Simultaneous Determination of Six Coumarins in Rat Plasma and

Subscriber access provided by UNIV OF DURHAM

Bioactive Constituents, Metabolites, and Functions

Simultaneous Determination of Six Coumarins in Rat Plasma and Metabolites Identification of Bergapten in Vitro and in Vivo Man Liao, Gengshen Song, Xiaoye Cheng, Xinpeng Diao, Yupeng Sun, and Lan-tong Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05637 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 17, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43

Journal of Agricultural and Food Chemistry

Simultaneous Determination of Six Coumarins in Rat Plasma and Metabolites Identification of Bergapten in Vitro and in Vivo Man Liao a#, Gengshen Song b#, Xiaoye Cheng a, Xinpeng Diao a, Yupeng Sun a, Lantong Zhang a* a

Department of Pharmaceutical Analysis, School of Pharmacy, Hebei

Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei 050017, P. R. China. b

*

Beijing Youcare Kechuang Pharmaceutical Technology Co., Ltd. Corresponding author:

Tel: +86-311-86266419. Fax: +86-311-86266419. E-mail address: [email protected] (L.T. Zhang). #

The authors contributed equally to this work.

1

ACS Paragon Plus Environment


ABSTRACT: Coumarins are abundant in Umbelliferae and Rutaceae plants possessing varied pharmacological activities. The objectives of this study are to develop and validate method for determination of six coumarins in rat plasma by liquid chromatography coupled with tandem mass spectrometry (LC-MS) and identify the metabolites of bergapten by ultra high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS), respectively. Data-dependent acquisition mode (DDA) was applied to trigger enhanced product ion (EPI) scans by analyzing multiple reaction monitoring (MRM) signals. An efficient data processing method ‘key product ions (KPIs)’ were used for rapid detection and identification of metabolites as an assistant tool. The time to reach the maximum plasma concentration (Tmax) for the six compounds ranged from 1 h to 6 h. A total of 24 metabolites of bergapten were detected in vitro and in vivo. The results could provide a basis for absorption and metabolism of coumarins.

KEY WORDS: Coumarins, bergapten, pharmacokinetic, UHPLC-Q-TOF-MS, metabolites

2


Page 2 of 43

Page 3 of 43


1

INTRODUCTION

2

Coumarins, which have the basic structure of benzo-[alpha]-pyrone, possess

3

excellent activities, including anti-inflammatory,1 anti-cancer,2 anticoagulant3

4

properties and effects on cardiovascular disease.4 They are abundant in Umbelliferae

5

and Rutaceae plants, such as Cnidium monnieri (L.) Cuss,5 Peucedanum ostruthium

6

(L.) Koch,6 Citrus paradise Macf.7 and Citrus aurantium L.8

7

Six coumarins, xanthotoxin, isopimpinelline, bergapten, imperatorin, osthole and

8

isoimperatorin are abundant in Cnidii Fructus (the dried ripe fruits of Cnidium

9

monnieri (L.) Cuss9). Plenty of investigations on their pharmacological effects have

10

proved that they possess anti-mutagenic,10 antitumor,11 antiarrhythmic12 and anti-

11

inflammatory13 activities. Moreover, they have an inhibitory effect on the central

12

nervous system14 and regulatory effects on the endocrine system.15 Coumarins

13

extracted from Cnidii Fructus have been granted more attention for their application

14

for insect control on crops in recent years.16-20 Therefore, it is essential to investigate

15

on the dynamic process in vivo in order to understand their pharmacological

16

mechanisms and for their safety. Bergapten, as the representative furocoumarin, has

17

been found in high concentration in most Umbelliferae plants and its ability to induce

18

apoptosis has also been shown. Panno ML et al., through flow cytometric analysis,

19

proved that bergapten was part of membrane signalling pathways which were able to

20

address apoptotic responses in both breast cancer cells with exposure to UV.21 Lin BH

21

et al. demonstrated the inhibitory effect of bergapten on nasopharyngeal carcinoma

22

cells CNE-2 and HONE-1 by CCK-8 in vitro,22 and Lee YM et al. have investigated

23

on the regulatory effect of bergapten on the proliferation and apoptosis of in HCC (J5)

24

cells by morphological analysis, cell viability assay, cell-cycle analysis and DNA

3



25

analysis in vitro.23 Hence, new insights are needed on the metabolism of bergapten for

26

increasing its clinical value.

27

In previous studies, Li YB et al. only developed a method for pharmacokinetic

28

study of osthole based on HPLC-UV,24 and Li J et al. developed a simultaneous

29

quantitation of osthole, bergapten and isopimpinellin in rat plasma and tissues by LC–

30

MS/MS after administration of Cnidii Fructus extract.25 The determination for

31

pharmacokinetic parameters of coumarins in Cnidii Fructus can reflect the absorption

32

and metabolism characteristics of them. To our knowledge, there is no report yet

33

about metabolism of bergapten in rats. Only Marumoto S et al. found that

34

biotransformation of bergapten incubated by glomerella cingulata was hydrolysis of

35

ester bond.26 Prototype compounds undergo rapid metabolism after administration,

36

and metabolites might exert higher biological activity or reduce the toxicity compared

37

with the prototype compounds.27

38

In the present study, we developed and validated an accurate, specific and sensitive

39

ultra high performance liquid chromatography coupled with electrospray ionisation

40

tandem mass spectrometry (UPLC-ESI-MS) method to determine xanthotoxin,

41

isopimpinelline, bergapten, imperatorin, osthole and isoimperatorin in rat plasma for

42

the pharmacokinetic profile. This was the first investigation on pharmacokinetic study

43

of six bioactive coumarins in Cnidii Fructus. UHPLC-Q-TOF-MS with high

44

resolution and wide mass range was applied to metabolite identification of bergapten.

45

The efficient data processing method ‘KPIs’ was used for rapid screening of

46

metabolites from biological samples preliminarily. It could avoid filtering metabolites

47

by molecular formula one by one. Based on chromatographic behaviors,

48

fragmentation patterns, accurate mass measurement and relevant biotransformation

49

pathways, a total of 24 metabolites (15 phase I and 9 phase II metabolites) were 4


Page 4 of 43

Page 5 of 43


50

investigated in rat liver microsomes (RLMs), rat plasma, urine, bile and feces. The

51

results would help to explain the process of major coumarins in Cnidii Fructus in vivo

52

and provide technical support for the pharmacological mechanism investigation of

53

new plant medicine.

54

MATERIALS AND METHODS

55

Chemicals and Reagents. Xanthotoxin (14012444) was purchased from Shanghai

56

Tauto Biotech Co., Ltd. Isopimpinelline (121023), bergapten (120816), imperatorin

57

(110826-201214), osthole (110822-200407), isoimperatorin (110827-200407) and

58

sulfamethoxazole (100025-200904) were obtained from National Institutes for Food

59

and Drug Control. The purities of them were greater than 98% by HPLC analysis.

60

RLMs were laboratory-made,28 and the protenin concentration was determined by the

61

Lowry method.29 RLMs were stored at -80 °C until used. Phosphate buffer saline

62

(PBS), β-Nicotin-amide adenine dinucleotide phosphate (NADPH), UDP-D-

63

glucuronide trisodium salt (UDPGA) and alamethicin were purchased from Solarbio

64

Science Co., Ltd. (Beijing, China). Sodium carboxymethyl cellulose (CMC-Na),

65

magnesium chloride hexa-hydrate and other chemicals were of analytical grade

66

(Tianjin Chemical Corporation, China). Methanol and acetonitrile purchased from

67

Merck Company (Germany) were of HPLC grade. And HPLC grade of formic acid

68

and acetic acid were obtained from Diamond Technology Incorporation. Purified

69

water was purchased from Wahaha (Hangzhou Wahaha Group Co., Ltd.). Cnidii

70

Fructus was collected from Hebei province and further confirmed by Professor Jiping

71

Duan (Hebei Institute for Food and Drug Control, Shijiazhuang, China). The voucher

72

specimens were preserved in the Laboratory of Pharmaceutical Analysis, School of

73

Pharmacy, Hebei Medical University (voucher No.CF-20160911-001).

5



74

UPLC-ESI-MS condition. Chromatographic separation of analytes was performed

75

on a Waters I-Class UPLC system (USA) with a Waters ACQUITY BEH C18 column

76

(50 mm×2.1 mm, 1.7 µm). The column temperature was maintained at 40 °C. The

77

mobile phase was composed of acetonitrile (A) and 0.1% acetic acid aqueous solution

78

(B) basing on a gradient elution of 30-50% A at 0-1.3 min, 50-80% A at 1.3-2.2 min,

79

80% A at 2.2-3.5 min, 80-30% A at 3.5-4.0 min, 30% A at 4.0-5.0 min, and the re-

80

equilibration time was 1 min. The flow rate was 0.3 mL/min. The sample injection

81

volume was 2 µL.

82

Determination was achieved by a Waters Xevo TQ-S system which possessed a

83

hybrid triple quadrupole linear ion trap mass spectrometer equipped with StepWaveTM

84

sources. The capillary voltage and source offset were set at 2.0 kV and 50 V on

85

positive electrospray mode (sFigure 1), respectively. The temperatures of source and

86

desolvation were 150 °C and 500 °C, and the flow rates of desolvation and cone gas

87

were 800 L/Hr and 150 L/Hr, respectively. Multi-reactions monitoring (MRM)

88

technology of triple-quadrupole tandem mass spectrometer was applied for

89

quantitative analysis.

90

UHPLC-Q-TOF-MS condition. The condition was as described in Sun YP et.al

91

(2018)30 with the modification: column temperature was set at 40 °C for greater

92

chromatographic separation. It was proved that the peak responses and shapes of

93

analytes were optimized with the mobile phase using a mixture 0.1% formic acid-

94

water (A) and acetonitrile (B). The optimized gradient elution for bergapten was as

95

follows: 0-20 min, 10-50% B; 20-21 min, 50-95% B; 21-23 min, 95% B. The program

96

maintained for 3 min at the initial gradient concentration for column balance before

97

next injection. The flow rate was set at 0.30 mL/min, and the injection volume was 5

98

µL. 6


Page 6 of 43

Page 7 of 43


99

The parameters of the MS/MS detector were as follows in positive ESI mode: ion

100

spray voltage (IS): 5500 V, turbo spray temperature: 550 °C, nebulizer gas (Gas1): 55

101

psi, heater gas (Gas2): 55 psi, curtain gas: 35 psi, declustering potential (DP): 60 V.

102

The collision energy spread (CES) was set to 35±15 eV which has been optimized for

103

observing better MS/MS spectra. For the DDA criteria, the parameter was set over 50

104

cps counts to perform a full scan, and the ten most intensitive fragment ions of each

105

compound were acquired. Moreover, multiple mass defect filter (MMDF) and

106

dynamic background subtraction (DBS) which are the superior techniques supplied by

107

AB Sciex software can screen the mass spectra entirely and conduct the product ion

108

scan to avoid omission of minor compounds. Therefore, they were applied to trigger

109

the DDA function in the study.30 MMDF window was set to ±40 mDa, and the mass

110

range was set to ±50 Da around each filter template. Simultaneously, calibration

111

delivery system was acquired for calibrating mass numbers online. Analyst® TF 1.7

112

software (AB Sciex) was applied for data acquisition.

113

Preparation of Cnidii Fructus extract. The preparation of Cnidii Fructus extract

114

was as described in Zhang ZY et.al (2014)31 with some changes. The percentage of

115

solvent was 75%. The proportions of three times extraction were 1:20, 1:20 and 1:10

116

(w/v). The final concentration of Cnidii Fructus extract was 1.0 g/mL of the raw

117

Cnidii Fructus material equivalently. The contents of xanthotoxin, isopimpinelline,

118

bergapten, imperatorin, osthole and isoimperatorin in the extract were 840.4±3.7,

119

1889.0±11.9, 958.7±4.5, 4401.3±28.5, 8800.7±51.4, 37.7±0.6 µg/mL determined by

120

the external standard method, respectively.

121

Preparation of standard solution and quality control (QC) Samples. The stock

122

solutions of xanthotoxin, isopimpinelline, bergapten, imperatorin, osthole and

123

isoimperatorin were prepared at 0.2 mg/mL in methanol and stored at –80 °C. The IS 7



124

solution (500.0 ng/mL) was prepared with sulfamethoxazole in methanol. A series of

125

working standard solutions of six compounds were conducted by dilution of stock

126

solutions with methanol to obtain the desired concentrations and stored at 4 °C before

127

injection.

128

For the calibration standards, the proper amounts of working solutions were

129

separately added to 0.5-mL microcentrifuge tubes and dried under nitrogen at 40 °C.

130

An aliquot (100 µL) of drug-free rat plasma was added to each microcentrifuge tube

131

and vortex-mixed to produce the following concentrations at 0.50, 1.00, 2.50, 12.50,

132

25.0 and 250.0 ng/mL in plasma. The QC samples were independently prepared in the

133

same method as calibration standards to produce three concentrations: 1.0 ng/mL (low

134

QC), 12.5 ng/mL (medium QC) and 250.0 ng/mL (high QC). The calibration

135

standards and QC samples were prepared freshly before analysis.

136

Preparation of plasma samples. Plasma samples (100 µL) were spiked with 400

137

µL methanol containing 100.0 ng/mL sulfamethoxazole (IS). The mixture was vortex-

138

mixed and centrifuged at 13,697g for 10 min. Clear supernatant (50 µL) was

139

transferred into vials, and 2 µL supernatant was injected into the UPLC-ESI-MS

140

system for analysis. All prepared samples were kept in an auto-sampler at 4 °C until

141

injection.

142 143

Method validation. In accordance with the regulatory guidelines,32 a full validation was performed for this assay in rat plasma.

144

Assay specificity.31 By comparing peak responses of blank plasma, plasma spiked

145

with analytes and IS and plasma samples after administration of Cnidii Fructus extract

146

from six different rats, potential interferences were investigated from endogenous

147

compounds.

8


Page 8 of 43

Page 9 of 43


148

Linearity, LLOQ and LOD.31 The plasma standard curves were determined by six

149

concentration levels, and each concentration was determined in duplicate on 3

150

separate days. The standard curves which were exhibited in the form of y = ax+b

151

(1/x2 weighted) were protracted by the plot of peak-area ratios of six compounds

152

versus IS against concentrations of the calibration standards.

153

LLOQ (S/N=10) and LOD (S/N=3) were investigated on dilution of standard

154

solutions to achieve the minimum concentration for reliably quantification and

155

detection of analytes, respectively. The acceptable precision should be lower than

156

20%, and the accuracy should be within ±20%.

157

Carryover effect. Carryover was evaluated by injecting a blank plasma sample

158

immediately after double injections of the highest calibration concentration (ULOQ).

159

Blank plasma sample measurements were required to be below 20% of the LLOQ and

160

5% of the IS.

161

Accuracy and precision. The intra-day precision, inter-day precision and accuracy

162

were evaluated by the analysis of six repeated samples at three different QC samples

163

(low, medium and high concentration levels) on the same day and continuous analysis

164

of duplicate samples on six days, respectively. The precision was showed as the

165

relative standard deviation (RSD, %), and the accuracy was calculated as the relative

166

error (RE, %) between calculated and actual concentrations. The acceptance criteria

167

were ≤15% for precision and ±15% for the accuracy. The reinjection reproducibility

168

was measured by the reinjection of all QC samples from an accepted precision-

169

accuracy batch during validation.

170

Extraction recovery and matrix effect. Six blank matrices from different donors

171

were collected to prepare different concentrations of QC samples. The extraction

172

recoveries were measured by comparing the peak areas detected from blank plasma 9



173

spiked with standard substances before extraction to those after extraction. The matrix

174

effects were evaluated by comparing the peak areas of the analytes and IS in pre-

175

treated blank plasma in which standard substances dissolved with equivalent amounts

176

of the analytes or IS.

177

Dilution reliability.33 Six blank plasma replicates of spiked plasma sample were

178

added moderate standard analytes to achieve 10-fold and 25-fold higher than high

179

concentration of QC. Then, the samples were diluted to high concentration of QC

180

with blank plasma and taken 100 µL for preparation samples to analyze with

181

undiluted calibration standards. The accuracy and precision were required to be within

182

the acceptable limits (±15%).

183

Stability.31 Six replicate QC samples were exposed at room temperature for 4 h and

184

24 h for determining post-preparation stability and short-term stability, respectively.

185

Long-term stability tests and freeze–thaw stability tests were evaluated by storing the

186

samples at -20 °C after 30 days and treating samples with three freeze (-20 °C)–thaw

187

(room temperature) cycles separately. Besides, extracted sample stability tests were

188

investigated by placing the extracted samples at room temperature for 24 h.

189

Pharmacokinetic study in rat plasma. Male Sprague-Dawley rats (Certificate No.

190

SCXK 2013-1-003, weighing from 240 g to 260 g) were provided by the

191

Experimental Animal Center of Hebei Medical University (Shijiazhuang, China). All

192

experiments on animals followed the both guidelines of the Committee on the Care

193

and Use of Laboratory Animals in our laboratory and experimental animal

194

management committee of Hebei Medical University in China. The rats were fed in

195

an environmentally managed room (temperature: 22±2 °C, humidity: 50±5%; 12-h

196

dark and light cycle) for 1 week before being used. The rats were fasted for 12 h but

197

with access to water before intragastric administered Cnidii Fructus extract. Six 10


Page 10 of 43

Page 11 of 43


198

healthy male Sprague-Dawley rats were intragastric administrated 6.0 mL/kg extracts

199

respectively. Blood samples were collected at 0.08, 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 6,

200

9, 12, 24 and 30 h from the fossa orbitalis vein after intragastric administration. The

201

blood samples were collected in heparinized tubes and centrifuged at 1522g for 10

202

min. Then the plasma layers were immediately moved to clean tubes and stored at -

203

20 °C before analysis.

204

The pharmacokinetic analysis of concentration-time data in non-compartmental

205

mode was conducted by Drug and Statistics (DAS) 2.0 software (Mathematical

206

Pharmacology Professional Committee of China, Shanghai, China).21 The time to

207

maximum concentration (Tmax) and maximum plasma concentration (Cmax) were

208

gotten intuitively from the observed data. The area under the plasma concentration–

209

time curve (AUC0–t) was observed by the trapezoidal rule. Based on AUC0–t, the

210

AUC0–∞ values were conducted by taking the value of Ct×k-1 into it. The elimination

211

half-life (T1/2) was calculated basing on the equation of T1/2=0.693/k. The elimination

212

rate constant (k) was evaluated by least-square regression of the logarithmic

213

transformation of the last four detected points of plasma standard curves.31

214

Microsomal incubation

215

Phase I metabolism. A typical incubation mixture34,35 was carried out in a 0.1

216

mol/L phosphate buffer (pH 7.4) containing 25.0 µmol/L bergapten, 0.4 mg rat liver

217

microsomal protein, 3.3 mmol/L MgCl2 and 2.0 mmol/L β-NADPH in the final

218

volume 200 µL. The percent of organic solvent must be limited in 1% (v/v) in the

219

final incubation. Then, the mixture was pre-warmed for 5 min at 37 °C and added

220

NADPH to initiate the system. The incubation time was 60 min under 37 °C. Ice-cold

221

ethyl acetate was used to end reaction and extract metabolites. After centrifugation at

222

21,380g for 10 min, the organic phase layer was retained and dried under nitrogen gas. 11



223

The residues were reconstituted in 200 µL 90% acetonitrile, and the suspension was

224

filtered with a 0.22 µm polytetrafluoroethylene (PTFE) membrane filter. Moreover,

225

blank samples were incubated without bergapten, and control samples were incubated

226

without NADPH generating system under the same condition.

227

Phase II metabolism. The incubation mixture contained 25.0 µmol/L bergapten,

228

0.4 mg rat liver microsomal protein, 3.3 mmol/L MgCl2, 2.0 mmol/L UDPGA and

229

25.0 µg/mL alamethicin in 50.0 mmol/L Tris-HCl buffer (pH 7.4) with a final volume

230

of 200 µL. The organic solvent did not exceed 1% (v/v) in the incubation. The mixture

231

was preincubated for 15 min at 37 °C and initiated by the addition of UDPGA.

232

Reaction termination was achieved after 60 min at 37 °C by adding 0.2 mL ice-cold

233

acetonitrile. The methods for centrifugation and filtration were the same as described

234

in phase I metabolism. Blank samples were incubated without bergapten, and control

235

samples were incubated without UDPGA under the same treatment as test sample.

236

Animals and drug administration. Eighteen male Sprague-Dawley rats were

237

randomly divided into six groups with three rats per group (Group 1, 2&3, blank

238

groups; Group 4, 5&6, experimental groups). The animals were the same batch as

239

these in the part of pharmacokinetic study and fed in the same environment as above.

240

All the animals were fasted for 12 h but free access to water before the experiment.

241

Bergapten powders was dissolved in 0.6% (10.0 mg/mL) CMC-Na solution, and a

242

dose of 50 mg/kg body weight was intragastric administrated to the experimental

243

groups (Group 4, 5&6). An equivalent CMC-Na solution without bergapten was

244

administration to the blank groups.

245 246

Bio-sample collection and pretreatment. Each group was housed individually in metabolic cage for sample collection.

12


Page 12 of 43

Page 13 of 43


247

Blood samples were collected from posterior orbital venous in heparinized tubes

248

before (0 h) and at 0.33, 0.5, 1, 1.5, 2, 3, 6, 9, 12 h after intragastric administration.

249

Then the samples were centrifuged at 1522g for 5 min to obtain the plasma. Each rat’s

250

samples from all time points were mixed together.

251

Three rats were immediately anesthetized with urethane (at the dose of 1.0 g/kg) by

252

intraperitoneal injection and fixed on a wooden plate after administered bergapten. An

253

abdominal incision was made, and the bile duct was fixed for collection. Bile samples

254

were collected for 24 h.

255

Urine and feces samples were collected for 48 h after administration and free

256

access to purified water. The blank group was administered equivalent water with the

257

same treatment as the test samples.

258

Three milli-litter (mL) of plasma was treated with 4-fold acetonitrile to precipitate

259

proteins and vortexed for 10 min. 3 mL of bile, 3 mL of urine and 0.5 g feces were

260

vortexed for 10 min with 3-fold ethyl acetate for three times and combined all

261

supernatant. Then the mixtures were centrifuged at 21,380g for 10 min, and the

262

organic phase was removed under nitrogen gas. The blank biological samples were

263

treated with the same method as the other samples. All samples were stored at -80 °C.

264

Before analysis, the residues were dissolved in 90% acetonitrile (600 µL) completely,

265

and the supernatant was filtered through a 0.22 µm PTFE membrane filter.

266

Analytical strategy. The analytic strategy was divided into five steps: First, on-line

267

data were acquired using full-scan, and an efficient MMDF and DBS-dependent data

268

acquisition method which held the ability of capturing the low-level metabolites

269

clearly were adopted to get MS and MS/MS data. Second, a special assistant tool

270

KPIs was used as markers for metabolites detection and identification. Fragment ion

271

m/z 91.0555 was selected as the diagnostic ion for observing relevant metabolites. 13



272

Third, a series of data mining tools included in MetabolitePilotTM software 2.0 (AB

273

Sciex) such as EIC and TIC were used for post-acquisition data processing. The

274

chemical libraries including possible metabolic pathways, chemical structures and

275

sample files of bergapten were set up for searching possible metabolites. Fourth,

276

unlikely metabolites were deducted from the blank sample. The structures of

277

remaining metabolites were identified by analysis of the chromatographic behaviors,

278

protonated ions and MS/MS fragment ions. The mass error should be limited in ±5

279

ppm. The last step was distinguishing the isomers by Clog P which was calculated by

280

ChemBioDraw Ultra 14.0 software. The values of Clog P varied inversely with the

281

retention time of metabolites.

282

RESULTS AND DISCUSSION

283

Results of pharmacokinetic study

284

Specificity. There were no significant interferences from endogenous components

285

detected in blank plasma. The retention time of xanthotoxin, isopimpinelline,

286

bergapten, imperatorin, osthole, isoimperatorin and IS were 1.49, 1.65, 1.74, 2.35,

287

2.50, 2.60 and 0.90 min, respectively. Total-ion MRM chromatograms and

288

representative extracted MRM chromatograms of xanthotoxin, isopimpinelline,

289

bergapten, imperatorin, osthole, isoimperatorin and IS of blank plasma, blank plasma

290

spiked with them and 3 h sample plasma after intragastric administration of Cnidii

291

Fructus extract were shown in Figure 1. The chemical structures and mass

292

spectrometry information were exhibited in sFigure 2.

293

Linearity, LLOQs and LODs. The calibration standard curves, LLOQs and LODs

294

of the analytes were showed in sTable 1. The correlation coefficient of standard

295

curves (r2) ranged from 0.9983 to 0.9998 with good linearity. The precision and

296

accuracy of LLOQ were lower than 15.0%. 14


Page 14 of 43

Page 15 of 43


297 298

Carryover effect. Carryover amount was shown to be negligible (≤0.45%) and conformed to the regulations.

299

Accuracy and precision. The results of intra-day precision, inter-day precision and

300

accuracy of six analytes at low, medium and high concentration levels were listed in

301

Table 1. The intra-day and inter-day precisions (RSD, %) were within 6.2% and 5.1%,

302

respectively. The intra-day and inter-day accuracies (RE, %) of these analytes were

303

ranged from −7.5% to 5.0% and −5.0% to 5.1%, respectively.

304

Extraction recovery and matrix effect. The mean extraction recoveries of six

305

analytes were ranged from 72.3% to 88.7% recorded in the Table 2. The results of

306

matrix effect implied that there were no obvious matrix effect for xanthotoxin,

307

isopimpinelline, bergapten, imperatorin, osthole, isoimperatorin and IS shown in

308

Table 2. The recovery rate and the matrix effect of IS was 85.7% and 99.8%,

309

respectively.

310

Dilution reliability. The accuracy (RE, %) and the precision (RSD, %) were all

311

within the range from −15% to 15%. These results fulfilled the acceptance criteria,

312

which indicated that plasma samples with concentrations exceeding the ULOQ could

313

be measured reliably after appropriate dilution. The data were shown in sTable 2.

314 315

Stability. The results of stability under different conditions were shown in sTable 3 and sTable 4. The analytes held good stability under the above storage conditions.

316

Pharmacokinetic study. The concentrations of the six coumarins in the plasma

317

were calculated by putting peak areas into the standard curve equations. The mean

318

plasma concentration–time profiles of the analytes were developed by LC–MS/MS

319

method and shown in Figure 2, and the pharmacokinetic parameters were listed in

320

Table 3.

15



Page 16 of 43

321

According to the pharmacokinetic test results, six analytes were rapidly absorbed

322

after intragastric administration of Cnidii Fructus extract in rat plasma. The highest

323

concentration and the lowest concentration among the six coumarins detected in

324

plasma were osthole and isoimperatorin, respectively. Compared with the content

325

detected in the extract, there might be a positive relationship between the

326

concentrations of analytes in the extract and that in the rat plasma. In general, the

327

elimination rates (Ke) of xanthotoxin, isopimpinelline, bergapten, imperatorin, osthole

328

and isoimperatorin were ranged from 0.1066 to 0.2502. The T1/2 values for them were

329

3.4, 5.7, 4.2, 3.9, 2.8 and 6.5 h, respectively. Osthole holds the shortest T1/2 and the

330

fastest elimination rate. Besides, there were significant differences in Tmax which

331

could be divided into two groups. Xanthotoxin, isopimpinelline, bergapten,

332

imperatorin and isoimperatorin were constituted to one group which achieved the

333

peak concentrations within 3 h. Osthole held the longest Tmax at 6 h specially which

334

indicated

335

pharmacokinetic parameters in vivo. All of the six analytes were absorbed rapidly and

336

had the similar elimination rate.

the

slowest

absorption.

The

six

analytes

possessed

distinctive

337

Compared with previous investigations on pharmacokinetics of Cnidii Fructus,24,25

338

the values of Cmax, Tmax, AUC0-t and AUC0-∞ presented some differences. However the

339

values of T1/2 and Ke were essentially in agreement. The major influence factors

340

contributed to the difference might be rat species, the content of coumarins and the

341

interaction between active conponents in the extract. Besides, different detecting

342

instruments and feeding environments were also the important causes.

343

Results of metabolites identification of bergapten

344

Mass spectral fragmentation of bergapten. To efficiently identify metabolites,

345

MS spectrum, MS/MS fragmentation pathways and retention time in the 16


Page 17 of 43


346

chromatogram were observed by bergapten standard. The chemical structure was

347

shown in Figure 3. Following the present chromatographic and mass conditions, the

348

reference bergapten was eluted at 14.33 min and yielded [M+H]+ ion at m/z 217.0507.

349

The major MS/MS product ions were m/z 217.0507, 202.0270, 174.0319, 146.0365,

350

118.0419 and 91.0555, which were yielded by the first loss of methyl and then

351

successive losses of CO.36 The cleavage pathway of bergapten was exhibited in

352

sFigure 3.

353

Characterization of metabolites by UHPLC-Q-TOF-MS. The extracted ion

354

chromatograms of KPI (KPI= m/z 91.0555) filtering to hunt metabolites of bergapten

355

in bio-samples were shown in sFigure 4. The potential metabolites were observed by

356

comparison of the extracted ion chromatograms (EICs) of normal samples with the

357

EICs of blank samples (as shown in Figure 4). The most possible molecular formulas

358

of metabolites were detected by different rules, including mass accuracy limited in ±5

359

ppm, the nitrogen rule, the isotopic pattern and the double-bond equivalents (DBE).

360

Then the tentative chemical structures of them were identified by MS/MS

361

fragmentation and common metabolic pathways (as shown in sTable 5). A total of 24

362

metabolites of bergapten were detected, including 15 phase I metabolites and 9 phase

363

II metabolites. The detailed information of them was listed in Table 4. Representative

364

MS/MS spectra of M12, M16, M19 and M23 were shown in Figure 5, and the

365

proposed metabolic pathways of bergapten in vitro and in vivo were summarized in

366

Figure 6. The structural inference process of representative metabolites which hold

367

higher amount or were conducted by characteristic metabolic reactions were

368

summarized as follows.

369

Phase I metabolites. M11 (tR=4.94 min) and M12 (tR=6.88 min) both showed

370

protonated ion at m/z 251.0556, implying that they were isomers of the molecular 17



371

formula C12H10O6. They were 34 Da (H2O2) higher than M0. The product ions at m/z

372

233, 205 and 175 were observed by losses of H2O, CO and CH2O consecutively.

373

Therefore, M11 and M12 were tentatively identified as the oxydrolysis metabolite of

374

M0.25 Oxydrolysis reaction refers to that the compound undergoes further hydrolysis

375

reaction at double bond of carbon based on mono-oxidation.37 The values of Clog P of

376

C-2’-C- 3’ oxydrolysis metabolite and C-3-C- 4 oxydrolysis metabolite were -0.5212

377

and 0.1216 calculated by ChemBioDraw Ultra 14.0, respectively. According to the

378

polarity rule, M11 was identified as C-2’-C- 3’ oxydrolysis metabolite, and M12 was

379

identified as C-3-C- 4 oxydrolysis metabolite.

380

M16 was eluted at 8.01 min and presented an accurate protonated ion [M+H]+ at

381

m/z 235.0603 (elemental composition of C12H10O5), which was 18 Da higher than that

382

of M0. The characteristic ion of M16 was m/z 164.0392, resulting from the hydrolysis

383

reaction located at C-1’ and C-2’ of m/z 146 from M0.37 The other main product ions

384

were m/z 220.0339 and m/z 164.0392.

385

The protonated ion at m/z 249.0393 (M17) with molecular formula speculated to

386

be C12H8O6 was eluted at 8.35 min, which was 32 Da (2O) higher than that of M0.

387

Compared with m/z 118.0419 from M0, the diagnostic ion m/z 150.0305 was 32 Da

388

higher than it. It indicated that the oxidation positions of M0 might be C-3’and C-8 or

389

C-4 and C-8.

390

M18 and M24 were the isomeric metabolites with the molecular formula

391

C13H10O5, which were eluted at 9.83 min and 14.49 min, respectively. They both

392

showed the protonated ion [M+H]+ at m/z 247, indicating addition of 30 Da (CH2O)

393

from M0. M24 was detected the fragment ions at m/z 162.0250 and m/z 134.0281,

394

which was 16 Da higher than m/z 146.0363 and m/z 118.0418 from M0, respectively.

395

From above-mentioned, the methoxylation was inferred to add at C-8. Compared with 18


Page 18 of 43

Page 19 of 43


396

the current reference, M24 was confirmed as isopimpinellin. Further major fragment

397

ion of M18 was m/z 232.0355, which was 30 Da higher than that of M0. In

398

consideration of steric hindrance, the methoxylation position might be C-2’or C-3’.

399

M19, M21 and M22 were the isomeric metabolites with the molecular formula

400

C12H8O5, suggesting that 16 Da (O) higher than that of M0. They were eluted at 10.00

401

min, 10.79 min and 12.69 min respectively and showed the protonated ion [M+H]+ at

402

m/z 233. Further major MS/MS framentation showed fragment ions at m/z 190, 162

403

and 134. The characteristic ion at m/z 134 indicated that mono-oxidation position

404

might be C-3’, C-4 or C-8 compared with the fragment ion m/z 118.0419 from M0.

405

The values of ClogP of C-3’, C-4 and C-8 mono-oxidation metabolites were

406

calculated to be 1.6813, 2.1618 and 2.4492. According to the normal rule ‘the greater

407

the value, the smaller the polarity, the shorter the retention time’, M19, M21 and M22

408

were investigated as C-3’, C-4 and C-8 mono-oxidation metabolites of M0,

409

respectively.

410

The protonated metabolite at m/z 219.0660 (M23) with molecular formula

411

C12H10O4 was eluted at 14.30 min. There were representative fragment ions at m/z

412

204. 0321, 176.0336 and 148.0428, which exhibited a neutral addition of 2 Da (2H)

413

compared with those of M0. It indicated that hydrogenation position of M0 might be

414

located at C-2’ and C-3’.37

415

Phase II metabolites. M1 and M2 were the isomeric metabolites with the

416

molecular formula C18H18O11, suggesting that 176 Da (O) higher than that of M16.

417

M1 and M2 was eluted at 3.18 min and 3.44 min and showed the protonated ions at

418

m/z 411.0920 and m/z 411.0918, respectively. The distinctive fragment ions at m/z

419

235.0503 and m/z 217.0491 were observed indicating the successive loss of C6H8O6

420

and H2O from M1. M2 held the same fragment pattern as M1. Therefore, we deduced 19



421

the M1 and M2 were the glucuronic acid conjugated products of M16. The glucuronic

422

acid conjugation position might be C-2’ hydroxyl or O-1’. Compared the values of

423

Clog P of them, M1 and M2 were identified as C-2’ hydroxyl glucuronic acid

424

conjugated metabolite and O-1’ glucuronic acid conjugated metabolite, respectively.

425

The summary of metabolites of bergapten. There were 13 metabolites detected in

426

vitro and 23 metabolites detected in vivo, including 13 metabolites (M3, M7, M11,

427

M12 and M16-M20) in RLMs, 13 metabolites (M4, M7, M10, M13-M15 and M18-

428

M24) in plasma, 16 metabolites (M1-M11, M13, M15, M18, M20 and M23) in bile,

429

18 metabolites (M1-M11, M13, M15, M18-M21 and M23) in urine and 17

430

metabolites (M1-M13, M15, M16, M20 and M24) in feces. According to the

431

biotransformation of bergapten in this study, we inferred that oxidation and

432

glucuronide conjugation might be the main metabolic pathways of furanocoumarins.

433

However, there were some differences between four metabolic pathways. By

434

comparison of metabolic profile, bergapten held more metabolically active in bile,

435

urine and feces samples than that in RLMs and blood sample. The main

436

biotransformation pathways were hydrolysis, hydrogenation and glucuronide

437

conjugation in vivo. Mono-oxidation, di-oxidation and oxydrolysis were the major

438

metabolic pathways in RLMs.

439

Mono-oxidation and ester hydrolysis metabolite (M7) and demethylation

440

metabolite (M20) were recognized as important metabolites which could be found in

441

all biological samples. The di-oxidized metabolite M17 was only detected in RLMs

442

samples which indicated that succession oxidation might relate to expose to for a

443

short period of time. The sulfate conjugation metabolites (M13 and M15) and

444

glucuronide conjugation metabolites (M1, M2, M4, M5, M6, M8 and M9) were only

20


Page 20 of 43

Page 21 of 43


445

observed in vivo. We inferred that the major reason was the absence of specific

446

enzymes for conjugation in microsomal incubation in RLMs.

447

In conclusion, the UPLC-ESI-MS/MS method was developed for simultaneous

448

determination of xanthotoxin, isopimpinelline, bergapten, imperatorin, osthole and

449

isoimperatorin in rat plasma in positive ionization mode with higher accuracy and

450

specificity after intragastric administration of Cnidii Fructus extract firstly. The

451

excellent precision, accuracy, extraction recovery, selectivity and sensitivity showed

452

the suitability of this method for pharmacokinetic study. A powerful UHPLC-Q-TOF-

453

MS method was conducted to detect and identify metabolites of bergapten, which

454

could qualitatively elucidate the clear mechanism of its transformation. Twenty-four

455

metabolites of bergapten including 13 in vitro and 23 in vivo were identified or

456

tentatively identified. In this work, pharmacokinetics study of six bioactive coumarins

457

would provide a basis for deeper investigation of the absorption mechanism of Cnidii

458

Fructus, and metabolic pathways of bergapten is meaning for metabolic profile of

459

furanocoumarins in vitro and in vivo.

460 461

ABBREVIATIONS USED

462

LC-MS, liquid chromatography coupled with tandem mass spectrometry; UHPLC-Q-

463

TOF-MS, ultra high performance liquid chromatography coupled with quadrupole

464

time of flight mass spectrometry; DDA, data-dependent acquisition mode; EPI,

465

enhanced product ion; MRM, multiple reaction monitoring; KPIs, key product ions;

466

UPLC-ESI-MS, ultra high performance liquid chromatography coupled with

467

electrospray ionisation tandem mass spectrometry; RLMs, rat liver microsomes; PBS,

468

Phosphate buffer saline; NADPH, β-Nicotin-amide adenine dinucleotide phosphate;

469

UDPGA, UDP-D-glucuronide trisodium salt; CMC-Na, sodium carboxymethyl 21



470

cellulose; MMDF, multiple mass defect filter; DBS, dynamic background subtraction;

471

QC, quality control; PTFE, polytetrafluoroethylene; DBE, double-bond equivalents.

472 473

ACKNOWLEDGMENTS

474

All of the authors are grateful for the support from Department of Pharmaceutical

475

Analysis of school of Pharmacy in Hebei Medical University.

476 477

FUNDING

478

The work received financial support from the National Natural Science Foundation of

479

China (No. 81473180).

480 481

CONFLICT OF INTEREST

482

The authors have declared no conflict of interest.

483 484 485 486

SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website.

487

The relevant results of method validation of pharmacokinetic study and the

488

extracted ion chromatograms of KPI (KPI= m/z 91.0555) filtering to hunt metabolites

489

of bergapten

490 491 492 493 494 22


Page 22 of 43

Page 23 of 43


495

REFERENCES

496

(1) Tuan Anh, H.L.; Kim, D.C.; Ko, W.; Ha, T.M.; Nhiem, N.X.; Yen, P.H.; Tai,

497

B.H.; Truong, L.H.; Long, V.N.; Gioi, T.; Hong Quang, T.; Minh, C.V.; Oh, H.; Kim,

498

Y.C.; Kiem, PV. Anti-inflammatory coumarins from Paramignya trimera. Pharm.

499

Biol. 2017. 55(1), 1195-1201.

500 501

(2) Jarząb, A.; Grabarska, A.; Skalicka-Woźniak, K.; Stepulak, A Pharmacological features of osthole. Postepy. Hig. Med. Dosw. 2017, 71(0), 411-421.

502

(3) Bouckaert, C.; Zhu, S.; Govers-Riemslag, J.W.P.; Depoorter, M.; Diamond,

503

S.L.; Pochet, L. Discovery and assessment of water soluble coumarins as inhibitors of

504

the coagulation contact pathway. Thromb. Res. 2017, 157, 126-133.

505

(4) Liu, J.C.; Wang, F.; Xie, M.L.; Cheng, Z.Q.; Qin, Q.; Chen, L.; Chen, R.

506

Osthole inhibits the expressions of collagen I and III through Smad signaling pathway

507

after treatment with TGF-β1 in mouse cardiac fibroblasts. Int. J. Cardiol. 2017, 228,

508

388-393.

509

(5) Chen, D.; Wang, J.; Jiang, Y.; Zhou, T.; Fan, G.; Wu, Y. Separation and

510

determination of coumarins in Fructus cnidii extracts by pressurized capillary

511

electrochromatography using a packed column with a monolithic outlet frit. J. Pharm.

512

Biomed. Anal. 2009, 50(5), 695-702.

513

(6) Vogl, S.; Zehl, M.; Picker, P.; Urban, E.; Wawrosch, C.; Reznicek, G.; Saukel,

514

J.; Kopp, B. Identification and Quantification of Coumarins in Peucedanum

515

ostruthium (L.) Koch by HPLC-DAD and HPLC-DAD-MS. J. Agric. Food Chem.

516

2011, 59 (9), 4371-4377. 23



517

(7) Melough, M.M.; Vance, T.M.; Lee, S.G.; Provatas, A.A.; Perkins, C.; Qureshi,

518

A.; Cho, E.; Chun, O.K. Furocoumarin Kinetics in Plasma and Urine of Healthy

519

Adults Following Consumption of Grapefruit (Citrus paradisi Macf.) and Grapefruit

520

Juice. J. Agric. Food Chem. 2017, 65 (14), 3006-3012.

521

(8) Siskos, E.P.; Mazomenos, B.E.; Konstantopoulou, M.A. Isolation and

522

Identification of Insecticidal Components from Citrus aurantium Fruit Peel Extract. J.

523

Agric. Food Chem. 2008, 56 (14), 5577-5581.

524 525 526 527

(9) Chinese Pharmacopoeia Commission. The Pharmacopoeia of the People's Republic of China, Part I. China Medical Science Press, Beijing, China, 2015. (10) Marumoto, S.; Oda, Y.; Miyazawa, M. Antigenotoxic activity of naturally occurring furanocoumarins. Environ. Mol. Mutagen. 2011, 52(8), 646-657.

528

(11) Hung, C.M.; Kuo, D.H.; Chou, C.H.; Su, Y.C.; Ho, C.T.; Way, T.Z. Osthole

529

Suppresses Hepatocyte Growth Factor (HGF)-Induced Epithelial-Mesenchymal

530

Transition via Repression of the c-Met/Akt/mTOR Pathway in Human Breast Cancer

531

Cells. J. Agric. Food Chem. 2011, 59 (17), 9683-9690.

532

(12) Zhang, S.Y.; Meng, L.; Gao, W.Y.; Song, N.N.; Jia, W.; Duan, H.Q. Advances

533

on biological activities of coumarins. Chin. J. Chin. Mater. Med. 2005, 30(6), 410-

534

413.

535

(13) Yang, Y.; Zheng, K.; Mei, W.; Wang, Y.; Yu, C.; Yu, B.; Deng, S.; Hu, J.

536

Anti-inflammatory and proresolution activities of bergapten isolated from the roots of

537

Ficus hirta in an in vivo zebrafish model. Biochem. Biophys. Res. Commun. 2018,

538

496(2), 763-769.

24


Page 24 of 43

Page 25 of 43


539 540

(14) He, J.; Feng, M.L.; Liu, X.; He, L.L; Wu, Y.P.; Niu, Y.Y. The Sedative and Hypnotic Action of Fructus Cnidii Extracts. Shanxi. J. TCM. 2007, 23(5), 61-62.

541

(15) Wang, W.L.; Yu, Z.M.; Ju, D.H.; Zang, L.S.; Wang, Z.; Yang, L.X.; Zhao,

542

H.X. Advances in chemical and pharmacological studies of cnidium fruit. Chin. J.

543

Basic Med. Tradit. Chin. Med. 2001, 17(6), 704-706.

544

(16) Song, P.P.; Zhao, J.; Liu, Z.L.; Duan, Y.B.; Hou, Y.P.; Zhao, C.Q.; Wu, M.;

545

Wei, M.; Wang, N.H.; Lv, Y.; Han, Z.J. Evaluation of antifungal activities and

546

structure-activity relationships of coumarin derivatives. Pest. Manag. Sci. 2017, 73(1),

547

94-101.

548

(17) Caboni, P.; Saba, M.; Oplos, C.; Aissani, N.; Maxia, A.; Menkissoglu-Spiroudi,

549

U.; Casu, L.; Ntalli, N. Nematicidal activity of furanocoumarins from parsley against

550

Meloidogyne spp. Pest. Manag. Sci. 2015, 71(8), 1099-1105.

551

(18) Mira, A.; Alkhiary, W.; Shimizu, K. Antiplatelet and Anticoagulant Activities

552

of Angelica shikokiana Extract and Its Isolated Compounds. Clin. Appl. Thromb.

553

Hemost. 2017, 23(1), 91-99.

554

(19) Schulzová, V.; Babička, L.; Hajšlová, J. Furanocoumarins in celeriac from

555

different farming systems: a 3-year study. J. Sci. Food. Agric. 2012, 92(14), 2849-

556

2854.

557

(20) Huang, Y.Q.; Li, G.Z.; Chen, A.L.; Gao, J.M.; Feng, J.T.; Zhang, X.

558

Separation and identification of insecticidal active components in Ligusticum. J. N.W.

559

Sci-Tech Uni. Agri. Fore. 2004, 32(z1), 54-56.

560

(21) Panno, M.L.; Giordano, F.; Palma, M.G.; Bartella, V.; Rago, V.; Maggiolini,

561

M.; Sisci, D.; Lanzino, M.; De Amicis, F.; Andò, S. Evidence that bergapten,

562

independently of its photoactivation, enhances p53 gene expression and induces

25



563

apoptosis in human breast cancer cells. Curr. Cancer Drug Targets. 2009, 9(4), 469-

564

481.

565

(22) Lin, B.H.; Ma, X.J.; Wan, S.W.; Wei, S.S.; Zhang, X.; Zhou, K.Y.; Bergapten

566

Induces Apoptosis of Nasopharyngeal Carcinoma Cells. Cancer. Res. Prev. Treat.

567

2014, 41(11), 1163-1170.

568

(23) Lee, M.; Wu, T.H.; Chen, S.F.; Chung, J.G. Effect of 5-methoxypsoralen (5-

569

MOP) on cell apoptosis and cell cycle in human hepatocellular carcinoma cell line.

570

Toxicol. In Vitro. 2003, 17(3), 279-287.

571

(24) Li, Y.B.; Meng, F.H.; Xiong, Z.L.; Liu, H.; Li, F.M. HPLC Determination and

572

Pharmacokinetics of Osthole in Rat Plasma after Oral Administration of Fructus

573

Cnidii Extract. J. Chromatogr. Sci. 2005, 43(8), 426-429.

574

(25) Li, J.; Ma, B.; Zhang, Q.; Yang, X.J.; Sun, J.J.; Tang, B.W.; Cui, G.B.; Yao,

575

D.; Liu, L.; Gu, G.Y.; Zhu, J.W.; Wei, P.; Ouyang, P.K. Simultaneous determination

576

of osthole, bergapten and isopimpinellin in rat plasma and tissues by liquid

577

chromatography–tandem mass spectrometry. J. Chromatogr. B. 2014, 970, 77-85.

578

(26) Marumoto, S.; Miyazawa, M. Biotransformation of Bergapten and

579

Xanthotoxin by Glomerella cingulata. J. Agric. Food Chem. 2010, 58 (13), 7777-7781.

580

(27) Zhang, X.; Yao, Y.; Lou, Y.; Jiang, H.D.; Wang, X.W.; Chai, X.J.; Zeng, S.

581

Metabolism of ebracteolata compound B studied in vitro with human liver

582

microsomes HepG2 cells and recombinant human enzymes. Drug. Metab. Dispos.

583

2010, 38(12), 2157-2165.

584

(28) Liang, Y.T.; Huang, H.B.; Liu, T. Establish A Method to Evaluate Activity of

585

Rat Liver Microsomal CYP3A1 that Inhibited in vitro. Pharm. Today. 2011, 21(1),

586

13-16.

26


Page 26 of 43

Page 27 of 43


587 588

(29) Hartree, E.F. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 1972, 48(2), 422-427.

589

(30) Sun, Y.P.; Li, L.; Liao, M.; Su, M.; Wan, C.C.; Zhang, L.T.; Zhang, H.L. A

590

systematic data acquisition and mining strategy for chemical profiling of Aster

591

tataricus rhizoma (Ziwan) by UHPLC-Q-TOF-MS and the corresponding anti-

592

depressive activity screening. J. Pharm. Biomed. Anal. 2018, 154, 216-226.

593

(31) Zhang, Z.Y.; Jia, P.P.; Zhang, X.X.; Zhang, Q.Y.; Yang, H.T.; Shi, H.; Zhang,

594

L.T. LC-MS/MS determination and pharmacokinetic study of seven flavonoids in rat

595

plasma

596

Ethnopharmacol. 2014, 158, 66-75.

597 598

after

oral administration

of

Cirsium

japonicum DC.

extract.

J.

(32) US Department of Health and Human Services. Food and Drug Administration. Guidance for industry: Bioanalytical method validation, 2013.

599

(33) Wang, X.Y.; Li, W.; Ma, X.H.; Yan, K.J.; Chu, Y.; Han, M.; Li, S.M.; Zhang,

600

H.C.; Zhou, S.P.; Zhu, Y.H.; Sun, H.; Liu. C.X.; Identification of a major metabolite

601

of danshensu in rat urine and simultaneous determination of danshensu and its

602

metabolite in plasma: application to a pharmacokinetic study in rats. Drug Test. Anal.

603

2015, 7(8), 727-736.

604

(34) Yuan, L.; Jia, P.P.; Sun, Y.P.; Zhao, C.C.; Zhi, X.R.; Sheng, N.; Zhang, L.T.

605

Study of in vitro metabolism of m-nisoldipine in human liver microsomes and

606

recombinant cytochrome P450 enzymes by liquid chromatography-mass spectrometry.

607

J. Pharmaceut. Biomed. Anal. 2014, 97, 65-71.

608

(35) Yoon, Y.J.; Kim, K.B.; Kim, H.; Characterization of benidipine and its

609

enantiomers’ metabolism by human liver cytochrome P450 enzymes. Drug. Metab.

610

Dispos. 2007, 35(9), 1518-1524.

27



Page 28 of 43

611

(36) Wang, X.G.; Yang, H.J.; Wang, S.S.; Ma, Y.; Li, D.F.; Zhang, Y.; Liu, Y.P.;

612

Xu. H.Y. Fragmentation pathways of five furocoumarine using line ion trap with

613

orbitrapmass spectrometry. Chin. J. Chin. Mater. Med. 2015, 40(7), 1334-1341. 

614

(37) Song, G.S.; Jin, M.M.; Du, Y.F.; Cao, L.; Xu. H.J.; UPLC-QTOF-MS/MS

615

based screening and identification of the metabolites in rat bile after oral

616

administration

of

imperatorin.

J.

Chromatogr.

28


B.

2016,

1022,

21-29.

Page 29 of 43


FIGURE CAPTIONS Figure 1. Representative exact-ion MRM chromatograms of xanthotoxin (l), isopimpinelline (2), bergapten (3), imperatorin (4), osthole (5), isoimperatorin (6) and sulfamethoxazole (IS) .A) blank plasma, (B) blank plasma spiked with the six analytes and IS, and (C) 3 h sample plasma after intragastric administration of Cnidii Fructus extract. Figure

2.

Mean plasma concentration–time profiles of xanthotoxin (A),

isopimpinelline (B), bergapten (C), imperatorin (D), osthole (E) and isoimperatorin (F) in six rats after intragastric administration of Cnidii Fructus extract. (The error of each data point was within ± 5.0%.) Figure 3. Chemical structure of bergapten. Figure 4. Extacted ion chromatograms of the 24 metabolites of bergapten. Figure 5. MS/MS spectra and major proposed fragmentation patterns of M12 (A), M16 (B), M19 (C), M23 (D). Figure 6. Proposed major metabolic pathways of bergapten (1.Oxidation, 2.Hydrogenation, 3.Hydrolysis, 4.Methylation, 5.Demethylation, 6.Oxydrolysis, 7.Loss of –OCH3, 8.Glucuronide Conjugation, 9.Sulfate Conjugation, ‘*’ means that the probable metabolic sites of bergapten metabolites).

29



Page 30 of 43

Table 1. The Intra-and Inter-day Accuracy and Precision of Xanthotoxin, Isopimpinelline, Bergapten, Imperatorin, Osthole and Isoimperatorin in Rat Plasma at Low, Medium, High Concentration Levels (n=6)

Compounds

Intra-day(n=6)

Spiked Measured concentration concentrationa (ng/mL) (ng/mL) Xanthotoxin 1.0 1.0±0.0 12.5 12.5±0.5 250.4 251.3±5.1 Isopimpinelline 1.0 1.1±0.1 12.5 12.5±0.8 240.3 250.6±5.4 Bergapten 1.0 0.9±0.1 12.5 12.4±0.4 250.6 251.4±5.3 Imperatorin 1.0 1.0±0.0 12.3 12.4±0.4 245.1 250.0±4.7 Osthole 1.0 1.0±0.0 12.4 12.5±0.3 248.8 248.3±5.8 Isoimperatorin 1.0 1.1±0.1 12.5 12.3±0.4 250.3 248.0±4.2 a Mean ± standard deviation.

Inter-day(n=6)

Accuracy (RE, %)

Precision (RSD, %)

Measured concentrationa (ng/mL)

Accuracy (RE, %)

Precision (RSD, %)

-5.0 -0.6 0.3

2.7 3.9 2.1

1.0±0.0 12.4±0.4 249.6±5.4

-2.5 -1.1 -0.3

2.6 2.9 2.2

5.0 0.2 0.6

4.8 6.2 2.2

1.0±0.1 12.8±0.4 245.5±5.0

-2.5 2.6 -1.4

5.1 3.3 2.1

-7.5 -1.2 0.3

5.4 3.5 2.1

1.1±0.0 12.8±0.3 247.5±4.2

5.0 1.7 -1.2

2.3 2.1 1.7

2.6 1.4 2.0

2.5 3.1 1.9

1.0±0.1 12.4±0.3 242.6±5.15

5.1 1.0 -1.0

4.9 2.5 2.1

-2.5 0.6 -0.2

2.6 2.2 2.4

1.1±0.0 12.6±0.4 245.0±4.2

5.0 1.2 -1.5

2.4 3.0 1.7

5.0 -2.0 -0.9

4.8 3.1 1.7

1.0±0.0 12.9±0.5 251.8±5.8

-5.0 3.2 0.6

4.8 3.1 2.3

30


Page 31 of 43


Table 2. The Mean Extraction Recoveries and Matrix Effect of Xanthotoxin, Isopimpinelline, Bergapten, Imperatorin, Osthole and Isoimperatorin in Rat Plasma (n = 6)

Mean extraction recoverya (%)

Matrix effecta (%)

Components Low

Medium

High

Low

Medium

High

Xanthotoxin

72.3±3.1

83.1± 3.0

78.9±2.1

82.3±3.5

85.1±5.0

99.6±2.3

Isopimpinelline

79.1±4.1

86.2±3.0

86.2±1.8

95.8±3.5

96.9±3.2

98.5±2.1

Bergapten

75.4±4.0

79.3±2.3

78.1±3.0

100.2±4.1

99.8±3.3

97.8±2.1

Imperatorin

73.1±4.1

86.2±3.0

83.9±2.5

100.1±3.3

97.6±2.8

94.7±3.2

Osthole

79.1±5.7

82.9±2.9

88.7±2.0

100.1±3.0

98.9±2.5

99.5±2.8

Isoimperatorin

78.4±3.9

82.3±3.1

88.7±3.0

99.3±2.8

98.9±2.7

97.8±3.7

a Mean ± standard deviation.

31



Page 32 of 43

Table 3. Pharmacokinetic Parameters of Xanthotoxin, Isopimpinelline, Bergapten, Imperatorin, Osthole and Isoimperatorin in Rat Plasma After Intragastric Administration of Cnidii Fructus Extract (n = 6)

Analytes

Cmaxa (ng/mL)

Tmax (h)

T1/2a (h)

Kea (1/h)

AUC0-ta (ng·h/mL)

AUC0-∞a (ng·h/mL)

Xanthotoxin

951.2±3.7

2.00

3.40±0.11

0.2038±0.0066

5466.2±120.2

5469.7±124.1

Isopimpinelline

716.9±4.0

3.00

5.69±0.18

0.1218±0.0039

4821.0±111.0

4866.1±113.3

Bergapten

490.8±2.0

3.00

4.21±0.12

0.1646±0.0047

3154.8±98.4

3163.3±99.9

Imperatorin

642.6±3.8

1.00

3.87±0.07

0.1791±0.0032

2555.7±60.9

2560.9±63..0

Osthole

5634.0±11.3

6.00

2.77±0.05

0.2502±0.0045

61323.3±690.2

61393.9±695.2

Isoimperatorin

75.5±2.9

1.50

6.50±0.07

0.1066±0.0011

356.4±13.7

364.6±13.0

a Mean ± standard deviation.

32


Page 33 of 43


Table 4. UHPLC-Q-TOF-MS Retention Times and Fragment Ions of Metabolites of Bergapten in Vitro and in Vivo Compound ID

Retention time (min)

Formula

Calculated m/z [M+H]

Experimental m/z [M+H]

Source

Error (ppm)

Fragment ions

Potential pathway -

M0

14.33

C12H8O4

217.0495

217.0500

2.30

202.0270,174.0319,146.0365, 118.0419,91.0555

M1

3.18

C18H18O11

411.0922

411.0920

-0.49

253.0503,217.0491,192.0412, 91.0553

M2

3.44

C18H18O11

411.0922

411.0918

-0.97

253.0505,217.0489,192.0413, 174.0888,91.0555

M3

3.74

C12H10O7

267.0499

267.0503

1.50

221.0448,178.0260,150.0310, 91.0548

M4

3.78

C18H20O12

429.1028

429.1024

-0.93

253.1045,217.0655,192.0407, 91.0553

M5

3.80

C18H18O12

427.0871

427.0865

-1.40

251.0550,233.0445,205.0494, 175.0386

M6

3.83

C18H20O11

413.1078

413.1075

-0.73

237.0756,219.0650,203.0336, 176.0456

M7

3.85

C12H12O6

253.0707

253.0698

-3.56

220.0396,203.0335,217.0668, 192.0411,91.0666

M8

4.12

C18H18O12

427.0871

427.0864

-1.64

251.0548,233.0441,205.0490, 175.0385,91.0650

M9

4.45

C17H14O10

379.0660

379.0652

-2.11

203.0335,147.0436,91.0662

33


Hydrolysis, Glucuronide Conjugation Hydrolysis, Glucuronide Conjugation Mono-oxidation, Oxydrolysis Mono-oxidation, Ester hydrolysis, Glucuronide Conjugation Glucuronide Conjugation, Oxydrolysis Hydrogenation, Hydrolysis, Glucuronide Conjugation Mono-oxidation, Ester hydrolysis Glucuronide Conjugation, Oxydrolysis Demethylation, Glucuronide Conjugation

RLMs

P B

U

F

+

+ +

+

+

-

- +

+

+

-

- +

+

+

+

- +

+

+

-

+ +

+

+

-

- +

+

+

-

- +

+

+

+

+ +

+

+

-

- +

+

+

-

- +

+

+


Page 34 of 43

Table 4. continued Compound ID

Retention time (min)

Formula

Calculated m/z [M+H]

Experimental m/z [M+H]

Error (ppm)

Fragment ions 219.0299,119.0103,91.0565

Potential pathway

Source RLMs

P B

Demethylation, Oxydrolysis

-

+ +

+

+

Oxydrolysis

+

- +

+

+

Oxydrolysis

+

- -

-

+

-

+ +

+

+

-

+ -

-

-

-

+ +

+

+

Hydrolysis

+

- -

-

+

Di-oxidation Mono-oxidation, Methylation

+

- -

-

-

+

+ +

+

-

Mono-oxidation

+

+ -

+

-

Demethylation

+

+ +

+

+

Mono-oxidation

+

+ -

+

-

Mono-oxidation

+

+ -

-

-

Hydrogenation Mono-oxidation, Methylation *: Identification by reference standards. RLMs: Rat Liver Microsomes; P: Plasma; B: Bile; U: Urine; F: Feces. +: detected; -: not detected.

+

+ +

+

-

+

+ -

-

+

M10

4.65

C11H8O6

237.0394

237.0385

-3.80

M11

4.94

C12H10O6

251.0550

251.0556

2.39

M12

6.88

C12H10O6

251.0550

251.0556

2.39

M13

7.17

C12H8O8S

313.0013

313.0015

0.64

233.0445,217.0118,175.0493, 91.0566

M14

7.27

C11H6O4

203.0339

203.0335

-1.97

175.0440,159.0443,147.0439, 119.0494

M15

7.28

C11H6O7S

282.9907

282.9909

0.71

203.0347,91.0543

M16

8.01

C12H10O5

235.0601

235.0603

0.85

M17

8.34

C12H8O6

249.0394

249.0394

0.00

M18

9.83

C13H10O5

247.0601

247.0602

0.40

M19

10.00

C12H8O5

233.0445

233.0435

-4.29

M20

10.25

C11H6O4

203.0339

203.0336

-1.48

M21

10.79

C12H8O5

233.0445

233.0450

2.15

M22

12.69

C12H8O5

233.0445

233.0444

-2.15

233.0448,205.0500,175.0395, 147.0436,119.0494,91.0555 233.0448,205.0500,175.0436, 149.0238,119.0494,91.0555

M23

14.30

C12H10O4

219.0652

219.0660

3.65

220.0364,202.0339,164.0392, 91.0556 150.0305,107.0496,91.0559 232.0355,217.0134,189.0179, 91.0554 190.0183,162.0238,134.0364, 91.0554 175.0447,147.0435,119.0495, 91.0563 190.0183,162.0238,134.0364, 91.0552 190.0259,162.0314,134.0354, 91.0550 204.0321,176.0366,148.0428

M24*

14.49

C13H10O5

247.0601

247.0595

-2.43

162.0250,134.0281,91.0558

34


Monooxidation,Sulfate Conjugation Mono-oxidation, Loss of –OCH3 Demethylation, Sulfate Conjugation

U

F

Page 35 of 43


Figure 1 35



Figure 2

36


Page 36 of 43

Page 37 of 43


Figure 3

37



Figure 4

38


Page 38 of 43

Page 39 of 43


Figure 4

39



Figure 5

40


Page 40 of 43

Page 41 of 43


Figure 5

41



Figure 6

42


Page 42 of 43

Page 43 of 43


Table of Contents (TOC) Graphic

43


Simultaneous Determination of Six Coumarins in Rat Plasma and

Recommend Documents