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Mol. Nutr. Food Res. 2015, 59, 843–852 843
DOI 10.1002/mnfr.201400791
RESEARCH ARTICL E
Identification and characterization of [6]-shogaol from
ginger as inhibitor of vascular smooth muscle cell
proliferation
Rongxia Liu1,2,ElkeH.Heiss
1, Nadine Sider1, Andreas Schinkovitz3, Barbara Groblacher3,
Dean Guo2, Franz Bucar3, Rudolf Bauer3, Verena M. Dirsch1and Atanas G. Atanasov1
1Department of Pharmacognosy, University of Vienna, Vienna, Austria
2Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory
for TCM Standardization Technology, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai, P. R. China
3Institute of Pharmaceutical Sciences, Department of Pharmacognosy, Karl-Franzens-University Graz, Graz,
Austria
Received: November 3, 2014
Revised: January 16, 2015
Accepted: January 16, 2015
Scope: Vascular smooth muscle cell (VSMC) proliferation is involved in the pathogenesis
of cardiovascular disease, making the identification of new counteracting agents and their
mechanisms of action relevant. Ginger and its constituents have been reported to improve
cardiovascular health, but no studies exist addressing a potential interference with VSMC
proliferation.
Methods and results: The dichloromethane extract of ginger inhibited VSMC proliferation
when monitored by resazurin metabolic conversion (IC50 =2.5 g/mL). The examination of ma-
jor constituents from ginger yielded [6]-shogaol as the most active compound (IC50 =2.7 M).
In the tested concentration range [6]-shogaol did not exhibit cytotoxicity toward VSMC and
did not interfere with endothelial cell proliferation. [6]-shogaol inhibited DNA synthesis and
induced accumulation of the VSMC in the G0/G1cell-cycle phase accompanied with activa-
tion of the nuclear factor-erythroid 2-related factor 2 (Nrf2)/HO-1 pathway. Since [6]-shogaol
lost its antiproliferative activity in the presence of the heme oxygenase-1 (HO-1) inhibitor tin
protoporphyrin IX, HO-1 induction appears to contribute to the antiproliferative effect.
Conclusion: This study demonstrates for the first time inhibitory potential of ginger con-
stituents on VSMC proliferation. The presented data suggest that [6]-shogaol exerts its antipro-
liferative effect through accumulation of cells in the G0/G1cell-cycle phase associated with
activation of the Nrf2/HO-1 pathway.
Keywords:
Ginger / Heme oxygenase-1 / Nrf2 / [6]-Shogaol / Vascular smooth muscle cell
Correspondence: Dr. Atanas G. Atanasov, Department of Phar-
macognosy, Faculty of Life Sciences, University of Vienna, Al-
thanstrasse 14, A-1090 Vienna, Austria
E-mail: atanas.atanasov@univie.ac.at
Abbreviations: BrdU, bromodeoxyuridine; EBM, endothelial cell
basal medium; FCS, fetal calf serum; HO-1, heme oxygenase-1;
HUVEC, human umbilical vein endothelial cells; LDH, lactate de-
hydrogenase; MEF, mouse embryonic fibroblasts; Nrf2, nuclear
factor-erythroid 2-related factor 2; PDGF, platelet-derived growth
factors; PI, propidium iodide; SAR, structure-activity-relationship;
VSMC, vascular smooth muscle cell; WT, wild type
1 Introduction
Ginger, Zingiber officinale Roscoe (Zingiberaceae), is widely
used as a spice in foods and beverages, and has also a long
history of use in traditional medicine for the treatment of
inflammation, rheumatic disorder, indigestion, vomiting,
and fever, among others [1–4]. Many pharmacological activ-
ities have been reported for this plant and its major pungent
principles, including anti-inflammatory, anti-tumorigenic,
anti-apoptotic, anti-hyperglycemic, cancer-chemopreventive,
anti-lipidemic, and anti-emetic effects [4–9].
Additional corresponding author: Dr. Elke H. Heiss
E-mail: elke.heiss@univie.ac.at
C2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in
any medium, provided the original work is properly cited.
844 R. Liu et al. Mol. Nutr. Food Res. 2015, 59, 843–852
Cardiovascular disease is the number one cause of death
in the world, mainly elicited by atherosclerosis together with
hypertension [10]. Aberrant and accelerated vascular smooth
muscle cell (VSMC) proliferation not only contributes to ini-
tial atherosclerotic plaque formation but also to restenosis
(pathological renarrowing of the vessel lumen) after surgical
interventions like percutaneous transluminal coronary an-
gioplasty or bypass surgery. To overcome restenosis, drug-
eluting stents have been developed, aiming at inhibiting
VSMC growth by the release of antiproliferative substances.
The most prominent drugs used in drug-eluting stents so
far have been paclitaxel (a microtubules stabilizing agent)
and sirolimus (a mTOR inhibitor). These compounds, how-
ever, exhibit a number of unresolved drug-related issues such
as impaired reendothelialization and delayed thrombosis in-
duction [11,12], which makes the discovery of novel effective
compounds and molecular mechanisms suppressing VSMC
proliferation highly relevant.
Plant derived natural products proved to be an excellent
resource for the identification of new lead compounds [13].
While ginger is reported to possess vasoprotective effects
[14, 15], its impact on VSMC proliferation in particular has
not been studied so far. In this study, we therefore examined
the antiproliferative potential of ginger extract and some
of its major active components ([6]-gingerol, [6]-shogaol,
zingerone, [6]-paradol, and rac-[6]-dihydroparadol) in VSMC,
and characterized in more detail the cellular mode of action
of the most active identified compound, [6]-shogaol.
2 Materials and methods
2.1 Chemicals and reagents
Primary rat aortic VSMC were purchased from Lonza
(Braine-L’Alleud, Belgium) and the human umbilical
vein endothelial cells (HUVECtert) were provided by
Dr. Hannes Stockinger (Medical University of Vienna, Aus-
tria) [16]. Wild type (WT) and isogenic nuclear factor-erythroid
2-related factor 2 (Nrf2)–/– mouse embryonic fibroblasts
were kindly provided by Dr. T. Kensler, University of
Pittsburgh, USA. Platelet-derived growth factors (PDGF)-BB
was supplied from Bachem (Weilheim, Germany). The heme
oxygenase-1 (HO-1) inhibitor tin protoporphyrin IX dichlo-
ride was from Enzo Life Sciences (Lausen, Switzerland). [6]-
Parodol and rac-[6]-dihydroparadol were isolated as described
[17]. All other used reagents were from analytical grade and
obtained from Sigma-Aldrich (Vienna, Austria). The anti-HO-
1 antibody was from Stressgene (purchased via Enzo, Lausen,
Switzerland), the anti-actin antibody [mouse anti-actin,
monoclonal (Clone: C4); #69100] was from mpbio (Eschwege,
Germany), and the secondary horseradish-peroxidase-
coupled antibodies came from Cell Signaling (Heidelberg,
Germany).
2.2 Ginger extraction
Root material of Zingiber officinale (Zingiberaceae) was pur-
chased from PLANTASIA (Oberndorf, Austria). A voucher
sample is kept at the University of Graz, Department of
Pharmacogonosy under reference number 730149. Ground
root material (3.17 g) was mixed with 0.97 g diatomaceous
earth (Dionex Corporation, Sunnyvale, CA, USA) before
being extracted with HPLC grade dichloromethane using an
Accelerated Solvent Extractor (ASE 200, Dionex Corporation,
Sunnyvale, CA, USA). Extraction conditions were as follows:
Temperature: 44⬚C, cell preheating time: 1 min, pressure:
68.9 bar, static time: 5 min, flush volume: 150%. Three
extraction cycles were performed yielding 168 mg of dry
extract (5.3%) after solvent evaporation.
2.3 Cell culture
VSMC were cultivated in DMEM–F12 (1:1) supplemented
with 20% fetal calf serum (FCS), 30 g/mL gentamicin, and
15 ng/mL amphotericin B. VSMC with passage number be-
tween 6 and 14 were used in this study. HUVEC were grown
in endothelial cell basal medium (EBM) supplemented with
10% FCS, 100 U/mL penicillin, 100 g/mL streptomycin,
1% amphotericin B and EBMTM SingleQuotsR, containing re-
combinant human epidermal growth factor, hydrocortisone,
gentamicin sulfate, amphotericin B and 0.4% bovine brain
extract. Mouse embryonic fibroblasts (MEF) were grown in
DMEM supplemented with 10% FCS, 100 U/mL penicillin,
and 100 g/mL streptomycin.
2.4 Resazurin conversion assay
VSMC were seeded in 96-well plates at 5 ×103cells/well.
After 24 h, cells were serum-starved for 24 h to render them
quiescent. Quiescent cells were pretreated for 30 min with
ginger extract, compounds, or vehicle (0.1% DMSO) as indi-
cated, and subsequently stimulated for 48 h with PDGF-BB
(20 ng/mL). To measure the number of metabolically active
VSMC by resazurin conversion [18, 19], cells were washed
with PBS and incubated in serum-free medium containing
10 g/mL resazurin for 2 h. Total metabolic activity was
measured by monitoring the increase in fluorescence at a
wavelength of 590 nm using an excitation wavelength of
535 nm in a 96-well plate reader (Tecan GENios Pro).
HUVECtert cells [16, 20] were seeded in 96-well plates at
5×103cells/well. After 24 h, HUVECtert cells were treated
with compounds or vehicle (0.1% DMSO) as indicated and
incubated for 48 h. Then, cells were washed with PBS and in-
cubated in culture medium containing 10 g/mL resazurin
for 2 h. The detection step is performed as described above.
2.5 Crystal violet biomass staining
VSMC were seeded in 96-well plates at 5 ×103cells/well.
Twenty-four hours later, cells were serum starved for 24 h
C2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
Mol. Nutr. Food Res. 2015, 59, 843–852 845
to render them quiescent. Quiescent cells were pretreated
for 30 min with ginger extract, compounds, or vehicle (0.1%
DMSO) as indicated and subsequently stimulated for 48 h
with PDGF-BB (20 ng/mL). To determine the total biomass
by crystal violet staining, cells were then incubated in 100 L
of crystal violet staining solution (0.5% crystal violet, 20%
methanol) for 15 min and then washed with ddH2O. After
drying, 100 L EtOH/Na-citrate solution were added (EtOH:
0.1M Na-citrate =1:1) and the absorbance of the samples was
measured at 595 nm in a 96-well plate reader (Tecan sunrise).
2.6 5-Bromo-2-deoxyuridine (BrdU) incorporation
assay
VSMC were seeded in 96-well plates at 5 ×103cells/well.
Twenty-four hours later, cells were serum starved for 24 h to
render them quiescent. Quiescent cells were pretreated for
30 min with compounds, or vehicle (0.1% DMSO) as
indicated and subsequently stimulated with PDGF-BB
(20 ng/mL). To estimate de novo DNA synthesis in VSMC
[21, 22], BrdU was added 2 h after PDGF stimulation, and
the incorporation amount was determined 22 h afterwards
according to the manufacturer’s instructions (Roche Diag-
nostics).
2.7 Assessment of cytotoxicity
VSMC were seeded in 96-well plates at 5 ×103cells/well.
Twenty-four hours later, cells were serum starved for 24 h
to render them quiescent. Quiescent cells were pretreated
for 30 min with compounds, or vehicle (0.1% DMSO) as
indicated, and subsequently stimulated for 24 h with PDGF-
BB (20 ng/mL). Loss of cell membrane integrity as a sign
for cell death can be quantified by the release of the soluble
cytosolic protein lactate dehydrogenase (LDH) [20, 23]. For
this, the supernatant of the treated cells was assessed for LDH
activity. For estimation of the total LDH, identically treated
samples were incubated for 45 min in the presence of 1%
Triton X-100. The released and total LDH enzyme activity was
measured for 30 min at the dark in the presence of 4.5 mg/mL
lactate, 0.56 mg/mL NAD+, 1.69 U/mL diaphorase, 0.004%
w/v BSA, 0.15% w/v sucrose, and 0.5 mM 2-p-iodophenyl-3-
nitrophenyl tetrazolium chloride. The enzyme reaction was
stopped with 1.78 mg/mL oxymate and the absorbance was
measured at 490 nm. Potential effects on cell viability were
estimated as percentage of extracellular LDH enzyme activity.
The cytotoxic natural product digitonin (100 g/mL) was used
as a positive control.
2.8 Cell-cycle analysis
VSMC were seeded in 12-well plates at 1 ×104cells/well.
Twenty-four hours later, cells were serum starved for 24 h
to render them quiescent. Quiescent cells were preincu-
bated with [6]-shogaol (10 M) or vehicle (1% DMSO) for
30 min. PDGF-BB (20 ng/mL) was added, and 16 h later
cells were trypsinized, washed once with PBS, and resus-
pended in a hypotonic propidium iodide (PI) solution con-
taining 0.1% v/v Triton X-100, 0.1% w/v sodium citrate,
and 50 g/mL PI. After incubation at 4⬚C overnight, PI-
stained nuclei were analyzed by flow cytometry (excitation
488 nm, emission 585 nm; FACScalibur; BD Biosciences,
Germany).
2.9 Immunoblot analysis
Cells (MEF or VSMC) were seeded onto six-well plates
((3–4) ×105cells/well). Twenty-four hours later, MEF were
treated with vehicle (0.1% DMSO) or [6]-shogaol at the indi-
cated concentrations for 20 h; VSMC were serum starved for
another 24 h, then quiescent cells were pretreated for 30 min
with vehicle (0.1% DMSO) or [6]-shogaol as indicated and
subsequently stimulated for 20 h with PDGF-BB (20 ng/mL).
Then, cells were lysed and protein extracts were subjected
to SDS-PAGE electrophoresis and immunoblot analysis as
described [20, 22]. Proteins were visualized using enhanced
chemiluminescence reagent and an LAS-3000 luminescent
image analyzer (Fujifilm) with AIDA software (Raytest) for
densitometric evaluation.
2.10 Cell counting
VSMC and HUVECtert were seeded and treated as described
in chapter 2.4 (“Resazurin conversion assay”). Cell numbers
were determined at different time points (with time point
zero corresponding to the treatment with the solvent vehicle,
0.1% DMSO) upon trypan blue staining with a ViCell counter
(Beckman Coulter, Brea, CA).
2.11 Statistical analysis
Statistical analysis was performed by analysis of variance
/Bonferroni test or by ttest (when comparing just two ex-
perimental groups). The number of experiments is given in
the figure legends, and a probability value <0.05 was con-
sidered significant. All tests were performed using GraphPad
PRISM software, version 4.03.
3 Results
3.1 Ginger extract and its major bioactive
components inhibit VSMC proliferation
For evaluating whether ginger contains compounds able
to inhibit PDGF-induced proliferation of VSMC, a
dichloromethane extract of Z. officinale roots was applied
at different concentrations (0.3–30 g/mL; Fig. 1), and the
total amount of metabolically active cells was measured
C2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
846 R. Liu et al. Mol. Nutr. Food Res. 2015, 59, 843–852
Figure 1. Metabolic activity of VSMC exposed to different con-
centrations of ginger extract. Quiescent VSMC were pretreated
with the indicated concentrations of ginger dichloromethane ex-
tract or an equal volume of the solvent vehicle (0.1% DMSO)
for 30 min, and then stimulated with 20 ng/mL PDGF-BB for
48 h. The metabolic activity was determined at the end of
the stimulation period by the resazurin conversion method. All
values are normalized to the signal obtained from the PDGF-
stimulated, vehicle (0.1% DMSO)-treated cells. The data groups
represent means ±SD from three independent experiments (n.s.,
not significant; ***p<0.001; *p<0.05; analysis of variance
(ANOVA)/Bonferroni).
after 48 h by the resazurin conversion method [18, 19]. The
extract suppressed VSMC proliferation concentration depen-
dently with an IC50 of 2.5 g/mL. The highest tested con-
centration (30 g/mL) decreased the signal to the basal level
of the untreated growth-arrested cells (Fig. 1). To investi-
gate which compounds from ginger are potentially mediat-
ing this antiproliferative effect, five major bioactive ginger
compounds, [6]-gingerol, [6]-shogaol, zingerone, [6]-paradol,
and rac-[6]-dihydroparadol (Fig. 2) were tested. Among these
five compounds, [6]-shogaol showed best antiproliferative ac-
Figure 2. Structures of the major ginger components studied in
this work.
tivity with an IC50 of 2.7 M in the resazurin conversion
assay (Table 1 and Fig. 3A). [6]-gingerol, [6]-paradol, and
rac-[6]-dihydroparadol, also inhibited VSMC proliferation but
less potently than [6]-shogaol (with IC50s in the range 5.3–
13.2 M; Table 1), while zingerone showed no activity up to
100 M. The resazurin conversion method is based on the
Ta b l e 1 . Comparison of the activity of a ginger extract and several major pure compounds in VSMC and HUVECtert. Antiproliferative effects
were evaluated by the resazurin metabolic conversion assay or crystal violet biomass staining. Cell death was evaluated by a
colorimetric LDH activity assay as described in details in the “Materials and methods” section. The data shown represent means
of three to five independent experiments each in triplicate or quadruplet. IC50 were determined by the GraphPad Prism software
version 4.03 (GraphPad Software Inc., USA). ANOVA/Bonferroni analysis was used for evaluation of the statistical significance
(n.s. indicates p>0.05)
VSMCs HUVECs
Resazurin metabolic Crystal violet Cell death Resazurin metabolic
conversion (IC50,g/mL or M) biomass staining (IC50,M) (LDH release) conversion
DCM extract 2.51 g/mL — — —
[6]-Gingerol 13.2 M 8.77 n.s. (up to 100 M) n.s. (up to 30 M)
[6]-Shogaol 2.72 M 1.13 n.s. (up to 10 M) n.s. (up to 10 M)
Zingerone >100 M— ——
[6]-Paradol 5.3 M 3.34 n.s. (up to 30 M) n.s. (up to 30 M)
rac-[6]-Dihydroparadol 10 M 4.11 n.s. (up to 30 M) n.s. (up to 30 M)
—: not detected.
n.s.: no significant difference.
DCM, dichloromethane.
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Mol. Nutr. Food Res. 2015, 59, 843–852 847
Figure 3. Effect of [6]-shogaol on VSMC proliferation and cell
death. Quiescent VSMC were pretreated with the indicated con-
centrations of [6]-shogaol or an equal volume of the solvent vehi-
cle (0.1% DMSO) for 30 min, and then stimulated with 20 ng/mL
PDGF-BB. Cell proliferation was quantified by the resazurin con-
version method (A) and crystal violet biomass staining (B). Cell
death was estimated by measuring the cell membrane integrity
through quantification of the percentage of the cytosolic enzyme
lactate dehydrogenase (LDH) detected inside cells and in the ex-
tracellular medium (C). The cytotoxic natural product digitonin
(100 g/mL) was used as a positive control in the cell death assay
(C). The data groups represent means ±SD from three indepen-
dent experiments (n.s., not significant; ***p<0.001; **p<0.01;
*p<0.05; ANOVA/Bonferroni).
Figure 4. HUVECtert viability in the presence of [6]-shogaol. HU-
VECtert were treated with the indicated concentrations of [6]-
shogaol or equal volume of the solvent vehicle (0.1% DMSO) for
48 h. Cell numbers were estimated at the end time point by the
resazurin conversion method. All values are normalized to the
signal obtained from vehicle (0.1% DMSO)-treated cells. The an-
tiproliferative natural product paclitaxel (1 M)wasusedaspos-
itive control. The data groups represent means ±SD from three
independent experiments (n.s., not significant; ***p<0.001;
ANOVA/Bonferroni).
metabolic conversion of resazurin, which generally correlates
well to the cell number but could be potentially sensitive to
redox-active chemicals or treatments modulating the cellular
metabolic capacity. To confirm the antiproliferative effects of
the investigated compounds with a method independent of
cell metabolism and redox reactions, we quantified total cel-
lular biomass by crystal violet staining. The obtained results
were in line with the data from the resazurin conversion assay
(Table 1 and Fig. 3B). To further assure that the decreased
VSMC number upon treatment with test compounds is not
due to cytotoxicity, we also quantified cell death by measur-
ing LDH inside cells and in cell supernatants. No significant
changes in cell viability were detected in the investigated con-
centration range (Table 1 and Fig. 3C).
As vascular health also depends on a functional and intact
endothelium, an optimal vasoprotective compound inhibits
VSMC activation, but does not interfere with endothelial
cell viability. We therefore investigated the impact of the
four most active compounds on endothelial viability using
metabolic activity as readout. In contrast to paclitaxel (1 M),
none of the four ginger compounds that clearly influence
VSMC proliferation showed a negative effect on endothelial
cells when used in the same concentration range, (Table 1,
Fig. 4). Interestingly, paclitaxel had an even stronger anti-
proliferative effect in endothelial cells (IC50 =4 nM) than
in VSMC (IC50 =108 nM; not shown). To assure that the
observed differences in the potency of ginger compounds
and paclitaxel in VSMC and endothelial cells are not due to
different growth rates between the two cell types, we have
determined the doubling times of the two cell types under
the used experimental conditions. Cell counting revealed
comparable proliferation rates for the PDGF-BB stimulated
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848 R. Liu et al. Mol. Nutr. Food Res. 2015, 59, 843–852
Figure 5. Effect of [6]-shogaol on cell-cycle distribution and
DNA synthesis of VSMC. Quiescent VSMC were stimulated with
20 ng/mL PDGF-BB for 20 h, and cell proliferation was quantified
by the BrdU incorporation in newly synthesized DNA in the pres-
ence of the indicated treatments (A); or the cells were incubated
with [6]-shogaol (10 M) or equal volume of the solvent vehicle
(0.1% DMSO) for 30 min, then stimulated with 20 ng/mL PDGF-BB
for 16 h, followed by PI nuclear staining and flow cytometry
analysis (B). The data groups represent means ±SD from three in-
dependent experiments (n.s., not significant; ***p<0.001; **p<
0.01; *p<0.05; ANOVA/Bonferroni (A), 2-tailed paired t-test (B)).
VSMC (cell doubling time 30.1 h) and the HUVECtert (cell
doubling time 33.1 h).
Based on the observed promising antiproliferative profile
of action on the VSMC, we have chosen the most potent iden-
tified compound, [6]-shogaol, for further mechanistic studies.
3.2 [6]-Shogaol leads to accumulation of VSMC in
G0/G1
In order to examine the mechanism of action of [6]-shogaol
in VSMC, we first investigated whether it blocks DNA
synthesis by quantification of BrdU incorporation in treated
cells. [6]-shogaol potently blunted PDGF-stimulated DNA
synthesis in a concentration-dependent manner exhibiting
an IC50 of 3.0 M, which is in the same range as the IC50
values obtained in the resazurin conversion and the crystal
violet assay (Fig. 5A). In line with the observed inhibition
of DNA synthesis, [6]-shogaol (10 M) counteracted the
PDGF-induced transition of VSMC from the G0/G1cell-cycle
phase to the G2/M phase, as revealed by flow cytometry
analysis of PI stained nuclei (Fig. 5B).
3.3 [6]-Shogaol blocks VSMC proliferation by
Nrf2-dependent HO-1 induction
In a consequent experiment the molecular events underly-
ing the observed cell-cycle arrest in VSMC upon shogaol
exposure were investigated. Among other bioactivities, [6]-
shogaol is reported to be an activator of nuclear factor E2
related factor 2 (Nrf2) [24, 25]. Nrf2 is a transcription fac-
tor that is activated by a vast variety of stressors including
oxidative insults, inadequate nutrient supply or electrophilic
agents. Activated Nrf2 then launches a transcriptional pro-
gram that mainly aims at detoxification and cellular stress
resistance including cell-cycle control and metabolic adapta-
tion (e.g. reviewed in [26, 27]). Activated Nrf2 and elevated
expression and activity of the Nrf2-target gene HO-1 have
been linked with reduced VSMC proliferation and preven-
tion of cardiovascular disease [28–31]. We therefore were
prompted to analyse whether Nrf2 activation and subsequent
HO-1 induction could possibly account for the growth arrest
observed in shogaol-treated VSMC. For this, HO-1 expres-
sion in proliferating VSMC upon [6]-shogaol exposure was
evaluated. Concentrations of 3 and 10 M [6]-shogaol signif-
icantly elevated HO-1 levels compared to vehicle treated cells
(Fig. 6A). Moreover, using WT and isogenic Nrf2-/- fibrob-
lasts, a strictly Nrf2-dependent increase of HO-1 upon [6]-
shogaol treatment (Fig. 6B) was observed, confirming Nrf2
activation by [6]-shogaol and excluding involvement of other
transcription factors in the HO-1 induction in fibroblasts and
most likely also in VSMC. In order to prove causality be-
tween the [6]-shogaol-induced HO-1 induction and prolifer-
ation stop in VSMC we made use of the HO-1 inhibitor tin
protoporphyrin IX. As shown in Fig. 6C and D, [6]-shogaol
loses its antiproliferative influence on VSMC in the presence
of the HO-1 inhibitor. These findings indicate that [6]-shogaol
induces HO-1 that then contributes to a reduced proliferation
rate in VSMC.
4 Discussion
The present study reveals for the first time the inhibition
of VSMC proliferation by a ginger extract and four ma-
jor ginger constituents exhibiting IC50 values from 2.72 to
13.2 M. [6]-shogaol, the most potent identified inhibitor,
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Mol. Nutr. Food Res. 2015, 59, 843–852 849
Figure 6. [6]-Shogaol induces HO-1 in Nrf2-dependent manner and HO-1 inhibition abolishes the antiproliferative effect of [6]-shogaol.
(A) Quiescent VSMC were pretreated with the indicated concentrations of [6]-shogaol or an equal volume of the solvent vehicle (0.1%
DMSO) for 30 min, and then stimulated with 20 ng/mL PDGF-BB for 20 h before their lysates were subjected to immunoblot analysis
for HO-1 and actin as a loading control. Representative blots of three performed independent biological experiments (with one technical
replicate) with consistent results are depicted. The bar graph shows compiled densitometric data of all performed experiments (mean ±
SD, n.s., not significant; **p<0.01; *p<0.05; ANOVA/Bonferroni). (B) WT and Nrf2−/−mouse embryonic fibroblasts were treated with
[6]-shogaol (10 or 3 M) for 24 h before their lysates were subjected to immunoblot analysis for HO-1 and actin as a loading control.
Representative blots are shown, the bar graph depicts compiled densitometric analyses from three biological replicates with one technical
replicate each (mean ±SD, n.s., not significant; ***p<0.001; ANOVA/Bonferroni). (C and D) Quiescent VSMC were pretreated with or
without the HO-1 inhibitor SnPP (10 M) for 1 h, then treated with [6]-shogaol (3 M) or an equal volume of the solvent vehicle (0.1%
DMSO) for 30 min, and then stimulated with 20 ng/mL PDGF-BB. Cell proliferation was determined by the resazurin conversion method.
The data groups represent mean ±SD from three independent experiments (n.s., not significant; ***p<0.001; ANOVA/Bonferroni).
induces growth stop associated with accumulation of cells in
G0/G1and induction of HO-1 expression.
Ginger has been widely used as a culinary spice as well
as in traditional oriental medicine for centuries. In recent
years ginger attracted increasing attention due to a variety
of newly described bioactivities promoting its use as a safe
and effective medicinal plant [1,2,4, 32]. The nonvolatile pun-
gent components, such as gingerols, shogaols, paradols, and
zingerone, have been reported to be responsible for many of
the reported biological effects of the plant [33–35]. In fresh
ginger, gingerols are the major pungent components, with [6]-
gingerol being the most abundant. Gingerols are not stable,
and their dehydration may yield large amounts of shogaols
during prolonged ginger storage. Zingerone also cooccurs
with shogaols in stored ginger. Shogaols may undergo reduc-
tion to form paradols that are also present in ginger. Shogaols
are minor components in fresh ginger, while predominant
pungent constituents in the dried ginger, with the ratio of
[6]-shogaol to [6]-gingerol being around 1:1 in dried ginger
[32–34,36,37]. In commercially available ground ginger pow-
ders the amount of [6]-shogaol was reported to be in the range
of 353–1459 g/g [36, 38]. After a single oral administration
of ginger oleoresin (300 mg/kg) to rats, the peak plasma
concentration of [6]-shogaol was reported to be 0.11 g/L
(equalling 0.40 M) [39]. It is important to note that
the anti-proliferative effect of [6]-shogaol on VSMC was
C2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
850 R. Liu et al. Mol. Nutr. Food Res. 2015, 59, 843–852
observed at higher concentrations in our study, with the IC50s
being 2.72 M, 1.13 M, and 3.0 M upon quantification of
metabolic activity (Fig. 3A), total biomass (Fig. 3B), and DNA
synthesis (Fig. 5A), respectively. Therefore, although the here
described anti-proliferative potential of [6]-shogaol might be
of relevance upon local application (e.g. as a coating agent in
drug-eluting stents), the question of whether acute or chronic
oral administration of ginger could result in beneficial anti-
restenotic effects requires further research.
Since all investigated ginger constituents are struc-
turally related (Fig. 2), a comparison of their IC50 values
(Table 1) allows deducting a structure-activity relationship
(SAR). All compounds possess a vanillyl moiety (4-hydroxy-
3-methoxyphenyl) and an alkyl side chain, as shown in
Fig. 2. They differ in the substitution pattern of the side
chain at positions C3 or C5. [6]-shogaol, which was most ac-
tive against PDGF-induced VSMC proliferation, possesses an
␣,-unsaturated ketone at C3. [6]-paradol, only differing from
[6]-shogaol by a saturation of the double bond, showed lower
activity. [6]-gingerol, differing from [6]-paradol by having an
additional hydroxy substituent at C5, showed even less po-
tency. The activity of rac-[6]-dihydroparadol with a hydroxyl
group at C3 was in a similar range as that of [6]-gingerol.
Zingerone, also having a carbonyl group at C3, but a short-
ened and therefore less lipophilic alkyl side chain, showed
little activity. Having all this in mind, the presence of an
␣,-unsaturated ketone and the length of the alkyl side-chain
seems to have significant impact on the extent of the observed
antiproliferative activity. Interestingly, the investigated com-
pounds showed a similar SAR when being investigated for
anti-oxidative and anti-inflammatory effects [40], cytotoxicity
and apoptosis induction in human promyelocytic leukemia
(HL-60) cells [41], and protection of neuronal cells from -
amyloid insult [42].
Noteworthy, the Michael system of the ␣,-unsaturated
carbonyl type is involved in Nrf2 activation by [6]-shogaol and
other small molecules [25, 43]. Moreover, HO-1 induction
in VSMC by [6]-shogaol appears to be dependent on Nrf2
suggesting that the capacity of activating Nrf2/HO-1 is the
basis for the observed SAR between the compounds.
Activation of Nrf2/HO-1 signaling also interferes with
VSMC migration (e.g. [44]) and cholesterol accumulation in
macrophages (e.g. [45]), as well as prevents endothelial dys-
function (e.g. [46]) and inflammation (e.g. [47]). These fea-
tures make the hub an attractive target for the prevention of
atherosclerosis by influencing different steps in the etiology
of the disease. Analyzing whether [6]-shogaol could exert such
pleiotropic vasoprotection by activation of Nrf2/HO-1 may
therefore represent an interesting subject of future research.
At this point it should be noted that although the causal-
ity between HO-1 induction and inhibited proliferation was
demonstrated, it cannot be excluded that also other factors
might be involved in the observed antiproliferative activity
of [6]-shogaol. Likewise, inhibition of STAT3 or activation of
PPAR␥were reported for [6]-shogaol [48, 49] and could also
contribute to the cell-cycle arrest in VSMC [22, 50].
Despite its potent effect on VSMC, [6]-shogaol did ob-
viously not impair endothelial viability and proliferation
(Fig. 4). Reendothelialization is a key step toward success-
ful vascular healing after therapeutic interventions such as
angioplasty or bypass surgery [51, 52]. Currently, the only
clinical available treatment for restenosis is drug-eluting stent
coated with rapamycin or paclitaxel [53]. Both drugs not
only inhibit VSMC, but also endothelial cells, causing im-
paired reendotheliazation and lengthening the healing of the
wounded vessels [54, 55]. Our results on the apparent prefer-
ential activity of ginger compounds towards VSMC (Table 1)
may therefore inspire the future search for novel stent coating
agents. Interestingly, [6]-shogaol exhibited its potent antipro-
liferation effect on VSMC without affecting the proliferation
of endothelial HUVECtert cells.
In conclusion, the present study examines for the first
time the inhibitory potential of ginger and its constituent
[6]-shogaol towards VSMC proliferation. Furthermore, the
structure-function relationship of several closely related gin-
ger constituents is characterized and it is demonstrated that
[6]-shogaol exerts its antiproliferative effect through accu-
mulation of cells in G0/G1associated with the activation of
Nrf2/HO-1 pathway.
This work was supported by European Union Seventh Frame-
work Programme (EU-FP7) Marie Curie Fellowship, by Vienna
University “Back-to-Research Grant” program (to R. Liu), and
by the Austrian Science Fund (FWF): S10704/S10705 (NFN-
project “Drugs from Nature Targeting Inflammation”), P25971-
B23 and P23317-B11.
The authors have declared no conflict of interest.
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