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Bioactive Constituents, Metabolites, and Functions
Metabolic profiles of ginger, a functional food, and its representative pungent compounds in rats by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry Liangliang He, Zi-Fei Qin, Mengsen Li, Zilin Chen, Chen Zeng, Zhihong Yao, Yang Yu, Yi Dai, and Xin-Sheng Yao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03600 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 6, 2018
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Journal of Agricultural and Food Chemistry
Metabolic profiles of ginger, a functional food, and its representative pungent compounds in rats by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry
Liangliang He†,‖, Zifei Qin†,‡,‖, Mengsen Li†,§, Zilin Chen†,#, Chen Zeng†,#, Zhihong Yao*,†,‡, Yang Yu†,‡, Yi Dai†,‡, Xinsheng Yao*,†,‡,#
†
College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China;
‡
Guangdong Provincial Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs
Research, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China; §
Guangzhou Research and Creativity Biotechnology Co. Ltd, Guangzhou, 510663, P. R.
China; #
‖
Guangzhou Xiangxue Pharmaceutical Co. Ltd, Guangzhou, 510663, P. R. China;
These authors contributed equally to this work.
*Correspondence authors. Associate Prof. Zhihong Yao, Tel: (086) 20-85221767, Fax: (086) 20-85221559 E-mail:
[email protected];
[email protected]; Prof. Xinsheng Yao, Phone: (086) 20-85225849, Fax: (086) 20-85221559 E-mail:
[email protected] 1
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ABSTRACT: Ginger, a popular functional food, has been widely used throughout the world for
2
centuries. However, its metabolic behaviors remain unclear, which entails an obstacle to further
3
understanding of its functional components. In this study, the metabolic profiles of ginger in rats were
4
systemically investigated by UPLC-Q/TOF-MS. The results included the characterization of 92
5
components of ginger based on the summarized fragmentation patterns and self-building chemical
6
database. Furthermore, four representative compounds were selected to explore the typical metabolic
7
pathways of ginger. Consequently, 141 ginger-related xenobiotics were characterized, following the
8
metabolic spots of the pungent phytochemicals were summarized. These findings indicated that the
9
in vivo effective components of ginger were mainly derived from [6]-gingerol and [6]-shogaol.
10
Meanwhile, hydrogenation, demethylation, glucuronidation, sulfation and thiolation were their major
11
metabolic reactions. These results expand our knowledge about the metabolism of ginger, which will
12
be important for discovering its functional components and the further mechanism research.
13 14
KEYWORDS: Ginger, metabolic profiles, pungent compound, UPLC-Q/TOF-MS, functional
15
component
16 17 18 19 20 21 22 23 2
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Nowadays, natural diets have drawn considerable attention from both the general public and the
26
scientific community owing to their various health benefits. In particular, ginger, the rhizome of
27
Zingiber officinale Roscoe (ZO), has been used as a widely consumed food in daily life for thousands
28
of years in various regions of the world.1 Due to its health-promoting effects (including
29
antioxidation,2,3 antitumor,4,5 antidiabetic,6,7 and anti-inflammatory8,9), ZO has been developed into
30
various kinds of functional foods, such as health beneficial beverages, dietary candy and flavored
31
teas. Meanwhile, these beneficial effects have also stimulated the increasing interest in its effective
32
components. It is considered that the pungent phytochemicals (mainly gingerols, shogaols and their
33
derivatives) are the characteristic and principal constituents in ZO and responsible for most of its
34
beneficial effects.10,11
35
One of the necessary factors to elucidate the mode of action underlying the beneficial effects of
36
ZO is to understand the absorption, disposition, metabolism, and excretion of its main components in
37
vivo.12 Previous pharmacokinetic studies showed that the pungent components in ZO exhibited poor
38
oral bioavailability with only small amounts of prototypes entering the systemic circulation.13-15 This
39
indicated that metabolites played an important role in the beneficial effects of ZO. To date, the in vivo
40
metabolic studies related to ZO have only been focused on its single compounds, such as [6]-, [8]-,
41
or [10]-shogaol or [6]-gingerol, and most of these studies were focused on the analysis of metabolites
42
in urine with little consideration of metabolites in plasma, feces and bile.16-22 Nevertheless, as a
43
complex chemical mixture, the health benefits of ZO are not necessarily the result of a single
44
component, but may be the results of a large group of multiple components working together to
45
perform the health care functions. Consequently, it is essential to systemically characterize the
46
metabolic profiles of ZO in vivo to explore the functional components that are associated with the 3
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multiple health benefits. However, it is noted that the pungent compounds in ZO show similar
48
structural features: for example, shogaols can be regarded as the dehydrated products of gingerols.
49
This phenomenon increases the difficulty of studying the metabolic profiles of ZO, because it cannot
50
be determined whether the in vivo metabolites originate from the bio-transformed products of
51
shogaols or gingerols. Therefore, it is also necessary to perform the in vivo metabolic studies of its
52
representative pungent chemicals to explore the typical metabolic pathways, as supplement for the in
53
vivo metabolic characteristics of ZO.
54
Recently, ultra-performance liquid chromatography coupled with quadrupole time-of-flight
55
tandem mass spectrometry (UPLC-Q/TOF-MS) has been widely introduced as an efficient analytical
56
technique for rapid screening and identifying components in complex samples. Although the studies
57
about identification of main chemical components in ZO have been performed previously by UPLC-
58
Q/TOF-MS,
59
unidentified, which posed a significant obstacle to further study of its in vivo metabolic profiles and
60
characteristics. Hence, there is a need to systemically characterize the chemical components of ZO as
61
a basis for studies of its in vivo metabolism.
23-25
there still had several limitations. For instance, a series of peaks remained
62
In this study, to systemically reveal the metabolic profiles and characteristics of ZO in vivo, a
63
four-step approach based on UPLC-Q/TOF-MS was applied. Briefly, the process was as follows: (A)
64
to establish a chemical compounds database of ZO by literature review; (B) to investigate the
65
chemical profiles of ZO by UPLC-Q/TOF-MS; (C) to perform the metabolic pathway studies of
66
representative pungent phytochemicals; (D) to characterize the ZO-related xenobiotics in vivo and
67
summarize the metabolic characteristics. Through these results, this paper enhances our
68
understanding of the in vivo metabolic fate of ZO, which will be helpful for revealing the in vivo
69
functional components of ZO, and provide a solid basis for further studies on its functional 4
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mechanism.
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MATERIAL AND METHODS
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Chemicals and Reagents. Dried ginger (No. 120942-201510) was obtained from the Guangdong
73
Institute for Food and Drug Control in China and taxonomically identified by Prof. Guangxiong Zhou
74
who works in the College of Pharmacy of Jinan University in China. Reference standards (Purity >
75
96%) of 3S,5S-octahydrocurcumin, 3R,5S-octahydrocurcumin, [6]-gingerol, 4’-methoxyl-[6]-
76
gingerol, 5-methoxyl-[6]-gingerol, [8]-gingerol, [6]-shogaol, [10]-gingerol, [6]-dehydrogingerdione,
77
[8]-shogaol, 4-dehydro-[8]-gingerol, [8]-dehydrogingerdione, [10]-shogaol, 4-dehydro-[10]-gingerol,
78
[10]-dehydrogingerdione, [12]-shogaol, 4-dehydro-[12]-gingerol were isolated and identified in our
79
laboratory (Table S4). Their 13C NMR and HRMS data were also listed in the Supporting Information.
80
Other chemicals and materials were all analytical grade.
81
Sample preparation. Dried ginger (1.0 g) was crushed and extracted with 10 mL of 70% (v/v)
82
aqueous methanol for 30 min by ultrasonic treatment at room temperature. After centrifugation at
83
13225 g for 10 min, an aliquot (2 μL) of supernatant was injected into the UPLC-Q/TOF-MS for
84
analysis. For animal administration, ZO (80 g) were extracted three times (each for 1 h) with 800 mL
85
of 70% (v/v) aqueous ethanol under heating reflux. All of the extract solutions were combined and
86
evaporated to approximately 80 mL at 40 °C under reduced pressure. Subsequently, the extract
87
solutions were freeze-dried (FreeZone Plus 6 L, Labconco, USA) for 48 h, and the powder was added
88
to distilled water to bring the final concentration to 1.0 g/mL (equivalent to the weight of ginger), and
89
the extracts were then stored at -4 °C before use.
90
Animal and Drug Administration. SPF-grade male Sprague-Dawley rats (220 ± 20) g were
91
provided by Medical Laboratory Animal Center of Guangdong Province. The rats were allowed to
92
acclimate for 7 days. After that, they were divided into three groups: ZO group, pure pungent 5
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compound groups ([6]-gingerol, [6]-shogaol, [6]-dehydrogingerdione and [10]-gingerol) and blank
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group. Thereafter, all rats were individually kept in stainless steel metabolic cages. The ZO extract
95
was administered to the rats (n=6) intragastrically at 2.0 g/kg (ginger weight / rat weight) for three
96
consecutive days. Each pungent compound was suspended in corn oil and was administered to the
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rats (n=4) at 40 mg/kg. Water was administered intragastrically to the rats of the control group in the
98
same way. The experimental protocol was approved by the Ethics Review Committee for Animal
99
Experimentation of Jinan University (NO. 20160919095739). All procedures were in accordance with
100
the Guide for the Care and Use of Laboratory Animals (National Institutes of Health).
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Biological Samples Collection. 26,27 Plasma samples. In the ZO group (n = 4), blood samples
102
(3 mL) were collected from hepatic portal vein into heparinized tubes at 30, 60, 120 and 240 min,
103
respectively. In each pure compound group (n = 2), blood samples were obtained in the same manner
104
at 60 and 120 min, respectively. Each group of samples was then centrifuged at 15521 g for 10 min
105
at 4 °C and combined to produce the pooled plasma.
106
Bile samples. The rats (n = 2) were anesthetized by intraperitoneal injection of 10% aqueous
107
chloral hydrate after last intragastric administration. Under light anesthesia, polyethylene tubing was
108
inserted into the common bile duct for the collection of bile samples for 0 - 4 h from the ZO group
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and 0 - 2 h for each pure compound group.
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Urine and fecal samples. The rats (n = 4) were housed in stainless steel metabolic cages and
111
provided free access to water. Separate samples were collected during the periods of 0 - 12 h, 12 - 24
112
h, 24 - 36 h and 36 - 48 h after intragastric administration of ZO or each pure pungent compound.
113 114 115
Blank samples were collected in the same way. All the biological samples were stored at -80 °C before analysis. Pretreatment of biological samples. All the biological samples were then thawed at room 6
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temperature. The pooled plasma sample (6 mL) from the ZO group was processed by 18 mL
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acetonitrile to precipitate the protein. After centrifuging at 15521 g for 10 min, the supernatant was
118
transferred to an EP tube and then evaporated to dryness at room temperature under nitrogen gas. The
119
residue was reconstituted in 6 ml water and loaded on a pretreated SPE column. After being washed
120
with 5% methanol (6 mL), the cartridge was eluted using 6 mL of methanol. The methanol eluate was
121
then collected and dried at room temperature under nitrogen gas. The residue was reconstituted in
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200 μL methanol. An aliquot of 4 μL was injected into the UPLC-Q/TOF-MS. The plasma samples
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from the pure pungent compound groups and blank group were processed in the same way.
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The urine samples were centrifuged and combined at 2325 g for 10 min, and the supernatant was
125
then dried under nitrogen gas at room temperature. The residue was reconstituted in 6 mL water. The
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fecal samples (1.0 g) were dried in air and then extracted with 10 mL methanol in an ultrasonic bath
127
for 60 min. After centrifugation at 2325 g for 10 min, the supernatant was dried under nitrogen gas at
128
room temperature. The residue was reconstituted in 6 mL water. Subsequently, the urine (6 mL), bile
129
(2 mL) and fecal samples (6 mL) were loaded on preconditioned SPE columns, and then treated in
130
the same way as the plasma samples. Aliquots of 2 μL of the urine, bile and fecal samples were
131
injected into the UPLC-Q/TOF-MS.
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UPLC-Q/TOF-MS Analysis. The UPLC analysis was performed on an AcquityTM UPLC 1-
133
Class system equipped with a binary solvent system, an automatic sample manager and photodiode
134
array detector (Waters Corporation). The chromatographic separation was achieved on a BEH RP C18
135
column (2.1×100 mm, 1.7 μm) at a temperature of 40 °C. The mobile phases consisted of Water (A)
136
and Acetonitrile (B), both including 0.1% formic acid (v/v). The solvent was delivered at a flow rate
137
of 0.4 mL/min using a gradient elution program as follow: 5% B from 0-0.5 min, 5-80% B from 0.5-
138
15 min, 80-85% B from 15-18 min, 85-95% B from 18-21 min, 95-100% B from 21-22 min. The 7
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injection volume was 4 μL, and the detection wavelength was 280 nm.
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The UPLC system was coupled to a hybrid quadrupole orthogonal time-of-flight (Q-TOF)
141
tandem mass spectrometer equipped with electrospray ionization (SYNAPTTM G2 HDMS, Waters,
142
Manchester, U.K.). The operating parameters were as follows: capillary voltage of 3 kV (ESI+) or -
143
2.5 kV (ESI-), sample cone voltage of 30 V (ESI+) or 40 V (ESI-), extraction cone voltage of 4 V,
144
source temperature of 100 °C, desolvation temperature 300 °C, cone gas flow of 50 L/h and
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desolvation gas flow of 800 L/h. Argon was used as collision gas for CID in both MSE and MS2 mode.
146
The full scan mass range was 50-1200 Da in both positive and negative mode. Leucine enkephalin
147
was used as an external reference at a constant flow of 5 μL/min by the LockSpray™ process, and
148
the data were centroided during acquisition.
149
Data processing. Data analysis was performed by MassLynx (V4.1, Waters Corporation, Milford,
150
MA, USA). The prediction rules of elemental composition were as following: the maximum tolerance
151
of the mass defect filter was set at 5 ppm; the relative intensity was set at 5%; the degree of
152
unsaturation was set at a range 5-15, and the atom numbers of carbon, hydrogen, oxygen, nitrogen
153
and sulfur were set at ranges of 0-60, 0-80, 0-30, 0-5 and 0-2, respectively. Blank samples were used
154
as controls for comparison with the analytic samples, and mass defect filter technology was used to
155
analyze the in vivo xenobiotics as published previously. 27-29
156
RESULTS
157
Establishment of chemical compounds database. The chemical information database of
158
compounds isolated from ZO was mainly obtained by retrieval from SciFinder, Web of Science and
159
CNKI. As a result, a total of 203 compounds (72 pungent phytochemicals, 51 diarylheptanoids, 37
160
terpenes and 42 others) were summarized and sorted (Table S1 and Figure S2).
161
Identification and characterization of chemical profiles in ZO. The analysis strategy for the 8
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characterization of the chemical profiles of ZO is described in the Supporting Information (Figure
163
S1). The proposed fragmentation patterns were summarized based on the reference standards (Figure
164
S3). Accordingly, 92 compounds (including 57 pungent phytochemicals, 27 diarylheptanoids and 8
165
others) were identified or tentatively characterized (Table S2 and Figure 1) based on the characteristic
166
fragmentation patterns and self-building chemical database. The detailed analytical processes of
167
pungent chemicals and diarylheptanoids are listed in the Supporting Information.
168
Metabolic pathways of representative pungent compounds. A preliminary analysis of the
169
xenobiotics of ZO in rats was carried out before selecting the representative compounds. Based on
170
the results of the ZO-related xenobiotics in vivo, four abundant (about 60% of the total content of ZO
171
extract) and characteristic pungent compounds in ZO with different structural types, including [6]-
172
gingerol (6G), [6]-shogaol (6S), [6]-dehydrogingerdione (6D) and [10]-gingerol (10G), were selected
173
to investigate their metabolic pathways. The elucidation of their related metabolites was as follows.
174
Characterization of metabolites of 6G. A total of 62 6G-related metabolites were identified in
175
rats (Table 1). Glucuronidation and sulfation were the main metabolic pathways of 6G in the rat
176
plasma, bile and urine, whereas phase I metabolites were the main forms of 6G in the feces (Figure
177
S6). The proposed metabolic pathways of 6G are presented in Figure 2A. The mass spectra of all 6G-
178
related metabolites and their proposed fragmentation pathways are listed in the Supporting
179
Information.
180
Phase I metabolites. M84, M92, M125, M157 and M165 showed the same retention times and
181
mass behaviors as the chemicals in ZO (Table S2). Hence, they were identified as (3R, 5S)-[6]-
182
gingerdiol, (3S, 5S)-[6]-gingerdiol, [6]-gingerdione, [6]-shogaol and 4-dehydro-[6]-gingerol,
183
respectively.
184
the daughter ion at m/z 99.081 was considered to be the diagnostic ion, which suggested the presence
17-18
M96 showed the same molecular formula of C17H26O4 as that of 6G. In addition,
9
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of a carbonyl on C5. Hence, M96 was identified as 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-5-
186
decanone, which was also confirmed by the reference standard. Furthermore, the mass of M77 was
187
14.016 Da (CH2) less than that of 6G, indicating that M77 was a 3´-O-demethylated 6G. Similarly,
188
M68 and M74 were determined to be isomers of 3´-O-demethylated [6]-gingerdiol. M145 and M155
189
showed deprotonated ions at m/z 263.165, which were 12.000 Da (C) less than that of 6S, which
190
indicated that they were isomers of the hydrogenated and demethylated products of 6S. However, we
191
could not determine the detailed sites (C=C or C=O) of the hydrogenation.
192
Phase II metabolites. Glucuronidated conjugates. M46 and M51 both showed an obvious loss of
193
176.032 Da from the ion at m/z 469.207 to the ion at m/z 293.174, which suggested that they were
194
glucuronide conjugation products of 6G.17,30-31 Likewise, M36 was identified as mono-glucuronide
195
of M96 owing to the presence of the diagnostic ion at m/z 99.081. Similarly, M1,17 M2, M26, M29,
196
M30, M32, M34, M37, M39, M48, M58, M63, M73, M90, M94, M99, M105, M107, M108 and
197
M113 were also identified as mono-glucuronidated derivatives due to a characteristic neutral loss of
198
176.032 Da. In addition, M20 (C28H39O16) exhibited the mother ion at m/z 631.224 ([M-H]-) and
199
fragmentation ions at m/z 455.194 ([M-H-GluA]-) and 279.159 ([M-H-2GluA]-), which indicated the
200
presence of two glucuronides. Hence, M20 was determined as a di-glucuronidated metabolite of 3´-
201
O-demethyl-6G. Similarly, M67 was identified as a di-glucuronidated metabolite of dehydrated 6G.
202
Sulfated conjugates. M148 eluted at 9.67 min with an ion at m/z 373.132 ([M-H]-), which was
203
79.957 Da (SO3) higher than that of 6G. The daughter ions at m/z 293.175 ([M-H-SO3]-) and 273.04
204
([M-H-C6H12O]-) both resulted from the deprotonated ion, which suggested that M148 was a sulfated
205
conjugate of 6G.32 Similarly, M115 and M131 were identified as sulfate conjugates of
206
dehydrogenated 6G, and M98 was identified as a sulfate conjugate of dehydrogenated M96 following
207
the ion at m/z 175.076 ([M+H-SO3-H20-C6H12O]+). M7, M11 and M18 showed the same 10
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deprotonated ions at m/z 389.127, which was 15.995 Da (O) higher than that of M148, and they were
209
therefore determined to be isomers of the sulfate conjugates of hydroxylated 6G.
210
Thiolated conjugates. M72 and M75 exhibited the same [M+H]+ ion at m/z 456.206
211
(C22H34NO7S), which was 161.015 Da higher than that of 6G. In the MS/MS analysis, the daughter
212
ions at m/z 396.184 ([M+H-H2O-C2H2O]+), 379.157 ([M+H-H2O-C2H2O-NH3]+) and 333.153
213
([M+H-H2O-C3H5O3-NH3]+) were produced from the mother ion at m/z 456.206, indicating the
214
presence of an N-acetylcysteine (NAC) moiety. Furthermore, the product ions at m/z 219.047 and
215
193.032 suggested that the NAC was conjugated to the C5´ of 6G. Accordingly, they were identified
216
as isomers of 5´-NAC-6G. Similarly, M69 and M71 were identified as isomers of 5´-NAC-[6]-
217
gingerdiol. M111 and M134 both showed the [M+H]+ ion at m/z 440.211, which was 18.011 Da (H2O)
218
lower than that of M69 and M71. In the MS/MS analysis, a series of ions at m/z 317.156, 303.142,
219
285.131, 233.063 and 215.053 were detected, which indicated the presence of a hydroxyl on the
220
carbon chain. Hence, M111 was identified as the dehydrated product of 5´-NAC-[6]-gingerdiol.
221
However, the ions of M134 at m/z 219.048 and 193.031 suggested that M134 contained the carbonyl
222
on carbon chain. M106 and M117 both showed the [M+H]+ at m/z 442.226, which was 2.016 Da (2H)
223
higher than that of M111. Thus, they were tentatively characterized as isomers of a reduced product
224
of M111. Compared to M106 and M117, M97 exhibited the same molecular formula of C22H36NO6S.
225
However, the fragments of M97 were totally different from those of M106 and M117. Obvious ions
226
at m/z 261.184, 177.090, 163.075 and 137.060 were detected, which indicated that the NAC was
227
conjugated to the carbon chain.
228
Characterization of metabolites of 6S. A total of 54 metabolites were identified in rats after
229
intragastric administration of 6S (Table 1 and Figure S7). The metabolic pathway for the main
230
metabolites is presented in Figure 2B. Unlike 6G, the mercapturic acid pathway was the major 11
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metabolic route for 6S in rats. The mass spectra of the main 6S-related metabolites and their proposed
232
fragmentation pathways are listed in the Supporting Information.
233
Phase I metabolites. M144, M159, M167 and M169 were identified as phase I metabolites of
234
6S. Based on their retention times and mass behaviors, they were tentatively identified as the
235
metabolites of 3´-O-demethylated and di-hydrogenated 6S, di-hydrogenated 6S, hydrogenated 6S and
236
the dehydrated metabolite of M144, respectively.20
237
Phase II metabolites. Thiolated conjugates. M49 showed the [M+H]+ ion at m/z 584.264, which
238
was 307.084 Da higher than that of 6S, which indicated that M49 might be a glutathione (GSH)
239
conjugate of 6S. A series of ions at m/z 509.232 (loss of glycine), 455.222 (loss of pyroglutamic acid)
240
and 277.180 (loss of GSH) were detected in the MS/MS analysis. On the basis of previously published
241
fragmentation behaviors,20 M49 was identified as a metabolite of hydrogenated 5-GSH-6S. M42 and
242
M50 showed the same [M+H]+ ion at m/z 586.280, which was 2.016 Da (2H) higher than that of M49.
243
These metabolites were identified as isomers of reduced products on carbonyl of M49.22 Similarly,
244
M23 and M38 were determined to be the cysteinylglycine (Cys-Gly) conjugate and cysteine (Cys)
245
conjugate of di-hydrogenated 6S, respectively.21,22 M86 / M97 and M100 / M103 were two pairs of
246
isomers. They were identified as di-hydrogenated isomers and hydrogenated isomers of 5-NAC-6S,
247
respectively.21,22 M13 and M15 exhibited the same [M+H]+ ions at m/z 416.211, which were 15.995
248
Da (O) higher than that of M38. The fragmentation ions at m/z 398.200 ([M+H-H2O]+), 311.169
249
([M+H-C3H7NO3]+), and 261.185 ([M+H-H2O-C3H7NO3S]+) indicated that there was one hydroxyl
250
group which was conjugated to the Cys. Hence, M13 and M15 were tentatively identified as isomers
251
of hydroxylated M38. Likewise, M41 and M45 were tentatively characterized as isomers of
252
hydroxylated products of di-hydrogenated 5-NAC-6S.
253
In vivo, both the N-acetylcysteine and the cysteine conjugates act as substrates of cysteine S12
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conjugate β-lyase, a mainly renal and hepatic enzyme that cleaves the S-C bond in the cysteinyl
255
moiety, thereby releasing a thiolated metabolite. 20 This product can be further S-methylated by thiol
256
S-methyltransferase to form methylthiol-conjugated metabolites.20 In this experiment, M146 and
257
M161 showed a loss of 48.003 Da (SCH4) in the MS/MS analysis. Comparison of their fragmentation
258
behaviors with that of previous report suggested that they were the methylthiol-conjugated
259
metabolites.20 M161 was eluted at 10.61 min with the precursor [M+H]+ ion at m/z 327.199. The ions
260
at m/z 309.189 [M+H-H2O]+ and m/z 261.185 [M+H-H2O-SCH3]+ were observed in its MS/MS
261
spectrum. Thus, M161 could be regarded as a methylthiol-conjugated metabolite of hydrogenated
262
6S.21,22 M146 was determined to be a demethylated product of M161 on the basis that its precursor
263
ion was 14.016 Da (CH2) lower than M161. M57 and M65 exhibited the same [M+H]+ ion at m/z
264
343.194, which was 15.995 Da (O) higher than that of M161, and the ions at m/z 325.183 ([M+H-
265
H2O]+) and 261.186 ([M+H-2H2O-SCH2]+) indicated that there was one hydroxyl group, which was
266
conjugated to the C of SCH3. Hence, M57 and M65 were tentatively identified as isomers of
267
hydroxylated M161. Similarly, M31 and M35 were determined to be isomers of the demethylated
268
products of M57 or M65.
269
Thiolated and glucuronidated conjugates. M55 and M59 showed the same [M+H]+ ion at m/z
270
616.243. The ions at m/z 440.211 ([M+H-GluA]+) and m/z 277.180 ([M+H-GluA-NAC]+ suggested
271
the presence of one glucuronide and one NAC. Hence, M55 and M59 were identified as isomers of
272
the NAC conjugates of glucuronidated 6S.18 Similarly, M12, M16, M54 and M61 were identified as
273
thiol-conjugates of glucuronidated and hydrogenated 6S.18 For M102, M110, M118, M120 and M128,
274
neutral losses of 176.032 Da and 48.003 Da were detected, which obviously suggested the presence
275
of one glucuronide and one SCH3.21 These metabolites might be the result of continued metabolism
276
of M16 and M24 under the action of S-conjugate β-lyase and thiol S-methyltransferase. 13
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Characterization of metabolites of 6D. A total of 38 6D-related metabolites were identified in
278
rats (Table 1 and Figure S8). The proposed metabolic pathways of the main metabolites are presented
279
in Figure 2C. Among them, 20 metabolites showed the same retention time and fragments as the
280
metabolites of 6G and 6S. The mass spectra of the main 6D-related metabolites and their proposed
281
fragmentation pathways are listed in Supporting Information.
282 283
Phase I metabolites. Five metabolites, including M84, M92, M96, M101 and M166, were identified as phase I products of 6D, and all of them were also detected as the metabolites of 6G.
284
Phase II metabolites. Phase II metabolism was the main means of eliminating 6D, whereas 15
285
characterized phase II metabolites were also identified in the metabolites of 6G or 6S. In addition,
286
M3, M4, M8, M9, M17, M22, M25, M43, M60 and M121 were determined to be mono-glucuronides
287
with a significant neutral loss of 176.032 Da. Among these, M60 and M121 were identified as the
288
isomers of glucuronidated 6D. M8 was identified as a mono-glucuronide of demethylated and
289
hydrogenated 6D. M43 showed the [M-H]- ion at m/z 481.171, which was 15.995 Da (O) higher than
290
that of M60 and M121. This suggested that M43 was a mono-glucuronide of hydroxylated 6D, and
291
hydroxylation might occur at the position of C9.16,17 Ultimately, M9 was identified as a mono-
292
glucuronide of dehydrated M43. M17 and M22 showed the same [M+H]+ ion at the m/z 657.203. The
293
ions at m/z 481.170 ([M+H-GluA]+) and 305.138 ([M+H-2GluA]+) suggested that they contained two
294
glucuronides. Furthermore, the fragmentation ion at m/z 305.139 was 2.016 Da (H2) lower than the
295
fragmentation ion at m/z 307.154 of M43. Hence, M17 and M22 were identified as isomers of di-
296
glucuronide conjugates of hydroxylated and dehydrogenated 6D. M25 showed the [M+H]+ ion at m/z
297
632.238, and the ions at m/z 456.205 ([M+H-GluA]+) and 451.195 ([M+H-H2O-NAC]+) suggested
298
the presence of one glucuronide and one NAC. Furthermore, the ions at m/z 161.060, 137.060 and
299
99.081 indicated the presence of a glucuronide bound to the benzene ring and a hydroxyl group on 14
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C5. Hence, M25 was identified as a NAC conjugate of hydroxylated M90. Similarly, M3 and M4
301
were identified as a Cys conjugate and a Cys-Gly conjugate of hydroxylated M90, respectively.
302
M139 showed the [M+H]+ ion at m/z 394.168, which was 121.020 Da higher than that of
303
dehydrated 6D, and ions at m/z 244.101 ([M+H-C9H10O2]+), which suggested the presence of a Cys
304
on the carbon chain. Ions at m/z 280.064 and 99.081 indicated that the Cys was bound to the C3.
305
Hence, M139 was identified as a Cys conjugate of dehydrated 6D. Similarly, M104 was tentatively
306
identified as a Cys-Gly conjugate of dehydrated 6D.
307
Characterization of metabolites of 10G. A total of 28 metabolites were identified in rats after
308
intragastric administration of 10G, including 13 phase I metabolites and 16 phase II metabolites
309
(Table 1 and Figure S9). The proposed metabolic pathways of the 10G-related metabolites are shown
310
in Figure 2D. The mass spectra of the main 10G-related metabolites and their proposed fragmentation
311
pathways are listed in the Supporting Information.
312
Phase I metabolites. M168, M171, M179, M186, M189 and M191 showed the same retention
313
times and mass behaviors as components in ZO (Table S2). Hence, they were identified as 3R,5S-
314
[10]-gingerdiol, 3S,5S-[10]-gingerdiol, [10]-gingerdione, [10]-shogaol, 4-dehydro-[10]-gingerol and
315
[10]-dehydrogingerdione, respectively.19 M190 showed the precursor [M+H]+ ion at m/z 357.240,
316
which was 2.016 Da (2H) higher than that of M186, and the ions at m/z 317.249, 163.076 and 137.060
317
indicated that the carbon group on C3 was hydrogenated. Hence, M190 was identified as 3-
318
hydrogenated [10]-shogaol. M156 and M158 showed the same [M+H]+ ion at m/z 339.254, which
319
was 14.016 Da (CH2) lower than that of [10]-gingerdiol. Consequently, they were identified as the
320
isomers of demethylated metabolites of [10]-gingerdiol. Similarly, M163 and M180 were the
321
demethylated metabolites of 10G and [10]-shogaol, respectively.
322
Phase II metabolites. Glucuronidated conjugates. M40, M47, M52, M93, M114, M129, M140, 15
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M143, M162, M165 and M172 were identified as mono-glucuronides with a characteristic neutral
324
loss of 176.032 Da. Among them, M140 and M143 were determined to be isomers of mono-
325
glucuronides of 10G.31 M114, M129, M162, M165 and M172 were determined to be mono-
326
glucuronides of dehydrogenated [10]-gingerol, [10]-gingerdiol, methylated [10]-gingerol, [10]-
327
shogaol and hydrogenated [10]-shogaol, respectively. M40 had the [M+Na]+ ion at m/z 565.262,
328
which was 15.995 Da (O) higher than that of M140 and M143. Therefore, M40 was identified as a
329
hydroxylated product of mono-glucuronidated 10G. Similarly, M93 was determined to be a
330
hydroxylated metabolite of M114. M47 and M52 were identified as isomers of hydroxylated M93.
331
Characterization of ZO-related xenobiotics in rat biological samples. After intragastric
332
administration of the ZO extract, a total of 141 xenobiotics were identified (Table 1). The main
333
xenobiotics are shown in Figure 3. Among these, 35 xenobiotics were determined to be prototypes,
334
and 97 xenobiotics showed the same mass behavior as those of metabolites of 6G, 6S, 6D and 10G.
335
In additional to the xenobiotics mentioned above, another 21 ZO-related xenobiotics were detected,
336
including 17 glucuronides, 3 thiol-conjugates and 1 glucuronidated thiol-conjugate.
337
M5 and M6 had the same [M-H]- ion at m/z 441.176, which was 176.032 Da higher than that of
338
[4]-gingerol. Hence, they were identified as isomers of mono-glucuronidated [4]-gingerol. Likewise,
339
M33, M85, M87, M89, M91, M124, M133, M141, M150, M151, M153, M160, M164, M170 and
340
M173 were also considered to be glucuronic acid derivatives. In addition, M28 eluted at 5.68 min
341
with [M+H]+ at m/z 600.259, which was 307.084 Da higher than that of 6G, and a series of ions at
342
m/z 525.225, 471.218 and 453.205 suggested that M28 was a GSH-conjugate. The ions at m/z 193.032
343
indicated that GSH was conjugated to the C5´. Hence, M28 was identified as a metabolite of 5´-GSH-
344
6G. Similarly, M27 was identified as a metabolite of 5´-Cys-6G, while M147 was identified as a
345
metabolite of 5-NAC-[8]-shogaol on the basis of the ions at m/z 177.091 and 137.060. M14 showed 16
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the [M+H]+ ion at m/z 576.248. The ions at m/z 400.215 ([M+H-GluA]+) and 261.185 ([M+H-GluA-
347
Cys-H2O]+) tentatively indicated that M14 was a Cys conjugate of glucuronidated and hydrogenated
348
6S.
349
DISCUSSION
350
A total of 141 ZO-related xenobiotics in rats were identified by UPLC-Q/TOF-MS, including 63
351
in plasma, 72 in bile, 51 in urine and 57 in feces (Table 1). Among these, nearly 68% of the xenobiotics
352
were derived from the four representative pungent compounds, and nearly 60% of the xenobiotics
353
were derived from 6S and 6G. However, only one diarylheptanoid was detected in the rat biological
354
samples due to the low content of these compounds in ZO, as shown in Figure 1. Generally, only the
355
prototypes or related metabolites in blood with a sufficiently high exposure in target organs for a
356
limited period of time can be regarded as the potential functional components with therapeutic
357
benefits.33 These results indicated that the pungent phytochemicals, especially 6G and 6S, could be
358
the main functional components in ZO.
359
Among the 141 xenobiotics, 89 phase II metabolites mainly derived from glucuronidation,
360
sulfation and thiolation were identified in rat biological samples (Table 1). The results suggested that
361
the compounds in ZO are mainly eliminated via phase II metabolism in vivo, and the liver should be
362
the main metabolic site on the basis that 65 phase II metabolites were identified in the bile sample.
363
Furthermore, glucuronides were the most abundant phase II metabolites in rats. Normally,
364
glucuronidation is a detoxification mechanism because glucuronides are not pharmacologically
365
bioactive due to their extremely high polarity and rapid excretion from the body.31 A previous study
366
showed that the glucuronidated 6S greatly reduced its cytotoxicity on human colon cancer cells,
367
which indicated that the phenolic hydroxyl group of 6S played an important role in its cell cytotoxicity
368
activity. 33 Furthermore, a series of glucuronidated metabolites (M34, M37, M39, M46, M51, M90, 17
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M94 and M108), mainly derived from 6G and 6S (Figure 3), were found to be present at high levels
370
in plasma. Preliminary pharmacokinetic studies showed that 6G (Cmax=1.33 μg/mL at 0.083 h) and
371
6S (Cmax=14.50 μg/mL at 0.67 h) were rapidly absorbed and eliminated in plasma.34,35 At the same
372
time, they also underwent massive phase II glucuronidation in the liver and intestine.34,35 UGTs 1A9
373
(CLint = 26.01 μl/min/mg) and 2B7 (CLint = 29.67 μl/min/mg) were the main contributors to the
374
glucuronidation of 6G, whereas UGTs 1A6 and 2B7 were the main enzymes for the glucuronidation
375
of 6S.31,36 These experiments indicated that the poor bioavailability of 6G and 6S was related to the
376
glucuronidation by UGTs. On the other hand, studies also reported that 6G was a significant inhibitor
377
(Ki ≤ 10 μM) of UGT 2B7 (Ki = 5.2 μM), and 6S had similar effects on UGT 1A7 (Ki = 0.05 μM) and
378
2B7 (Ki = 3.4 μM).37,38 These results indicated that there was a high possibility of drug-drug
379
interaction between ZO and the drugs such as morphine, zidovudine, naloxone, and others whose
380
main metabolic pathways was catalyzed by UGT 1A7 and UGT 2B7.
381
Furthermore, a total of 19 thiol-conjugated metabolites were also found in vivo after intragastric
382
administration of the ZO extract. To the best of our knowledge, the conjugation of thiol group at the
383
C5´ position of the pungent compounds in ZO found in this study was a previously unknown
384
metabolic spot. Furthermore, four GSH conjugates (M28, M42, M49 and M50) were also identified
385
in vivo for the first time in this study after intragastric administration of ZO extract. Evidence has
386
been gathered about the important role played by GSH in the detoxification and protection from
387
oxidative injuries. Recently, it has been reported that dietary electrophiles can modify the cysteine
388
residues in Keap1 to activate the transcription factor Nrf2, and then the excessive production of GSH
389
is stimulated to detoxify carcinogens and electrophilic substances.39-41 Researchers found that after
390
treatment with 6S (an electrophilic compound in ZO), the level of GSH in HCT-116 cells decreased
391
and then returned to the basal level within 8 h and subsequently increased further to a level 2.5-fold 18
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higher than the basal level at 24 h.22 From the facts, we deduced that the detoxification42 and
393
antioxidant activity2-3 of ZO may be related to the presence of GSH-conjugates.
394
In addition, 36 prototypes and 17 phase I metabolites were identified in rat biological samples
395
after intragastric administration of the ZO extract. As mentioned early, the pungent phytochemicals
396
in ZO usually showed similar structural features, which meant that the mutual transformation among
397
the prototypes in vivo was possible. For example, 6D could be remarkably transformed into 6G in
398
vivo (Figure 2D), and 6G could be further converted to 6S and [6]-gingerdiols (Figure 2A). This
399
phenomenon was important for the beneficial functions of ZO as these prototypes have been reported
400
to possess various health-promoting effects, such as antioxidation, anti-inflammatory, antitumor, and
401
hematopoietic effects.18-19,
402
relatively high exposure in rat biological samples, whereas 6S could not be detected after its
403
intragastric administration. In addition to the contribution of 6G biotransformation, this phenomenon
404
could also be related to the interaction of multiple components in ZO, which suggested that the health
405
benefits of ZO are the result of common effects of its multiple components. Furthermore, previous
406
study also showed that M159 and M167 both produce measurable antiproliferative activity in H-1299
407
and HCT-116 cancer cells.20 These findings provided evidence that some pungent chemicals in ZO
408
continue to exhibit some health-promoting effects after in vivo being metabolized, which also
409
provided helpful information that can act as a reference for nutraceutical developments of ZO. In
410
addition, most phase I metabolites and prototypes could be detected in the rat feces. Currently,
411
scientists are increasingly concerned about the role of the intestinal microbiota. It is made up of 1013
412
- 1014 microorganisms and shows at least 100 times as many as genes as human genome. Evidence
413
has shown that the poor oral bioavailability components influence intestinal dysfunction through
414
effects of their prototypes or their secondary metabolites in the intestinal tract.44 It was previously
43
Meanwhile, in the ZO-related xenobiotics, 6S was found with a
19
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415
reported that the pungent ginger constituents exhibited a range of biological bioactivities but showed
416
poor bioavailability.13-15 It is suspected that intestinal microflora may play a key role. Therefore,
417
more attention should be paid to the xenobiotics in the feces, and further research on intestinal
418
microbiota should be carried out.
419
In conclusion, 92 chemical components in ZO and 141 xenobiotics in rats were identified or
420
tentatively characterized based on a four-step approach. The metabolic spots of the pungent
421
phytochemicals of ZO in vivo were also summarized as follows: (1) For the aromatic ring, phase II
422
metabolism was the main reaction, in which glucuronidation and sulfation mainly occurred at the
423
phenolic hydroxyl group of C4’ position. In addition, the conjugation site of thiolation reactions at
424
C5´ position of the pungent compounds in ginger was reported for the first time. Furthermore, the
425
phase I metabolic modification of demethylation could also be detected at C3’ position of the aromatic
426
ring. (2) For the aliphatic chain, phase I metabolism including desaturation, reduction or dehydration
427
between C3 and C5 position was considered to be the main metabolic route for pungent chemicals.
428
In addition, the phase II metabolic modification of thiolation at the C5 position was important for the
429
elimination of the shogaols. The pungent chemicals underwent massive phase I and phase II
430
metabolism. In particular, there was a general tendency that the pungent chemicals were metabolized
431
into highly polar metabolites (glucuronidation, sulfation and thiolation) that are eliminated and
432
excreted from the rat organism. Taken together, the results of this study lead to a better understanding
433
of the biotransformation of ZO in vivo and provide important information on the functional
434
components of ZO.
435
ABBREVIATIONS USED
436
ZO, Zingiber officinale Roscoe; UPLC-Q/TOF-MS, ultra-performance liquid chromatography
437
coupled with quadrupole time-of-flight tandem mass spectrometry; BPI, base peak intensity; 6G, [6]20
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gingerol; 6S, [6]-shogaol; 6D, [6]- dehydrogingerdione; 10G, [10]-gingerol; NAC, N-acetylcysteine;
439
GSH, glutathione; Cys-Gly, cysteinylglycine; Cys, cysteine.
440
ACKNOWLEDGMENT
441
This work was financially supported by National Major Scientific and Program of Introducing
442
Talents of Discipline to Universities (B13038), National Natural Science Foundation of China
443
(81630097), National Natural Science Foundation of China (81774219) and Guangdong Provincial
444
Science and Technology Project (2016B090921005).
445
ASSOCIATED CONTENT
446
Supporting Information. The analytical strategy for identifying compounds in ginger. The
447
detailed analytical process of pungent phytochemicals and diarylheptanoids. The detailed information
448
of matrix effects and glucuronidation assays, as well as the optimization of ginger extract preparation.
449
Tables of the self-building chemical database and UPLC-Q/TOF-MS data of the identified
450
compounds in ginger, as well as the listed of the reference standards. Structures of the compounds in
451
the Self-building database and the determined chemicals in ginger by UPLC-Q/TOF-MS. Figures of
452
the fragmentation patterns for the pungent phytochemicals in ginger based on reference standards.
453
The mass spectra of the main in vivo xenobiotics and their corresponding proposed fragmentation
454
pathways. The 13C NMR and HRMS data of all reference standards.
455
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456
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coupled with quadrupole time-of-flight tandem mass spectrometry analysis. J. Pharm. Biomed.
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Anal. 2015, 112, 23-35.
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27. Geng, J.-l.; Dai, Y.; Yao, Z.-h.; Qin, Z.-f.; Wang, X.-l.; Qin, L.; Yao, X.-s., Metabolites profile
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of Xian-Ling -Gu-Bao capsule, a traditional Chinese medicine prescription, in rats by ultra
536
performance liquid chromatography coupled with quadrupole time-of-flight tandem mass
537
spectrometry analysis. J. Pharm. Biomed. Anal. 2014, 96, 90-103.
538
28. Zhang, X.; Yin, J.; Liang, C.; Sun, Y.; Zhang, L., UHPLC-Q-TOF-MS/MS Method Based on
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Four-Step Strategy for Metabolism Study of Fisetin in Vitro and in Vivo. J. Agric. Food. Chem.
540
2017, 65, 10959-10972.
541
29. Wang, K.; Chai, L.; Feng, X.; Liu, Z.; Liu, H.; Ding, L.; Qiu, F., Metabolites identification of
542
berberine in rats using ultra-high performance liquid chromatography/quadrupole time-of-flight
543
mass spectrometry. J. Pharm. Biomed. Anal. 2017, 139, 73-86.
544 545
30. Pfeiffer, E.; Heuschmid, F. F.; Kranz, S.; Metzler, M., Microsomal hydroxylation and glucuronidation of 6 -gingerol. J. Agric. Food. Chem. 2006, 54, 8769-8774.
546
31. Wu, Z.; Liu, H.; Wu, B., Regioselective glucuronidation of gingerols by human liver microsomes
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and expressed UDP-glucuronosyltransferase enzymes: reaction kinetics and activity correlation
548
analyses for UGT1A9 and UGT2B7. J. Pharm. Pharmacol. 2015, 67, 583-596.
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32. Yu, Y.; Zick, S.; Li, X.; Zou, P.; Wright, B.; Sun, D., Examination of the Pharmacokinetics of Active Ingredients of Ginger in Humans. AAPS J. 2011, 13, 417-426. 33. Wang, X.; Studies on Serum Pharmacochemistry of Traditional Chinese Medicine. World Science Technology-Modernization of Traditional Chinese Medicine. 2002, 4, 1-4. 25
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553
34. Wang, W.; Li, C.-Y.; Wen, X.-D.; Li, P.; Qi, L.-W., Plasma pharmacokinetics, tissue distribution
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and excretion study of 6-gingerol in rat by liquid chromatography-electrospray ionization time-
555
of-flight mass spectrometry. J. Pharm. Biomed. Anal. 2009, 49, 1070-1074.
556
35. Asami, A.; Shimada, T.; Mizuhara, Y.; Asano, T.; Takeda, S.; Aburada, T.; Miyamoto, K.; Aburada,
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M., Pharmacokinetics of 6-shogaol, a pungent ingredient of Zingiber officinale Roscoe (Part I).
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J. Nat. Med. 2010, 64, 281-287.
559 560
36. Wang, P.; Zhao, Y.; Zhu, Y.; Sang, S., Glucuronidation and its impact on the bioactivity of 6 shogaol. Mol. Nutr. Food Res. 2017, 61.
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37. Ji, Y.; Yu, Y., In Vitro-In Silico Determination of the Inhibition of 6-Shogaol Towards Phase II
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Drug-Metabolizing Enzymes (DMEs). Latin American Journal of Pharmacy 2016, 35, 1686-1691.
563
38. Liu, Y.; Tian, Z., Wang, J.; Shao, Y.; Wang, X.; XU, W.; Lu, J.; Comprehensive Understanding
564
of
the
Inhibition
Profile
of
6-Gingerol
Towards
Various
Isoforms
of
UDP-
565
Glucuronosyltransferases (UGTs). Latin American Journal of Pharmacy 2016, 35, 1042-1045.
566
39. Higgins, L. G.; Kelleher, M. O.; Eggleston, I. M.; Itoh, K.; Yamamoto, M.; Hayes, J. D.
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Transcription factor Nrf2 mediates an adaptive response to sulforaphane that protects fibroblasts
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in vitro against the cytotoxic effects of electrophiles, peroxides and redox-cycling agents. Toxicol.
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Appl. Pharmacol. 2009, 237, 267-280.
570 571
40. Juge, N.; Mithen, R. F.; Traka, M., Molecular basis for chemoprevention by sulforaphane: a comprehensive review. Cell. Mol. Life Sci. 2007, 64, 1105-1127.
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41. MacLeod, A. K.; McMahon, M.; Plummer, S. M.; Higgins, L. G.; Penning, T. M.; Igarashi, K.;
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Hayes, J. D., Characterization of the cancer chemopreventive NRF2-dependent gene battery in
574
human keratinocytes: demonstration that the KEAP1-NRF2 pathway, and not the BACH1-NRF2
575
pathway, controls cytoprotection against electrophiles as well as redox-cycling compounds. 26
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576
Journal of Agricultural and Food Chemistry
Carcinogenesis 2009, 30, 1571-1580.
577
42. Egwurugwu, J. N.; Ufearo, C. S.; Abanobi, O. C.; Nwokocha, C. R.; Duruibe, J. O.; Adeleye, G.
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S.; Ebunlomo, A. O.; Adetola, A. O.; Onwufuji, O., Effects of ginger (Zingiber officinale) on
579
cadmium toxicity. African Journal of Biotechnology 2007, 6, 2078-2082.
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43. Semwal, R. B.; Semwal, D. K.; Combrinck, S.; Viljoen, A. M., Gingerols and shogaols: Important nutraceutical principles from ginger. Phytochemistry 2015, 117, 554-568.
582
44. Zhang, R.; Cao, H.; Gilbert, S.; Vallance, J.; Steinbrecher, K.; Zhang, D.; Eluri, M.; Shroyer, N.;
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Denson, L.; Han, X.; Yao, X.; Moriggl, R.; Chen, H., Natural compound methyl protodioscin
584
protects against intestinal inflammation through modulation of intestinal immune responses.
585
Pharmacol Res Perspect 2015, 3, e00118.
27
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Journal of Agricultural and Food Chemistry
Page 28 of 45
Table 1. UPLC-Q/TOF-MS data of the xenobiotics of ZO and four representative pungent phytochemicals in rat biological samples. No.
Time
Selected ion
Elemental composition
Measured mass
Mass (ESI+) MS/MS or MSE fragmentations error
(ESI-) MS/MS or MSE fragmentation
identification
6G
M1
3.70
[M-H]-
C23H34O11
485.2023
-0.2
487.2177, 469.2037, 311.1866, 293.1754, 275.1660, 177.0917
175.0243
6G+OH+GluA
PUB PUB
P
3.75
[M-H]-
C23H31O11
483.1866
0.4
485.2023, 467.1930, 291.1597, 179.0708, 137.0602
6G+OH-2H+GluA
PUB U
P
M3
3.89
[M+H]+
C28H43N2O13S 647.2486
-1.9
451.1959, 275.1638, 177.0910, 163.0770, 161.0595, 137.0602, 645.2321 99.0808
6D→6G-H2O+2H+OH+GluA+Cys+Gly
U
M4
4.19
[M+H]+
PU
M2
6D
6S
C26H40NO12S
590.2271
2.2
451.1951, 275.1647, 161.0599, 137.0598, 99.0815
588.2095
6D→6G-H2O+2H+OH+GluA+Cys
-
C21H29O10
441.1761
-1.4
465.1730, 249.1495,177.0910, 163.0754, 137.0602
265.1436, 175.0249
[4]-Gingerol+GluA
PBU
C21H29O10
441.1761
-0.5
465.1746, 249.1483, 177.0916, 163.0755, 137.0598
265.1440, 175.0235
[4]-Gingerol+GluA
PBU
C17H25O8S
389.1270
1.5
M5
4.30
[M-H]
M6
4.45
[M-H]-
M7
4.45
[M-H]
-
M8
4.48
[M-H]-
C22H29O10
453.1761
-1.5
413.1239, 391.1419, 373.1316, 355.1211, 311.1866, 293.1745, 375.1117, 315.0540, 301.0385, 273.0436, 6G+OH+Sul 275.1641, 179.0715, 177.0910, 137.0602 259.0279, 235.0972, 221.0823, 187.0065 477.1747, 279.1611, 261.1488, 179.0715, 161.0603, 137.0603 175.0250 6D-CH2+2H+GluA
4.51
[M-H]-
C23H27O10
463.1604
-1.3
487.1582, 289.1453, 271.1326, 139.0751, 137.0599
287.1288, 175.0235
M10
4.60
[M-H]
-
C17H23O8S
387.1114
1.0
411.1103
373.0957, 315.0536, 273.0445, 235.0959, 6G-2H+OH+Sul 187.0065
M11
4.61
[M-H]-
C17H25O8S
389.1270
-1.0
413.1243, 391.1417, 373.1320, 355.1219, 311.1872, 275.1637, 309.1702, 273.0432, 259.0269, 179.0714 6G+OH+Sul 179.0710, 177.0916, 137.0603
M12
4.79
[M+H]+
C28H45N2O12S 633.2693
-1.7
457.2381, 439.2262, 293.1559, 261.1848, 179.0492, 177.0904, 455.2213, 175.0248 162.0230, 144.0114, 137.0603, 116.0165
6S+4H+GluA+Cys+Gly
U
M13
4.86
[M+H]+
C20H34NO6S
416.2107
-0.7
398.2003, 311.1690, 261.1850, 177.0910, 163.0755, 137.0603
309.153, 277.1808
6S+4H+OH+Cys
F
M14
4.97
[M+H]+
C26H42NO11S
576.2479
-0.9
400.2151, 261.1855, 177.0907, 163.0768, 137.0603
574.2313
[6]-Gingerdiol-OH+Cys+GluA
5.03
[M+H]+
C20H34NO6S
416.2107
1.2
398.2009, 311.1674, 261.1855, 177.0920, 163.0750, 137.0605
277.1817
6S+4H+OH+Cys
F
M16
5.04
[M-H]-
C26H40NO11S
574.2322
-3.5
598.2289, 576.2474, 400.2150, 382.2039, 261.1846, 177.0905, 398.2021, 175.0240, 113.0233 163.0755, 137.0601
6S+4H+GluA+Cys
PU
M17
5.10
[M+H]+
C29H37O17
657.2031
1.8
481.1711, 305.1392, 177.0547, 137.0600
655.1899, 479.1576
6D-2H+OH+2GluA
M18
5.11
[M-H]-
C17H25O8S
389.1270
-0.3
413.1248, 373.1305, 355.1215, 273.0438
309.1698, 273.0438, 259.0266, 179.0714, 6G+OH+Sul 135.0448
P
M19
5.12
[M-H]-
C16H23O7S
359.1164
-0.3
383.1132, 343.1219, 281.1754, 263.1639,137.0600, 123.0450
279.1588, 243.0323, 163.0765
6G-CH2+Sul
U
M20
5.30
[M-H]-
C28H39O16
631.2238
3.5
655.2181, 633.2401, 457.2054, 281.1741,
455.1943, 279.1587, 121.0290
6G-CH2+2GluA
U
5.38
-
C23H31O11
483.1866
2.8
507.1831, 309.1701, 291.1596, 273.1491, 137.0605
307.1530, 175.0240,113.0233
6S+2OH+GluA
479.1567
6D-2H+OH+2GluA
M9
M15
M21
[M-H]
+
M22
5.42
[M+H]
C29H37O17
657.2031
-0.3
481.1702, 305.1382, 137.0603
M23
5.56
[M+H]+
C22H37N2O6S
457.2372
3.5
261.1860, 163.0760, 137.0599
5.60
[M+H]+
C26H40NO10S
558.2373
-0.2
382.2055, 261.1857, 177.0904, 137.0597
5.64
[M+H]+
C28H42NO13S
632.2377
-0.9
456.2053, 451.1949, 275.1643, 177.0909, 161.0598, 137.0600, 630.2222 99.0814
M24 M25
10G ZO
556.2211
28
ACS Paragon Plus Environment
UB
U B
6D-2H+GluA
PB B U
B
U
P
P
B U
U PU
6S+4H+Cys+Gly
U
6S+4H-H2O+Cys+GluA
U'F
6D→6G-H2O+2H+OH+GluA+NAC
U
U
B
Page 29 of 45
Journal of Agricultural and Food Chemistry
M26
5.65
[M-H]-
C22H29O10
453.1761
1.3
477.1748, 455.1916, 279.1589, 181.0860, 123.0452
277.1444, 175.0248, 113.0230
6G-CH2-2H+GluA
M27
5.66
[M+H]+
C20H32NO6S
414.1948
-0.5
396.1851, 293.1755, 275.1636, 177.0907, 137.0600
412.1798
6G+Cys
B
5.68
+
[M+H]
C27H42N3O10S 600.2591
-2.0
525.2250, 507.2143, 471.2178, 453.2054, 275.1641, 193.0319
598.2402, 467.1903, 451.1940,
[6]-Gingerol+GSH
B
M29
5.71
[M-H]-
C22H33O10
457.2074
-0.7
481.2038, 459.2248, 283.1896, 265.1800, 247.1695, 163.0765, 281.1759, 175.0243, 113.0233 123.0442
6G-CH2+2H+GluA
U
M30
5.75
[M-H]-
C23H35O10
471.2230
2.3
495.2197, 473.2389, 297.2067, 279.1945, 261.1847, 177.0913, 295.1903, 175.0241, 113.0233 163.0755, 137.0605
6G+2H+GluA
B
M31
5.77
[M+H]+
6S-CH2+4H+OH+SCH3
M28
C17H29O4S
329.1787
0.3
311.1676, 247.1706, 163.0760, 149.0589, 137.0600, 123.0446
-
327.1631, 281.1749, 263.1653
C22H33O10
457.2074
-0.6
481.2028, 459.2217, 283.1903, 265.1789, 247.1691, 163.0766, 281.1761, 175.0240, 113.0233 123.0441
6G-CH2+2H+GluA
U
PBU B F
M32
5.78
[M-H]
M33
5.85
[M-H]-
C23H35O10
471.2230
2.3
495.2197, 473.2389, 297.2067, 279.1945, 261.1847, 177.0913, 295.1900, 175.0241, 113.0233 163.0755, 137.0605
[6]-Gingerdiol+GluA
M34
5.92
[M-H]-
C23H35O10
471.2230
-0.3
495.2202, 473.2387, 297.2046, 279.1958, 261.1851, 177.0902, 295.1900, 175.0248, 113.0233 163.0750, 137.0611
6G+2H+GluA
M35
5.97
[M+H]+
C17H29O4S
329.1787
0.4
311.1672, 247.1706, 163.0755, 149.0589, 137.0606, 123.0443
6S-CH2+4H+OH +SCH3
M36
5.98
[M-H]-
C23H33O10
469.2074
0.4
493.2045, 471.2240, 295.1910, 277.1799, 163.0758, 137.0604, 293.1740, 175.0252, 113.0233 99.0811
6D+4H+GluA
B
B
M37
6.00
[M-H]-
C23H35O10
471.2230
-0.7
495.2200, 473.2373, 297.2042, 279.1971, 261.1855, 177.0927, 295.1924, 175.0243 163.0752, 137.0610
6G+2H+GluA
PU
PU
M38
6.03
[M+H]+
C20H34NO5S
400.2158
-1.0
293.1579, 382.2050, 261.1847, 177.0911, 163.0753, 137.0599
398.2007
6S+4H+Cys
M39
6.05
[M-H]-
C22H31O10
455.1917
-0.8
457.2056, 281.1749, 263.1649, 163.0754, 137.0603, 123.0245
175.0239, 121.0288
6G-CH2+GluA
M40
6.05
[M+Na]+
C27H42O11Na
565.2625
-1.4
367.2489, 349.2383, 331.2269, 177.0913, 137.0603
541.2648
10G+OH+GluA
M41
6.07
[M+H]+
C22H36NO7S
458.2212
-0.4
261.1855, 177.0904, 163.0756, 162.0219, 137.0603
456.2063
6S+4H+OH+NAC
U'F
M42
6.09
[M+H]+
C27H44N3O9S
586.2798
-2.6
511.2460, 457.2356, 439.2263, 261.1852, 163.0753, 137.0601
584.2627
6S+4H+GSH
B
M43
6.12
[M-H]-
C23H29O11
481.1710
-2.9
505.1678, 307.1541, 289.1440, 177.0557, 137.0600
6D+OH+GluA
M44
6.13
[M+Na]+
C15H22O4Na
289.1416
-1.7
249.1484, 231.1390, 193.0864, 179.0708, 177.0916, 163.0757, 137.0601
[4]-Gingerol
M45
6.15
[M+H]+
C22H36 N O7 S 458.2212
2.4
261.1855, 177.0904, 163.0754, 162.0225, 137.0602
M46
6.16
[M-H]-
C23H33O10
469.2074
1.1
493.2037, 471.2223, 453.2123, 277.1794, 179.0709, 177.0912, 293.1738, 175.0241, 113.0233 137.0598
6G+GluA
M47
6.22
[M+Na]+
C27H40O12Na
579.2417
3.1
381.2277, 363.2167, 345.2081, 177.0910, 137.0599
555.2437, 361.2009
10G-2H+2OH +GluA
M48
6.25
[M-H]-
C22H33O9
441.2125
-0.5
465.2114, 443.2286, 267.1967, 137.0602, 123.0441
265.182, 175.0239, 121.0288, 113.0216
6G-CH2-H2O+4H+GluA
M49
6.27
[M+H]+
C27H42N3O9S
584.2642
-3.8
509.2316, 455.2224, 437.2122, 420.1820, 352.1951, 308.0911, 582.2461 277.1802, 233.0585, 179.0490, 177.0904, 163.0764, 162.0227, 144.0117, 137.0604, 116.0170
263.1642
456.2061
29
ACS Paragon Plus Environment
U
PBU P
PUB PU
PBU F
F
PBU UF
U
PBU B
B
PU P
6S+4H+OH+NAC
6S+2H+GSH
F
U'F PU
PBU
PBU P
P B
B
Journal of Agricultural and Food Chemistry
Page 30 of 45
M50
6.30
[M+H]+
C27H44N3O9S
586.2798
2.6
511.2472, 457.2385, 439.2250, 422.2002, 354.2093, 337.1811, 584.2669 308.0911, 293.1559, 261.1857, 179.0500, 177.0904, 163.0763, 162.0228, 144.0117, 137.0608, 116.0162
6S+4H+GSH
M51
6.30
[M-H]-
C23H33O10
469.2074
0.4
493.2050, 471.2232, 453.2114, 295.1907, .277.1798, 179.0715, 293.1750, 175.0238 177.0916, 137.0601
6G+GluA
M52
6.30
[M+Na]+
C27H40O12Na
579.2417
-2.2
381.2276, 363.2171, 345.2067, 177.0912, 137.0608
555.2431, 379.2111, 361.2025
10G-2H+2OH+GluA
M53
6.46
[M-H]-
C17H19O7S
367.0851
2.2
391.0834, 369.0998, 289.1439, 177.0552, 137.0605
287.1292, 151.0760
6D-2H+Sul
M54
6.46
[M-H]-
C28H42NO12S
616.2428
-1.0
640.2433, 618.2594, 442.2263, 424.2151, 261.1856, 137.0603
440.2112, 175.0240
6S+4H+GluA+NAC
B
+
B
B
PUB PU
PBU PU
U
M55
6.54
[M+H]
C28H42NO12S
616.2428
-0.2
440.2113, 277.1800, 164.0384, 137.0603, 122.0273
614.2283, 162.0237
6S+2H+GluA+NAC
B
M56
6.55
[M-H]-
C16H23O6S
343.1215
-1.1
163.0751, 137.0600
261.1487, 139.1126, 121.0291,
6S+2H-CH2+Sul
F
F
6.55
[M+H]+
C18H31O4S
343.1944
-1.5
325.1847, 261.1859, 177.0916, 163.0760, 137.0598
6S+4H+OH+SCH3
F
F
M58
6.55
[M-H]-
C22H31O9
439.1968
1.4
463.1935, 247.1708, 147.0811
M59
6.60
[M+H]+
C28H42NO12S
616.2428
0.6
440.2110, 277.1800, 164.0384, 137.0605, 122.0273
614.2283,
6S+2H+GluA+NAC
M60
6.62
[M+H]+
C23H31O10
467.1917
-0.4
291.1591, 177.0554, 137.0596
465.1756, 289.1439, 149.0608
6D +GluA
M61
6.64
[M-H]-
C28H42NO12S
616.2428
0.6
640.2411, 618.2576, 442.2253, 424.2172, 295.1724, 261.1843, 440.2117, 175.0452 164.0384, 163.0760, 137.0607, 130.0488, 122.0276
6S+4H+GluA+NAC
PU
U
M62
6.65
[M-H]-
C16H23O6S
343.1215
0.5
163.0757, 137.0601
261.1488, 139.1125, 121.0297
6S+2H-CH2+ Sul
UF
F
M63
6.72
[M-H]-
C23H31O10
467.1917
1.7
491.1898, 469.2086, 293.1753, 275.1641, 137.0602, 99.0805
291.1605, 175.0244, 113.0233
6G-2H+GluA
M64
6.74
[M+H]+
C23 H34 N O9 468.2234
-1.7
292.1916, 275.1656, 178.0867, 156.1387, 137.0601, 99.0808
466.2061
6D-H2O+2H+NH3+GluA
M65
6.75
[M+H]+
C18H31O4S
343.1944
0.3
325.1832, 261.1859, 177.0916, 163.0758, 137.0600
M66
6.76
[M-H]-
C28H40NO12S
614.2271
-0.3
638.2237, 616.2416, 440.2095, 277.1804, 137.0599
162.0244
6S+2H+GluA+NAC
M67
6.76
[M-H]-
C29H39O15
627.2289
-0.5
651.2271, 453.2144, 277.1806, 177.0914, 151.0762, 137.0602
451.1949, 175.0244
6G-H2O+2GluA
U
M68
6.77
[M+H]+
C16H27O4
283.1902
3.9
265.1780, 247.1695, 163.0757, 149.0595, 123.0450
6G-CH2+2H
F
UF
M69
6.80
+
[M+H]
C22H36NO7S
458.2212
0.4
422.2002, 317.1588, 293.1559, 291.1429, 259.1699, 193.0331, 456.2049, 162.0226, 135.0447 162.0233, 130.0507
6G+2H+NAC
UBF
BUF
M70
6.81
[M-H]-
C22H32NO9S2
518.1518
0.2
520.1679, 440.2106, 277.1804, 137.0603
6S+2H+Sul+NAC
M71
6.90
[M+H]+
C22H36NO7S
458.2212
1.1
422.2002, 317.1556, 293.1559, 291.1429, 259.1687, 193.0331, 456.2057 162.0228, 130.0504
6G+2H+NAC
UBF
BUF
M72
6.94
[M+H]+
C22H34NO7S
456.2056
0.7
438.1950, 420.1820, 396.1839, 379.1572, 333.1531, 219.0475, 454.1902, 162.0222 193.0315, 162.0219, 130.0511
6G+NAC
UBF
BU
M73
7.00
[M-H]-
C23H31O9
451.1968
-0.4
475.1942, 453.2112, 277.1792, 137.0611
275.1647, 193.0870
6G-H2O+GluA
PBF
M74
7.02
[M+H]+
C16H27O4
283.1902
-2.5
265.1790, 247.1704, 163.0762, 149.0598, 123.0451
281.1759
6G-CH2+2H
F
UF
M75
7.09
[M+H]+
C22H34NO7S
456.2056
2.4
438.1954, 420.1855, 396.1839, 333.1531, 219.0464, 193.0320, 454.1892, 162.0222 169.0318, 162.0224, 130.0496
6G+NAC
UBF
BU
M76
7.24
[M-H]-
C22H32NO9S2
518.1518
0.2
520.1679, 440.2093,355.1235, 277.1816, 137.0602
6S+2H+Sul+NAC
M57
Iso-6G-CH2-H2O+2H+GluA
PB B U
B
162.0239
30
ACS Paragon Plus Environment
B
UF
6S+4H+OH+SCH3
162.0242
PBU
F
F
PU
U
B
B
Page 31 of 45
Journal of Agricultural and Food Chemistry
M77
7.27
[M+Na]+
C16H24O4Na
303.1572
2.3
281.1768, 263.1644, 165.0546, 163.0755, 137.0602, 123.0440
279.1601
6G-CH2
M78
7.35
[M+H]+
C21H34NO6S
428.2107
1.2
410.1996, 247.1696, 137.0601, 123.0441
426.1955
6S-CH2+4H+NAC
M79
7.44
[M-H]-
C16H21O7S
357.1008
2.8
381.0944, 179.0709, 177.0920, 163.0752, 137.0601, 123.0441, 277.1440, 259.0283, 243.0327, 163.0756 6G-CH2-2H+Sul
U
M80
7.45
[M-H]-
C24H35O10
483.2230
1.7
507.2211, 309.2063, 291.1951, 165.0904, 151.0614, 137.0600
PU
7.51
[M+H]+
C24H33N5O3
440.2662
0.9
177.0917, 163.0757, 137.0597, 136.0625
M82
7.66
[M+Na]+
C17H26O6SNa
381.1348
-2.4
341.1414, 261.1856, 141.1274, 137.0599
M83
7.68
[M-H]-
C24H35O10
483.2230
1.7
991.4520, 507.2202, 309.2066, 277.1807, 177.0904, 137.0604, 307.1913, 175.0240, 113.0233
6G+CH3+GluA
PUB
BU
M84
7.69
[M+Na]+
C17H28O4Na
319.1885
-2.8
279.1955, 261.1849, 177.0910, 163.0756, 137.0604
295.1911
3R, 5S-[6]-Gingerdiol
PUF UF
PUF
M85
7.70
[M-H]-
C23H35O9
455.2281
0.7
479.2262, 457.2441, 439.2332, 281.2115, 263.2011, 137.0602
279.1961, 175.0251
[6]-Shogaol+4H+GluA
M81
+
307.1899, 175.0242, 113.0233
6G+CH3+GluA
UF
UF F U U
[8]-Zingerine
F
357.1376, 275.1648, 207.0689, 139.1128 6S+2H+Sul
U
PB
M86
7.76
[M+H]
C22H36NO6S
442.2263
-1.4
424.2148, 261.1844, 177.0904, 163.0747, 164.0384, 137.0599, 440.2112 130.0511, 122.0273, 84.0459
6S+4H+NAC
M87
7.80
[M-H]-
C22H33O9
441.2125
-1.8
443.2290, 249.1852, 137.0602, 123.0446
265.1815, 175.0250, 113.0230
[6]-Gingerdiol-CH2-OH+GluA
M88
7.83
[M+H]+
C26H42NO7S
512.2682
-0.8
494.2576, 331.2282, 177.0913, 137.0604
510.2531
10G+NAC
M89
7.90
[M+H]+
C25H39O10
499.2543
4.2
323.2232, 305.2117, 177.0914, 137.0607
321.2076, 175.0248, 113.0233
[8]-Gingerol+GluA
7.91
[M-H]-
C23H31O9
451.1968
2.4
475.1944, 453.2138, 277.1807, 137.0600
275.1660, 175.0236, 113.0233
6S+GluA
M91
7.95
[M-H]-
C25H37O10
497.2387
2.4
521.2363, 499.2543, 323.2221, 305.2113, 177.0912, 137.0607
321.2091, 175.0248
[8]-Gingerol+GluA
M92
7.99
[M+Na]+
C17H28O4Na
319.1885
-1.3
297.2059, 279.1958, 261.1847, 163.0754, 137.0602
8.01
[M+Na]
+
C27H40O11Na
563.2468
-0.4
365.2319, 347.2212, 329,2112, 137.0600
M94
8.02
[M-H]-
C23H33O9
453.2125
2.4
477.2086, 455.2271, 437.2168, 279.1949, 261.1842, 177.0913, 277.1810, 175.0249, 113.0233 163.0759, 155.1429, 137.0601
6G-H2O+2H+GluA
M95
8.05
[M-H]-
C23H35O9
455.2281
1.1
479.2249, 457.2434, 439.2324, 281.2115, 263.2006, 137.0602
279.1960, 175.0251, 113.0233
6S+4H+GluA
M96 a
8.07
[M+Na]+
C17H26O4Na
317.1729
-0.3
295.1916, 277.1802, 163.0755, 137.0604, 99.0810
293.1767
6G+2H-2H (Iso-6G)
PBU PBUF
M97
8.08
[M+H]+
C22H36NO6S
442.2263
-0.2
424.2155, 261.1842, 177.0904, 163.0754, 164.0384, 137.0602, 440.2119, 162.0232 130.0502, 122.0273
6S+4H+NAC
B
M98
8.08
[M-H]-
C17H23O7S
371.1164
-1.1
175.0761, 151.0758, 137.0606, 99.0809
291.1602, 155.1073
Iso-6G-2H+Sul
U
8.18
[M-H]
-
C22H31O9
439.1968
-0.9
463.1952, 265.1804, 137.0604, 123.0445
263.1653, 175.0238, 113.0233
6S-CH2+2H+GluA
PB
8.21
[M+H]+
C22H34NO6S
440.2107
-2.0
317.1556, 277.1799, 259.1697, 177.0915, 164.0384, 163.0753, 438.1956, 162.0231 137.0600, 122.0273
6S+2H+NAC
[M+Na]+
C17H26O4Na
317.1729
0.3
277.1796, 259.1687, 179.0706, 177.0908, 137.0600
[6]-Gingerol
C24H37O9S
501.2158
-1.6
525.2124, 503.2325, 485.2206, 327.1994, 309.1884, 261.1851, 325.1835, 175.0235 177.0914, 163.0759, 137.0606
6S+4H+GluA+SCH3
PB
PB
BUF
BUF
M90
M93
M99 M100
M101 a 8.22
-
3S,5S-[6]-Gingerdiol 539.2499
8.23
[M-H]
M103
8.27
[M+H]+
C22H34NO6S
440.2107
2.0
317.1556, 277.1802, 259.1707, 177.0919, 164.0384,163.0753, 137.0602, 122.0273
438.1961, 162.0229
6S+2H+NAC
M104
8.29
[M+H]+
C22H31N2O6S
451.1903
0.4
337.0816, 301.1222, 203.0480, 137.0605
449.1753
6D-OH+Cys+Gly
ACS Paragon Plus Environment
B PB P PB
PBU BU
PBU
PBU PB
UF
F
PUF
10G-2H+OH+GluA
M102
31
BF
B B
PB
PB PBU
PBU PBU
BUF
PBUF F
PB
PBU
BUF
BUF
PUF PBUF
UBF
PBUF
Journal of Agricultural and Food Chemistry
Page 32 of 45
M105
8.33
[M-H]-
C22H31O9
439.1968
0.7
463.1953, 265.1793, 137.0601, 123.0440
6S-CH2+2H+GluA
PB
M106
8.35
[M+H]+
C22H36NO6S
442.2263
0.5
424.2142, 406.2067, 382.2060, 365.1783, 319.1747, 293.1575, 440.2107 287.1480, 193.0314, 169.0314, 162.0217, 130.0503, 84.0449
6G-OH+2H+NAC
B
M107
8.40
[M-H]-
C23H31O10
467.1917
0.6
469.2072, 293.1739, 275.1658, 177.0921, 175.0755, 137.0604, 291.1611, 175.0240 99.0810
Iso-6G-2H+GluA
PB
PBU
M108
8.48
[M-H]-
C23H33O9
453.2125
1.1
931.4293, 477.2104, 455.2282, 279.1957, 177.0913, 137.0601
6S+2H+GluA
PB
PB
+
263.1642, 175.0255, 113.0233
277.1804, 175.0249, 113.0233
M109
8.51
[M+Na]
C18H30O4Na
333.2042
-2.4
293.2113, 275.2010, 191.1076, 177.0915, 151.0761
M110
8.55
[M+HH2O]+
C23H35O8S
471.2053
0.0
247.1705, 137.0605, 123.0444
M111
8.59
[M+H]+
C22H34NO6S
440.2107
0.5
422.2002, 398.1998, 381.1743, 363.1630, 335.1676, 317.1560, 438.1948 309.1524, 303.1424, 285.1313, 233.0634, 215.0527, 193.0319, 189.0368, 162.0222, 130.0494
6G-OH+NAC
M112
8.61
[M+Na]+
C26H34O9Na
513.2102
0.2
431.2092, 371.1856, 247.1339, 193.0857, 167.0705, 137.0604
3,5-Diacetoxy-1-(4-hydroxy-3,5dimethoxyphenyl)-7-(4-hydroxy-3methoxyphenyl)-heptane
M113
8.62
[M-H]-
C22H31O8
423.2018
-1.4
447.1995, 425.2175, 249.1853, 231.1742, 147.0816, 133.0652
247.1689, 175.0246
6G-CH2O-OH+GluA
M114
+
PB
B
PB
PB P
F BF
B
P
P
PB
[M+Na]
C27H40O10Na
547.2519
-1.5
349.2387, 331.2273, 177.0915, 137.0597
175.0240
10G-2H+GluA
M115
8.70
[M-H]-
C17H23O7S
371.1164
1.1
177.0914, 137.0603
291.1606, 155.1074, 121.0295
6G-2H+Sul
U
M116
8.71
[M+H]+
C18H29O3S
325.1837
1.2
307.1719, 277.1810, 179.0704, 177.0910, 137.0599, 131.0894
6G-H2O+SCH3+2H
B
M117
8.76
+
[M+H]
C22H36NO6S
442.2263
-1.4
424.2142, 406.2038, 382.2052, 365.1790, 347.1663, 319.1747, 440.2112 293.1559, 287.1480, 193.0324, 169.0328, 162.0221, 130.0501
6G-OH+2H+NAC
B
M118
8.77
[M-H]-
C24H35O9S
499.2005
0.6
523.1990, 501.2160, 325.1838, 307.1729, 277.1797, 137.0597, 323.1682, 275.1646, 175.0237, 113.0233 6S+2H+GluA+SCH3 131.0893
M119
8.81
[M+Na]+
C18H30O4Na
333.2042
-1.2
293.2107, 275.2014, 191.1075, 177.0913, 151.0759
3S,5S-methyl-[6]-gingerdiol
8.82
[M-H]-
C24H35O8S
483.2053
0.2
507.2029, 485.2208, 309.1888, 261.1855, 137.0601, 131.0889
6S+4H-H2O+SCH3+GluA
M121
8.85
[M+H]+
C23H31O10
467.1917
1.3
291.1597, 177.0550, 137.0602
6D+GluA
PUB
M122
8.87
[M+H]+
C17H26NO3
292.1913
0.3
275.1647, 178.0864, 156.1377, 137.0598, 99.0808
6D-H2O+2H+NH3
UF
8.90
+
[M+H]
C26H37N5O3
468.2975
0.4
177.0922, 163.0761, 151.0763, 137.0604, 136.0628
[10]-Zingerine
M124
8.90
[M-H]-
C23H29O9
449.1812
3.1
473.1794, 451.1949, 275.1643, 177.0548, 137.0600
M125
8.91
[M+Na]+
C17H24O4Na
315.1572
-1.6
275.1648, 177.0917, 137.0606, 99.0810
[6]-Gingerdione
M126
8.94
[M+H]+
C23H38NO6S2
488.2141
-2.3
470.2027, 380.1898, 317.1556, 293.1578, 291.1438, 259.1687, 193.0310, 162.0225, 130.0504
6S+4H+SCH3+NAC
M127
8.95
[M-H]-
C17H23O7S
371.1164
1.9
177.0547, 145.0287, 137.0597
M128
8.96
[M-H]-
C24H35O8S
483.2053
0.2
507.2022, 485.2193, 449.1994, 309.1880, 261.1860, 137.0601, 131.0889
6S+4H-H2O+SCH3+GluA
M129
9.02
[M-H]-
C27H43O10
527.2856
-2.3
551.2832, 529.3013, 353.2691, 335.2578, 317.2473, 177.0917, 351.2535, 175.0240, 113.0233 137.0604
10G+2H+GluA
M123
U
3R,5S-methyl-[6]-gingerdiol 487.1989, 469.1915, 311.1700, 297.1538, 6S-CH2+4H+SCH3 +GluA 245.1536, 175.0242
8.69
M120
PBU
465.1779, 289.1439, 149.0607
273.1486, 135.0441, 121.0284
291.1594, 155.1072, 149.0603
32
ACS Paragon Plus Environment
B
B BF
BF
PB
PB P
PB
F
[6]-Shogaol-2H+GluA
6D +2H+Sul
PB
PBU UF
U BF
B
U PB P'B
PB
Page 33 of 45
Journal of Agricultural and Food Chemistry
M130 a 9.05
[M+Na]+
C18H28O4Na
331.1885
0.3
291.1949, 193.0873, 191.1068, 151.0758
M131
9.05
[M+Na]+
C17H24O7SNa
395.1140
3.0
373.1319, 355.1208, 293.1763, 137.0606, 99.0806
M132
9.05
[M+H]+
C17H24NO3
290.1756
0.0
248.1664, 177.0551, 140.1068, 137.0597
6D-H2O+NH3
M133
9.08
[M-H]-
C27H43O11
527.2856
2.5
551.2831, 529.3018, 353.2694, 335.2585, 317.2477, 177.0916, 351.2532, 175.0244 163.0761, 137.0599
[10]-Gingerol+GluA
M134
9.09
[M+H]+
C22H34NO6S
440.2107
-2.0
422.2000, 398.1979, 381.1743, 363.1646, 335.1665, 317.1570, 309.1507, 291.1404, 219.0485, 193.0314, 169.032, 162.0226, 130.0497
6S+2H+NAC
BF
M135
9.15
[M-H]-
C16H25O3
265.1804
-0.4
249.185, 123.0466
247.1703, 135.044
6G-CH2-OH+2H
F
M136
9.22
[M-H]-
C23H33O9
453.2125
1.6
279.1974, 261.1854, 137.0602
435.2014, 277.1815
6S+2H-H2O+GluA
PBUF
PB
M137
9.30
[M+H]+
C23H36NO6S2
486.1984
1.2
438.1955, 420.1830, 396.1839, 379.1569, 333.1524, 309.1509, 291.1410, 219.0474, 193.0325, 169.0323, 162.0223, 130.0499
6S+2H+SCH3+NAC
BF
B
M138
9.30
[M+Na]+
C19H30O5Na
361.1991
-0.6
321.2053, 261.1850, 163.0755, 137.0600
3-acetoxy-[6]-gingerdiol or 5-acetoxy-[6]-gingerdiol
M139
9.33
[M+H]+
C20H28NO5S
394.1688
-1.5
280.0641, 244.1014, 146.0276, 99.0808
392.1521, 289.1447
M140
9.37
[M-H]-
C27H41O10
525.2700
-0.9
527.2856, 509.2727, 351.2534, 333.2420, 177.0911, 137.0600
507.2597, 349.2384, 175.0242, 113.0233 10G+GluA
M141
9.40
[M-H]-
C25H35O9
479.2275
-0.2
503.2248, 305.2108, 177.0915, 137.0601
303.1970, 175.0249, 113.0234
[8]-Shogaol+GluA
9.41
[M-H]
-
C17H25O7S
373.1321
1.6
397.1289, 137.0604
293.1739, 259.0282
6D+4H+Sul
M143
9.46
[M-H]-
C27H41O10
525.2700
0.4
527.2847, 509.2741, 351.2532, 333.2422, 177.0912, 137.0605
507.2595, 349.2381, 175.0244, 113.0233 10G+GluA
M144
9.47
[M-H]-
C16 H25O3
265.1804
3.0
533.3842, 289.1767, 249.1850, 123.0444
247.1706, 135.0446
6S+4H-CH2
F
M145
9.53
[M-H]-
C16H23O3
263.1647
2.3
287.1608, 265.1787, 247.1694, 149.0609, 123.0451
141.1274, 121.0290
6G-H2O-CH2+2H or Iso-6G-H2O-CH2+2H
UF
+
C17H29O3S
313.1837
-1.6
295.1729, 247.1696, 163.0751, 149.0602, 123.0445
311.1690, 265.1809
6S-CH2+4H+SCH3
+
M142
M146
9.60
[M+H]
4’-methoxyl-[6]-gingerol 371.1174, 357.1020, 291.1608, 277.1439, 6G-2H+Sul 155.1075, 121.0295
P U UF PB BF
F
P
6D-H2O+2H+Cys
BF
PU PB
[M+H]
C24H38NO6S
468.2420
-0.2
305.2118, 177.0912, 137.0603
466.2254, 162.0223
[8]-Shogaol+2H+NAC
9.67
[M-H]-
C17H25O7S
373.1321
2.9
397.1288, 277.1794, 137.0599
293.1753, 273.0450
6G+Sul
PUF PU
M149
9.73
[M+Na]+
6G+CH2
U
-0.6
309.2069, 277.1805, 179.0704, 177.0910, 163.0760, 137.0604
PB UF UF F B
U
M150
9.88
[M-H]
C25H35O10
495.2230
-1.2
519.2211, 321.2058, 177.0913, 137.0606, 127.1119
[8]-Gingerdione+GluA
B
M151
9.94
[M-H]-
C29H43O10
551.2856
-1.1
575.2817, 553.3013, 377.2697, 359.2588, 331.2263, 177.0919, 347.2220, 175.0247, 113.0240 137.0602
[12]-Gingerdione+GluA
PB
M152 a 9.96
[M+Na]+
C18H28O4Na
331.1885
-1.5
309.2055, 277.1799, 179.0704, 177.0907, 163.0755, 137.0602
307.1913
6G+CH2
M153
[M-H]-
C25H37O9
481.2438
2.5
505.2415, 307.2290, 137.0607
305.2111, 175.0247, 169.1600, 113.0240
[8]-Shogaol+2H+GluA
M154 a 10.04
[M+Na]+
C19H30O4Na
345.2042
0.0
305.2109, 287.2015, 179.0703, 177.0910, 137.0599
M155
[M-H]-
C16H23O3
263.1647
3.0
287.1611, 265.1793, 247.1694, 149.0600, 123.0440
10.02
10.06
319.1918, 175.0244, 113.0233
UF
F
9.66
331.1885
PB P
M148
C18H28O4Na
BU B
M147
-
BF
U
PB
[8]-Gingerol 141.1275, 121.0293
33
ACS Paragon Plus Environment
6G-H2O-CH2+2H or Iso-6G-H2O-CH2+2H
U
PU F
UF
Journal of Agricultural and Food Chemistry
M156
10.22
M157 a 10.37 M158
[M+H]+
C20H35O4
339.2535
4.1
321.2437, 303.2319, 149.0604, 137.0601, 123.0442
[M+Na]+
C17H24O3Na
299.1623
-2.3
277.1805, 137.0601
+
337.2376
Page 34 of 45
10G-CH2+2H [6]-Shogaol
10.47
[M+H]
C20H35O4
339.2535
-1.2
321.2437, 303.2307, 149.0603, 123.0440
337.2379
10G-CH2+2H
M159
10.49
[M+Na]+
C17H28O3Na
303.1936
0.8
263.2013, 247.1696, 137.0603
279.1954
6S+4H
M160
10.50
[M-H]-
C27H39O9
507.2594
-1.8
531.2540, 333.2416, 137.0605
331.2274, 175.0248, 113.0236
C21H32O3+GluA
10.61
[M+H]+
C18H31O3S
327.1994
0.9
309.1887, 261.1848, 163.0753, 137.0602, 131.0889
325.1826
6S+4H+SCH3
M161
-
F PUBF
F PBUF
F UF
F UF PB
F
M162
10.73
[M-H]
C28H43O10
539.2856
2.8
563.2832, 333.2439, 137.0598
363.2521, 175.0240, 113.0239
10G+CH2+GluA
B
B
M163
10.76
[M+Na]+
C20H3 O4Na
359.2198
-3.1
337.2371, 319.2266, 301.2177, 179.0705, 163.0753, 123.0442
335.2223
10G-CH2
F
F
10.89
[M-H]-
C29H45O10
553.3013
-1.6
577.2999, 555.3162, 379.2860, 361.2746, 177.0909, 137.0602
377.2699, 175.0245, 113.0230
[12]-Gingerol+GluA
M165
10.90
[M-H]
-
C27H39O9
507.2594
-1.2
531.2584, 333.2435, 137.0602
331.2278, 175.0243, 113.0233
10G-H2O+GluA
M166
11.04
C17H23O3
275.1647
0.0
179.0706, 177.0916, 137.0599, 99.0810
291.1606
4-Dehydro-[6]-gingerol
M167
11.05
[M+HH2O]+ [M+Na]+
C17H26O3Na
301.1780
-3.0
279.1955, 137.0601
277.1809
6S+2H
M168
11.11
[M+Na]+
C21H36O4Na
375.2511
1.3
353.2709, 335.2579, 317.2473, 177.0910, 163.0746, 151.0750, 351.2539 137.0602
[10]-Gingerdiol
M169
11.17
[M+Na]+
C16H24O2Na
271.1674
-1.1
249.1841, 231.1747, 137.0603, 123.0448
247.1704
6S-CH2-H2O+4H
M170
11.28
[M-H]-
C29H45O10
553.3013
2.0
577.2994, 379.2848, 361.2751, 177.0912, 137.0601
377.2700, 175.0245, 113.0230
[12]-Gingerol+GluA
M171
11.38
[M+Na]+
C21H36O4Na
375.2511
0.8
353.2693, 335.2584, 317.2468, 177.0902, 163.0751, 151.0750 137.0600
351.2540
[10]-Gingerdiol
U'F
M172
11.49
[M+Na]+
C27H42O9Na
533.2727
1.1
335.2589, 137.0602
509.2747, 333.2442, 175.0247
10G-OH+GluA
PB
C27H41O9
509.2751
-2.4
533.2728, 335.2574, 137.0599
333.2432, 197.1903, 175.0241, 113.0241 [10]-Shogaol+2H+GluA
M164
M173
11.53
[M-H]
-
B PB U
UF
PB UF
U'F
UF UF
F B
PB PB
M174 a 11.63
[M+Na]+
C21H34O4Na
373.2355
-3.2
333.2417, 315.2321, 179.0708, 177.0910, 137.0598
349.2377
[10]-Gingerol
M175 a
11.69
[M+Na]+
C17H22O4Na
313.1416
-0.1
291.1597, 177.0552, 137.0601
289.1450, 149.0617
[6]-dehydrogingerdione
M176 a 12.01
[M+Na]+
C19H28O3Na
327.1936
-0.3
305.2121, 137.0603
[8]-Shogaol
+
C21H34NO3
348.2539
0.0
212.2019, 170.1911, 137.0600
10G-H20+NH2
UF
F
+
F
M177
12.08
[M+H]
PBUF PBUF UP
BUF P
M178
12.16
[M+H]
C21H34NO3
348.2539
-2.0
212.2019, 170.1911, 137.0599
10G-H20+NH2
UF
M179
12.18
[M+Na]+
C21H32O4Na
371.2198
0.5
331.2271, 177.0922, 155.1429, 137.0600
347.2230
[10]-Gingerdione
U
M180
12.54
[M-H]-
C20H29O3
317.2119
0.6
319.2269, 181.1590, 123.0445
195.1753, 121.0287
10G-H20-CH2
F
F
M181
12.72
[M-H]-
C21H33O6S
413.1998
-1.9
397.2039, 317.2489, 315.2312, 137.0603
333.2446, 331.2267, 79.9566
10G-H20+2H+Sul
F
PBF
M182
13.10
[M+Na]+
C23H38O4Na
401.2668
1.5
361.2725, 177.0911, 137.0604
M183
13.16
[M+Na]
+
C22H36O4Na
387.2511
2.6
365.2678, 333.2429, 179.0707, 177.0907, 137.0598
M184
13.17
[M+Na]+
C20H32O3Na
343.2249
-1.2
321.2423, 303.2314, 137.0598, 123.0442
[12]-Gingerol
319.2278, 197.1905, 121.0293
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P
10G+CH2
UF
10G--CH2-H2O+2H
F
F
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Journal of Agricultural and Food Chemistry
13.41
[M+Na]+
C27H38O4Na
449.2668
-1.8
427.284, 371.2209, 137.0600, 179.0724, 177.0546, 135.1167
M186 a 13.48
[M+Na]+
C21H32O3Na
355.2249
-1.2
333.2431, 177.0920, 137.0601
+
C27H38O4Na
449.2668
0.0
427.2839, 399.2512, 371.2244, 291.2318, 275.1639, 277.2159, 179.0718, 177.0543, 137.0604, 135.1183
(E/Z) neral acetal of 1,6-didehydro-[6]gingerdiol isomer 2
F
(E/Z) neral acetal of 1,6-didehydro-[6]gingerdiol isomer 3
F
M185
331.2280
M187
13.54
[M+Na]
M188
13.62
[M+Na]+
C27H38O4Na
449.2668
0.0
427.2843, 399.2519, 371.2210, 291.2318, 179.0726, 177.0542, 137.0599, 135.1174
M189 a 14.05
[M+Na]+
C21H32O4Na
371.2198
-0.8
349.2384, 331.2265, 177.0919, 155.1438, 137.0604
M190
[M+H]+
M191
14.09 a
C21H34O3Na
357.2406
-4.2
317.2479, 163.0764, 137.0602
+
(E/Z) neral acetal of 1,6-didehydro-[6]gingerdiol isomer 1
347.2230
149.0602
[10]- Shogaol
F PUF PBF
4-Dehydro-[10]-gingerol
U
F
10G-H2O+2H
UF
F
[10]-dehydrogingerdione
U
BUF
14.56
[M+Na]
C21H30O4Na
369.2042
0.3
347.2222, 177.0549, 137.0601
M192
14.58
[M+Na]+
C29H42O4Na
477.2981
0.8
455.3156, 371.2217, 179.0711, 177.0555, 137.0612, 135.1167
(E/Z) neral acetal of 1,6-didehydro-[8]gingerdiol isomer 1
F
M193
14.70
[M+Na]+
C29H42O4Na
477.2981
-1.3
455.3148, 399.2538, 319.2638, 305.2476, 303.1943, 179.0724, 177.0532, 137.0614, 135.1167
(E/Z) neral acetal of 1,6-didehydro-[8]gingerdiol isomer 2
F
M194
14.78
[M+Na]+
C29H42O4Na
477.2981
-1.3
455.3152, 399.2518, 319.2638, 305.2483, 303.1974, 179.0724, 177.0532, 137.0602, 135.1164
(E/Z) neral acetal of 1,6-didehydro-[8]gingerdiol isomer 3
F
M195
15.28
[M+Na]+
C27H42O4Na
453.2981
-0.7
431.3161, 279.1958, 261.1840, 177.0916, 163.0752, 137.0599, 135.1176
neral acetal-[6]-gingerdiol
PF
M196 a 15.35
C23H35O3
359.2586
-0.3
183.1761, 177.0916, 137.0598
4-Dehydro-[12]-gingerol
F
C31H46O4Na
505.3294
-1.4
483.346, 371.2233, 179.0709, 177.0556, 137.0606, 135.1174
(E/Z) neral acetal of 1,6-didehydro-[10]Gingerdiol isomer 1
PF
(E/Z) neral acetal of 1,6-didehydro-[10]gingerdiol isomer 2
PF
[12]-dehydrogingerdione
F
(E/Z) neral acetal of 1,6-didehydro-[10]gingerdiol isomer 3
PF
M197
15.71
[M+HH2O]+ [M+Na]+
M198
15.79
[M+Na]+
C31H46O4Na
505.3294
-2.6
483.3471, 455.3169, 427.2881, 347.2950, 331.2257, 179.0709, 177.0545, 137.0602, 135.11858
M199
15.81
[M+H]+
C23H35O4
375.2535
0.0
177.0557, 137.0601
15.87
[M+Na]+
C31H46O4Na
505.3294
-1.8
483.3463, 455.3181, 427.2881, 347.2971, 333.2777, 331.2277, 179.0710, 177.0541, 137.0601, 135.1173
M200
a
375.2517
373.2379, 223.1698, 149.0607
Mean that the compounds were identified with reference standards. 6G, 6S, 6D, 10G and ZO meant [6]-gingerol, [6]-shogaol, [6]-dehydrogingerdione, [10]-
gingerol and ginger, respectively. P, U, B and F represented rat plasma, urine, bile and fecal samples, respectively. GluA, Sul, Cys, Gly, NAC and GSH meant glucuronic acid, sulfation, cysteine, glycine, N-acetylcysteine and glutathione.
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Page 36 of 45
FIGURE CAPTIONS Figure 1. UPLC-PDA and BPI chromatograms of the Zingiber officinale (ZO) extract. (A) UV chromatogram of ZO; (B) (+) ESI-MS chromatogram of ZO; (C) (-) ESI-MS chromatogram of ZO chromatogram; (D) EICs of reference standards. 6G, 6S, 6D and 10G represented [6]gingerol, [6]-shogaol, [6]-dehydrogingerdione and [10]-gingerol, respectively. Figure 2. The proposed metabolic pathways of [6]-gingerol (A), [6]-shogaol (B), [6]-dehydrodingerdione (C) and [10]-gingerol (D) in rats. GluA, Cys, Gly, and NAC meant glucuronic acid, cysteine, glycine, and N-acetylcysteine. Figure 3. Extracted ion chromatograms (EICs) of Zingiber officinale related xenobiotics in rats. P, U, B and F represented rat plasma, urine, bile and fecal samples. Pos and Neg meant positive and negative ion mode. Figure 4. Metabolic soft spots of the pungent phytochemicals in Zingiber officinale.
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Figure 1. 37
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Figure 2A 38
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Journal of Agricultural and Food Chemistry
Figure 2B 39
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Figure 2C
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Journal of Agricultural and Food Chemistry
Figure 2D. Figure 2.
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Figure 3-1.
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Journal of Agricultural and Food Chemistry
Figure 3-2. Figure 3.
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Figure 4.
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Journal of Agricultural and Food Chemistry
TOC Graphic:
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