4758
R. Tundis et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4757–4760
spasmolytic, cholagogic, and anti-inflammatory
1
The hydrolysis of the rutinose unit of pectolinarin
(100 mg; 0.161 mmol) was performed with a solution
of H SO 0.04 N in ethanol 50% overnight at reflux.
2,13
drugs. No previous phytochemical or biological
studies on L. reflexa Desf. have been reported.
2
4
The reaction mixture was then neutralized with a solu-
tion of NaOH and extracted with ethyl acetate. This
extract was subjected to column chromatography over
silica gel 20–45 lm (CH Cl /MeOH 85:15), to afford
Fresh aerial parts of L. reflexa [1 kg, collected (February
2
003) in Calabria, Italy, voucher specimen P1001: Her-
barium of University of Calabria, CLU] were extracted
with methanol three times at room temperature. After
evaporation of the solvent under vacuum, the crude ex-
tract (80.82 g, 8.08%) was dissolved in methanol; upon
cooling, a solid precipitate was obtained by filtration
2
2
0
pectolinarigenin (5,7-dihydroxy-6,4 -dimethoxyflavone)
2
0,21
(42 mg, 0.134 mmol; 83%).
A solution of pectolinarin (150 mg; 0.241 mmol), tar-
trate (2 g) in water (15 mL) was set at pH 4 by the addi-
tion of 0.5 N aqueous HCl and heated at 40 ꢁC.
Naringinase (EC 3.2.1.21) (74 mg; Sigma N 1385) was
added the reaction mixture was stirred at 40 ꢁC for
2 h. The cloudy mixture was diluted with DMF
(
16 g). The methanolic extract was evaporated to dry-
ness (64 g, 6.4%) and an aliquot (10 g), after extraction
with n-hexane, was subjected to repeated column chro-
matography over silica gel (SiO ) 20–45 lm (CH Cl /
2
2
2
MeOH 95:5, 9:1, 7:3, 1:1), to afford two known iridoid
glycosides: antirrhide (15.5 mg) and antirrhinoside
(5 mL), neutralized with 10% aqueous NaHCO , and
3
(
D-glucopyranoside (7 mg). The solid precipitate was dis-
solved in H O, acidified with HCl (1 N), and extracted
2
with EtOAc, to give a residue and an EtOAc-soluble
fraction.
120 mg), pectolinarin (6 mg), and b-sitosterol 3-O-b-
extracted with n-butanol. After evaporation of the
organic layer, the dried residue was crystallized from
MeOH to yield pure pectolinarigenin-7-O-b-glucoside
2
2,23
(100.9 mg, 0.205 mmol; 85%; 266–268 ꢁC).
The protein-staining sulforhodamine B (SRB) assay,
developed by the National Cancer Institute for in vitro
The EtOAc-soluble fraction was subjected to column
chromatography over silica gel 20– 45 lm (CH Cl /
2
4,25
anticancer screening,
was used in this study to esti-
2
2
MeOH 85:15), to afford linariin (1.97 g) and a fraction
of 70 mg.
mate cell number indirectly by providing a sensitive in-
dex of total cellular protein content that is linear to
cell density.
This fraction was purified by HPLC [JASCO RP-18
2
(
50 · 20 mm i.d.; the elution solvents used were A
aqueous 0.01 M phosphoric acid) and B (100% MeOH)
with the following gradient: 5% B as initial condition;
0% B for 10 min; 70% B for 5 min; 80% B for 5 min;
and finally 100% in B for 5 min] to afford isolinariin A
30 mg) and isolinariin B (35 mg). The residue was crys-
Five cancer cell lines, large cell carcinoma COR-L23
(ECACC No.: 92031919), colorectal adenocarcinoma
Caco-2 (ATCC No.: HTB-37), amelanotic melanoma
C32 (ATCC No.: CRL-1585), hepatocellular carcinoma
HepG-2 (ECACC No.: 85011430), renal cell adenocarci-
noma ACHN (ATCC No.: CRL-1611), and one normal
cell line human fetal lung MRC-5 (ATCC No.: CCL-
171), were used in this experiment. The COR-L23,
C32, and ACHN cells were cultured in RPMI 1640
medium, while MRC-5, Caco-2, and HepG-2 cells were
cultured in DMEM. Both media were supplemented
with 10% fetal bovine serum, 1% L-glutamine, and 1%
penicillin/streptomycin. The cell lines were maintained
5
(
tallized from MeOH to afford pectolinarin (2.50 g;
Fig. 1). The structures of the compounds were deter-
mined on the basis of the spectral data (UV, IR, MS,
1
13
H NMR, and C NMR), identical with those previous-
9
ly described.
,14–18
To explore the structure–activity relationships for sever-
al flavonesÕ cytotoxicity, pectolinarin (100 mg) was con-
verted into a corresponding peracetylated derivative
with a mixture of Ac O (3 mL) and pyridine (2 mL) at
2
reflux overnight. The solid was washed with water and
dried. The product was recrystallized from aqueous eth-
at 37 ꢁC in a 5% CO atmosphere with 95% humidity.
2
The optimal plating density of each cell line was deter-
4
4
mined over a concentration range of 2 · 10 –5 · 10 to
ensure exponential growth throughout the experimental
period and to ensure a linear relationship between
absorbance at 540 nm and cell number where analyzed
by the SRB assay. For the assay, cells were detached
with 0.1% trypsin–EDTA to make a single-cell suspen-
sion, and viable cells were counted by trypan blue exclu-
sion in a hemocytometer and diluted with medium to
1
9
anol to give 103 mg of colorless crystals (119–121 ꢁC).
4
give a final concentration of 2–5 · 10 cells/well.
One hundred microliters per well of this cell suspension
was seeded in 96-well microtiter plates and incubated to
allow for cell attachment. After 24 h, the cells were treat-
ed with serial dilutions of pure compounds. Each com-
pound was initially dissolved in an amount of DMSO
and diluted further in medium to produce six concentra-
tions. One hundred microliters per well of each concen-
tration was added to the plates in six replicates to obtain
final concentrations of 0.25, 0.5, 1, 2.5, 5, 12.5, 25, and
Figure 1. Flavones isolated from L. reflexa.
Tundis, Rosa
