A. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 4300–4305
4305
the assay mixture consisted of 0.25 M potassium phos-
Acknowledgements
phate buffer (pH 6.5), liver microsomes (25 lg protein),
AC (concentration as in Fig. 2), liver cytosol (10–15 lg
protein), and water to make up the total volume of
The financial assistance from the Department of Bio-
technology (Govt. of India, New Delhi) and Council
of Scientific and Industrial Research (CSIR, New Delhi)
is gratefully acknowledged.
0
.8 ml. The contents of the tube (scaled up as per the
requirement) were preincubated for 10 min at 37 ꢁC.
The aliquots (0.8 ml portion) were removed periodically
into spectrophotometer cuvette containing CDNB and
GSH to make the final concentration 1 mM in a total
volume of 1 ml and GST activity was assayed by moni-
toring absorption at 340 nm as described by Habig
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1
7
1. Raj, H. G.; Parmar, V. S.; Jain, S. C.; Goel, S.; Singh, A.;
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2
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4
.1.5. TAase mediated biochemical action of acet-
oxycoumarins (AC). The rat liver microsomes (40 lg
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Fig. 3), 0.05 M phosphate buffer (pH 7.7), and water
to make upto 0.5 ml. The contents (scaled up as per
requirement) were preincubated at 37 ꢁC in a shaking
water bath. The aliquots (0.5 ml portion) were removed
periodically into spectrophotometer cuvette containing
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.1 mM EDTA, 36 mM cytochrome c, and 1 mM
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1
8
by monitoring the absorption at 550 nm. In the con-
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.1.6. Optimization of structures of 7,8-diacetoxy-4-
methylcoumarin (1), 7,8-diacetoxy-4-phenylcoumarin (2),
,7-diacetoxy-4-methylcoumarin (3), 5,7-diacetoxy-4-
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phenylcoumarin (4), 5,7-diacetoxy-4-methyl-3,4-dihydro-
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1
2
Inc., USA), which were optimized by the molecular
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9,20
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1
2
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uum Solvation model using the COSMO (Conductor
1
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2
2
like Screening Model) method as implemented in
MOPAC 97 in the CS MOPAC Pro version 5.0, with
dielectric constant taken as 78.5 for water at 298 K. In this
model, the solute molecule is embedded in a cavity con-
2
2
2
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1
1
2
3
structed from the overlapping van der Waals spheres
of the component atoms surrounded by a dielectric con-
tinuum of permitivity e. This is a good approximation
for solvents of high relative permitivity, such as water.