Original Paper 173
The material eluted from the Amberlite XAD-2 column with
methanol was further purified over a 50×8 cm column packed
with Sephadex LH-20 and eluted with CH2Cl2-MeOH (1:1), six
frs (J1 – J6) of 500 mL each being collected. Frs J1 – J3 containing
mainly alkaloids and other components exhibiting no UV ab-
sorption were combined with frs S1 – S3. Frs J4 – J6 (2 g) were sub-
jected to gel filtration on Sephadex LH-20 using 42 200-mL frs of
H2O-MeOH (1:1). Frs 14–22 afforded after recrystallization
from MeOH 260 mg of 7-glucotrifolin (7). Frs 28–31 yielded
45 mg of trifolin (3), frs 33–38 18 mg of hyperoside (4). Frs. 5–
12 on RP-HPLC on a C-18 Sphinx column (100×10 mm, flow rate
1.4 mL min–1) using MeOH-H2O (1:1) afforded 7 mg of robinin
(9, tr 12 min) and 5 mg at biorobin (8, tr 16 min).
phere containing 5% CO2. The cells were routinely kept in the
logarithmic growth phase at 0.1–0.9×106 cells/mL by dilution
with fresh medium at least every third day. Cells were counted
by a hematocytometer; the viability was always greater than
95% as assayed by the 0.025% trypan blue exclusion method.
Stock solutions of the flavonoids were further diluted with cul-
ture medium just before use. In all experiments the final concen-
tration of DMSO did not exceed 0.5% (v/v), a concentration
which is non-toxic to the cells.
Cytotoxicity assays were performed using a colorimetric 3-[4,5-
dimethylthiazol-2-yl-]-2,5 diphenyl-2H-tetrazolium bromide
(MTT) assay as described [15]. Briefly, 104 exponentially growing
cells were seeded on 96-well microculture plates with various
flavonoid concentrations (0.1–10 μM) in a 200 μL volume for
72 h. Surviving cells were detected based on their ability to me-
tabolize 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium
bromide (Sigma) into formazan crystals. The optical density at
570 nm was used as a measure of cell viability. Cell survival was
calculated as fraction of cells relative to control for each point:
Cell survival (%) = mean absorbance in treated cells/mean ab-
sorbance in control wells×100. Concentrations inducing 50% in-
hibition of cell growth (IC50) were determined graphically for
each experiment using the curve fitting routine of the computer
software Prism 2.0TM. (GraphPad) and the equation derived by
DeLean et al. Etoposide (Sigma) was used as a positive control
(IC50 = 0.32 0.02 mM in HL-60 cells, IC50 = 1.25 0.25 mM in
U937 cells, not determined in SK-MEL-1 cells).
General method for acetylation
Dry phenolic material was dissolved in the minimum volume of
pyridine. Twice the amount of acetic anhydride was added and
the solution was allowed to stand overnight at ambient temper-
ature. The mixture was diluted with H2O and extracted three
times with EtOAc. The extract was evaporated under vacuum
and the residue containing the polyacetate was further purified
by column chromatography over silica gel using hexane-EtOAc
as eluent. Mass spectra of the polyacetylated compounds, all
gums, are listed below.
Kaempferol tetraacetate (1a): EI-MS: m/z = 412 (M+ – C2H3O2, 23),
370 (M+ – 2 C2H3O2, 57) 286 (M+ - 4 C2H3O2, 100).
Quercetin pentaacetate (2a): HR-EI-MS: m/z = 513.0997, calcd. for
C25H20O2 + H+: 513.1033.
Trifolin heptaacetate (3a): HR-FAB-MS: m/z = 743.1784, calcd. for
C35H34O18 + H+: 743.1823.
Hyperoside octaacetate (4a): HR-FAB-MS: m/z = 823.1687, calcd.
for C37H36O20Na: 823.1698.
Supporting information
1H- and 13C-NMR spectra of the new compounds 3a, 3b, 4a, 7a-9a
as well as the previously unreported 13C-NMR spectra of com-
pounds 7, 8 and 9 are available as Supporting Information.
Glucotrifolin decaacetate (7a): HR-FAB-MS: m/z = 1030.2592,
calcd. for C47H50O26 :1030.2590.
Biorobin nonaacetate (8a): HR-FAB-MS: m/z = 972.2583, calcd.
for C45H48O24 :972.2536.
Results and Discussion
!
Robinin undecaacetate (9a): HR-FAB-MS: m/z = 1226.3344, calcd.
Kaempferol tetraacetate (1a), quercetin pentaacetate (2a), the
polyacetates 3a, 4a, the monoacetates 5 and 6 as well as the me-
thylated derivative 3b were screened for in vitro cytotoxicity
against the human myeloid leukemia HL-60 and U937 cell lines
and against the human melanoma SK-MEL-1 cell line. The re-
sults, given in concentrations causing 50% growth inhibition
for C55H63O30Na: 1226.3302, m/z = 1202.3344, calcd. for C55H62O30
1202.3326.
:
Methylation of trifolin
To a solution of 3 (14 mg) in DMSO (2 mL) was added aqueous
50% NaOH (2 mL) and 2 mL of methyl iodide. The mixture was
stirred at room temperature overnight, poured into H2O and ex-
tracted with ethyl acetate. The organic extract was dried over
Na2SO4, filtered and evaporated at reduced pressure. The residue
was purified by HPLC (SiO2, EtOAc:hexane 3:2, flow rate 2 mL/
min) to afford 10 mg of heptamethoxytrifolin 3b (TR 62 min) as a
"
(IC50), are summarized in ● Table 1. Polyacetates 1a, 2a, 3a and
4a inhibited growth and viability of human HL-60 and U937 cells
in a dose-dependent manner as determined by the 3-[4,5-dime-
thylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT dye re-
Table 1 Effect of flavonoid acetates on the growth of three human cell lines
gum.
HR-FAB-MS:
m/z = 569.1981,
547.2155 calcd.
for
C
28H34O11Na:569.1999, calcd. for C28H35O11 :547.2179.
Compound
IC50 (μM)
HL-60
U937
SK-MEL-1
Tumor cell growth assay
Human HL-60 and U 937 cells were obtained from the European
Collection of Cell Cultures (Salisbury, UK) and human SK-MEL-
1 melanoma cells (DSMZ No ACC 303) from the German Collec-
tion of Microorganisms and Cell Cultures, Braunschweig, Germa-
ny. HL-60 and U 937 were grown as described [15]. HL-60, U 937
and SK-MEL-1 cells were cultured suspended in RPMI 1640 (Sig-
ma) with L-glutamine cell culture medium supplemented with
10% heat-inactivated fetal bovine serum (FBS) medium and anti-
biotics (100 units/mL of penicillin and 100 μg/mL of streptomy-
cin) in 250 cm3 culture vessels at 37 8C in a humidified atmos-
1a
2a
3a
3b
4a
5
45
38
21
88
15
3
6
8
9
1
48 17
25 11
37
58
15
8
7
2
10
2
> 100
> 100
19
2
23 2
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
6
7a
Cells were cultured for 72 h. IC50 values were calculated as described in the
Materials and Merthods section. Data shown represent the mean SEM of 2 – 3
independent experiments with three determinations in each.
Díaz JGet al. Cytotoxic Activities of… Planta Med 2008; 74: 171–174