[10]-Gingerdiols as the major metabolites of [10]-gingerol in zebrafish embryos and in humans and their hematopoietic effects in zebrafish embryos

Article abstract of DOI:10.1021/jf401501s

Gingerols are a series of major constituents in fresh ginger with the most abundant being [6]-, [8]-, and [10]-gingerols (6G, 8G, and 10G). We previously found that ginger extract and its purified components, especially 10G, potentially stimulate both the

Full text of DOI:10.1021/jf401501s

Article
pubs.acs.org/JAFC
[10]-Gingerdiols as the Major Metabolites of [10]-Gingerol in
Zebrafish Embryos and in Humans and Their Hematopoietic Effects
in Zebrafish Embryos
Huadong Chen,† Dominique N. Soroka,† Jamil Haider,‡ Karine F. Ferri-Lagneau,‡ TinChung Leung,‡
and Shengmin Sang*,†
†Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, North Carolina
Research Campus, 500 Laureate Way, Kannapolis, North Carolina 28081, United States
‡Nutrition Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University,
North Carolina Research Campus, 500 Laureate Way, Kannapolis, North Carolina 28081, United States
ABSTRACT: Gingerols are a series of major constituents in fresh ginger with the most abundant being [6]-, [8]-, and [10]-
gingerols (6G, 8G, and 10G). We previously found that ginger extract and its purified components, especially 10G, potentially
stimulate both the primitive and definitive waves of hematopoiesis (blood cell formation) in zebrafish embryos. However, it is
still unclear if the metabolites of 10G retain the efficacy of the parent compound toward pathological anemia treatment. In the
present study, we first investigated the metabolism of 10G in zebrafish embryos and then explored the biotransformation of 10G
in humans. Our results show that 10G was extensively metabolized in both zebrafish embryos and humans, in which two major
metabolites, (3S,5S)-[10]-gingerdiol and (3R,5S)-[10]-gingerdiol, were identified by analysis of the MSn spectra and comparison
to authentic standards that we synthesized. After 24 h of treatment of zebrafish embryos, 10G was mostly converted to its
metabolites. Our results clearly indicate that the reductive pathway is a major metabolic route for 10G in both zebrafish embryos
and humans. Furthermore, we investigated the hematopoietic effect of 10G and its two metabolites, which show similar
hematopoietic effects as 10G in zebrafish embryos.
KEYWORDS: [10]-gingerol, metabolism, hematopoiesis, zebrafish embryos, human urine
gingerols, varying side chain lengths of gingerols may influence
their biological efficacies. For example, [10]-gingerol displayed
stronger quenching ability of DPPH radicals than [6]-gingerol,
[6]-shogaol, and curcumin in a study comparing activities of
twenty-nine phenolic compounds isolated from root bark of
fresh ginger.9 In the same study, [10]-gingerol exhibited higher
inhibition of lipid peroxidation of rat brain homogenates than
quercetin.9 Earlier research on isolated components of dried
rhizomes of ginger showed that [10]-gingerol exhibited higher
toxicity than [4]-, [6]-, and [8]-gingerols against human A549,
SK-OV-2, SK-MEL-2, and HCT15 tumor cells.10 In particular,
we studied the hematopoiesis promoting effects of ginger
extract and its components [6]-, [8]-, and [10]-gingerol, and
[6]-, [8]-, and [10]-shogaol in zebrafish embryos, in which 10G
showed higher activities among these gingerols and shogaols.
We also demonstrated that treatment with 10G promoted
hematopoietic recovery from phenylhydrazine-induced anemia
in zebrafish embryos,11 while the efficacy of its metabolites is
still unknown.
INTRODUCTION
■
Ginger, the rhizome of Zingiber officinale, has been used
worldwide not only as a spice but also as a useful crude drug in
traditional Chinese medicine. Recently, there has been a surfeit
of scientific research to validate the use of ginger for symptoms
such as inflammation, cancer, nausea and vomiting during
pregnancy, sprains, muscular aches, cramps, constipation,
hypertension, fever, and infectious diseases, as well as rheumatic
and gastrointestinal effects.1,2 The fresh rhizome of ginger
contains a rich source of biologically active constituents
including volatile oils (1% to 3%) and nonvolatile pungent
oleoresin components.3 A variety of active components were
identified in the oleoresin of ginger including gingerols, a series
of homologues with varied unbranched alkyl chain lengths with
the most abundant being [6]-, [8]-, and [10]-gingerol (6G, 8G,
and 10G, respectively)4−6 (Figure 1).
Gingerols have been found to possess many distinct
pharmacological and physiological activities. For example, [6]-
gingerol has been reported to significantly and dose-depend-
ently restore renal functions, reduce lipid peroxidation, and
enhance the levels of reduced glutathione and activities of
superoxide dismutase and catalase against cisplatin-induced
oxidative stress and renal dysfunction in rats.7 With our
collaborators, we have found that [6]-gingerol was more
effective than curcumin, a known cancer preventive agent from
Curcuma longa L., in inhibiting 12-O-tetradecanoylphorbol 13-
acetate (TPA)-induced tumor promotion in mice.8 Although
many reports demonstrate similar bioactivities among different
The pharmacokinetics of [10]-gingerol have been examined
in humans and in rats.3,12−14 For example, Zick et al. reported
that, 1 h after oral administration of 2.0 g of ginger extracts
(containing 24.4 mg of 10G), free 10G and its glucuronidated
Received: April 4, 2013
Revised: May 13, 2013
Accepted: May 14, 2013
Published: May 14, 2013
© 2013 American Chemical Society
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MATERIALS AND METHODS
■
Materials. 10G was purified from ginger extract in our laboratory.16
Analytical TLC plates, dimethyl sulfoxide (DMSO), and CDCl3 were
purchased from Sigma (St. Louis, MO). HPLC-grade solvents and
other reagents were obtained from VWR Scientific (South Plainfield,
NJ). HPLC-grade water was prepared using a Barnstead Nanopure
water purification system (Waltham, MA). Liquid chromatography/
mass spectrometry (LC/MS)-grade water and MeOH were obtained
from Thermo Fisher Scientific (Waltham, MA). Ginger tea bags for
human experiment were bought from the local supermarket, and the
level of [10]-gingerol was 4.66 mg/100 g according to our previous
reports.17
Biotransformation of 10G in Zebrafish Embryos. Zebrafish
embryos were staged and maintained according to NCCU IACUC
guidelines. In general, fifty zebrafish embryos at the 10−11 h
postfertilization (hpf) stage were incubated at 28.5 °C in 0.3×
Danieau’s solution (19.3 mM NaCl, 0.23 mM KCl, 0.13 mM MgSO4,
0.2 mM Ca(NO3)2, 1.7 mM HEPES, pH 7.0) with or without 5 μg/
mL 10G. At 24 hpf, embryos were dechorionated manually and the
chorions were carefully removed one by one from the culture medium,
so that we did not significantly change the volumes of the solutions. At
28−30 hpf, zebrafish embryos were harvested for analysis.
Zebrafish Embryo Sample Preparation. Acetonitrile (300 μL)
with 0.2% acetic acid was added to 50 zebrafish embryos. Samples
were homogenized for 90 s by an Omni Bead Ruptor Homogenizer
(Kennesaw, GA) and then centrifuged at 17000g for 10 min. The
supernatant (250 μL) was collected and diluted 5 times for HPLC and
LC−MS analysis.
Synthesis of the Two Major Metabolites of 10G. NaBH4 (57
mg, 1.5 mmol) was added to a solution of 10G (210 mg, 0.6 mmol) in
methanol at 0 °C. After stirring at 0 °C for 2 h, the reaction medium
was neutralized with a diluted acetic acid solution (0.1 M) and
extracted with ethyl acetate (10 mL × 3). Combined organic layers
were concentrated under reduced pressure. The residue was purified
by preparative HPLC to produce the required compounds M1 (44
mg) and M2 (84 mg).
Separation of the Isomers (M1 and M2) Using Preparative
HPLC. Waters preparative HPLC systems with 2545 binary gradient
module, Waters 2767 sample manager, Waters 2487 autopurification
flow cell, Waters fraction collector III, dual injector module, and 2489
UV/visible detector were used to separate M1 and M2. A Phenomenex
Gemini-NX C18 column (250 mm × 30.0 mm i.d., 5 μm) was used
with a flow rate of 20.0 mL/min. The wavelength of the UV detector
was set at 230 nm. The injection volume was 0.5 mL for each run. The
mobile phase consisted of solvent A (H2O) and solvent B (MeOH).
The mixtures of M1 and M2 were injected to the preparative column
and eluted with an isocratic elution (80% B) for 20 min. M1 and M2
were eluted at 16.5 and 16.8 min, respectively.
Human Urine Samples. The Institutional Review Board approved
the protocol for human experimentation through the Protection of
Human Subjects in Research (11-0092) at North Carolina Agricultural
& Technical State University. Three healthy male volunteers (30−40
years old, weighing 60−80 kg, nonsmokers) participated in the study.
The subjects did not consume ginger or ginger products for at least 3
days before the experiment. They had the same breakfast, lunch, and
dinner during the experiment. The first urine sample was collected just
before breakfast, which included 200 mL of two reconstituted ginger
tea bags (18 g/bag), and the rest were at 0−2, 2−4, 4−6, 6−9, 9−12,
and 12−24 h thereafter. The urine samples were stored at −80 °C
before analysis.
Figure 1. Chemical structures of [10]-gingerol and its major
metabolites M1 and M2.
and sulfated metabolites were detected in human plasma with
peak concentrations of 9.5 2.2 ng/mL, 370 190 ng/mL,
and 18.0
6.0 ng/mL, respectively, and very low
concentrations (2−3 ng/mL) of 10G glucuronide and sulfate
were found in human colon tissues.12 This study concluded that
the only nonconjugated ginger components detected in the
plasma were 10G and [6]-shogaol, thereby warranting further
validation of the pharmacological efficacy of these com-
pounds.12 Additionally, Wang et al. reported that only free
10G was detected in plasma of rats up to four hours after oral
administration of 300 mg/kg ginger oleoresin, with no
detectable glucoronide or sulfate conjugates between zero and
seven hour harvests.13
Given the short apparent half-life of [10]-gingerol, studies of
its biotransformation are necessary. However, limited data have
been reported on the metabolism of 10G, which may influence
its bioactivity in vivo. Furthermore, significant differences in
metabolic pathways among different species may exist. The
various metabolites and their concentrations can potentially
affect in vivo bioactivities and toxicities. For example, coumarin
is metabolized to 7-hydroxy coumarin in human and in mouse.
However, in the surrogate rat model, coumarin is transformed
to a carcinogenic epoxide, a drastic consequence of differential
metabolism in seemingly similar mammals.15 This proven
rationale of interspecies variation in terms of metabolic
behavior was part of the impetus to investigate the
biotransformation of 10G in both zebrafish embryos and
humans. As our previous study showed, 10G positively
promotes hematopoiesis (blood cell formation) in zebrafish
embryos.11 The current study therefore investigates the
metabolism of 10G in zebrafish embryos and in humans to
compare its biotransformation in these two models, and
examines the hematopoietic effects of the metabolites of
[10]-gingerol.
Human Urine Sample Preparation. Enzymatic deconjugation
was performed as described previously with slight modification.18 In
brief, duplicate human urine samples were prepared in the presence of
β-glucuronidase (250 U) and sulfatase (3 U) for 24 h at 37 °C and
then extracted twice with ethyl acetate. The ethyl acetate fraction was
dried under vacuum, and the solid was resuspended in 200 μL of 80%
aqueous methanol with 0.1% acetic acid for further LC/MS analysis.
HPLC Analysis. An HPLC-ECD (ESA, Chelmsford, MA)
consisting of an ESA model 584 HPLC pump, an ESA model 542
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Figure 2. HPLC-ECD chromatograms of [10]-gingerol (10G) (A), (3S,5S)-[10]-gingerdiol (B), and (3R,5S)-[10]-gingerdiol (C) standards, and
lysate of 10G treated zebrafish embryos (D).
autosampler, an ESA organizer, and an ESA CoulArray detector
coupled with two ESA model 6210 four sensor cells, was used in our
study. A Gemini C18 column (150 mm × 4.6 mm, 5 μm; Phenomenex,
Torrance, CA) was used for chromatographic analysis at a flow rate of
1.0 mL/min. The mobile phases consisted of solvent A (30 mM
sodium phosphate buffer containing 1.75% acetonitrile and 0.125%
tetrahydrofuran, pH 3.35) and solvent B (15 mM sodium phosphate
buffer containing 58.5% acetonitrile and 12.5% tetrahydrofuran, pH
3.45). The gradient elution had the following profile: 20% B from 0 to
3 min; 20−55% B from 3 to 11 min; 55−60% B from 11 to 12 min;
60−65% B from 12 to 13 min; 65−100% B from 13 to 40 min; 100%
B from 40 to 45 min; and 20% B from 45.1 to 50 min. The cells were
then cleaned at a potential of 1000 mV for 1 min. The injection
volume of the sample was 10 μL. The eluent was monitored by the
Coulochem electrode array system (CEAS) with potential settings at
200, 250, 300, 350, 400, 450, and 500 mV. Data for Figure 2 was from
the channel set at 350 mV of the CEAS.
LC/APCI-MS Method. LC/MS analysis was carried out with a
Thermo-Finnigan Spectra System, which consisted of an Accela high-
speed MS pump, an Accela refrigerated autosampler, and an LTQ
Velos ion trap mass detector (Thermo Electron, San Jose, CA)
incorporated with atmospheric-pressure chemical ionization (APCI)
interfaces. A Gemini C18 column (150 × 4.6 mm i.d., 5 μm;
Phenomenex, Torrance, CA) was used for separation at a flow rate of
0.7 mL/min. The column was eluted with 100% solvent A (5%
aqueous methanol with 0.2% acetic acid) for 1 min, followed by linear
increases in B (95% aqueous methanol with 0.2% acetic acid) to 50%
from 1 to 5 min, to 85% from 5 to 10 min, isocratic elution with 85%
for 15 min, then to 100% from 25 to 33 min, and then with 100% B
from 33 to 40 min. The column was then re-equilibrated with 100% A
for 5 min. The LC eluent was introduced into the APCI interface. The
positive ion polarity mode was set for the APCI source with the
voltage on the APCI interface maintained at approximately 4.5 kV.
Nitrogen gas was used as the sheath gas and auxiliary gas. Optimized
source parameters, including APCI capillary temperature (330 °C),
vaporizer temperature (400 °C), sheath gas flow rate (45 units),
auxiliary gas flow rate (20 units), and tube lens (50 V), were tuned
using authentic 10G. The collision-induced dissociation (CID) was
conducted with an isolation width of 2 Da and normalized collision
energy of 35 for MS2 and MS3. Default automated gain control target
ion values were used for MS−MS3 analyses. The mass range was
measured from 100 to 1000 m/z. Data acquisition was performed with
Xcalibur version 2.0 (Thermo Electron, San Jose, CA, USA).
1
Nuclear Magnetic Resonance (NMR) Analysis. H, 13C, and
two-dimensional (2D) spectra were acquired on a Bucker AVANCE
600 MHz instrument (Bruker, Inc., Silberstreifen, Rheinstetten,
Germany). All compounds were analyzed in CDCl3. 1H and 13C
NMR data of 10G, M1, and M2 are listed in Table 1.
Hematopoietic Effects of 10G and Its Metabolites (M1 and
M2) in Zebrafish Embryos. Transgenic Zebrafish and Embryos.
Zebrafish (Danio rerio) AB transgenic strains Tg(gata1:dsRed)19 were
maintained in an Aquaneering fish housing system with 14 h light/10 h
dark cycle. The fish embryos were maintained at 28.5 °C in 0.3×
Danieau’s solution (19.3 mM NaCl, 0.23 mM KCl, 0.13 mM MgSO4,
0.2 mM Ca(NO3)2, 1.7 mM HEPES, pH 7.0) containing 30 μg/mL
phenylthiourea (PTU) to inhibit pigmentation. Zebrafish embryos
were washed, dechorionated, and anesthetized before observations and
fluorescence video imaging for analysis.
Fluorescent Microscopy. Olympus MVX10 MacroView fluores-
cence microscope (Olympus, Center Valley, PA) equipped with
Hamamatsu C9300-221 high-speed digital CCD camera (Hamamatsu
City, Japan) were used for fluorescence video microscopy. Tg-
(gata1:dsRed) fluorescent embryos were anesthetized in tricaine (0.168
mg/mL) and imaged at 5 dpf (days postfertilization). MetaMorph TL
for Olympus software (Olympus, Center Valley, PA) was used for
image acquisition and analysis.
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Table 1. δH (600 MHz) and δC (150 MHz) NMR Spectral Data of 10G, M1, and M2 (CDCl3, δ in ppm and J in Hz)
10G
M1
M2
δH multi (J)
2.63 m
δC
δH multi (J)
δC
δH multi (J)
δC
a
1
2
31.88
2.51 m
2.62 m
1.65 m
1.74 m
3.88 m
1.54 m
31.94 a
2.52 m
2.61 m
1.65 m
1.74 m
3.80 m
1.48 m
31.38
2.73 m
2.63 m
2.73 m
45.43
39.37
39.98
3
4
211.48
49.35
68.96
42.37
72.46
42.75
2.39 dd (9.1, 17.4)
2.46 dd (2.7, 17.4)
3.92 m
5
6
67.68
36.47
3.86 m
1.36 m
1.43 m
1.19 m
1.32 m
1.18 m
1.18 m
1.18 m
1.18 m
1.17 m
1.19 m
0.78 t (7.0)
69.61
37.51
3.76 m
1.36 m
73.43
38.27
1.30 m
1.40 m
7
1.16 m
25.45
25.77
1.19 m
1.29 m
1.18 m
1.18 m
1.18 m
1.18 m
1.17 m
1.19 m
0.78 t (7.0)
25.32
1.30 m
8
1.16 m
29.57 a
29.54 a
29.54 a
29.30 a
29.27 a
22.67
29.62 b
29.61 b
29.56 b
29.31 b
31.89 a
22.68
29.59 a
29.59 a
29.55 a
29.31 a
31.88
9
1.16 m
10
11
12
13
14
1′
2′
3′
4′
5′
1.16 m
1.16 m
1.16 m
1.16 m
22.67
0.78 t (7.0)
14.11
14.11
14.11
132.64
111.00
146.46
143.97
114.40
120.73
55.87
133.90
111.01
146.46
143.73
114.31
120.86
55.87
133.85
111.05
146.45
143.72
114.29
120.88
55.87
6.58 s
6.61 s
6.62 s
6.72 d (8.0)
6.56 d (8.0)
3.77 s
6.73 d (8.0)
6.59 d (8.0)
3.77 s
6.73 d (8.0)
6.59 d (8.0)
3.77 s
6′
OMe
a
The same letter a or b indicates that the assignments are interchangeable.
Comparison of Erythropoiesis-Stimulating Activity for 10G and
and HMBC) spectra as well as by comparing with literature
data.21 One product showed the molecular formula C21H36O4
Metabolites. The effects of 10G and its metabolites were compared by
their abilities to increase the production of erythrocytes during
recovery in an acute hemolytic anemia zebrafish model.11 Phenyl-
hydrazine (PHZ) (0.7 μg/mL) was added into 0.3× Danieau’s embryo
medium at 28 hpf. The PHZ was washed 3 times extensively with
embryo medium at 48 hpf. Then, embryos were incubated with 10G or
metabolites (1.0 μg/mL). On day 5 (5 dpf), embryos were
anesthetized and fluorescent erythrocytes from the transgenic
Tg(gata1:dsRed) embryos were imaged at the dorsal aorta of the tail
region (see the box of the cartoon in Figure 5a). Videos of circulating
erythrocytes within the dorsal aorta were analyzed with about 24−30
embryos per group by counting the number of Tg(gata1:dsRed)
fluorescent cells entering/exiting the video (100 frames in 1 s; 327 μm
distance). The relative number of erythrocytes was normalized by the
measured blood flow. Data are presented as mean SEM. p values
were determined by using Student’s t test. p < 0.01 was considered
statistically significant. One-way ANOVA was used to determine the
difference among groups.
+
based on positive APCI-MS at m/z 317 [M + H − 2H2O]
1
and its H and 13C NMR data (Table 1). Its molecular weight
was 2 mass units higher than that of 10G, indicating that it
might be the product from keto reduction of 10G. In addition
to the distinguishable resonance for a methoxyl group (δH 3.77,
1
3H, s), its H NMR spectrum (Table 1) also indicated the
presence of a 1,3,4-trisubstituted phenyl group [δH 6.73 (1 H, J
= 8.0 Hz); 6.61 (1 H, s); and 6.59 (l H, d, J = 8.0 Hz)] and a
methyl group (δH 0.78, 3H, t, J = 7.0 Hz). Its 13C NMR
spectrum (Table 1) displayed 21 carbon resonances, which
were classified by HMQC experiments as two methyls, 11
methylenes, five methine, and three quaternary carbons. The
aforementioned NMR data implied that the structure of this
product was closely related to that of 10G. The only difference
was its C-3 being assigned as an oxymethine (δH 3.88, 1H, m;
δC 68.96) instead of the expected ketone carbonyl (δC 211.48)
in 10G (Table 1). This was confirmed by the HMBC (Figure
3) correlations of H-1/C-3, H-2/C-3, and H-3/C-5. Therefore,
we confirmed that it is the keto reduced product of 10G, [10]-
gingerdiol. The other product showed molecular formula
C21H36O4 based on positive APCI-MS at m/z 317 [M + H −
RESULTS
■
Synthesis and Structure Elucidation of the Metabo-
lites of 10G in Zebrafish Embryos. After 18−19 h
incubation of 10G with zebrafish embryos, three major
metabolites (M1, M2, and one unknown) were detected by
HPLC chromatography (Figure 2). Previously, we reported
that [6]-gingerol was metabolized to two major metabolites,
isomeric [6]-gingerdiols.20 Since 10G and 6G are different only
in side chain length, we speculated that they shared a similar
biotransformation pathway. 10G was reduced by NaBH4 and
separated by preparative HPLC (as described in Materials and
Methods) to give two products. Their structures were
+
1
2H2O] and its H and 13C NMR data (Table 1). 2D NMR
spectra (HMQC and HMBC) indicated that these two
products possess the same planar structure (Figure 3). Since
a new chiral center was formed, the difference between these
two products arose at the configuration at C-3. After comparing
the NMR data of these two products with those of the two
known [6]-gingerdiols, (3R,5S)-gingerdiol and (3S,5S)-ginger-
1
established by analyzing the H, 13C, and 2D NMR (HMQC
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diol,20,21 we identified these two products as (3S,5S)-[10]-
gingerdiol and (3R,5S)-[10]-gingerdiol, respectively.
Metabolites M1 and M2 showed the same retention times as
those of the synthetic (3S,5S)-[10]-gingerdiol and (3R,5S)-
[10]-gingerdiol, respectively, indicating that M1 and M2 were
(3S,5S)-[10]-gingerdiol and (3R,5S)-[10]-gingerdiol, respec-
tively. We further confirmed this by tandem mass spectrometry,
which indicated that M1 and M2 had almost the same mass
fragments as those of the synthetic (3S,5S)-[10]-gingerdiol and
(3R,5S)-[10]-gingerdiol, respectively (Figure 4). Therefore, M1
and M2 were identified as (3S,5S)-[10]-gingerdiol and (3R,5S)-
[10]-gingerdiol, respectively. Besides these two identified
metabolites, there remains one unidentified metabolite at the
retention time of 38.9 min (Figure 2D), for which we are
unable to elucidate the structure at the current stage.
Metabolism of 10G in Human. While 10G was
transformed into two isomeric metabolites, (3S,5S)-[10]-
gingerdiol and (3R,5S)-[10]-gingerdiol, in zebrafish embryos,
the metabolism of 10G in humans was still unclear. Thus, we
Figure 3. Significant HMBC (H → C) correlations of (3S,5S)-[10]-
gingerdiol (M1) and (3R,5S)-[10]-gingerdiol (M2).
Figure 4. LC−MS2 (positive ion) spectra of the lysate collected from [10]-gingerol (10G) treated zebrafish embryos and urinary sample collected
from humans after drinking ginger tea (A). LC−MS2 (positive ion) spectra of authentic (3S,5S)-[10]-gingerdiol (B) and (3R,5S)-[10]-gingerdiol
(C).
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Figure 5. Chemical-induced anemia zebrafish model. A representative picture of zebrafish embryo showing the flow of erythrocytes in the dorsal
aorta in the tail region (A). Representative fluorescent images of Tg(gata1:dsRed) for control, [10]-gingerol (10G), M1, and M2 (B). Red dashed
rectangle indicates the flow of erythrocytes in the dorsal aorta.
Figure 6. Erythropoiesis-stimulating activity of [10]-gingerol (10G) and its metabolites M1 and M2 in zebrafish embryos. The relative number of
erythrocytes was compared at 5 day postfertilization embryos during hematopoietic recovery after phenylhydrazine (PHZ)-induced anemia in
zebrafish embryos. The relative number of erythrocytes is indicated as mean SEM. n is number of embryos per group. p value from Student’s t test.
pursued an experiment in which urine samples were collected
from three healthy human subjects that were given ginger tea to
drink. We then searched the urine samples for the two potential
10G metabolites, (3S,5S)-[10]-gingerdiol and (3R,5S)-[10]-
gingerdiol, using LC/MS, and confirmed that 10G was
extensively metabolized in humans in a similar manner as in
zebrafish embryos, with the same major metabolites (Figure 4).
10G and Its Metabolites M1 and M2 Can Stimulate
Erythropoiesis. We previously developed a chemical-induced
anemia zebrafish model11 in which zebrafish embryos
developed acute hemolytic anemia and the condition was
reversible. This model is feasible for testing the erythrocyte-
stimulating activity of compounds such as 10G and its
metabolites M1 and M2 (Figure 5). Here we used PHZ to
create acute anemia in zebrafish as the anemic control (17.93
1.44). The addition of 10G promoted the production of
erythrocytes during the hematopoietic recovery (25.57 + 2.08
relative number of erythrocytes/unit time). In addition, both
metabolites M1 and M2 exhibited a similar erythrocyte-
stimulating activity in promoting the recovery from anemia
condition, 27.16 2.75 and 23.03 1.49 relative number of
erythrocytes/unit time, respectively (Figure 6). All 10G, M1,
and M2 activities were higher than the control with statistical
significance (p < 0.01).
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M1 and M2 display similar activity to 10G, it is likely that they
exert their efficacies via this mechanistic pathway.
DISCUSSION
■
Previously we reported that ginger extract and its purified
components, especially 10G, potentially stimulated both the
primitive and definitive waves of hematopoiesis in zebrafish
embryos.11 In order to clarify whether 10G itself or its
converted metabolites showed this potent effect on hema-
topoiesis in zebrafish embryos, we first investigated metabolism
of 10G in zebrafish embryos and then in humans upon the
consumption of ginger tea containing 10G. Our results show
that 10G is extensively metabolized in zebrafish embryos
through a reductive pathway, and its two isomeric metabolites,
(3S,5S)-[10]-gingerdiol (M1) and (3R,5S)-[10]-gingerdiol
(M2), were identified by LC/MS analysis as well as comparison
with synthesized standards. In addition, our results showed that
10G gives a similar metabolic profile in humans as it did in
zebrafish embryos. That is, the two major reductive metabolites,
(3S,5S)-[10]-gingerdiol and (3R,5S)-[10]-gingerdiol, were also
found in human urine. To our knowledge, this is the first study
on the metabolism of [10]-gingerol in zebrafish embryos and in
humans.
Stereochemical configuration is a fundamental aspect of
molecular structure. Substrate stereoselectivity may occur in
enzyme-mediated catalysis by virtue of the innate asymmetry of
the active site. Product stereoselectivity may also arise when
new chiral centers are introduced during an enzymatic reaction,
because enzymes may specifically stabilize only one of the
possible transition states for a given reaction. In this study, we
separated the diastereomers of [10]-gingerdiol to give the
major metabolites. We observed that there were two dominant
peaks (M1 and M2) in human urine compared to only one
major peak (M1) in zebrafish embryos. We found that M1 had
slightly higher activity in induction of erythropoiesis than M2,
indicating that stereochemistry had some influence on
biological efficacy. Additionally, M1 and M2 are the reducing
products of ketone in 10G. It is reported that ketones can be
reduced by carbonyl-reducing enzymes, which are grouped into
two large protein superfamilies: the aldo-keto reductases
(AKRs) and the short-chain dehydrogenases/reductases
(SDRs).22 It is possible that the compositions of the
carbonyl-reducing enzymes in zebrafish embryos and in
humans are different, giving rise to the observed mismatched
products. Substrate and product stereoselectivity by specific
carbonyl-reducing enzymes remains to be fully explored, yet
could help to further elucidate the biotransformation of 10G.
Erythropoiesis, the process by which red blood cells
(erythrocytes) are produced, is conserved in humans, mice,
and zebrafish.23 Anemia is a common blood disorder and is
characterized by a decreased number of erythrocytes, which we
previously found that it would be abrogated by the treatment
with 10G.11 Both M1 and M2 had similar erythropoiesis-
stimulating activity on zebrafish embryos as that of 10G, and
M1 showed slightly higher activity than 10G. Our previous
study confirmed that 10G could promote the expression of
gata1 in erythroid cells and increase the expression of
hematopoietic progenitor markers cmyb and scl. We also
demonstrated that 10G could promote hematopoietic recovery
from acute hemolytic anemia in zebrafish. We displayed that
10G treatment during gastrulation resulted in an increase of
bmp2b and bmp7a expression and their downstream effectors,
gata2 and eve1. At later stages 10G can induce bmp2b/7a, cmyb,
scl, and lmo2 expression in the caudal hematopoietic tissue. As
In conclusion, results from this study are important for
understanding the metabolism of [10]-gingerol in zebrafish
embryos and in humans and provide useful information that
may act as a reference for nutraceutical developments toward
treating anemia with ginger. Knowledge of the metabolism of
10G may help in understanding the mechanism of action and
therapeutic effects of ginger extract in hematopoiesis. Even
though studies have shown that zebrafish and mammals have
common genetic pathways and regulators during hematopoi-
esis, whether ginger has the hematopoietic effect in human is a
topic for future study.
AUTHOR INFORMATION
Corresponding Author
*Phone: 704-250-5710. Fax: 704-250-5709. E-mail: ssang@
ncat.edu or shengminsang@yahoo.com.
■
Funding
This work was partially supported by grants CA138277 (S.S.)
from the National Cancer Institute and CA138277S1 (S.S.)
from National Cancer Institute and Office of Dietary
Supplement of National Institutes of Health.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
10G, [10]-gingerol; APCI, atmospheric-pressure chemical
ionization; hpf, hours postfertilization; HPLC, high-perform-
ance liquid chromatography; LC/MS, liquid chromatography/
mass spectrometry
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