P. Rawat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 228–233
233
19. Indian Council of Medical Research (ICMR), Collaborating Centres, New Delhi,
Flexible dose open trial of Vijayasar in case of newly-diagnosed non-insulin-
dependent diabetes mellitus. Indian J. Med. Res. 1998, 108, 24.
20. Rawat, P.; Kumar, M.; Sharan, K.; Chattopadhyay, N.; Maurya, R. Bioorg. Med.
Chem. Lett. 2009, 19, 4684.
Control
13
22
24
16
19
600
500
400
300
200
100
0
21. Sato, S.; Naito, Y.; Aoki, K. Carbohydr Res 2007, 342, 913.
22. Spectral data of 22: 1H NMR: (CD3OD, 300 MHz) dH 7.22 (4H, m, H-2, 3, 5, 6),
7.15 (1H, m, H-4), 5.94 (1H, s, H-50), 4.87 (1H, d, J = 9.0 Hz, H-100), 3.98 (1H, t,
J = 8.9 Hz, H-200), 3.39 (1H, m, H-300), 3.40 (2H, m, H-400, 500), 3.83 (1H, m, H-600a),
3.70 (1H, m, H-600b), 3.34 (2H, m, H- ), 2.94 (2H, t, J = 7.2 Hz, H-b).13C NMR:
a
(CD3OD, 75 MHz) dC 206.5 (C@O), 165.9 (C-40), 165.0 (C-20), 164.1 (C-60), 143.2
(C-1), 129.6 (C-2,6), 129.6 (C-3, 5), 126.9 (C-4), 105.5 (C-10), 104.4 (C-30), 96.0
(C-50), 82.6 (C-300), 80.0 (C-500), 76.1 (C-100), 73.1 (C-200), 71.6 (C-400), 62.6 (C-600),
47.1 (C-
23. Spectral data of 24: 1H NMR: (CD3OD, 300 MHz) dH 7.83 (1H, d, J = 16.0 Hz, H-b),
a
), 32.2 (C-b). ESIMS: m/z 421 [M+Na]+.
7.62 (2H, d, J = 7.2 Hz), 7.42 (1H, d, J = 16.0 Hz, H = a), 7.39 (2H, d, J = 7.2 Hz,
H-3, 5), 6.12 (1H, s, H-50), 4.85 (1H, d, J = 9.0 Hz, H-100), 3.97 (1H, t, J = 8.9 Hz,
H-200), 3.37 (1H, m, H-300), 3.40 (2H, m, H-400, 500), 3.83 (1H, m, H-600a), 3.81 (9H,
Time (min)
s, 3OCH3) 3.70 (1H, m, H-600b), 2.98 (1H, s, H-1000), 1.33 (6H, d, J = 6.9 Hz, H-2000
,
Figure 4. Effect of compounds 13, 16, 19, 22 and 24 (at 25 mg/kg) on the blood
3000). ESIMS: m/z 525 [M+Na]+.
24. Hwang, S. L.; Yang, B. K.; Lee, J. Y.; Kim, J. H.; Kim, B. D.; Suh, K. H.; Kim, D. Y.;
Kim, M. S.; Song, H.; Park, B. S.; Huh, T. L. Biochem. Biophys. Res. Commun. 2008,
371, 289.
glucose levels of the streptozotocin-induced diabetic rats at various time intervals.
STZ model. Compounds 11, 22 and 24 lowered the blood glucose to
around 23.8%, 26.5% and 33.6% after 24 h on STZ model, which is
comparable to standard drug metformin. Further study on lead
optimization and mechanism of action is in progress.
25. Methodology of glucose uptake
L6 cell culture: Stock culture of L6 myoblasts were maintained in DMEM
supplemented with 10% (v/v) FBS, streptomycin (200
lg/ml), and penicillin G
(100 g/ml) under an atmosphere of 5% CO2/95% humidified air at 37 °C. For
l
differentiation into myotubes, cells were reseeded in 24-well plate (approx.
35,000 cells/well) containing DMDE media (10% FBS) for overnight culture.
When cells were nearly confluent, 10% FBS containing DMDE media were
replaced with DMDE containing 2% FBS and cells were maintained for seven to
eight days for differentiation into myotubes. Medium was changed every 48 h
prior to use in experiments.
Acknowledgements
Manmeet Kumar, Preeti Rawat are thankful to the CSIR, New
Delhi, India for Senior Research Fellowship and SAIF division, CDRI,
for spectral data.
Glucose uptake assay by L6 rat muscle cells
Measurements of radio labeled 2-deoxyglucose uptake were carried out with
some modification in previously described method of Hwang et al.23
Differentiated L6 mature myotubes cultured on 24-well plates were treated
References and notes
with the desired concentration of test compounds (10 lM or 50 lM) for 18 h.
After that cells washed three-times with Krebs–Ringer N-(2-hydroxye
thyl)piperazine-N-2-ethanesulfonic acid (HEPES) buffer saline. For glucose
uptake measurement, cells were incubated in HEPES buffer saline (HBS)
containing 3 lCi radio-labeled 2-deoxy-[3H]-D-glucose and 20 lM unlabeled
2-deoxyglucose for 15 min. The reaction was terminated by three quick washes
with ice-cold HEPES buffer saline. Non-specific uptake was determined in the
presence of 10 lM cytochalasin B. Cells were lysed in 0.1 NaOH and cell-
associated radioactivity was determined by liquid scintillation counter and
results were expressed as cpm/well as compared to controls.
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27. Evaluation for antihyperglycemic activity in streptozotocin-induced diabetic
rats Male albino rats of Sprague–Dawley strain of blood glucose level between
60 and 80 mg/dl (8–10 weeks of age body weight 140 20 g) were selected for
this study. Streptozotocin was dissolved 0.1 M citrate buffer pH 4.5, and
calculated amount of the fresh solution was injected to overnight fasted rats
(60 mg/kg) intraperitoneally. Blood glucose was checked 48 h later by
glucometer by using glucostrips and animals showing blood glucose values
between 140 and 270 mg/dl were selected and divided into groups of five
animals each. Rats of experimental groups were administered suspension of
the desired test samples orally (made in 1.0% gum acacia) at a dose of 25 mg/kg
body weight. Animals of control group were given an equal amount of 1.0%
gum acacia. Control group was taken as 100%. A sucrose load of 2.5 g/ kg of
body weight was given after 30 min of drug administration. After 30 min of
post-sucrose load, blood glucose level was again checked at 1, 2, 3, 4, 5, 6 and at
24 h, respectively. Comparing the AUC of experimental and control groups
determined the percent antihyperglycemic activity. Statistical analysis was
made by Dunnett’s test (Prism Software).