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A. Waghmare et al. / European Journal of Medicinal Chemistry 46 (2011) 3581e3589
5.4. NMR experiments
intervals i.e. after 1 h and 2 h of incubation. Sperm motility inhi-
bition was expressed as a percentage scale.
NMR experiments were recorded on BRUKER AVANCE 500 and
700 MHz NMR spectrometers. NOESY (Nuclear Overhauser effect
spectroscopy) spectra were recorded at 400 ms mixing time using
standard pulse program [40]. 31P and 13C NMR experiments were
carried out with a relaxation delay of 2 s and broadband proton
decoupling. The NMR data was processed with Bruker Topspin 2.1.
External standard used was TSP and inorganic phosphate for
referencing 13C and 31P NMR spectra, respectively.
5.7.3. Metabolic profile measurement
The 13C NMR experiments were carried out on Bruker 500 MHz
spectrometer using 25,000 Hz spectral width, 60ꢁ flip angle and 2s
relaxation delay with power gated broadband proton decoupling.
Dulbecco buffer with 10% D2O was used for NMR field-frequency
locking. Glucose labeled with 13C at C-1 position was used as the
substrate for glycolytic reaction. 13C NMR spectra monitored as
a function of time show a decrease in glucose signal (substrate
consumption) intensity and an increase in lactate signal (buildup)
intensities. The progress of glycolysis has been monitored by
directly measuring glucose consumption and lactate production
with time [13,44e46]. The effect of nifedipine, its analogues and
sulfasalazine on the metabolism of sperm cells was compared by
monitoring the lactate signal build up with time and thus esti-
mating their glycolytic inhibitory potency.
5.5. Sample preparation for NMR and DSC experiments
Multilamellar vesicles (MLV) were prepared using standard
procedure [41] wherein the desired quantity of DPPC and synthe-
sized molecules were dissolved in chloroform. The solvent was
then evaporated with a stream of nitrogen gas so as to deposit
a thin film on the walls of the container. The last traces of the
solvent were removed using vacuum for 2 h. The lipid film was
hydrated with the required amount of D2O; this was then incubated
for half an hour in a water bath at 50 ꢁC with repeated vortexing.
The lipid concentrations were maintained at 100 mM for the NMR
and 50 mM for the DSC experiments. Unilamellar vesicles (ULV)
were prepared by sonicating the above dispersions with a Branson
sonicator (Model 450) at 50% duty cycles till the solution was
optically clear.
All data were analysed by Anova and Bonferroni tests and
expressed as mean ꢃ SEM (n ¼ 6). A value of P < 0.05 was
considered statistically significant.
Acknowledgements
The authors thank Unichem and IPCA laboratories for the gift
sample of nifedipine and sulfasalazine respectively. The National
facility for High Field NMR located at TIFR is gratefully acknowl-
edged for their invaluable support.
5.6. Determination of MLV-drug (nifedipine, its analogues and
sulfasalazine) binding
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method [42]. Optical density of 100
molecules was measured using spectrophotometer at a wavelength
range of 220e400 . MLVs were prepared by varying lipid concen-
tration systematically from 0.25 mg/ml to 2.0 mg/ml and fixed drug
concentration of 100 M. The resulting solutions were incubated
mM solution of the drug
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placed on a slide and covered with a cover glass (18 mm ꢂ 18 mm)
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evaluated twice. Motility data has been collected at two time