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ZIEL Research Center of Nutrition and Food Sciences, Department of Biochemistry, Technical Universtiy of
Munich, Gregor-Mendel-Str. 2, Freising 85350, Germany
b
Pharmaceutical Institute, Department of Pharmaceutical Biology, Christian-Albrechts-University of Kiel,
Gutenbergstrasse 76, Kiel 24118, Germany
c
BioActive Food GmbH (HV), Am Ihlsee 36a, Bad Segeberg 23795, Germany
A R T I C L E
I N F O
A B S T R A C T
Article history:
Extracts and flavonoids from onion are described as having anti-diabetic activities. We here
demonstrate that an onion extract and individual flavonoids thereof diminish glucose uptake
mediated by the intestinal glucose transporter SGLT1 when expressed in oocytes and studied
2015
in mouse intestinal segments in vitro. Strongest inhibition of SGLT1 was observed for quercetin-
4-O-glucoside (Q4glc) in oocytes but with only moderate inhibition in jejunal segments of
Available online
mice. An oral glucose tolerance test (OGTT) performed in obese/hyperglycaemic mice revealed that the onion extract to reduce blood glucose increases significantly. However, an
Keywords:
OGTT performed in healthy volunteers after administration of the onion extract failed to
Onion
reveal an effect on glucose and insulin levels. Despite its capability to inhibit intestinal glucose
Anti-hyperglycaemic
uptake via SGLT1 in vitro and in mice in vivo, the onion extract did not alter blood glucose
SGLT1
levels during an OGTT in human volunteers and this may predominantly be due to a dosing
Type 2 diabetes
effect.
Quercetin-4-O-glucoside
Quercetin
* Corresponding author. Molecular Nutrition Unit, Technical University Munich, Gregor-Mendel-Strasse 2, 85350 Freising-Weihenstephan,
Germany. Tel.: +49 8161713401; fax: +49 8161713999.
E-mail address: hannelore.daniel@tum.de (H. Daniel).
Abbreviations: ANOVA, analysis of variances; iAUC, incremental area under the curve; GLUT2, glucose transporter 2; IGT, impaired glucose
tolerance; BW, body weight; Q4glc, quercetin-4-O-glucoside; Q3,4diglc, quercetin-3,4-O-diglucoside; HPLCDAD/ESIMS2, high performance liquid chromatographyelectrospray ionization tandem mass spectrometry; OGTT, oral glucose tolerance test; SGLT1, sodiumdependent glucose co-transporter 1; T2DM, type 2 diabetes mellitus
http://dx.doi.org/10.1016/j.jff.2015.06.037
1756-4646/ 2015 Elsevier Ltd. All rights reserved.
118
1.
Introduction
2.
2.1.
2.2.
Characterization and quantification of onion
polyphenols by HPLCDAD and HPLCESIMS2
One milligram of dried onion extract was dissolved in 1 mL of
methanol (70%) and sonicated for 30 min. The extract was centrifuged (10 min, 20,200 g at 20 C) before separation and
analysis was performed by HPLCDAD (waters e2695 separation module, Alliance; PDA waters 2998). Chromatographic
separation was performed on an analytical Phenonenex RPC18 column (Aqua 5 m, 125 , 250 4.6 mm) with a security
guard cartridge (AQ C18, 4.0 3.0 mm) at 30 C. Solvent A consisted of water/trifluoroacetic acid (TFA) (99.99/0.01, v/v) and
solvent B of acetonitrile (ACN)/TFA (99.99/0.01, v/v); gradient
elution: 010 min 100% A, 1040 min down to 50% A, 40
50 min down to 0% A, 5051 min up to 100% A, 5160 min 100%
A; flow rate 1 mL/min. Identification and quantification were
performed by co-chromatography of external standards. Calibration curves were used in the range of 5300 g/mL. UV
spectra for flavonols and the aglycones were recorded at 330 nm
and 360 nm, respectively.
Flavonoids were characterized by HPLCESIMS2 using a
Bruker Esquire-LC with an ESI interface in a negative ionization modus (Bruker Daltonics, USA). Fragmentation was carried
out in the automatic mode by which the most abundant ions
were fragmented (MS2). HPLC conditions were performed as described earlier, but TFA was replaced by 0.1% acetic acid (Agilent
1100 series HPLC system, Agilent 1100 UV/VIS, Agilent Technologies, Germany). For determination of the molecular weight
of compounds, a scan from m/z 50 to 1600 was used. Nitrogen was used as nebulizing gas at pressure of 40 psi and the
flow rate was adjusted to 1 mL/min. The temperature and the
2.3.
2.4.
Electrophysiological studies in oocytes
expressing hSGLT1
The experimental procedure was carried out as described previously by Schulze et al. (2014). Apparent kinetic constants Km
and Imax (nA) and IC50 values were determined as described
by Kottra and Daniel (2007). Dixon-type experiments were
carried out at 60 mV.
2.5.
Uptake experiments in oocytes expressing hGLUT2
with radio-labeled glucose
Since the glucose transporter 2 (GLUT2) is a non-electrogenic
transporter, all studies on hGLUT2 expressed in oocytes were
performed using 3H-labelled 2-desoxy-D-glucose (2-DG), a GLUT2
specific substrate which is not metabolized. Four days after
cRNA injection, 510 oocytes were incubated in 250 L of Barths
solution (pH 7.4) containing 1 mM non-labelled 2-DG and 1.8 M
3
H-labelled 2-DG [2 Ci/mL] in the presence or absence of the
compound to be tested. After 30 min incubation on a shaking
incubator at RT, incubation medium was removed, oocytes were
washed 3 times in ice-cold Barths solution and lysed individually in scintillation vials by addition of 100 L 10% SDS and
agitation for 1 h at RT. After mixing with 3 mL scintillation cocktail (Rotiscint Eco plus, Roth, Germany) the signals were
detected in a liquid scintillation counter (Perkin Elmer Wallac
GmbH, Freiburg,Germany). Results were expressed as percentage of 2-DG per oocyte per 30 min.
2.6.
119
Animals
2.7.
Glucose uptake studies employing everted
jejunal rings
Twelve week old male C57BL/6N mice, kept on a standard chow,
were fasted for 6 hours. Afterwards mice were anaesthetized
with isoflurane (Baxter, Unterschleiheim, Germany) and killed
by cervical dislocation. The experimental procedure was carried
out as described previously by Schulze et al. (2014).
2.8.
2.9.
Subjects
120
2.10.
Study design
2.11.
2.12.
Statistical analysis
Identification
tr (min)
max (nm)
[M-H]
(m/z)
MS2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Quercetin-triglucoside
Quercetin-diglucoside
Quercetin-triglucoside
Quercetin-triglucoside
Quercetin-3,4-O-diglucoside
Isorhamnetin-diglucoside
Quercetin-diglucosid
Quercetin-diglucosid
Quercetin-glucosid
Quercetin-diglucosid
Quercetin-4-O-glucosid
Kaempferol-glucosid
Isorhamnetin-glucosid
Quercetin-glucosid
Quercetin-glucosid
Quercetin
Kaempferol
Isorhamnetin
22.8
24.4
26.2
26.7
26.9
27.4
28.6
29.1
29.5
31
31.4
31.9
32.2
33.5
34.7
36
39.2
39.7
250/345
253/355
252/364
265/345
265/345
251/345
249/364
254/364
254/356
252/360
252/364
266/363
252/364
250/360
252/361
254/364
265/364
254/364
787
625
787
787
625
639
625
625
463
625
463
447
477
463
463
301
285
315
927 [2MH]
301 (324)
927 [2MH]
301 (162)
301 (162)
3.
Results
3.1.
Identification and characterization of onion
polyphenols by HPLCDAD and HPLCESIMS2
Flavonols in the onion extract were analyzed by HPLCDAD and
HPLCESIMS2 as described in Section 2. In total, up to 20 flavonol derivatives could be identified (Table 1, Fig. 1). The total
amount of flavonoids was calculated to be equivalent to
18.8 mg/100 mg onion dry mass. HPLC analysis of individual
components without prior hydrolysis of the extract identified quercetin-derivatives as the most abundant compounds,
with Q4glc (9.5 mg/100 mg dry mass; compound 11), Q3,4diglc
(5.4 mg/100 mg dry mass; compound 5) and the aglycon quercetin (5.4 mg/100 mg dry mass; compound 16) itself as the main
component (Fig. 1, Table 1). Additional quercetin-glucosides were
also found but only in trace amounts. Other flavonols detected were isorhamnetin and kaempferol as well as derivatives
thereof, but those all had 30 to 50-times lower concentrations than the quercetin derivatives (Fig. 1, Table 1).
3.2.
Inhibition of human SGLT1 expressed Xenopus
laevis oocytes by onion extracts and flavonols from onion
using TEVC
To exclude that the test compounds by themselves are substrates of human SGLT1, extracts were first tested for their ability
to generate a transport current in the absence of any specific
121
3.3.
Inhibition of human GLUT2 expressed in cRNAinjected Xenopus laevis oocytes by the onion extract and
quercetin derivatives
There is some evidence that at high luminal glucose concentration GLUT2 may translocate from an intracellular pool into
Fig. 1 HPLC chromatogram of the onion extract. Detection at 330 nm. Peak identities are numbered in Table 1.
122
Fig. 2 Inhibition of the -MDG-induced transport currents (A) in the presence of increasing concentrations of the onion
extract at different membrane potentials, -MDG(1) means the measurement of -MDG-induced transport currents before
the exposition to onion extract; -MDG(2) means the measurement of -MDG-induced transport currents after the
exposition to onion extract; (B) at 60 mV in the presence of flavonoids identified in the onion extract. All compounds were
added in a concentration of 100 M. The IC50 values were calculated at a membrane potential of 60 mV. (C) Dixon plot
analysis for inhibition of -MDG-mediated currents by the onion extract or (D) by Q4glc. The crossing point of regression
lines above the abscissa shows that the inhibition is of competitive type. The Ki value determined from the plot is
231.3 98.4 g/mL for the onion extract and 0.11 0.04 mM for Q4glc. Mean values of 46 oocytes SEM. Statistical
differences were evaluated by one-way ANOVA with Dunnetts posthoc test. *p < 0.05, **p < 0.01, ***p < 0.001.
the apical membrane allowing on top of SGLT1 larger quantities of glucose to be absorbed. We therefore studied whether
the onion extract and its constituents can also inhibit human
GLUT2 when expressed in oocytes. As shown in Fig. 3, the onion
3.4.
The onion extract and Q4glc inhibit SGLT1 in
murine jejunal tissues
Uptake experiments in everted jejunal rings were carried out
using 1 mM -MDG supplemented with 0.3 Ci/mL [ 14 C]radiolabelled -MDG either in the absence or in the presence
of increasing concentrations of the onion extract or Q4glc. The
apparent Km for -MDG uptake into the mouse tissue was
6.5 1.1 mM (n = 4) and affinity was thus considerably lower
than in oocytes. The onion extract (Fig. 4A) inhibited -MDG
uptake significantly dose-dependent with a calculated EC50 value
of 2.6 0.9 mg/mL. Q4glc showed only moderate inhibition of
SGLT1 in mouse intestinal rings with a decrease of -MDG
uptake by 25% at a concentration of 1 mM and 35% at 2 mM
but this failed to reach statistical significance (Fig. 4B).
Fig. 3 Inhibition of 2-DG uptake in oocytes expressing
hGLUT2 in the presence of 0.25 mg/mL onion extract (OE)
or 100 M of the major onion flavonols. Mean values of 46
oocytes SEM. Statistical differences were evaluated by
one-way ANOVA with Dunnetts posthoc test. *p < 0.05,
**p < 0.01, ***p < 0.001.
3.5.
The onion extract but not Q4glc diminish
postprandial glucose response in obese C57BL/6N mice
C57BL/6 mice were fed a high-fat diet for 12 weeks which led
to a weight gain of 12 g (41.3 0.8 g vs. 29.0 0.3 g, p < 0.0001)
123
Fig. 4 Dose-dependent inhibition of -MDG uptake into murine everted jejunal rings by onion extract (A) or Q4glc (B).
Mean values of 35 mice SEM. Statistical differences were evaluated by one-way ANOVA with Dunnetts posthoc test.*
p < 0.05, **p < 0.01, ***p < 0.001.
3.6.
In healthy young men, administration of the onion
extract did not cause changes in postprandial glucose and
insulin levels nor urinary glucose excretion
Fifteen healthy young men were subjected to an OGTT with
or without prior administration of 3.1 g onion extract. Similar
to the findings in lean mice, the changes () in postprandial
venous blood glucose and insulin response or in iAUC (Table 2)
were not significantly different at any time in the same volunteers with or without the onion extract (Fig. 6A, B). Since
kidney tubular cells express SGLT1 and SGLT2 we also monitored glucose excretion over 24 hours but the onion extract did
not show any significant changes in urinary glucose output
when compared to glucose alone (Fig. 6C).
4.
Discussion
4.1.
4.2.
Interaction of the onion extract and contained
polyphenols with the glucose transporters in vitro
Although inhibition of glucose transporters by onion extracts
has never been studied, the interaction of quercetin and Q4glc
with SGLT1 has been demonstrated in several in vitro studies
(Ader, Block, Pietzsch, & Wolffram, 2001; Cermak, Landgraf, &
Wolffram, 2004; Kottra & Daniel, 2007; Kwon et al., 2007). Quercetin only modestly inhibited hSGLT1 in oocytes in line with
previous findings (Kottra & Daniel, 2007) although other studies
failed to show any significant effect of quercetin on glucose
uptake via SGLT1 (Cermak et al., 2004; Kwon et al., 2007; Song
et al., 2002). The strongest inhibition amongst the onion flavonols tested was observed with Q4glc, indicating that a glucose
moiety at this position increases SGLT1 binding affinity. This
124
4.3.
Fig. 5 Effect of a single dose of Q4glc or onion extract coadministered with glucose (60 mg) on rise in blood glucose
concentration in 20 week old male C57BL/6N mice at the
end of the 12-week feeding trial. (A) Control diet, (B) high
fat diet. (C) IAUCs calculated over a period of 120 min. Data
are shown as mean of n = 10 mice SEM. Statistical
difference was evaluated with two-way-ANOVA with
repeated measures for one factor (time) and multiple
comparison posthoc test (TukeyKramer test) for (A) and (B)
or by use of one-way ANOVA without repeated measures
(C) using SAS. *p < 0.05, **p < 0.01.
125
Table 2 . Postprandial venous blood glucose and plasma insulin incremental area under the curve (iAUC) in healthy
young men after an OGTT with 75 g glucose (reference) or after an OGTT with prior ingestion (30 min) of 3.1 g onion
extract.
Glucose iAUC (mg/dL)
Time (min)
Reference
Onion extract
p-Value
Reference
Onion extract
p-Value
015
030
045
060
090
0120
0180
167.3 13.8
567.8 50.6
1224.8 108.9
1854.8 178.5
2678.3 299.6
3075.8 383.9
3354.75 485.1
174.8 16.4
504.8 59.7
1033.5 117.3
1622.3 173.7
2582.3 270.4
3158.3 388.3
3689.25 523.0
0.66
0.36
0.13
0.2
0.73
0.83
0.48
63.2 13.8
1167.2 212.3
3959.8 623.3
7406.1 1029.6
12,698.0 1643.0
15,353.8 1893.0
16,880.2 1991.7
144.9 48.1
1110.0 261.6
3306.1 742.6
6210.7 1355.9
11,596.8 2079.1
14,818.8 2282.5
16,636.6 2344.0
0.09
0.68
0.12
0.1
0.33
0.68
0.87
All values are means SEM, n = 15. Significant differences in venous blood glucose and plasma insulin iAUCs were evaluated by one-wayANOVA with repeated measures and multiple comparison posthoc test (TukeyKramer test).
126
5.
Conclusions
Acknowledgements
The authors thank the Bundesministerium fr Bildung und
Forschung (BMBF) (0315371B) for funding these studies.We thank
Barbara Gelhaus, Pia Rder and Ronny Scheundel for excellent technical assistance and the participants of the human
study for their compliance. Furthermore, we would like to thank
Adelmar Stamfort for statistical support.We thank Ulrich Girreser
and Sven Wichmann for excellent support with the LCMS analysis. The authors have declared no conflict of interest.
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