Metabolic profiles of ginger, a functional food, and its representative

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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

ACS Paragon Plus Environment


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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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INTRODUCTION

25

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.

71

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

94

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

97

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).

101

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

109

and 0 - 2 h for each pure compound group.

110

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

117

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

122

200 μL methanol. An aliquot of 4 μL was injected into the UPLC-Q/TOF-MS. The plasma samples

123

from the pure pungent compound groups and blank group were processed in the same way.

124

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

126

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.

132

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



139

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injection volume was 4 μL, and the detection wavelength was 280 nm.

140

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

145

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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208

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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231

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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254

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



Page 14 of 45

277

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


Page 15 of 45


300

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



Page 16 of 45

323

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


Page 17 of 45


346

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



Page 18 of 45

369

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


Page 19 of 45


392

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



Page 20 of 45

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


Page 21 of 45


438

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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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

547

and expressed UDP-glucuronosyltransferase enzymes: reaction kinetics and activity correlation

548

analyses for UGT1A9 and UGT2B7. J. Pharm. Pharmacol. 2015, 67, 583-596.

549 550 551 552

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

554

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,

557

M., Pharmacokinetics of 6-shogaol, a pungent ingredient of Zingiber officinale Roscoe (Part I).

558

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.

561

37. Ji, Y.; Yu, Y., In Vitro-In Silico Determination of the Inhibition of 6-Shogaol Towards Phase II

562

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.

567

Transcription factor Nrf2 mediates an adaptive response to sulforaphane that protects fibroblasts

568

in vitro against the cytotoxic effects of electrophiles, peroxides and redox-cycling agents. Toxicol.

569

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.

572

41. MacLeod, A. K.; McMahon, M.; Plummer, S. M.; Higgins, L. G.; Penning, T. M.; Igarashi, K.;

573

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


Page 27 of 45

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Carcinogenesis 2009, 30, 1571-1580.

577

42. Egwurugwu, J. N.; Ufearo, C. S.; Abanobi, O. C.; Nwokocha, C. R.; Duruibe, J. O.; Adeleye, G.

578

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.

580 581

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.;

583

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



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


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


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


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


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


B

UF

6S+4H+OH+SCH3

162.0242

PBU

F

F

PU

U

B

B

Page 31 of 45


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


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


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


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


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


6G-H2O-CH2+2H or Iso-6G-H2O-CH2+2H

U

PU F

UF


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

34


P

10G+CH2

UF

10G--CH2-H2O+2H

F

F

Page 35 of 45


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


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

+


347.2230

149.0602

[10]- Shogaol

F PUF PBF


U

F

10G-H2O+2H

UF

F


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


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


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


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


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


PF


F


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.

35



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.

36


Page 37 of 45


Figure 1. 37



Figure 2A 38


Page 38 of 45

Page 39 of 45


Figure 2B 39



Figure 2C

40


Page 40 of 45

Page 41 of 45


Figure 2D. Figure 2.

41



Figure 3-1.

42


Page 42 of 45

Page 43 of 45


Figure 3-2. Figure 3.

43



Figure 4.

44


Page 44 of 45

Page 45 of 45


TOC Graphic:

45


Metabolic profiles of ginger, a functional food, and its representative

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