Food Research International 44 (2011) 1597–1603

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Food Research International

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Isolation of quinone reductase (QR) inducing agents from ginger rhizome and their in
vitro anti-inflammatory activity
Feng Li a,b, Yongli Wang a, Kirk L. Parkin b, Viriya Nitteranon b, Jin Liang a, Wenjian Yang a, Ying Li b,
Guodong Zhang b, Qiuhui Hu a,⁎
a
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People's Republic of China
b
Department of Food Science, University of Wisconsin-Madison, 1605 Linden Drive, Madison, WI 53705, United States

a r t i c l e i n f o a b s t r a c t

Article history: To investigate the potential activity of ginger rhizome extracts to induce quinone reductase (QR), we
Received 27 February 2011 performed bioactivity-guided fractionation using a murine hepatoma cell (Hepa 1c1c7) bioassay. Anti-
Accepted 8 April 2011 inflammatory effects were then studied utilizing lipopolysaccharide (LPS)-stimulated mouse macrophage
(RAW 264.7) cells. An ethyl acetate-partitioned fraction from ethanolic extract, rich in both QR inducing
Keywords:
potency and anti-inflammatory activity, was subjected to repeated silica gel column and preparative thin
Ginger rhizome
Quinone reductase
layer chromatography to yield three compounds. The three isolated compounds were [6]-shogaol, 1-dehydro-
[6]-Dehydroshogaol [6]-gingerdione and hexahydrocurcumin. [6]-Dehydroshogaol, a minor component in ginger rhizome, was
Inflammation chemically synthesized and used for comparison in the subsequent bioassay based on its excellent QR
inducing potency. Results showed that [6]-dehydroshogaol had the highest ability to induce QR activity
(CD = 9.23 ± 0.22 μM), followed by 1-dehydro-[6]-gingerdione (CD = 13.24 ± 0.45 μM), and then hexahy-
drocurcumin (CD = 68.81 ± 3.90 μM). Increasing QR activity in induced cells was associated with elevated
expression of NQO-1 protein as confirmed by Western blot. [6]-Dehydroshogaol, [6]-shogaol and 1-dehydro-
[6]-gingerdione were also potent inhibitors of nitric oxide (NO) synthesis in activated macrophages. Their IC50
values ranged from 5.80 ± 1.27 to 25.06 ± 4.86 μM. Hexahydrocurcumin exhibited the weakest inhibitory
effect (IC50 = 304.76 ± 54.80 μM). These findings contribute to our theoretical understanding of the potential
beneficial effects of consuming ginger as food and/or dietary supplement.
© 2011 Elsevier Ltd. All rights reserved.

1. Introduction tion enzymes through the antioxidant response element (ARE) by

Cancer chemoprevention has been defined as the use of dietary
and pharmacological intervention with specific natural or synthetic
agents, designed to prevent, suppress, or reverse the process of
carcinogenesis before the development of malignancy (Hong & Sporn
1997). Recently, increasing evidence based on in vitro and in vivo
investigations have been linked to a decreased cancer risk with the
consumption of certain dietary photochemicals (Bachmeier et al.
2010; Russo 2007). Among the multiple mechanisms underlying
the chemopreventive potential of these constituents, xenobiotic-
transforming and antioxidant defense systems are widely viewed as
affording protection from cancer through the detoxification of
potential carcinogens and mitigation of oxidative stress (Calabrese
et al. 2008). The induction of phase II enzymes was proposed as a
major mechanism of cellular protection against the toxic and reactive
chemical species (Begleiter et al. 1997), and neoplastic effects of
carcinogens (Wattenberg 1992). The elevation of phase II detoxifica-
⁎ Corresponding author. Tel./fax: + 86 25 84399086.
E-mail address: qiuhuihu@njau.edu.cn (Q. Hu).
dietary factors is considered more beneficial than coordinative
induction of both phase I (cytochrome P450s) and phase II enzymes.
Other ARE-encoded enzymes include glutathione and glucuronide
conjugating enzymes, multidrug resistance protein, antioxidant
defense agents such as hemeoxygenase-1 (HO-1), thioredoxin
reductase, sirtuins, glutamate cysteine ligase, and several heat shock
proteins (Russo 2007). Talalay (1989) and Wattenberg (1992) have
reported that induction of phase II detoxification enzymes, which can
modulate the threshold for chemical carcinogenesis and thereby
increase cellular resistance to carcinogen exposure, was correlated
with protection against chemical-induced carcinogenesis in animal
models (Song et al. 1999). This was demonstrated in the stage of
promotion as well as initiation (Gills et al. 2006). The regulatory
regions of inducible ARE genes are activated upon binding of the
nuclear factor E2-related protein 2 (Nrf2) transcription factor. Nuclear
translocation of Nrf2 has been shown to be essential in the up-
regulation of these protective genes as a response to oxidative stress,
electrophiles, and some phytochemicals (Eggler et al. 2008).
QR (NAD(P)H: quinone reductase, EC1.6.99.2), one of the
important components of phase II detoxification enzyme systems,
catalyzes the two-electron reductions of a variety of quinone

0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.04.010
1598 F. Li et al. / Food Research International 44 (2011) 1597–1603
compounds. It thereby protects cells against mutagenicity and
carcinogenicity that would otherwise result from free radicals and
toxic oxygen metabolites generated by the one-electron reductions
promoted by cytochromes P450 and other enzymes (Joseph et al.
1994). As a predominantly cytosolic FAD-containing flavoprotein, QR
is inducible in the cells of many tissues such as those from the liver,
lung, and colon (Wang et al. 1998). Measuring the induction of QR
activity may provide an efficient approach to searching the potential
chemopreventive phytochemicals from plants. This may lead to
further understanding the chemopreventive mechanisms behind
their actions. Many classes of plant extracts or phytochemicals have
been isolated and identified as potential chemopreventive agents
based on their potent QR-inducing properties. Examples include, but
are not limited to sulforaphane from broccoli, curcumin from
turmeric, organosulfur compounds from garlic and onion, and certain
withanolides from tomatillo (Chen & Kong 2005; Surh et al. 2008).
Ginger rhizome (Zingiber officinale Roscoe), is used worldwide both
as a spice and as a medicinal herb. Numerous studies demonstrated that
ginger or its extracts present some degree of pharmacological activity
including anti-inflammation, analgesic effect, anti-tumor and anti-
oxidation activity (Dugasani et al. 2010; Lantz et al. 2007). Ginger
contains a distinct phytochemical profile with more than 63 and 115
substances being identified from fresh and dry ginger, respectively
(Jolad et al. 2004; Jolad et al. 2005). Oral consumption of ginger
increases antioxidant levels and reduces oxidative damages in the liver
and kidney of rats (Kota et al. 2008). Ginger extracts, specially gingerol
and shogaol, exhibit antiallergenic effects by inhibiting the release of
inflammatory mediators and β-hexosaminidase form RHL-2H cells
(tumor analog of mast cells) without causing cytotoxicity (Chen et al.
2009). [6]-Gingerol and [6]-paradol induce apoptosis in different cancer
cell lines, and aqueous ginger extract inhibits proliferation and
angiogenesis of colonic adenocarcinoma cells (Brown et al. 2009). Our
preliminary study also indicated that [6]-dehydroshogaol, a minor
component in ginger rhizome, exhibited potent QR-inducing activity in
murine hepatoma Hepa 1c1c7 cells (Imm et al. 2010). Although several
pharmacological properties of ginger have been studied, there appear to
be few investigations focusing on QR-inducing activity of ginger extracts
or on the structure–activity relationship of isolated compounds. Thus, as
a part of our ongoing screening program to evaluate the QR-inducing
potentials of natural compounds, we performed a bioassay-guided
fractionation using murine hepatoma cells (Hepa 1c1c7) model. The
purpose of our study was to isolate potential phase II enzymes-inducing
components from ginger rhizome. These isolated components were then
subjected to both phase II enzymes induction and anti-inflammatory
activity using a lipopolysaccharide (LPS)-stimulated macrophages (RAW
264.7 cells) bioassay.
2. Materials and methods
2.1. Chemicals
Dulbecco's Modified Eagle's minimum essential medium (DMEM),
α-Minimum essential medium (MEM), trypsin–EDTA (0.25% trypsin
with EDTA-4Na), fetal bovine serum (FBS), and penicillin streptomy-
cin were obtained from Gibco (Grand Island, NY). Lipopolysaccharide
(LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (MTT) were obtained from Sigma-Aldrich Chemical Co.
(Milwaukee, WI). NAD(P)H: quinone oxidoreductase 1 (NQO-1) and
β-actin monoclonal antibodies and secondary antibodies were
purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). All
other chemicals were of analytical grade and obtained from Sigma
Chemical Co. (St. Louis, MO). [6]-Dehydroshogaol was chemically
synthesized in our lab by the condensation of vanillin with acetone
under aqueous NaOH generated dehydrozingerone, which was
condensed with hexanal using LiN(TMS)2 in anhydrous THF to obtain
[6]-dehydroshogaol (Wu et al. 1998).
2.2. Isolation and purification of ginger extracts
Market-fresh ginger rhizome (7.756 kg) was purchased from a
local grocer (Metcalfe Sentry Foods, Madison, WI) in April, 2010.
Ginger rhizome was transversely sliced into ~ 1–2 mm pieces and first
refluxed with 4 L ethyl acetate (EtOAc) for 4 h. The residue was
subjected to further sequential extraction by refluxing with ethanol,
and finally aqueous extraction at 60 °C for 4 h. For each extract,
solvents were removed by vacuum evaporation or lyophilization to
obtain dry matter. Preliminary QR inducing and anti-inflammatory
(inhibition of NO evolution) bioassay revealed that only ethanolic
extract had a low enough CD value (concentration required to double
QR activity) of ~ 7 μg/mL and IC50 value of ~20 μg/mL to warrant
further fractionation.
The ethanolic extract was suspended in H2O and partitioned with
EtOAc, and EtOAc-soluble fraction was found to be active and a
portion (10.07 g) was chromatographed on a silica gel column
(5.5 × 60 cm) using a step gradient of n-hexane-ethyl acetate (2–
100% of EtOAc). Collected material was pooled according to
segregation of material absorbing at 254 nm and finally to afford 14
fractions, F1–F14 (Fig. 1). Fraction F4 was resolved by flash silica gel
chromatography using 40–80% of CH2Cl2 in hexane to obtain
compound 1. Fractions F5 were loaded onto a silica gel column
eluting with 0–20% of EtOAc in CH2Cl2, and then preparative thin layer
chromatography (TLC) on silica gel using a developing solvent of 5%
EtOAc in benzene to obtain compound 2. Fraction F11 was subjected
to flash silica gel chromatograph using 30–60% EtOAc in petroleum
ether to afford 3 subfractions (F11-1, F11-2 and F11-3). The F11-3 was
repeatedly resolved on silica gel chromatography using 10–40% EtOAc
in CHCl3, and finally purified by reverse phase TLC to obtain
compound 3. Structures of these isolated compounds were analyzed
using NMR and MS by comparison with previously reported profiles.
NMR spectra were obtained on Varian Unity-Inova 400 (400 MHz)
spectrometer (Analytical Instrumental Center, UW-Madison, WI).
High resolution ESI-MS analyses were performed on Agilent ESI-TOF
Mass Spectrometer (Mass spectrometry/Proteomics Facility, Biotech-
nology Center, UW-Madison).
2.3. Cell culture
Murine hepatoma cells (Hepa 1c1c7) was obtained from American
Type Culture Collection (ATCC, Rockville, MD), and maintained in α-
Minimal Essential Medium (MEM) supplemented with 10% FBS
(treated with activated charcoal to remove any traces of endogenous
QR inducers), and 100 U/mL penicillin and 100 μg/mL of streptomycin
5.00 100
F4
F1
4.00 80
Absorbance (λ= 254 nm)
% EtOAc in Hexane
3.00 60
F11
F12
2.00 40
F5
F13
F3 F7
1.00 F2 F6 F8
F9 F10 F14 20
0.00 0
0 40 80 120 160 200 240 280
Fraction Number
Fig. 1. Silica gel chromatographic resolution of EtOAc-soluble layer of crude ginger
ethanolic extract by a gradient of hexane-ethyl acetate system (2–100% of EtOAc).
Fractions were collected according to absorbance at 254 nm. Step gradient was
indicated by a long dashed line.
 F. Li et al. / Food Research International 44 (2011) 1597–1603
(Gibco, Grand Island, NY). Mouse macrophage cells (RAW 264.7,
ATCC) were grown in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL of
streptomycin. Cells were grown in a humidified incubator at 37 °C and
5% CO2.
2.4. Quinone reductase (QR) inducing assay
A bioassay based on cultured murine hepatoma cells (Hepa 1c1c7)
was used to assess changes in QR activity as a biomarker for phase II
enzyme induction (Prochaska & Santamaria 1988). Hepa 1c1c7 cells
(5× 103 cells/well) were cultured in 96-well plates in MEM supple-
mented with 10% FBS, 100 U/mL penicillin and 100 μg/mL of strepto-
mycin for 24 h at 37 °C and 5% CO2. The medium was then replaced with
fresh medium containing serial dilutions of test fractions (0.98 to
62.5 μg/mL) or compounds (0.39 to 100 μM), and cells were incubated
for an additional 48 h. At the end of incubation, cells were lysed by
adding saturated digitonin aqueous solution and then incubated at 37 °C
for 30 min with gentle shaking. QR activity was determined by adding a
standard assay cocktail to each well and measuring the reduction of 3-
(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) by
absorbance at 490 nm over a 10-min reaction progress using a
Spectramax plus optical microtiter plate scanner (Molecular Devices,
Sunnyvale, VA). A duplicate plate was prepared for cell protein
(viability) assay using crystal violet stain and measuring absorbance
at 610 nm, which allowed calculation of relative QR activity in the
control and induced cells ([ΔAbs490nm for QR assay/Abs610nm for protein
content], with the ratio for control cells set to 1.0). Relative QR activity is
not reported in cases where loss of viability exceeded 50% relative to
control cells. The potency of the compounds was compared by their CD
values (concentration required to double QR activity).
2.5. Western blot analysis
Hepa 1c1c7 cells were cultured (1 × 106 cells/mL) in 6-well plates
for 24 h. The medium was replaced with fresh medium containing
serial dilutions of test compounds (1.875 to 30 μM) for another 24 h-
incubation. For the whole-cell extract preparation, cells were quickly
washed with ice-cold PBS and solubilized in 100 μL of RIPA buffer
(50 mM Tris-base, 150 mM NaCl, 0.1% Triton X-100 and 0.1% SDS)
with protease inhibitor (10×, Calbiochem, San Diego, CA). Superna-
tant was collected and protein concentration was estimated with Bio-
Rad DC protein assay reagent (Bio-Rad Laboratories, Hercules, CA)
using bovine serum albumin (BSA) as the standard. Total proteins
(50 μg) were separated by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) using an 8% polyacrylamide gel. The
proteins were then transferred onto nitrocellulose membrane. The
membrane was blocked with 5% skim milk in TBST buffer for 1 h at
20–21 °C. Afterwards, the membranes were probed with the primary
antibodies (1:5000) in 3% skim milk in TBST buffer for 2 h, and then
incubated with corresponding secondary antibodies (1:5000) conju-
gated with horseradish peroxidase (HRP) for 1 h. Blots were washed
three times with TBST between each step. The signal was visualized by
chemiluminescence detection system (Thermo Scientific, Rockford,
IL), and the membrane was exposed to X-ray film.
2.6. Nitric oxide (NO) assay
The nitrite accumulated in the culture medium was measured as
an indicator of NO production based on the Griess reaction. Mouse
macrophage cells (RAW 264.7) were seeded in DMEM phenol red-free
medium in 96-well plates at a density of 5 × 103 cells/well for 24 h.
Thereafter, the medium was replaced with fresh DMEM containing
either 0.5 μg/mL of LPS alone or LPS with serial dilutions of test
fractions (0.98 to 250 μg/mL) or compounds (0.49 to 125 μM), and the
cells were further incubated for 24 h. Culture supernatant (100 μL)
1599
was collected and mixed with equal volume of freshly prepared Griess
reagent [equal volumes of 1% (w/v) sulfanilamide in 5% (v/v)
phosphoric acid and 0.1% (w/v) naphtylethylene-diamine-HCl], and
absorbance was recorded at 542 nm to afford quantification of NO as
nitrite relative to a standard curve of sodium nitrite prepared in
DMEM. The level of isolates required to inhibit NO evolution by 50%
was defined as IC50 value and was interpolated from the dose
response results.
Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) assay (Denizot & Lang 1986).
Cells were seeded in 96-well plates in the absence and/or presence of
ginger compounds for 24 h as described in the preceding section. At
the end of incubation period, 100 μL of MTT dye solution (0.1 mg/mL
in PBS buffer) was added to each well and the plated was incubated
for 4 h at 37 °C. Then 200 μL of DMSO was added and incubated for
15 min under gentle shaking at 37 °C to dissolve/extract tetrazolium
dye. Relative cell viability was calculated by determining the
absorbance at 550 nm and untreated control cells were assigned a
relative viability of 100%.
2.7. Statistical analysis
All experiments were carried out in triplicate and data were
expressed as means ± standard deviation. A one-way analysis of
variance (ANOVA) was performed to calculate significant differences
in treatment means, and multiple comparisons of means were done
by the LSD (least significance difference) test using the statistical
software SAS 9.2 (SAS Institute Inc., Cary, NC). A probability value of
b0.05 was considered significant.
3. Results and discussion
3.1. Bioassay-guided fractionation
Preliminary data showed that the ethanol extract from ginger rhizome
contained more QR-inducing bioactive agents than the EtOAc extract and
aqueous extract (date not shown). We further fractionated the ethanol
extract into EtOAc-soluble and H2O-soluble fractions. The EtOAc-
partitioned layer exhibited higher potency in inducing QR activity in
cultured Hepa 1c1c7 cells with a CD value of 5.36±0.32 μg/mL. It also
suppressed the production of NO in LPS-stimulated RAW 264.7
macrophage cells with an IC50 of 7.79±0.24 μg/mL. No QR inducing or
anti-inflammatory activity was observed in the H2O layer with a dose
range of 0–1000 μg/mL. Thus, EtOAc-partitioned material was separated
on a silica gel column, to yield 14 major fractions, F1–F14, which was
eluted by a gradient of n-hexane-ethyl acetate (2–100% of EtOAc) (Fig. 1).
Table 1 shows the yields and relative potencies of each chromatographic
fraction in QR induction and NO inhibition bioassays. Among the 14 final
fractions, the most potent QR inducing effect was found in fractions F4, F7,
F10, F11 and F12-treated cells, as indicated by their CD values of 5.43±
0.89 μg/mL, 5.38±0.29 μg/mL, 8.02±0.63 μg/mL, 8.21±0.90 μg/mL and
7.76±0.55 μg/mL, respectively. The initial two lipophilic fractions (F1 and
F2) and the final polar fraction (F14), representing 1.1017 g (17.72%) and
0.3804 g (6.12%), had insignificant activity in inducing QR. For anti-
inflammatory bioassay, fractions F1, F2, F4, F6, F10 and F14 exhibited weak
NO inhibition (IC50 N 20 μg/mL). Interestingly, the original EtOAc parti-
tioned material showed comparable potency in inducing QR and
inhibiting NO evolution with some active fractions, suggesting the
possible synergistic effect among different fractions in both bioassays.
The QR induction in Hepa 1c1c7 cells has been suggested as an
effective and reliable biomarker for the screening of potential
chemoprotective compounds (Cuendet et al. 2006). Compounds
with CD values b10 μg/mL are considered as attractive opportunities
for further evaluation of potential as chemopreventive agents
(Cuendet et al. 2006). Based on their high QR-inducing potency, less
complicated TLC profiles and relative high abundance, the bioactive-
1600 F. Li et al. / Food Research International 44 (2011) 1597–1603

Table 1 O
1
Summary of yield, relative potencies of crude ginger extract and chromatographic 2’ 5 7 9
fractions in QR induction and nitric oxide (NO) inhibition bioassays. H3CO 1’
3
Extract/fractions Yield (g) CD# value (μg/mL) IC50⁎ value (μg/mL) 3’
4 6 8 10
2
EtOAc-EtOH 34.1861 5.36 ± 0.32 7.79 ± 0.24
H2O-EtOH 210.4802 N/A N/A
6’
HO 4’
F1 0.5727 25.58 ± 3.45a 39.57 ± 0.96a 5’
F2 0.5290 N/A 26.72 ± 4.98b
F3 0.5552 12.73 ± 0.76bcd 7.05 ± 0.04fg [6]-Dehydroshogaol
F4 0.5617 5.43 ± 0.89f 26.71 ± 2.91b
F5 0.1380 9.24 ± 0.39def 10.38 ± 1.25fg
F6 2.4881 11.44 ± 0.27dce 21.37 ± 2.86cde O O
1
F7 0.1175 5.38 ± 0.29f 6.30 ± 1.09g 2’ 7 9
F8 0.2571 9.01 ± 0.23def 19.20 ± 4.36de H3CO 1’
F9 0.1442 12.57 ± 1.01bcd 17.49 ± 0.57e 3 5
3’
F10 0.0363 8.02 ± 0.63ef 22.89 ± 0.14bcd 2 4 6 8 10
F11 0.1340 8.21 ± 0.90def 10.72 ± 1.55fg
F12 0.0717 7.76 ± 0.55ef 11.48 ± 2.88f 6’
HO 4’
F13 0.2321 12.37 ± 1.15bc 11.17 ± 2.00fg 5’
F14 0.3804 15.37 ± 3.12b 24.57 ± 0.44bc
1-Dehydro-[6]-gingerdione
Results were expressed as means ± SD of three determinations and means within the
same column followed by different letters indicated statistically different at P b 0.05.
#
CD, the concentration required to double QR activity. O
⁎ IC50, the concentration required to inhibit NO evolution by 50%; N/A, not available. 1 5
2’ 7 9
H3CO 1’
3
3’ 2 4 6 8 10
enriched fractions (F4, F5 and F11) were further purified with
repeated silica gel chromatography (see Materials and methods) to 6’
yield three compounds (Fig. 2). HO 4’
5’
Their structures were identified on the basis of NMR and MS [6]-Shogaol
spectroscopic methods and confirmed by comparison with those
reported in the literature. Compound 1 (9.2 mg) was obtained as
O OH
yellow crystal. 1H NMR (400 MHz, ClCD3), δ: 0.91 (3H, t, J = 6.8 Hz, H-
10), 1.33 (4H, m, H-8, H-9), 1.65 (2H, m, H-7), 2.37 (2H, m, H-6), 3.93 2’ 1 7 2”
H3CO 3’ 1’ 3” OCH3
(3H, s, OCH3), 5.62 (1H, s, H-4), 6.34 (1H, d, J = 16 Hz, H-2), 6.92 (1H, 3 5
1”
dd, J = 8, 4.8 Hz, H-5′), 7.02 (1H, d, J = 2 Hz, H-2′), 7.08 (1H, dd, J = 8, 2 4 6
2 Hz, H-6′), 7.52 (1H, dd, J = 16, 4.8 Hz, H-1); [M−H]−m/z: 289.1454 4’ 4”
6’ 6”
for C17H21O4 (Calc. 289.1445). These results were in agreement with HO OH
5”
reported spectral analysis and identified to be 1-dehydro-[6]- 5’
gingerdione (Charles et al. 2000). Compound 2 (6.7 mg) was obtained Hexahydrocurcumin
as pale yellow oil. 1H NMR (400 MHz, ClCD3), δ: 0.89 (3H, t, J = 6.8 Hz,
H-10), 1.26–1.31 (4H, m, H-8, H-9), 1.44 (2H, m, H-7), 2.19 (2H, m, H- Fig. 2. Chemical structures of ginger compounds evaluated in this study. [6]-
6), 2.85 (4H, m, H-1 and H-2), 3.87 (3H, s, OCH3), 6.09 (1H, dt, J = 16, Dehydroshogaol was chemically synthesized; 1-dehydro-[6]-gingerdione, [6]-shogaol
and hexahydrocurcumin were isolated from chromatographic fractions F4, F5 and F11,
1.6 Hz, H-4), 6.68 (1H, dd, J = 8, 2 Hz, H-6′), 6.71 (1H, d, J = 1.6 Hz, H-
respectively.
2′), 6.81(2H, m, H-5 and H-5′); [M−H]−m/z: 275.1646 for C17H23O3
(Calc. 275.1652). These spectral data were elucidated as [6]-shogaol
(Connell & Sutherland 1969). Compound 3 (3.9) was obtained as that many edible plants contain substantial quantities of potent phase
colorless needles. 1H NMR (400 MHz, CDCl3), δ: 1.63 (1H, dddd, J = 14.0, II enzyme inducers has stimulated great interest in utilizing such
10.1, 6.7, 4.3 Hz, H-6), 1.77 (1H, dddd, J = 14.0, 9.2, 9.2, 5.5 Hz, H-6), 2.51 plants, and their inducer components, to achieve protection against
(1H, dd, J = 17.4, 8 Hz, H-4), 2.57 (1H, dd, J = 17.4, 3.1 Hz, H-4), 2.59 cancer. QR, as a convenient target enzyme, provides a reasonable
(1H, ddd, J = 12.9, 9.2, 6.7 Hz, H-7), 2.71 (2H, t, J = 7.2 Hz, H-2), 2.72 biomarker for the estimation of the potential chemoprotective effect
(1H, ddd, J = 12.9, 10.1, 5.5 Hz, H-7), 2.82 (2H, t, J = 7.2 Hz, H-1), 3.85 of test agents against cancer initiation. This is primarily because of its
(3H,s, OCH3), 3.86 (3H,s, OCH3), 4.03 (1H, dddd, J = 9.2, 8.0, 4.3, 3.1 Hz, coordinate induction with other phase II enzymes, wide distribution
H-5), 5.53 (1H, s, OH), 5.55 (1H, s, OH), 6.64 (1H, dd, J = 8, 2 Hz, H-6′ or in mammalian tissues, large inducer response, and ease of measure-
H-6″), 6.66 (1H, d, J = 2 Hz, H-2′ or H-2″), 6.67 (1H, dd, J = 8, 2 Hz, H-6′ ment (Talalay 2000). The murine hepatoma cell Hepa 1c1c7, which
or H-6″), 6.70 (1H, d, J = 2 Hz, H-2′ or H-2″), 6.81 (2H, d, J = 8 Hz, H-5′ contains easily measurable inducible QR, that provides a reliable,
and H-5″); [M−H]−m/z: 373.1655 for C21H25O6 (Calc. 373.1656). These high-throughput system for detecting inducer of phase II enzymes,
data were consistent with those of the known compound hexahydro- and has been extensively utilized for screening inducers of phase II
curcumin, which also was obtained from acetone extract of Japanese enzymes.
commercial ginger (Yamagishi et al. 1972). All isolated compounds used Our previous study demonstrated that [6]-dehydroshogaol, a
for this study were checked by HPLC and were N98% pure. minor component present in ginger rhizome, possess comparable
QR inducing potency to the well-known cancer preventive agent,
3.2. QR inducing potency of ginger compounds curcumin (Imm et al. 2010). We therefore chemically synthesized this
compound and used it in the subsequent bioassay for comparison
Extensive evidence has demonstrated that phase II enzymes with other compounds isolated from fresh ginger.
provide a major mechanism by which cells combat the toxicities of Fig. 3 shows the induction of QR activity in Hepa 1c1c7 cells.
electrophiles and reactive oxygen species (ROS). Their induction is Typical dose-dependent curves (solid symbols) are shown for [6]-
highly effective for protecting cells against the toxic and neoplastic dehydroshogaol, 1-dehydro-[6]-gingerdione and hexahydrocurcumin
challenges of many types of carcinogens (Talalay 2000). The discovery in concentrations where cell viability was maintained at N50%. The
 F. Li et al. / Food Research International 44 (2011) 1597–1603 1601

lowest CD value was observed for [6]-dehydroshogaol, 9.23 ±0.22 μM, Table 2
indicating it has greatest induction of QR activity in this in vitro model Relative potencies of ginger compounds in QR induction and nitric oxide (NO)
inhibition bioassays.
(Table 2). The QR inducing activity of 1-dehydro-[6]-gingerdione was
lower than that of [6]-dehydroshogaol, but significantly higher than Compound CD# value (μM) IC50⁎ value (μM)
that of hexahydrocurcumin (P b 0.05), as indicated by its CD value of Hexahydrocurcumin 68.81 ± 3.90a 304.76 ± 54.80a
13.24 ± 0.45 μM. Hexahydrocurcumin exhibited the weakest QR 1-Dehydro-[6]-gingerdione 13.24 ± 0.45b 25.06 ± 4.86b
inducing potential with a highest CD value of 68.81 ± 3.90 μM [6]-Shogaol N/A 5.80 ± 1.27c
[6]-Dehydroshogaol 9.23 ± 0.22c 7.50 ± 0.44c
among three compounds (P b 0.05). The CD value of [6]-shogaol was
not available because only ~ 1.42-fold increase in QR relative activity Results were expressed as means ± SD of three determinations and means within the
occurred before cell viability dropped down below 50% (data not same column followed by different letters indicated statistically different at P b 0.05.
#
CD, the concentration required to double relative QR activity.
shown). Since [6]-dehydroshogaol and 1-dehydro-[6]-gingerdione ⁎ IC50, the concentration required to inhibit NO evolution by 50%; N/A, not available.
exhibited stronger potency in inducing QR bioassay, we further
investigated their effect on the expression of NQO-1 protein in Hepa
1c1c7 cells by Western blot. Both [6]-dehydroshogaol and 1-dehydro- compounds. Additional studies would be beneficial to elucidate the
[6]-gingerdione significantly increased the expression of NQO-1 molecular mechanisms (pathway) underlying the QR inducing
protein in induced cells in a dose-dependent manner (Fig. 4). The potency of these compounds.
increased expression of NQO-1 protein was consistent with elevation
of QR activity. 3.3. Inhibition of NO production in LPS-induced macrophages
Dinkova-Kostova et al. (2001) investigated the correlation be-
tween structure and inducer potency of a series of conjugated Nitric oxide (NO) is generated by nitric oxide synthase (cNOS and
benzylidene ketone Michael reaction acceptors of plant and synthetic iNOS; constitutive and inducible) in numerous mammalian cells and
origins. Although monofunctional inducers were assorted into nine tissues (Nathan & Xie 1994). It served as an important mediator
distinct chemical classes, all inducers can covalently modify sulfhydryl involved in the regulation of physiological and pathophysiological
groups by alkylation, oxidation or reduction. Many inducers also mechanisms in cardiovascular, nervous and immunological systems.
contained a Michael reaction acceptor group. The presence of However, NO increases and becomes cytotoxic to cells by interacting
hydroxyl substituent(s) at the ortho-position(s) on the aromatic with superoxide anions to produce peroxynitrite (ONOO−) during the
ring(s) dramatically enhanced the inducing potencies of some inflammatory process. The peroxynitrites induce oxidative damages
Michael reaction acceptor-containing inducers (Dinkova-Kostova & in host tissue and are potentially harmful to the cellular targets
Talalay 1999). Imm et al. (2010) reported that α, β-unsaturated (Sautebin 2000). Thus, the inhibitory activity of compounds on NO
carbonyl structure among a set of vanilloid compounds played a production in macrophage cells was tested as an indicator of potential
dominant role in QR inducing activity. Similar structure–bioactivity anti-inflammatory activity.
relationship was also observed in the present study. 1-Dehydro-[6]- Four compounds were able to inhibit NO production in LPS-
gingerdione possesses a single α, β-unsaturated carbonyl moiety stimulated macrophages in a dose-dependent fashion (Fig. 5). [6]-
coupled with a Michael reaction acceptor unit, and less potent than Shogaol and [6]-dehydroshogaol exhibited extremely high NO
the strongest inducer of [6]-dehydroshogaol, which share a common inhibitory effect with IC50 of 5.80 ± 1.27 μM and 7.50 ± 0.44 μM,
α, β-unsaturated carbonyl structure with two Michael addition respectively, while 1-dehydro-[6]-gingerdione showed high inhibitory
acceptor sites (Imm et al. 2010). The weakest QR inducing potency effect with an IC50 value of 25.06 ± 4.86 μM (Table 2). The lowest anti-
was found for hexahydrocurcumin, which is possibly due to its lack of inflammatory activity was observed in hexahydrocurcumin-treated
a Michael addition acceptor sites in its molecular structure, and its cells (IC50 = 304.76 ± 54.80 μM). The cytotoxicity of these four com-
inducing power being derived solely from the ortho-dioxyphenol pounds was evaluated in the presence or absence of LPS using MTT
functional unit. To the best of our knowledge, this appeared to be the
first comparison of the QR inducing activity of pure ginger
A μM)
[6]-Dehydroshogaol(μ

4 Control 1.875 3.75 7.5 15

Relative cell viability (protein)

1.00 NQO-1
3
Relative QR activity

β-actin
.75

2
.50
B 1-Dehydro-[6]-gingerdione (μM)
1
QR Viability, [6]-Dehydroshogaol .25 Control 3.75 7.5 15 30
QR Viability, Hexahydrocurcumin
QR Viability, 1-Dehydro-[6]-gingerdione
NQO-1
0 0.00
.1 1 10 100
Concentration (μM) β-actin

Fig. 3. Quinone reductase (QR) inducing assay and cell viability in cultured Hepa 1c1c7
cells treated with ginger compounds. Solid symbols represent QR induction and open
symbols represent viability responses. Horizontal dashed lines indicate intersection of
dose curves at CD value for QR induction (arrow toward left ordinate) and points where
50% loss in cell viability occurred (arrow toward right ordinate). The results were
expressed as means ± SD of triplicate tests.
Fig. 4. The expression of NQO-1 protein in cultured Hepa 1c1c7 cells treated with
(A) [6]-dehydroshogaol and (B) 1-dehydro-[6]-gingerdione. The cells were treated
with different concentrations of ginger compounds for 48 h. The whole-cell protein was
extracted and subjected to Western blot analysis for NQO-1 protein. The detection of β-actin
was performed in the same blot as a loading control.
1602
100
1-Dehydro-[6]-gingerdione
% Inhibition of NO evolution
Hexahydrocurcumin
[6]-Dehydroshogaol
75 [6]-Shogaol
50
25
0
1 10
Concentration (μM)
Fig. 5. Dose–response curves for ginger compounds in inhibiting NO production in LPS-
stimulated macrophage RAW 264.7 cells. Horizontal dashed line indicates intersection
of dose curves at 0% and 50% inhibition of NO evolution. The results were expressed as
means ± SD of triplicate tests.
assay, and N80% viability was maintained for all compounds over the
entire dose-range used in Fig. 5 (data not shown).
Chronic inflammation has been associated with a number of
human diseases including chronic obstructive pulmonary disease,
asthma and rheumatoid arthritis. While “conventional” treatments
have met with some success, patients suffering from diseases with
associated chronic inflammation are turning to alternatives for relief
of their symptoms or as prophylactic treatments (Jolad et al. 2005).
More and more research focuses on plant-derived supplements for
the purpose of treating or preventing chronic inflammatory diseases
(Saviranta et al. 2011).
Our results were consistent with the previous reports regarding
IC50 values for NO inhibition in activated macrophages. [6]-Shogaol
exhibited more potent anti-inflammatory than did [6]-, [8]- and [10]-
gingerols (Dugasani et al. 2010). The anti-inflammatory potency of
[6]-dehydroshogaol and [6]-shogaol was found to be comparable with
IC50 of 10.6 ± 2.1 μM and 11.5 ± 3.5 μM, respectively, significantly
more potent than [6]-gingerol and capsaicin (Imm et al. 2010). The
proposed mechanism behind [6]-shogaol inhibition of NO evolution
involves the down-regulation of inflammatory iNOS and COX-2 gene
expression in macrophage by inhibition the activation of NF-κB,
because of the critical role that NF-κB plays in the coordination of the
expressions of pro-inflammatory enzymes (Lappas et al. 2002). Taken
together, it appeared that the pattern of anti-inflammatory activity of
ginger compounds was structure-dependent. A similar correlation has
been previously reported on the inhibition of COX-2 enzyme and
protection of cells from β-amyloid insult (Kim & Kim 2004; Tjendraputra
et al. 2001). Recent studies suggested that α, β-unsaturated carbonyls
are very susceptible to nuceophilic addition reaction with thiols such as
gluthione. Moreover, NF-κB activation could be inhibited by the α, β-
unsaturated carbonyls group (Chinh et al. 2006). The presence of α, β-
unsaturated carbonyl might play a predominant role in exhibiting
antioxidant and anti-inflammatory properties. [6]-Dehydroshogaol, 1-
dehydro-[6]-gingerdione and [6]-shogaol may be related to the
sulfhydryl reactivity of α, β-unsaturated carbonyl with cysteines of
redox-sensing elements of inflammatory signaling pathway (Liu et al.
2008).
4. Conclusion
Although several pharmacological properties of ginger have been
studied, there seems to be relatively few investigations focusing on
the QR-inducing activity of ginger extracts or isolated compounds. In
this study, 1-dehydro-[6]-gingerdione, [6]-shogaol and hexahydro-
curcumin were isolated from ginger rhizome as potentially beneficial
F. Li et al. / Food Research International 44 (2011) 1597–1603
components related to anti-cancer activity. [6]-Dehydroshogaol, [6]-
shogaol and 1-dehydro-[6]-gingerdione exhibited potent anti-inflam-
matory activity. [6]-Dehydroshogaol and 1-dehydro-[6]-gingerdione
were also potent QR inducers. Structure–activity relationship analysis
demonstrated that the pattern of their activities was structure-
dependent, and that α, β-unsaturated carbonyl may play a predom-
inant role in exhibiting QR-inducing and anti-inflammatory proper-
ties. Further efforts should be made to elucidate the molecular
mechanism behind their QR-inducing properties and anti-inflamma-
tory effects in vivo, and to further investigate their structure–activity
relationships.
Acknowledgements
100
This study was supported by China Special Fund for Agro-scientific
Research in the Public Interest (Grant No. 200903018) and by the 111
Project of Education Ministry of P. R. China (Grant No. B07030). We thank
China Scholarship Council for providing a 1 year fellowship to Feng Li.
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