -induced hepatic injury [3], EtOH absorption-
4
inhibitory effects [4][5], and hypoglycemic activities [6–8]. Previously, our studies
revealed that the growth of H tumor on mice and A549 transplantable tumor in vitro
22
in nude mice could be inhibited by the total saponins of A. elata leaves [9][10]. To
further investigate the constituents and to screen bioactive compounds from its leaves,
a photochemical investigation on the leaves of this plant has now led to the isolation
and structure elucidation of three new triterpenes, 1–3, and four known triterpenes, 4–
7
. Known compounds were identified by detailed 1D- and 2D-NMR analyses,
comparison of their ESI-MS and spectral data with those reported in literature [11–
3] as congmuyenoside A (4), congmuyenoside B (5), congmuyanoside B (6), and
1
aralia-saponin VI (7). The cytotoxic effects of triterpenes 1–7 against human
hepatoma (HepG2), human ovarian carcinoma (SKOV3), and human lung epithelial
carcinoma cells (A549) were evaluated by means of the MTT (¼ 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The results indicated that all
the saponins inhibited the growth of HepG2, SKOV3, and A549 cells.
ꢀ
2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Results and Discussion. – Compound 1, named congmuyenoside C, was obtained as
white amorphous powder (MeOH). The molecular formula was determined as
þ
C H O from the [MþNa] signal at m/z 1159.5515 by HR-FAB-MS. The IR
54
88 25
ꢁ
1
spectrum indicated the presence of an ester C¼O group (1740 cm ) and an olefinic
ꢁ
1
1
moiety (1640 cm ). The H-NMR spectrum of 1 (Table 1) showed signals of six Me
groups (s at d(H) 0.89, 1.02 (6 H), 1.04, 1.15, and 1.77) and one olefinic H-atom (5.61
(
br. s)). The four typical downfield CH doublets at d(H) 4.97 (d, J¼7.6), 5.63 (d, J¼
7.9), 5.29 (d, J¼7.9), and 5.14 (d, J¼7.6) were assigned to the anomeric H-atoms of the
13
b-d-glucopyranosyl moieties. The C-NMR spectrum of 1 (Table 2) showed signals of a
pair of olefinic C-atoms (d(C) 122.4 and 145.2), four anomeric C-atoms (d(C) 103.6,
1
03.8, 104.0, and 105.2), and a C¼O group (d(C) 180.1). On acid hydrolysis, 1 yielded
only d-glucose as a sugar component by GC analysis using a hydrogen flame detector
after treatment with l-cysteine methyl ester hydrochloride in pyridine [14], and
cauiophyllogenin was obtained and identified as the aglycone on the basis of its NMR
data and TLC by comparison with authentic samples.
1
13
1
1
The H- and C-NMR signals of the aglycone part were assigned by HSQC, H, H-
COSY, and HSQC-TOCSY experiments. The connectivities within the aglycone were
deduced from the HMBCs (Fig.). In particular, the key long-range correlations
Me(24)/C(3), C(4), C(5), and C(23), Me(25)/C(1), C(5), C(9), and C(10), Me(26)/
C(7), C(8), C(9), and C(14), Me(27)/C(8), C(13), C(14), and C(15), and Me(29)/
C(19), C(20), C(21), and C(30) evidenced that aglycone of 1 is the triterpene
cauiophyllogenin.
The interglucosidic linkages were deduced from HMBC, HSQC-TOCSY, and ESI-
2
1
MS results. In the HMBC spectrum, the cross-peaks HꢁC(1) of Glc /C(2) of Glc ,
3
1
4
3
HꢁC(1) of Glc /C(3) of Glc , and HꢁC(1) of Glc /C(3) of Glc were observed. The
correlations from the anomeric H-atom of the glucosyl moiety at d(H) 4.97 to d(C)
8
3.0 could also be observed, which confirmed this unit to be at C(3). Furthermore, the
ꢁ
ꢁ
characteristic ion peaks at m/z 1135 ([MꢁH] ), 973 ([MꢁGlcꢁH] ), 811 ([Mꢁ
GlcꢁH] ), 649 ([Mꢁ3GlcꢁH] ), and 487 ([Mꢁ4GlcꢁH] ) in the negative-ion-
mode ESI-MS confirmed the above sugar linkages. In the HSQC-TOCSY spectrum,
ꢁ
ꢁ
ꢁ
2
1
1
the key correlations HꢁC(1) of Glc /C(2), C(3), C(4), and C(5) of Glc , HꢁC(1) of
2
2
3
Glc /C(2), C(3), C(4), and C(5) of Glc , HꢁC(1) of Glc /C(2), C(3), C(4), and C(5) of
3
4
4
Glc , HꢁC(1) of Glc /C(2), C(3), C(4), and C(5) of Glc established the interglucosidic
linkages. Thus, the structure of 1 was established as 3-O-{b-d-glucopyranosyl-(1!3)-b-

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
705

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
707
1
1
Figure. Key HMB (H!C) and H, H-COSY (—) correlations of 1
d-glucopyranosyl-(1!3)-[b-d-glucopyranosyl-(1!2)]-b-d-glucopyranosyl}caulophyl-
logenin.
Compound 2, named congmuyenoside D, was obtained as white amorphous powder
with a molecular formula C H O , based on the HR-FAB-MS (m/z 1305.6095 ([Mþ
6
0
98 29
þ
1
Na] )). The H-NMR spectrum of 1 (Tables 1 and 2) showed signals of six Me groups
(s at d(H) 0.86, 0.87, 0.88, 1.03, 1.08, and 1.16), an olefinic H-atom (d(H) 5.39 (br. s)),
and five anomeric CH doublets (d(H) 4.97 (d, J¼7.9), 5.12 (d, J¼7.9), 5.28 (d, J¼7.9 ) ,
1
3
5
.63 (d, J¼7.7), and 6.29 (d, J¼7.9 )) . The C-NMR spectrum (Table 1) exhibited 60 C-
atom signals that were attributed by a DEPTexperiment to six Me, 15 CH , and 31 CH
2
groups, as well as eight quaternary C-atoms, including two olefinic C-atoms (d(C) 122.8
and 144.1), five anomeric C-atoms (d(C) 103.7, 103.5, 103.9, 105.1, and 95.7), and an
ester C¼O group (d(C) 176.4).
On acid hydrolysis, 2 only yielded hederagenin as the aglycone and d-glucose as
sugar component, which were identified by comparison with authentic samples. On the
1
1
basis of the 2D-NMR ( H, H-COSY, HMQC, and HSQC-TOCSY) experiments, the
spin systems of the five sugar moieties were assigned as compiled in Table 2. In the
2
1
3
HMBC spectrum, cross-peaks HꢁC(1) of Glc /C(2) of Glc , HꢁC(1) of Glc /C(3) of
1
3
1
4
3
5
Glc , HꢁC(1) of Glc /C(3) of Glc , HꢁC(1) of Glc /C(3) of Glc , and HꢁC(1) of Glc /
C(28) were observed. Moreover, the significant fragment ions observed at m/z 1281
ꢁ
ꢁ
ꢁ
ꢁ
(
[MꢁH] ), 1119 ([MꢁGlcꢁH] ), 957 ([Mꢁ2GlcꢁH] ), 795 ([Mꢁ3GlcꢁH] ),
ꢁ
ꢁ
6
33 ([Mꢁ4GlcꢁH] ) and 471 ([Mꢁ5GlcꢁH] ) in the negative-ion-mode ESI-MS,
confirmed the linkages of the sugar units. Hence, 2 was determined as 3-O-{b-d-
glucopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-[b-d-glucopyranosyl-(1!2)]-b-d-
glucopyranosyl}hederagenin 28-O-b-d-glucopyranosyl ester.
Congmuyenoside E (3) was obtained as white amorphous powder. The molecular
formula was determined as C H O by positive-ion-mode HR-FAB-MS (m/z
6
0
98 29
þ
1
305.6090 ([MþNa] )). An acid hydrolysis of 3 only liberated echinocystic acid
[
12][13] as the aglycone and d-glucose, which was identified by GC analysis [14]. The
1
H-NMR spectrum revealed the presence of seven tertiary Me groups (d(H) 0.86, 1.01,
.05, 1.08, 1.12, 1.23, and 1.84) and an olefinic H-atom (d(H) 5.61 (br. s)). The C-NMR
13
1

7
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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
spectrum (Tables 1 and 2) showed 60 C-atom signals that were assigned to seven Me,
1
3 CH , 32 CH groups, and eight quaternary C-atoms by a DEPT experiment. In
2
1
addition to the above-mentioned signals, the H-NMR spectrum of 3 exhibited
resonances in the downfield region due to the five anomeric H-atoms at d(H) 4.82, 5.64,
5.36, 5.14, and 6.32 that correlated in the HMQC experiment with the corresponding C-
atoms at d(C) 104.9, 103.6, 104.1, 105.1, and 95.9, respectively. Along with mass spectra,
these data revealed the presence of five monosaccharide residues in 3, and the
anomeric configurations of the sugar moieties were determined as b on the basis of the
2
J(H,H) values (7.0–8.0 Hz). In the HMBC spectrum, the cross-peaks HꢁC(1) of Glc /
1
3
1
3
1
C(2) of Glc , HꢁC(1) of Glc /C(3) of Glc , HꢁC(1) of Glc /C(3) of Glc , HꢁC(1) of
4
3
5
Glc /C(3) of Glc , and HꢁC(1) of Glc /C(28) confirmed the interglucosidic linkages.
ꢁ
Moreover, the significant fragment-ion peaks observed at m/z 1281 ([MꢁH] ), 1119
ꢁ
ꢁ
ꢁ
(
[MꢁGlcꢁH] ), 957 ([Mꢁ2GlcꢁH] ), 795 ([Mꢁ3GlcꢁH] ), 633 ([Mꢁ4Glcꢁ
ꢁ ꢁ
H] ), and 471 ([Mꢁ5GlcꢁH] in the negative-ion-mode ESI-MS confirmed the
sugar linkages. Accordingly, compound 3 was identified as 3-O-{b-d-glucopyranosyl-
(
1!3)-b-d-glucopyranosyl-(1!3)-[b-d-glucopyranosyl-(1!2)]-b-d-glucopyranosyl}-
echinocystic acid 28-O-b-d-glucopyranosyl ester.
The cytotoxic activities of compounds 1–7 against HepG2, SKOV3, and A549 cell
lines were evaluated by MTT method [15–17]. As showed in Table 3, these compounds
showed only marginal cytotoxic activities against all three tested cell lines.
Table 3. Cytotoxicities of Compounds 1–7
Compound
IC50 [mm]a)
HepG-2
Skov3
A549
1
2
3
4
5
6
7
39.7ꢀ7.9
62.4ꢀ12.8
29.4ꢀ8.3
82.4ꢀ8.5
128.4ꢀ28.1
17.4ꢀ3.7
25.2ꢀ3.4
6.8ꢀ0.6
92.1ꢀ8.4
120.2ꢀ13.0
55.4ꢀ5.5
40.2ꢀ10.0
187.3ꢀ22.7
9.9ꢀ1.5
150.2ꢀ7.5
165.4ꢀ10.2
100.3ꢀ10.8
80.2ꢀ7.0
55.1ꢀ9.0
96.5ꢀ5.7
25.4ꢀ5.9
27.9ꢀ4.6
11.7ꢀ3.6
5
-Fluorouracilb)
16.1ꢀ4.6
a) MeanꢀSD, n¼3. ) The positive control, 5-fluorouracil, was purchased from Shanghai Parmaceutical
b
Industry Co. Ltd.
This work was financially supported by the International Science and Technology Cooperation
Project (No. 20072135) and the National Major Projects for Science and Technology Development
(
No. 2011ZX09102-001-018) from the Science and Technology Ministry of P. R. China.
Expermental Part
General. Column chromatography (CC): silica gel 60 (SiO ; 200–300 mesh; Merck, Germany).
2
HPLC: Waters Delta-600 pump and Waters 2414 refractive-index detector; TSK-gel ODS-120T column
(
0
10 mm, 40ꢂ300 mm, Tosoh, Japan). GC: Fuli-9790 hydrogen flame detector; DM-5 (0.25 mm, 30 mꢂ
ꢁ
1
.25 mm; Dikma, China). IR Spectra: Jasco FT/IR-230 infrared spectrometer; ˜n in cm . NMR Spectra:
JNM LA-500 spectrometer; d in ppm rel. to Me Si as internal standard, J in Hz. ESI-MS: Perkin-Elmer
4

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
709
SCIEX API III biomolecular mass analyzer; in m/z. HR-FAB-MS: JEOL JMS DX-302 mass
spectrometer; in m/z.
Plant Materials. The leaves of A. elata were collected in Daxingꢂanling, Heilongjiang Province, P. R.
China, in April 1998 and identified by Prof. Zhenyue Wang at Heilongjiang University of Chinese
Medicine, P. R. China. A voucher specimen (No. 19980006) is deposited with the Herbarium of
Heilongjiang University of Chinese Medicine, P. R. China.
Extraction and Isolation. The fresh leaves of A. elata (7.8 kg) were extracted with 70% EtOH under
reflux (3ꢂ30 l) for 2 h, and the combined extracts were filtered and evaporated in vacuo to a syrup,
which was suspended in H O (5 l), and extracted with petroleum ether (3ꢂ3 l). The aq. layer was
2
evaporated. The residue (830 g) was suspended in H O (2 l), and the suspension was subjected to AB-8
2
cross-linked polystyrene CC, sequentially eluted with H O, 30% EtOH, and 70% EtOH. The 70% EtOH
2
elution fraction was concentrated under vacuum to yield a syrup residue, and this crude residue (170 g)
was then subjected to CC (SiO ; CHCl /MeOH 10 :1!1:1 gradient) to give five fractions, Frs. 1–5.
2
3
CC (SiO ; CHCl /MeOH 10 :1!2 :1; and ODS; MeOH/H O 1:1!8 :2) of Fr. 2 (22 g) yielded
2
3
2
three yellow fractions, Frs. A –A (385, 385, and 430 mg, resp.), which were purified by prep. HPLC
1
3
(
3
TSK-gel ODS-120T column (10 mm, 40ꢂ300 mm, flow rate 8 ml/min; MeOH/H O 4 :6) to afford 1 (t
2
R
2.5 min; 95 mg) from Fr. A , 2 (t 36.8 min; 82 mg) from Fr. A , and 3 (t 40.3 min; 79 mg) from Fr. A .
1 R 2 R 3
Compound 4 (37.1 mg) was obtained from Fr. 3 after CC (ODS; MeOH/H O 4 :6!9 :1) and HPLC
2
(
MeOH/H O 45 :55; t 25.5 min). Fr. 4 was separated by HPLC (MeOH/H O 45 :55) to give compounds
2 R 2
5
3
3
(tR 27.4 min; 37.5 mg) and 7 (tR 30.4 min; 26.6 mg). Fr. 5 was separated by CC (ODS; MeOH/H O
2
:7!9 :1) and HPLC (MeOH/H O 45 :55) to furnish compounds 6 (t 24.5 min; 25.1 mg) and 7 (tR
2
R
0.9 min; 10.6 mg).
Acid Hydrolysis of 1–3. Compounds 1–3 (each 3 mg) were hydrolyzed in 7% aq. H SO (1.0 ml) for
2
4
1
2 h at 808. The mixtures were cooled and partitioned between CHCl (2.0 ml) and H O (2.0 ml). The aq.
3
2
layers were washed with CHCl (3ꢂ3.0 ml), neutralized with Ba(OH) , filtered, and evaporated under
3
2
reduced pressure. The residues were dissolved in pyridine (1.0 ml), and l-cysteine methyl ester
hydrochloride in pyridine (0.1m, 2.0 ml) was added. The mixtures were heated at 608 for 1 h. An equal
volume of Ac O was added under heating, and incubation was continued for 1 h. The acetylated
2
thiazolidine derivatives were analyzed by GC with a DM-5 column (30 mꢂ0.25 mm, 0.25 mm) using
authentic samples as standards. Temps. of both injector and detector were 2808. A temp. gradient system
was used for the oven, starting at 1608 and increasing up to 1958 at a rate of 58/min. The determination of
the sugar units in compounds 1–3 were achieved by comparison of tR values of peaks between the
hydrolysate and authentic sample of d-glucose (tR 18.7 min).
Congmuyenoside C (¼(3b,16a)-3-{[b-d-Glucopyranosyl-(1!2)-[b-d-glucopyranosyl-(1!3)-b-d-
glucopyranosyl-(1!3)]-b-d-glucopyranosyl]oxy}-16,23-dihydroxyolean-12-en-28-oic Acid; 3-O-{b-d-
Glucopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-[b-d-glucopyranosyl-(1!2)]-b-d-glucopyranosyl}-
2
0
caulophyllogenin; 1). White amorphous powder. [a]
D
¼ þ23.1 (c ¼ 0.3, pyridine). IR (KBr): 3400, 2920,
1 13
1
(
(
740, 1640, 1550, 1460, 1394, 1300, 1260, 1080, 1035, 995. H- and C- NMR: Tables 1 and 2. ESI-MS
ꢁ
ꢁ
ꢁ
ꢁ
neg.): 1135 ([MꢁH] ), 973 ([MꢁGlcꢁH] ), 811 ([Mꢁ2GlcꢁH] ), 649 ([Mꢁ3GlcꢁH] ), 487
ꢁ
þ
þ
[Mꢁ4GlcꢁH] ). HR-FAB-MS (pos.): 1159.5515 ([MþNa] , C H NaO ; calc. 1159.5512)
54
88
25
Congmuyenoside D (¼1-O-[(3b)-3-{[b-d-Glucopyranosyl-(1!2)-[b-d-glucopyranosyl-(1!3)-b-
d-glucopyranosyl-(1!3)]-b-d-glucopyranosyl]oxy}-23-hydroxy-28-oxoolean-12-en-28-yl]-b-d-glucopyr-
anose; 3-O-{b-d-Glucopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-[b-d-glucopyranosyl-(1!2)]-b-d-
2
0
glucopyranosyl}hederagenin-28-O-b-d-glucopyranosyl Ester; 2). White amorphous powder. [a]
¼
D
1
þ28.1 (c ¼ 0.3, pyridine). IR (KBr): 3400, 2920, 2850, 1740, 1660, 1550, 1460, 1380, 1080, 1030. H-
13
ꢁ
ꢁ
and C-NMR: Tables 1 and 2. ESI-MS (neg.): 1281 ([MꢁH] ), 1119 ([MꢁGlcꢁH] ), 957 ([Mꢁ
ꢁ
ꢁ
ꢁ
ꢁ
2
GlcꢁH] ), 795 ([Mꢁ3GlcꢁH] ), 633 ([Mꢁ4GlcꢁH] ), 471 ([Mꢁ5GlcꢁH] ). HR-FAB-MS
þ þ
(
pos.): 1305.6095 ([M þ Na] , C54
H88NaO25 ; calc. 1305.6091).
Congmuyenoside E (¼1-O-[(3b,16a)-3-{[b-d-Glucopyranosyl-(1!2)-[b-d-glucopyranosyl-(1!
3
)-b-d-glucopyranosyl-(1!3)]-b-d-glucopyranosyl]oxy}-16-hydroxy-28-oxoolean-12-en-28-yl]-b-d-glu-
copyranose; 3-O-{b-d-Glucopyranosyl-(1!3)-b-d-glucopyranosyl-(1!3)-[b-d-glucopyranosyl-(1!
2
)]-b-d-glucopyranosyl}echinocystic Acid 28-O-b-d-Glucopyranosyl Ester; 3). White amorphous
2
0
powder. [a] ¼ þ18.0 (c ¼ 0.25, pyridine). IR (KBr): 3400, 2860, 1738, 1628, 1450, 1394, 1310, 1210,
D

7
10
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1
13
ꢁ
ꢁ
1
9
040, 890. H- and C-NMR: Tables 1 and 2. ESI-MS (neg.): 1281 ([MꢁH] ), 1119 ([MꢁGlcꢁH] ),
ꢁ
ꢁ
ꢁ
ꢁ
57 ([Mꢁ2GlcꢁH] ), 795 ([Mꢁ3GlcꢁH] ), 633 ([Mꢁ4GlcꢁH] ), 471 ([Mꢁ5GlcꢁH] ). HR-
þ
þ
25
FAB-MS (pos.): 1305.6090 ([MþNa] , C H NaO ; calc. 1305.6091).
54
88
Cytotoxicity Assays. Test compounds were dissolved in DMSO, diluted to 2 mg/ml in RPMI 1640
medium with free fetal bovine serum (FBS), and stored at ꢁ208. The human tumor cell lines used in the
present study were supplied from the Tumor Hospital of Harbin Medical University. They were
maintained as adherent-type cultures under standard conditions. HepG-2, Skov3, and A549 cells were
cultured at 378 under a humidified atmosphere of 5% CO in RPMI 1640 medium supplemented with
2
1
0% FBS and subculture twice weekly to maintain continuous logarithmic growth. Cells were seeded into
3
(
aliquots/well) at a density of 8ꢂ10 cells/ml for 24 h. Serial drug dilutions were prepared in medium
immediately prior to each assay. Thereafter, aliquots of serial dilution of each test compound were added
parallel triplicate wells were set), and then the cells were incubated for 48 h in 5% CO incubator at 378.
(
2
The cell viability was assessed through a MTT conversion assay. Aliquots of MTT soln. (5 mg/ml in PBS)
were added to every well. Then, the plates were incubated for 4 h at 378. The medium was then
completely removed from the wells and replaced with 100 ml of DMSO. The OD of the dissolved
formazan dye was recorded at 540 nm using a microplate spectrophotometer (BMG Labtechnologies,
Fluostar Optima). The data were expressed as meanꢀstandard deviation. The standard Microsoft Excel
program was used for the calculation of the IC values.
50
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Received March 17, 2012