Iridoids from Fraxinus excelsior
Journal of Natural Products, 2010, Vol. 73, No. 1 5
was identified for 1 and 2 by comparison with retention time of authentic
Cell Culture and Transient Transfection. Human embryonic
kidney (HEK) cells were cultured in DMEM supplemented with 10%
fetal bovine serum (FBS), 1% penicillin streptomycin, and 2 mM
D-glucose (t ) 9.72 min) after treatment in the same manner with
R
Tri-Sil Z.
6
Cell Culture and Adipocyte Differentiation. For differentiation
assays, 3T3-L1 cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) and supplemented with 10% FBS at 37 °C in a 10%
L-glutamine. For experiments, 2 × 10 cells were grown in T-75 flasks
2
at 37 °C with 5% CO . In 24-well plates, 80 000 cells were seeded per
well to obtain 70-80% confluency, 24 h before transfection. Polyeth-
ylenimine (PEI)-based transfection was performed. The protocol was
applied for a 24-well plate transfection. Plasmid DNA (1 µg) was diluted
into 50 µL of 150 mM NaCl per well. PEI solution (2 µL) was diluted
into 50 µL of 150 mM NaCl for each well. The PEI solution was gently
added to the DNA solution, and after mixing, it was incubated at room
temperature for 15 to 30 min to permit the formation of PEI/DNA
complex. DMEM supplemented with 10% fetal bovine serum (FBS),
CO
2
cell incubator. Preadipocyte 3T3-L1 cells were grown in 12-well
plates until 2 days post-confluence. The differentiation was induced as
27,28
previously described
-isobutyl-1-methylxanthine (IBMX), and 0.25 mmol/L dexamethasone
DEX). Two days after induction, the IS/IBMX/DEX-containing
by addition of 1 mg/L insulin (IS), 0.5 mmol/L
3
(
medium was replaced with medium containing 1 mg/L IS. The medium
was subsequently replaced again with fresh culture medium (DMEM
supplemented with 10% FBS) after 2 days and then every other day
thereafter. To determine the roles of compounds in adipocyte dif-
ferentiation, different concentrations of individual compound were
added to the medium along with IS/IBMX/DEX (BE/IS/IBMX/DEX).
The compound-treated cells were assayed for their glucose uptake
activity 9-12 days after the initiation of induction.
1
% penicillin streptomycin, and 2 mM L-glutamin was taken out from
the transfection plate, and PEI/DNA complex was gently added to each
well. The wells were filled with unsupplemented DMEM. The cells
were transfected for 4 h, and after changing the medium with
supplemented (medium including derivatives) DMEM, the cells were
incubated for 2 days to allow luciferase protein expression. After 48 h,
cells were rinsed by 1% PBS and lysed with reporter lysis buffer. Plates
were shaken for 2 h and kept at -80 °C for 1 h. The luciferase activity
of cell lysates was measured with 50 µL of luciferase assay kit by
luminometer (Wallac 1420 Victor, Perform Hungaria KFT, Budapest,
Hungary). The results were normalized against ꢀ-gal as control.
Glucose Uptake Activity Assay. Glucose uptake activity was
3
analyzed by measuring the uptake of 2-deoxy-D-[ H]glucose as
2
7,28
described previously.
Briefly, confluent 3T3-L1 adipocytes grown
in 12-well plates were washed twice with serum-free DMEM and
incubated with 1 mL of the same medium at 37 °C for 2 h. The cells
were washed three times with Krebs-Ringer-Hepes (KRP) buffer and
incubated with 0.9 mL of KRP buffer at 37 °C for 30 min. Insulin, FE,
or compounds were then added and adipocytes incubated at 37 °C for
While FE was diluted 1:10 000 in H
2
O, compounds 2-5 and 8 were
dissolved in DMSO at 10 M and applied to the cell culture medium
supplemented DMEM). The cell culture experiments were conducted
three times independently and normalized to 100, corresponding to
-
4
(
1
5 min. Glucose uptake was initiated by the addition of 0.1 mL of
-
5
3
WY14, 643 × 10 M, a PPARR agonist used as positive control. The
activation of PPARR by FE, the isolated compounds, and the positive
control resulted in the expression of luciferase and consequent increment
of the luminescent signals, which were measured by spectrophotometry.
Results were expressed as the relative activation of PPARR proportional
to the luminescent signal emitted by the control conditions (DMSO).
Results are expressed as mean ( SD.
KRP buffer and 37 MBq/L 2-deoxy-D-[ H]glucose and 1 mmol/L
glucose as final concentrations. After 10 min, glucose uptake was
terminated by washing the cells three times with cold PBS. The cells
were lysed with 0.7 mL of 1% Triton X-100 at 37 °C for 20 min. The
radioactivity retained by the cell lysates was determined by a scintil-
lation counter. Assays were repeated at least once (n ) 2), and data
were analyzed by comparison of experimental samples of the same
treatment conditions as a group with negative control (untreated)
samples, positive (insulin-treated) samples, or experimental samples
with different treatment conditions. If values were below that of the
negative control, they were interpreted as due to inhibitory activity.
Adipocyte Differentiation Assay. Undifferentiated 3T3-L1 preadi-
pocytes were induced to differentiate into adipocytes as described above.
The degree of the differentiation of the cells induced by different agents
was evaluated by microscopic observation of lipid accumulation, as
well as by their glucose uptake activities at the end of the induction.
The glucose uptake assay was chosen and performed here for
determination of the degree of adipocyte differentiation on the basis
of the observation that differentiated adipocytes can be induced by
insulin to take up glucose, whereas undifferentiated preadipocytes
Acknowledgment. The authors wish to thank J. Kim, Y. Cao, and
Y. Liu, Edison Biotechnology Institute and Department of Biological
Sciences, Ohio University, for their help in performing the antidiabetic
and adiposity bioassays. We would also like to thank Dr. Guido F.
Pauli, the University of Illinois at Chicago, for valuable suggestions
and linguistic improvement of the manuscript.
Supporting Information Available: 1H and C NMR, COSY,
13
1
3
HMQC, HMBC, and ROESY spectra of 1 and 2. C NMR data for
compounds 6 and 8. This material is available free of charge via the
Internet at http://pubs.acs.org.
2
7,28
cannot.
In addition, a near-linear relationship was found between
References and Notes
the glucose uptake activity of differentiated adipocytes and the
triglyceride contents of cells (unpublished data). Glucose uptake and
adipocyte differentiation inhibition assays for each compound or fraction
were performed at least twice (n ) 2). The result was normalized and
expressed as percentage by considering the activities of the positive (1
nmol/mL of insulin) and negative control (MeOH) as 100% and 0%,
respectively. Results were reported as mean ( standard error of means
(
(
(
(
1) Hemmer, W.; Focke, M.; Wantke, F.; G o¨ tz, M.; Jarisch, R.; J a¨ ger, S.
Allergy 2000, 55, 923–930.
2) Eddouks, M.; Maghrani, M.; Lemhadri, A.; Ouahidi, M. L.; Jouad,
H. J. Ethnopharmacol. 2002, 82, 97–103.
3) Eddouks, M.; Maghrani, M.; Zeggwagh, N. A.; Haloui, M.; Michel,
J. B. J. Ethnopharmacol. 2005, 99, 49–54.
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Michel, J. B.; Eddouks, M. J. Ethnopharmacol. 2004, 91, 309-
316.
(
SEM). Data were analyzed by comparing compound-treated samples
with untreated negative control samples or with insulin-treated positive
samples using one-way ANOVA with Tukey’s post hoc test. Signifi-
cance level was set at p e 0.05.
PPAR Reporter Cell Lines. Preparation of Plasmids. MH100-
TK-LUC was utilized as luciferase reporter gene, and ꢀ-galactosidase
gene was used as internal control. PPARR and RXRR constructs and
(5) Eddouks, M.; Maghrani, M. J. Ethnopharmacol. 2004, 94, 149-
54.
6) Iossifova, T.; Kostova, I.; Evstatieva, L. N. Biochem. Syst. Ecol. 1997,
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(
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(
(
7) Kostova, I.; Iossifova, T. Fitoterapia 2007, 78, 85–106.
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Ecol. 2004, 32, 1069–1071.
ꢀ
-galactosidase vector were transfected with MH100-TK-LUC. In order
-
1
to equalize the DNA amount, the VDR vector plasmid was used.
All the plasmids contain the ampicillin resistance gene, which are
controlled by SV40 promotor, and all plasmids originate from the
Nuclear Hormone Receptor Research group of the Department of
Biochemistry and Molecular Biology, Debrecen, Hungary. DNA was
transformed into Escherichia coli DH5-R cells using heat shock
transformation. The plasmids were replicated in DH5-R E. coli grown
in Luria-Bertani (LB) medium supplemented with ampicillin (25 ng/
mL). Plasmid extraction was conducted via Wizard Prep Mini Column
purification kit.
(9) Damtoft, S.; Franzyk, H.; Jensen, S. R. Phytochemistry 1992, 31, 4197–
4201.
10) Shulman, A. I.; Mangelsdorf, D. J. N. Engl. J. Med. 2005, 353, 604–
(
(
(
6
15.
11) Evans, R. M.; Barish, G. D.; Wang, Y. X. Nat. Med. 2004, 10, 355–
61.
12) Lee, C. H.; Olson, P.; Evans, R. M. Endocrinology 2003, 144, 2201–
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(13) Bays, H.; Stein, E. A. Expert Opin. Pharmacother. 2003, 4, 1901–
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Bai, Naisheng
