4336
E. Kato, J. Kawabata / Bioorg. Med. Chem. Lett. 20 (2010) 4333–4336
9. Spectral data of 17: 1H NMR (270 MHz, CD3OD, rt): 8.19 (1H, s), 7.96 (1H, d,
J = 8.8 Hz), 7.37 (2H, d, J = 8.8 Hz), 6.97 (1H, d, J = 8.8 Hz), 6.84 (2H, d,
J = 8.8 Hz), 3.99 (1H, tt, J = 6.0, 6.2 Hz), 3.53 (2H, d, J = 6.0 Hz), 3.06 (2H, d,
J = 6.2 Hz) ppm; 13C NMR (67.5 MHz, CD3OD, rt): 178.6, 162.4, 158.7, 157.9,
154.7, 131.4, 126.1, 125.5, 124.3, 118.4, 116.2, 115.6, 114.0, 73.0, 67.1,
elevate glucose uptake, at least partially, by the PPAR
activity.14,15 Also several isoflavones are reported to possess a
PPAR
agonist activity.16 Therefore, it is readily accepted that the
c agonist
c
activity of 1 largely owes to its aglycon daidzein (19). Regarding
the importance of isoflavone, then what does the C-glucoside of
1 participates for? Although it is shown not participating in activity
itself in this experiment, from compounds contained in P. iobata, 1,
not 19 is the content reported to be active for glucose uptake
enhancement.17 Therefore, C-glucoside may give an advantage
when tested in vivo by giving solubility to scarcely water-soluble
isoflavone and raising the absorbability into the body.
In conclusion, we have synthesized puerarin (1) and several
derivatives to study their glucose uptake enhancing activity
against 3T3-L1 adipocyte. The structure responsible for the glucose
uptake enhancing activity was shown to be the isoflavone moiety
daidzein (19) and the C-glucoside of 1 was not much involved in
the activity. Although principal structure responsible for the activ-
ity of 1 was isoflavone moiety, the C-glucoside may involves in
physical properties of the compound and raises availability by live
body.
28.3 ppm; HR FD MS (positive): [M]+ Found m/z 328.0913, C18H16O6
þ
requires m/z 328.0947.
10. Yamamoto, N.; Sato, T.; Kawasaki, K.; Murosaki, S.; Yamamoto, Y. Anal.
Biochem. 2006, 351, 139.
11. Glucose uptake enhancing activity assay: The tested compounds were dissolved
in DMSO and diluted 10 times with deionized water to the appropriate
concentration resulting 10% DMSO aq solution. The sample solution was then
sterilized by passing through 0.2 lm filter. 3T3-L1 cells were cultured in 24- or
48-well plates following the standard procedure for differentiation and the
cells were used 8 days after inducing differentiation. Sample solution was
added to the differentiated 3T3-L1 cells, the control cells were treated with the
same amount of 10% DMSO aq and positive control cells were treated with
1 l .
M of rosiglitazone, a concentration enough to maximally activate PPARc 12
In every experiment, final concentration of DMSO was kept below 0.1%. After
four days of incubation, cells were rinsed with serum-free Dulbecco’s modified
Eagle’s medium (DMEM) and incubated for two hours in serum-free DMEM.
The cells were washed twice with Krebs-Ringer-Phosphate-HEPES (KRPH)
buffer (20 mM HEPES, 136 mM NaCl, 4.7 mM KCl, 1 mM NaH2PO4, 1 mM
MgSO4, 1 mM CaCl2, pH 7.4). After washing, the cells were incubated in KRPH
buffer with or without (blank) 100 nM insulin for 30 min. Then the cells were
incubated in KRPH buffer with 1 mM 2-deoxyglucose (2-DG) for 30 min. After
incubation, the cells were washed three times with saline and then 0.1 M
NaOH aq (150
lysate was frozen once and then heated at 90 °C for 40 min. The lysate was
neutralized by 1 M HCl aq and triethanolamine buffer (200 mM, pH 8.1, 50
for 24-well plate, 25 L for 48-well plate) was added. The amount of 2-DG or 6-
phospho-2-DG contained in the lysate was then measured by the modified
method of Yamamoto et al.10 Briefly, 50
L of the cell lysate or standard sample
containing various concentration of 2-DG, 50 L of assay cocktail (50 mM KCl,
0.5 mM MgCl2, 50
M resazurin sodium salt, 1.3 mM ATP, 0.1 mM NADP+, 12
units/mL hexokinase, 32 units/mL G6PDH, 4 units/mL diaphorase in 50 mM
triethanolamine buffer, pH 8.1) was mixed and incubated for 90 min. at 37 °C.
The fluorescence of resulting resorfin (Ex 530, Em 590) was measured by
microplate reader (Bio-tech Instruments Inc., Synergy™ MX) for quantification
of the amount of 2-DG uptake of the cells. Protein concentration of the lysate
was quantified by Quickstart protein assay (Bio-Rad Laboratories Inc.) with
BSA as the standard and the 2-DG uptake values were calculated as nmol/mgꢁ
protein. The blank amount was subtracted as a basal glucose uptake, and the
relative amount against the control value was calculated. Each sample was
tested at least three times of individual experiment and average value with
standard error is shown as a result.
lL for 24-well plate, 75 lL for 48-well plate) was added. Cell
Supplementary data
lL
l
Supplementary data associated with this article can be found, in
l
l
l
References and notes
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8. Spectral data of 10: 1H NMR (500 MHz, DMSO-d6, rt): 10.86 (1H, s, OH), 10.06
(1H, s, OH), 9.36 (1H, s, OH), 7.76 (2H, d, J = 8.7 Hz), 6.87 (2H, d, J = 8.7 Hz), 6.58
(1H, s), 6.32 (1H, s), 4.65 (1H, d, J = 9.9 Hz), 3.65 (1H, dd, J = 2.0, 12.0 Hz), 3.61
12. Lehmann, J. M.; Moore, L. B.; Smith-Oliver, T. A.; Wilkison, W. O.; Willson, T. M.;
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16. Chacko, B. K.; Chandler, R. T.; D’Alessandro, T. L.; Mundhekar, A.; Khoo, N. K. H.;
Botting, N.; Barnes, S.; Patel, R. P. J. Nutr. 2007, 137, 351.
(1H, dd, J = 9.1, 9.5 Hz), 3.54 (1H, dd, J = 4.6, 12.0 Hz), 3.3–3.2 (3H, m) ppm; 13
C
NMR (125 MHz, DMSO-d6, rt): 179.7, 165.7, 165.5, 158.9, 156.2, 145.7, 132.9,
123.2, 116.0, 109.7, 107.7, 103.1, 90.9, 81.2, 78.2, 74.3, 71.6, 69.þ6, 60.5 ppm; HR
FD MS (positive): [M]+ Found m/z 432.1040, C21HÞ20O10 requires m/z
432.1057.
17. Fan, L. L.; O’Keefe, D. D.; Powell, W. J., Jr. Acta Pharm. Sin. 1984, 19, 801.