Secoisolariciresinol-4ꢀ,4ꢀꢀ-diacetate (3). Compound 2 (0.054 g, 0.15 mmol) was treated with acetylchloride
(0.9 mL, 12.7 mmol). The reaction was carried out with stirring at room temperature for 12 h. The mixture was evaporated at
reduced pressure and separated using preparative chromatography on Silica Gel G plates (Analtech UniplateTM, USA).
The eluent was EtOAc:petroleum ether (2:3). Yield 0.042 g (63%). Mass spectrum (ESI, m/z, I , %, positive-ion mode):
rel
+
[M + H] 447.72 (100).
–1
IR spectrum (KBr, ꢄ, cm ): 3437 (OH), 2935, 2851 (CH –O–Ar), 1764 (C=O), 1605, 1511, 1466, 1420, 1370
3
(C –O), 1270, 1199, 1151, 1122, 1033 (C–O–C), 903 (Ar), 825 (Ar), 791, 737, 646, 602, 514, 465.
Ar
PMR spectrum (400.13 MHz, CDCl , ꢅ, ppm, J/Hz): 2.21 (2H, m, H-2), 2.30 (6H, s, COCH ), 2.58 (2H, dd, J = 13.8,
3
3
8.2, H-1ꢀa), 2.68 (2H, dd, J = 13.8, 6.1, H-1ꢀb), 3.54 (2H, dd, J = 8.6, 5.6, H-1a), 3.78 (6H, s, OCH ), 3.93 (2H, dd, J = 8.6, 6.5,
3
H-1b), 6.62 (2H, dd, J = 8.4, 1.8, H-6ꢀꢀ), 6.67 (2H, d, J = 1.8, H-2ꢀꢀ), 6.92 (2H, d, J = 8.4, H-5ꢀꢀ).
Determination of DNA-protector Activity. We used Phasmid DNA containing a cloned fragment of human
chromosomal DNA (38,000 bp) (obtained from Phasmid clone WI2-1799L7, which is part of human Phasmid library of clones
WIBR-2; source, Children’s Hospital Oakland Research Institute, BACPAC Resources Center, USA). pDNA was isolated
from a recombinant strain of E. coli using a QIAprep Spin Miniprep Kit (QIAGEN Ltd., Great Britain) according to the
manufacturer’s instructions. The concentration was measured with a Nanodrop 1000 spectrophotometer (Thermo Scientific,
USA) at 260 nm. The DNA in Tris-EDTA buffer was diluted beforehand several times with water. The stock solution of Tris-
EDTA buffer had concentration 0.184 ꢃg/ꢃL and was stored at –20°C until used.
Next, an Eppendorff tube was charged successively with Tris-HCl buffer (4 ꢃL, 0.1 M, pH 7.4) containing Tween 20
(0.05%), DNA (0.184 ꢃg/ꢃL), horseradish peroxidase (0.1 ꢃM), benzidine (25 ꢃM), and H O (1 mM). The mixture volume
2
2
was 20 ꢃL. The mixture was incubated for 60 min at 25°C, treated with bromphenol blue solution (6ꢇ, 4 ꢃL), placed (12 ꢃL)
on a track in agarose gel (0.9%) prepared with TAE-buffer (40 mM Tris-acetate and 1 mM EDTA, pH 7.4) containing ethidium
bromide (0.5 ꢃg/mL). Electrophoresis was carried out at 60 V for 1 h. The electrophoretic mobility of the DNAwas analyzed
using a trans-illuminator. The electrophoregrams were photographed. The emission intensity of the DNAbands was estimated
using the TotalLab V2.0 program (Nonlinear Dynamics Ltd., Great Britain).
Determination of Cytotoxic Activity. The sensitivity of leukemia cells to the test compounds was determined using
5
the MTT-test according to the literature method [12]. Cells were resuspended to a concentration of 2 ꢇ 10 cells/mL in RPMI-
1640 complete medium containing fetal calf serum (10%), penicillin (100 U/mL), streptomycin (100 ꢃg/mL), and L-glutamine
(2 mM). Cell suspension in medium (80 ꢃL) was distributed evenly into a 96-well plate among wells containing different
dilutions of test compounds in 20 ꢃL with each sample doubled, a row of wells with cell suspension (survival control), and
wells containing only RPMI complete medium (medium control). The SDG solution (2.9 mM) was prepared in distilled water.
Stock solutions (29 mM) of secoisolariciresinol and secoisolariciresinol-4ꢀ,4ꢀꢀ-diacetate were prepared in EtOH (96%). Etoposide
solution (33.9 ꢃM) was prepared in RPMI-1640 medium. Then compounds were diluted with physiological solution to
concentrations of 2.9, 1.45, 0.725, 0.29, 0.116, and 0.058 mM. The final concentrations of the compounds in the wells were
11.6, 23.3, 58, 145, 290, and 580 ꢃM, respectively. The EtOH concentration in the wells was less than 2%. Furthermore, a
cell-survival control in medium containing EtOH (2%) was set up. Plates were cultivated in a humid incubator with 5% CO
2
for 3 d at 37°C. Then, wells were treated with MTT (5 ꢃg/mL of RPMI-1640 medium) and held for 4 h under the same
conditions.
The formazan crystals that formed after incubation with MTT were dissolved in HCl solution (100 ꢃL, 2 N) in
isopropanol. The contents of each well were thoroughly mixed. The optical density of the solutions was determined at 540 nm
in the plate on a spectrophotometer (ELISA-reader, Sanofi Diagnostic Pastuer PR 2100, France). Cell survival (CS) was
determined using the formula:
CS(%) = (ODw – ODc)/(ODs – ODc) ꢇ 100%,
where ODw is the optical density of test wells; ODs, of survival-control wells; ODc, control wells without added cells and
EtOH.
The ODw value was the arithmetic average of two determinations for each dilution. Results were considered adequate
if ODs – ODc > 0.05.
Determination of Cytotoxic Activity Using Apoptosis Induction and Flow Cytometry. Reaction mixtures
5
(1000 ꢃL) containing a suspension of Raji cell line (5 ꢇ 10 cells/mL) in RPMI-1640 complete medium and test compounds
708