Chemical constituents of the leaves of glochidion obliquum and their bioactivity

Article abstract of DOI:10.1007/s12272-011-0305-y

A new flavonoid glycoside, globlin A (1), and eleven known compounds were isolated from methanolic extracts of the leaves of Glochidion obliquum. The structure of this new compound was established with a combination of 2D NMR techniques (COSY, NOESY, HMQC

Full text of DOI:10.1007/s12272-011-0305-y

Arch Pharm Res Vol 34, No 3, 383-389, 2011
DOI 10.1007/s12272-011-0305-y
Chemical Constituents of the Leaves of Glochidion obliquum and
Their Bioactivity
1
2,
3,
4
1
5
*
*
Tran Dinh Thang , Ping-Chung Kuo , Chun-Shu Yu , Yuh-Chiang Shen , Le Thi Mai Hoa , Tran Van Thanh ,
Yao-Haur Kuo4, Mei-Lin Yang6, and Tian-Shung Wu3,6
1Department of Chemistry, Vinh University, Vinh City, Nghean province, Vietnam, 2Department of Biotechnology,
3
National Formosa University, Yunlin 632, Taiwan, Department of Pharmacy, and Chinese Medicinal Research and
Development Center, China Medical University and Hospital, Taichung 404, Taiwan, National Research Institute of Chinese
Medicine, Taipei 112, Taiwan, Department of Pharmacognosy, Hanoi College of Pharmacy, Vietnam, and Department
of Chemistry, National Cheng Kung University, Tainan 701, Taiwan
4
5
6
(Received February 10, 2010/Revised August 5, 2010/Accepted August 24, 2010)
A new flavonoid glycoside, globlin A (1), and eleven known compounds were isolated from
methanolic extracts of the leaves of Glochidion obliquum. The structure of this new compound
was established with a combination of 2D NMR techniques (COSY, NOESY, HMQC and
HMBC) and HR-ESI-MS analyses. Chemical structures of the other known compounds were
identified by comparison of their spectroscopic and physical data with those reported in the
literature. Some of the isolates were examined for their bioactivities. Among the tested com-
pounds, rotundic acid (4) displayed significant cytotoxicity and anti-inflammatory activities.
Key words: Flavonoid glycoside, Euphorbiaceae, Cytotoxicity, Anti-inflammatory, DPPH
recent years, some lupane-type triterpenes from the
Glochidion genus have been reported to inhibit tumor
INTRODUCTION
Glochidion is a relatively large genus of the promotion and to be cytotoxic (Tanaka et al., 2004;
Euphorbiaceae family, comprising approximately 300 Puapairoj et al., 2005). Therefore, in the continuing
species that are distributed from Madagascar to the course of our integrated program aimed at new drug
Pacific Islands but mainly in tropical Asia. More than discovery, G. obliquum Decne. was selected as a
twenty species can be found in Vietnam, and most are target. In our investigation of the phytochemical
commonly used as folk medicine in the treatment of diversity of this plant, the structure of a new
influenza, dysentery, impaludism, rheumatoid arthritis, flavonoid, globlin A (1), was established using a
and dyspepsia (Delectis Florae Reipublicae Popularis combination of spectroscopic methods including 1D-
Sinicae Edita, 1999; Nguyen, 2003). These species and 2D-NMR and HR-ESI-MS. We also identified
produce a wide variety of metabolites including eleven known compounds, including five triterpenoids,
sesquiterpenoids (Xiao et al., 2007, 2008), triterpenoids two phenolics, one flavonoid, two steroids, and one
(Ganguly et al., 1966; Srivastava and Kulshreshtha, carbohydrate. We did this by comparison of their
1986, 1988; Puapairoj et al., 2005; Kiem et al., 2009), spectral and physical data with literature data. In the
steroids (Hui et al., 1970), flavonoids (Otsuka et al., present study we report the structure of this new
2001), alkaloidal glycosides (Otsuka et al., 2004), and compound and the cytotoxicity and anti-inflammatory
lignans (Takeda et al., 1998; Otsuka et al., 2000). In activities of all 12 isolates.
*These authors contributed equally to this work.
Correspondence to: Tian-Shung Wu, Department of Pharmacy,
MATERIALS AND METHODS
and Chinese Medicinal Research and Development Center,
China Medical University and Hospital, Taichung 404, Taiwan
General
Melting points for the 12 isolates were determined
using an Electrothermal IA-9200 melting point mea-
Tel: 886-6-2747538, Fax: 886-6-2740552
E-mail: tswu@mail.ncku.edu.tw
383

384
T. D. Thang et al.
suring apparatus without correction. UV spectra were used for fraction 3 as well. It was eluted with a step
recorded on an Agilent UV-VIS recording spectro- gradient elution of -hexane/acetone (15:1, 10:1, 7:1,
photometer. The IR spectrum was obtained, as KBr and 5:1) and led to the isolation of taraxerol (
n
3
)
discs, on a Hitachi 270-30 type spectrometer. Optical (132 mg; Sakurai et al., 1987). Purification of fraction
rotation was measured with a Jasco DIP-1000 KUY 5 by column chromatography with silica gel (eluted by
polarimeter. The electrospray ionization (ESI) mass
n-hexane/acetone (9:1)) afforded rotundic acid (4)
spectrum was determined using an Agilent 1200 LC- (12.90 g; Saimaru et al., 2007) and pedunculoside (
5)
MSD Trap spectrometer, and the HR-ESI-MS was com- (6.80 g; Wu et al., 2007).
pleted with the aid of the Bruker APEX II mass spec-
The n-butanol soluble residues (143 g) were subjected
1
trometer. H- and 13C-NMR, COSY, NOESY, HMQC, to silica gel column chromatography which was done
and HMBC spectra were recorded on the Bruker using step gradient elution with chloroform and in-
Avance-500 NMR spectrometer, using tetramethyl- creasing concentrations of methanol (0%, 5%, 10%, 20%,
silane (TMS) as the internal standard. Standard pulse 30%, 50%, 70%, 90%, and 100%) to afford nine fractions
sequences and parameters were used for the NMR (1-9). Silica gel column chromatography of fraction 2
experiments and all chemical shifts are reported in with the eluant mixtures of chloroform-methanol (19:1)
parts per million (ppm,
(CC) was done on silica gel (Kieselgel 60, 70-230 mesh
and 230-400 mesh, E. Merck). HPLC analysis of sugars 12-en-28-oic acid (
was done on a Shimadzu LC-10ATVP series pumping syringin ( ) (71 mg; Kiem et al., 2003). Separation of
system equipped with a Shimadzu SPD-6AV UV-Vis fraction 3 by silica gel column chromatography (eluted
spectrophotometric detector at 210 nm, a Cosmosil with chloroform-methanol (15:1)) yielded globlin A (
δ
). Column chromatography and step gradient with methanol afforded euphorginol
) (92 mg; Rasool et al., 1989), 2, 3, 24-trihydroxyurs-
) (324 mg; Jung et al., 2004), and
(
6
7
8
1
)
packed column with 5C18-AR-II Waters type (4.6
mm, 5 mm), and a Rheodyne injector.
× 250 (987 mg), bergenin (9) (145 mg; Taneyama et al., 1983),
and vitexin (10) (46 mg; Lin et al., 2000), successively.
Fraction 6 was subjected to column chromatography
with silica gel eluted with chloroform-methanol-water
Plant materials
The leaves of Glochidion obliquum (Euphorbiaceae) (9:1:0.05) and further purified by repeated silica gel
were collected from Nghe An, Vietnam, during May column chromatography to yield myo-inositol (11) (57
2008 and the plant materials were identified and mg; Breitmaier and Voelter, 1986) and
β-sitosteryl-3-O-
authenticated by Dr. Tran Huy Thai, Institute of Ecol- -D-glucopyranoside (12) (268 mg; Kuo and Yeh, 1997),
β
ogy and Biological Resources, Vietnamese Academy of respectively.
Science and Technology. A voucher specimen (Viet-
TSWu-20080505) was deposited in the Herbarium of Globlin A
the Institute of Ecology and Biological Resources, Colorless powder (MeOH); m.p. 244-246°C; [α D
Vietnamese Academy of Science and Technology, (c 1.0, MeOH); UV (MeOH)
max (log ) 330 (2.50), 276
(3.20), 216 (3.50) nm; IR (KBr) max 3406, 2927, 2360,
1687, 1660, 1605, 1556, 1511, 1497, 1366, 1304, 1248,
(1)
30
]
−69
λ
ε
Hanoi, Vietnam.
ν
1
1209, 1186, 1067, 1025 cm–1; H-NMR (Pyridine-d5
7.88 (2H, d, = 9.0 Hz, H-2' and -6'), 7.06
= 9.0 Hz, H-3' and -5'), 6.78 (1H, s, H-8), 6.76
perature, and the combined extracts were concentrat- (1H, s, H-3), 5.58 (1H, d, = 7.5 Hz, H-1"), 5.23 (1H,
ed under reduced pressure to give a deep brown syrup br s, H-1"'), 4.37 (1H, br d, = 11.0 Hz, H-6"), 4.24
(304 g). The crude extract was suspended in water (1H, dd, = 3.3, 1.5 Hz, H-2"'), 4.22-4.15 (3H, m, H-2",
and partitioned with -hexane, ethyl acetate, and -4", and -5"), 4.05-4.01 (2H, m, H-3"' and -5"'), 3.99
butanol, successively to afford -hexane (11 g), ethyl (3H, s, OCH3-7), 3.97 (1H, br d, = 11.0 Hz, H-6"),
acetate (129 g), -butanol (143 g), and water (21 g) 3.93 (1H, m, H-4"'), 3.91 (1H, d, = 9.5 Hz, H-3"), 3.78
fractions, respectively. Compounds soluble in ethyl (3H, s, OCH3-4'), 1.45 (3H, d,
= 6.0 Hz, CH3-6"'); 13C-
acetate were chromatographed over a silica gel col- NMR (Pyridine-d5, 125 MHz): 182.9 (C-4), 164.4 (C-
umn by gradient elution with -hexane and increasing 2), 163.1 (C-4'), 159.7 (C-7), 153.9 (C-9), 152.9 (C-5),
,
Extraction and isolation
The leaves of G. obliquum (6.3 kg) were powdered 500 MHz):
and soaked with methanol (20 L 3) at room tem- (2H, d,
δ
J
×
J
J
J
J
n
n-
n
J
n
J
J
δ
n
concentrations of acetone to afford eight fractions (1- 129.8 (C-6), 128.6 (C-2' and -6'), 123.1 (C-1'), 115.0 (C-
8). Fraction 2 was further subjected to silica gel 3' and -5'), 106.2 (C-10), 104.5 (C-3), 104.4 (C-1''),
column chromatography. It was eluted with
acetone (19:1) to yield -sitosterol ( ) (783 mg; Kuo and (C-2''), 73.6 (C-3'''), 72.2 (C-5''), 71.7 (C-2'''), 71.4 (C-3''),
Yeh, 1997). Silica gel column chromatography was 69.3 (C-5'''), 67.8 (C-6''), 56.9 (OCH3-7), 55.6 (OCH3-4'),
n-hexane/ 101.8 (C-1'''), 92.1 (C-8), 77.9 (C-4''), 77.4 (C-4'''), 75.4
β
2

Chemical Constituents of Glochidion obliquum
385
18.1 (C-6'''); ESI-MS m/z (%) 645 [M+Na]+ (100); HR- previously (Wang et al., 2006). BV2 cells (2
×
107 cells)
ESI-MS m/z 645.1791 [M+Na]+ (Calcd for C29H34O15Na, were suspended in lysis buffer [20 mM potassium
645.1795).
phosphate (pH 7.0), 1 mM ethylene-bis(oxyethylenenitrilo)
-tetraacetic acid (EGTA), and a protease inhibitor
cocktail (Roche Applied Science)]. The cell suspension
Acid hydrolysis of flavonoid glycosides
Compound
1
(10.0 mg) was refluxed at 100oC for 1 h was sonicated using five 10-s bursts at 30% power
with 2N HCl (5 mL). The acid hydrolysate was ex- followed by cooling on ice for 30 sec between each
tracted with ethyl acetate and evaporated to dryness sonication. The homogenate was centrifuged twice at
to yield a colorless amorphous solid, which, on re- 800
× g for 15 min to remove unbroken cells. Protein
crystallization from methanol, afforded 2.5 mg of 5,6- concentrations were determined using the Bradford
dihydroxy-7,4'-dimethoxyflavone (Horie et al., 1997). reagent (Bio-Rad). Fifty micrograms of cell homogenate
This compound was identified by mp, UV, IR, 1H-, 13C- were added to the wells of a bioluminescence plate.
NMR and mass spectral analysis. The sugars in the NADPH oxidase activity was measured in a 50 mM
aqueous layer were identified as D-glucose and D- phosphate buffer containing 1 mM EGTA, 150 mM
rhamnose, respectively, using HPLC analysis, by com- sucrose, and 10 µM lucigenin. Various drugs were tested
parison of their retention times and optical rotations including diphenyleneiodonium (DPI, an NADPH
with those of authentic samples as described in Shin oxidase inhibitor) and 0.1-50 µM of the test drugs. These
et al. (2003).
drugs were added to the wells of a bioluminescence plate
and incubated for 20 min at 37oC in the dark. O2
production was stimulated with 200 µM NADPH
In vitro cytotoxicity assay
All stock cultures of cells were grown in T-25 flasks. (Sigma-Aldrich), and chemiluminescence was monitored
Freshly trypsinized cell suspensions were seeded in 96- for 30 min, after which the area under the curve (AUC)
well microtiter plates at densities of 1500-7500 cells was calculated to represent ROS production by NADPH
per well with compounds added from DMSO-diluted oxidase (NOX activity) and expressed as relative light
stock. After 3 days in culture, attached cells were fixed units (RLU)/50 µg protein.
with cold 50% trichloroacetic acid and then stained
with 0.4% sulforhodamine B (SRB). The absorbance at
562 nm was measured using a microplate reader after
solubilizing the bound dye. The mean EC50 is the
Measurement of 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical scavenging capacity
Drugs were diluted with MeOH to yield a range of
concentration of agent that reduces cell growth by 50% concentrations (0.1-50 µM). DPPH (Sigma-Aldrich) solu-
under the experimental conditions and is the average tion (200 µL, final concentration: 200 µM in MeOH) was
from at least three independent determinations that added to 10 µL of each diluted sample in a 96-well
were reproducible and statistically significant. Four microplate, and the resulting solution was allowed to
human cancer cell lines, Daoy, Hep2, MCF-7, or HeLa, react for 30 min in the dark at ambient temperature. The
were used in the assay. Mitomycin C (5 µM, final absorbance at 517 nm was determined by a microplate-
concentration) and DMSO (0.3%, final concentration) spectrophotometer. Trolox (OXIS) was included as a
were used as positive and vehicle controls. Results reference (Lin et al., 2006).
were expressed as percent of cell growth for the DMSO
control (Scudiero et al., 1988).
RESULTS AND DISCUSSION
Air-dried, powdered leaves of G. obliquum were ex-
tracted with methanol at room temperature and con-
Microglial cell culture and measurements of
nitric oxide (NO)
The murine microglial cell lines (BV2) were cultured centrated to give a dark brown syrup. The methanol
in Dulbecco's modified Eagle's medium (Gibco) sup- extract was suspended in water and partitioned with
plemented with 10% fetal bovine serum (HyClone). hexane, ethyl acetate, and -butanol, successively to
Production of NO was measured by the Griess reagent afford -hexane, ethyl acetate, -butanol, and water
n-
n
n
n
as in a previous report (Wang et al., 2006). NO was soluble fractions, respectively. Further purification by
measured by the accumulation of nitrite in the culture a combination of conventional chromatographic tech-
medium in the presence of test drug (0.1-50
after stimulation with 0.5 µg/mL LPS (Sigma-Aldrich). known compounds were also identified from the ethyl
acetate and -butanol extracts including -sitosterol
), taraxerol ( ), rotundic acid ( ), pedunculoside ( ),
NADPH oxidase activity was measured as described euphorginol ( ), 2, 3, 24-trihydroxyurs-12-en-28-oic acid
µM) 24 h niques yielded a new compound, globlin A (1). Eleven
n
β
(2
3
4
5
Measurement of NADPH oxidase (NOX) activity
6

386
T. D. Thang et al.
(
(
7
), syringin (
11), and -sitosteryl-3-O-
structure of this new flavonoid glycoside, globlin A (
was established on the basis of 1D and 2D NMR and measurement. With the aid of 13C-NMR and DEPT
mass spectroscopic analyses. spectral analytical techniques, twelve carbon signals
Compound was isolated as an optically active col- were identified, including one methyl (
orless powder with an m.p. 244-246oC. The HR-ESI-MS oxymethylene (
67.8), eight oxymethine (
of
645.1791 [M+Na]+, corresponding to the pseudomole- carbons (
cular formula of C29H34O15Na. The UV absorption components of D-glucose (
maxima at 330, 276, and 216 nm were characteristic and 67.8) (Watanabe et al., 2003) and D-rhamnose (
8
), bergenin (
-D-glucopyranoside (12). The since they exhibited NOE crosspeaks with the H-8 (d
), 3.99) and H-3' ( 7.06) in the NOESY experimental
9), vitexin (10), myo-inositol to be located at positions C-7 and C-4', respectively,
β
β
1
δ
1
δ
18.1), one
δ 77.9, 77.4,
δ
1
displayed a sodium adduct ion peak at m/z 75.4, 73.6, 72.2, 71.7, 71.4, 69.3), and two anomeric
104.4 and 101.8). They were identified as
104.4, 77.9, 75.4, 72.2, 71.4,
δ
δ
δ
of a flavone skeleton (Mabry et al., 1970). The IR 101.8, 77.4, 73.6, 71.7, 69.3, and 18.1) (Burger et al.,
absorption bands at 3406, 1687, and 1660 cm-1 showed 1998) by comparison with literature values and the
the presence of a hydroxyl moiety, a carbon-carbon assistance of comprehensive COSY, NOESY, HMQC,
double bond, and hydrogen-bonded carbonyl groups, and HMBC spectroscopic examinations. The sugar frac-
respectively. In the 1H-NMR spectrum, a typical set of tion of acid hydrolysates of compound
1
was further
= 9.0 Hz) and 7.06 (2H, analyzed with HPLC by comparing their retention
= 9.0 Hz) were attributed to H-2', -6' and H-3', -5' times and optical rotations with those of authentic
of the para-substituted B-ring. Two singlets at 6.78 samples as described in Shin et al. (2003); the sugar
(1H, s) and 6.76 (1H, s) were assumed to be H-8 and moiety was confirmed as mentioned. In the HMBC
H-3 since they displayed 2J 3
-HMBC correlations spectrum, long range correlations from H-1" ( 5.58) to
with the carbon signals at 159.7 (C-7), 153.9 (C-9), C-6 ( 129.8); and H-1"' ( 5.13) to C-2"' ( 71.7), C-5"'
129.8 (C-6), 106.2 (C-10); and 182.9 (C-4), 164.4 (C-2), 69.3), and C-6" ( 67.8), established that the sugar
123.1 (C-1'), respectively. Two anomeric proton signals moiety, D-rhamnosyl-(1 6)-D-glucose, was attached
at d 5.58 (d, =7.5 Hz) and 5.24 (br s) suggested the at C-6 through the C–O linkage. The stereochemistry
presence of two sugar units. In addition, there were of the sugar moiety was established with reference to
oxygenated methine and methylene protons at 4.37 the relative coupling constants of protons, and the
(1H, br d, =11.0 Hz), 4.24 (1H, dd, =3.3, 1.5 Hz), configurations of glucose and rhamnose were deter-
4.22-4.15 (3H, m), 4.05-4.01 (2H, m), 3.97 (1H, br d, mined as and , respectively, according to the coupl-
=11.0 Hz), 3.93 (1H, m), and 3.91 (1H, d, =9.5 Hz), ing constants of the corresponding anomeric protons
which were identified as the proton signals of the H-1" (7.5 Hz) and H-1"' (~0 Hz). In addition, the methoxy
sugar moieties. Another upfield methyl doublet at proton signals at 3.99 (OCH3-7) and 3.78 (OCH3-4')
1.45 (3H, d,
=6.0 Hz) was the characteristic signal displayed 3J-HMBC correlations with the carbon
for the rhamnose fragment. Moreover, two methoxy signals at 159.7 (C-7) and 163.1 (C-4'), respectively,
groups at d 3.99 (s, 3H) and 3.78 (s, 3H) were assigned which indicated the substitution of two methoxy
A2B2 signals at
d,
δ 7.88 (2H, d, J
J
δ
,
J
δ
δ
δ
δ
δ
δ
(δ
δ
→
J
δ
J
J
J
β
α
J
δ
δ
J
δ
Fig. 1. Structure and significant NOESY and HMBC correlations of globlin (1).

Chemical Constituents of Glochidion obliquum
387
Table I. EC50 values of tested compounds against human
tumor cell lines
tion and NOX-dependent ROS production in micro-
glial cells. Inhibition of NOX and NOS were measured
by ROS and NO production, respectively, in the pre-
sence of 1-100 µM of drugs. DPI and L-NAME were
included as positive controls. Data were calculated as
50% inhibitory concentrations (IC50s) and expressed
as the mean ±S.E.M. from 3~6 experiments perform-
ed on different days using cells from different passages.
All tested compounds had IC50 values in the range of
39.5-98.4 µM, which were not quite as potent as L-
NAME (IC50 18.9 µM), a non specific NOS inhibitor
that inhibits LPS-induced NO production. These data
are shown in Table II. NOX is the major ROS-produc-
ing enzyme in activated inflammatory cells (Van den
Worm et al., 2001). Previous reports indicated that
compounds with anti-inflammatory activity are also
potent inhibitors of NOX activity (Liou et al., 2003; Lin
et al., 2006). Therefore, the effects of these isolates on
NOX activity in lysates of microglial cells were
ED50
Hep-2
(µ
M)
Tested
compounds
Daoy
MCF-7
HeLa
1
3
(
−
)
)
(
(−
−
)
)
(
(−
−
)
)
(−)
(−
(
−
)
4
24.67 ± 0.43 29.22 ± 1.00 29.43 ± 1.13
(
−
)
5
(
−
)
(
−
)
(
−
)
(
−
)
6
(
−
)
(
−
)
(
−
)
(
−
)
8
(
−
)
(
−
)
(
−
)
(
−
)
9
(
−
)
(
−
)
(
−
)
(
−
)
10
(
−
)
(
−
)
(
−
)
(−)
Mitomycin
C 0.30 ± 0.03 0.30 ± 0.003 0.21 ± 0.03 0.45 ± 0.03
(
−): ED50 > 40, µM
Table II. IC50 values (µM) of tested compounds on nitric
oxide synthase (NOS) and NADPH oxidase (NOX) activities
in murine microglial cells
Compounds
NOS
NOX
determined. Compounds
3 and 4 showed weaker
1
3
4
5
6
8
9
10
80.1±01.6
60.6±12.7
39.5±09.3
78.7±01.5
60.5±07.1
78.9±18.1
98.4±10.0
65.7±02.9
N.A.
52.2
80.9
N.A.
N.A.
N.A.
N.A.
N.A.
inhibition of NOX activity (IC50 values of 52.2 and
80.9 µM, respectively) than the specific NOX inhibitor
DPI (IC50 0.4 µM) (Table II). This suggests that NOX
might not be the direct target of these compounds. In
addition, the free radical-scavenging capacities of
these com pounds were examined in a cell-free DPPH
solution. Trolox, a vitamin E analogue included as a
positive control, displayed a stronger free radical-
scavenging effect than any of the compounds examined
(data not shown). Thus, none of these compounds
showed considerable free radical-scavenging activity.
18.9±03.2
(L-NAME)
0.4±0.2
(DPI)
Reference
N.A.: not effective.
ACKNOWLEDGEMENTS
groups attached at C-7 and -4'. The successive NOESY
and HMBC experiments completed the assignments
The authors would like to thank Dr. Tran Huy Thai,
of all the proton and carbon signals of
its chemical structure was established as 5,6-di- Academy of Science and Technology for plant identi-
hydroxy-7,4'-dimethoxyflavone 6-O- -D-rhamnosyl-(1 fication. The authors also wish to thank the National
6)- -D-glucose (Fig. 1) and given the trivial name of Basic Research Program in the Natural Sciences
globlin A.
Among these isolated compounds,
1 and therefore Institute of Ecology and Biological Resources, Vietnam
α
→
β
(Vietnam) and the National Science Council (Taiwan)
1,
3-6, and 8-10 for partial financial support of this research. We are
were examined for cytotoxicity against tumor cell lines also grateful to the National Center for High-perfor-
Daoy (human medulloblastoma), Hep-2 (human laryngeal mance Computing, Taiwan, for computer time and
carcinoma), MCF-7 (human breast adenocarcinoma), and facilities. This study was supported in part by the
HeLa (human cervical epitheloid carcinoma), as de- Taiwan Department of Health Clinical Trial and
scribed previously (Scudiero et al., 1988). The EC50 Research Center of Excellence (DOH100-TD-B-111-
values are summarized in Table I. Only compound
4
004).
exhibited moderate cytotoxicity with EC50 values of
24.67±0.43, 29.22±1.00, and 29.43±1.13 µM against
the Daoy, Hep-2, and MCF-7 tumor cell lines, respec-
tively. The anti-inflammatory potentials of compounds
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