Human Gastrointestinal Metabolism of the Cistanches Herba Water

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Human gastrointestinal metabolism of Cistanches Herba water extract in vitro: elucidation of the metabolic profile based on comprehensive metabolites identification in gastric juice, intestinal juice, human intestinal bacteria and intestinal microsome Yang Li, Ying Peng, Mengyue Wang, Peng-Fei Tu, and Xiaobo Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02829 • Publication Date (Web): 03 Aug 2017 Downloaded from http://pubs.acs.org on August 6, 2017

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Journal of Agricultural and Food Chemistry

Human gastrointestinal metabolism of Cistanches Herba water extract in vitro: elucidation of the metabolic profile based on comprehensive metabolites identification in gastric juice, intestinal juice, human intestinal bacteria and intestinal microsome Yang Li,† Ying Peng,† Mengyue Wang,† Pengfei Tu,‡ and Xiaobo Li*,†

†

School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China

‡

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical

Sciences, Peking University, Beijing 100191, China

Corresponding Author *(X.L.)

Phone:

+86-21-3420-4806.

Fax:

+86-21-3420-4804.

[email protected].

1

ACS Paragon Plus Environment

E-mail:


1

ABSTRACT: Cistanches Herba, is taken orally as a health food supplement and

2

medicinal plant in Asian countries. It consists of the stems of Cistanche deserticola

3

(CD) and C. tubulosa (CT). The gastrointestinal metabolism of the multi-components

4

contained in Cistanches Herba is crucial for the discovery of bioactive constituents.

5

This study aims to elucidate the comprehensive metabolic profile of Cistanches Herba

6

water extract by simulating human gastrointestinal metabolism in vitro using four

7

models - gastric juice, intestinal juice, human intestinal bacteria, and human intestinal

8

microsomes, independently and sequentially. A total of 35 and 18 metabolites were

9

characterized from CD and CT water extract. These metabolites were formed through

10

reduction, methylation, dimethylation, deglycosylation, de-caffeoyl, de-rhamnose,

11

dehydrogenation, and glucuronidation. The difference in metabolites of Cistanches

12

Herba water extract and single compounds, and the difference in metabolites of CD

13

and CT water extracts were caused by the oligosaccharides and polysaccharides in

14

Cistanches Herba.

15 16 17

KEYWORDS: Cistanches Herba, UPLC-Q-TOF-MS, gastrointestinal metabolism,

18

digestive juice, human intestinal bacteria, human intestinal microsome

19

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INTRODUCTION

21

Herbal medicines are often decocted with water, and the extracts derived from these

22

plants may be added to foods as additives or consumed directly as functional foods.1

23

After oral administration, their components inevitably go through gastrointestinal

24

metabolism before being absorbed from the alimentary tract into the blood stream to

25

exert biological activities. For example, intestinal bacteria metabolize ginsenosides to

26

produce deglycosylated metabolites that exert anticancer bioactivity after oral

27

administration.2,3 In fact, sequential biotransformation of multi-constituents in herbal

28

medicines is easily facilitated in the gastrointestinal environment, due to the presence

29

of enzymes in gastric juice, low pH in stomach, digestive enzymes from intestine,

30

metabolic enzymes from gut microbiota, and metabolic enzymes (including phase I

31

and phase II) from gut wall.4–6 These exciting findings give us reason to believe that

32

elucidation of the comprehensive metabolic profile of herbal medicine extracts will be

33

a significant step toward revealing its bioactive constituents and understanding their

34

pharmacological mechanisms.

35

Cistanches Herba is one of the most popular edible and medicinal plants that is

36

widely utilized as a health food supplement in China, Japan, and Southeast Asia.7–9

37

Since hundreds of years, Cistanches Herba has been used to treat overstrain-induced

38

impairment by preparing a tonic food from boiling it with mutton, potato or rice.7 It

39

could also be directly consumed with tea, soup, and wine, or used in several health

40

foods.10 The stems of Cistanche deserticola (CD) and C. tubulosa (CT) are recorded

41

as the two official sources of Cistanches Herba in Chinese Pharmacopoeia (2015

42

edition) and are used for the treatment of kidney deficiency, impotence, female

43

infertility, morbid leucorrhea, profuse metrorrhagia, and senile constipation.11,12

44

According to phytochemical evaluations, phenylethanoid glycosides (PhGs) such as

3



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echinacoside, acteoside, isoacteoside, and 2´-acetylacteoside are considered to be the

46

main active components and markers of Cistanches Herba.12,13 The metabolism of

47

these four individual PhGs by human intestinal bacteria has been investigated

48

previously in our research.14,15 However the gastrointestinal metabolic profile of

49

Cistanches Herba water extract, which is frequently administered orally, may be

50

absolutely different from that of the individual compounds due to competitive effects

51

and

52

polysaccharides and oligosaccharides existing in the water extract of Cistanches

53

Herba, are potential sources of prebiotics, which may affect the composition of

54

intestinal microbiota, and in turn influence the metabolic profile of Cistanches

55

Herba.17 Hence, it becomes extremely necessary to elucidate the human

56

gastrointestinal metabolism of the water extract of Cistanches Herba i.e. the stems of

57

CD and CT.

exposure

to

components

in

different

concentrations.6,16

Additionally,

58

Four in vitro incubation models including simulated gastric juice (GJ), simulated

59

intestinal juice (IJ), human intestinal bacteria (HIB), and human intestinal microsome

60

(HIM) were employed independently and sequentially to investigate the

61

bioconversion of Cistanches Herba water extract in the stomach, intestine, gut

62

microbiota, and gut wall. UPLC-Q-TOF-MS analysis was used for comprehensive

63

metabolites authentication and metabolic profile elucidation. Moreover, a

64

comparative analysis among the single compounds, water extract of Cistanches Herba

65

as well as water extracts of each CD and CT were carried out. Such a set up allows for

66

further understanding of the metabolism and active metabolites of Cistanches Herba,

67

and provides new insights for the study of bioactive constituents and pharmacological

68

mechanisms of herbal medicines.

69

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MATERIALS AND METHODS Chemicals and Reagents. Echinacoside was provided by Dr. Pengfei Tu’s

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laboratory,

73

2´-acetylacteoside, and cistanoside A were purchased from Sichuan Weikeqi

74

Biological Technology Co., Ltd. (Chengdu, China). Hydroxytyrosol, caffeic acid, 3,

75

4-dihydroxybenzenepropionic

76

3-phenylpropionic acid were purchased from Aladdin Industrial Inc. (Shanghai,

77

China). The purity of each component was determined to be >95% by HPLC-UV.

78

Pepsin,

Peking

University

pancreatin,

(Beijing,

acid,

β-NADP+,

China).

Acteoside,

3-hydroxyphenylpropionic

glucose-6-phosphate,

isoacteoside,

acid,

and

glucose-6-phosphate

79

dehydrogenase, D-saccharic acid-1, 4-lactone, and UDP-glucuronic acid (UDPGA)

80

were purchased from Sigma-Aldrich (St. Louis, MO, USA). CHAPS was purchased

81

from Aladdin Chemistry Co., Ltd (Shanghai, China). Pooled human intestinal

82

microsomes (Lot No. 1210440) were obtained from Sekisui XenoTech (Kansas City,

83

KS, USA). General anaerobic medium broth (GAM broth) was purchased from

84

Shanghai Kayon Biological Technology Co., Ltd. (Shanghai, China). HPLC-grade

85

acetonitrile was purchased from Merck (Darmstadt, Germany). Deionized water was

86

prepared by distilling water through a Milli-Q water purification system (Millipore,

87

Bedford, MA, USA). All of the other reagents and chemicals used were of analytical

88

grade.

89

Preparation of Cistanches Herba Water Extract. Dried stems of CD and CT

90

were collected from Alashan County (Inner Mongolia, China) and Hetian County

91

(Xinjiang, China) respectively. The voucher specimen samples were authenticated by

92

Prof. Xiaobo Li and deposited at the herbarium of the School of Pharmacy, Shanghai

93

Jiao Tong University (Shanghai, China).

94

Ten grams of homogeneous powdered CD and CT stems were each suspended in

5



95

100 mL of water, and refluxed with boiled water three times, each for 1 h at 100 °C.

96

The extracts were filtered using gauze, evaporated under vacuum at 65 °C and

97

lyophilized. The samples were stored at 4 °C and re-dissolved in 10 mL of sterile

98

water prior to use. The sterile water solutions of CD and CT samples were then

99

filtered through a 0.22 µm hydrophilic PTFE filter (ANPEL Laboratory Technologies

100

Inc., Shanghai, China), and the filtrates were collected in sterile tubes. For

101

UPLC-Q-TOF-MS analysis, an aliquot each of 0.5 mL of CD and CT water extract

102

was diluted to 10 mL with water, and then filtered through a 0.22 µm membrane.

103

Study of Cistanches Herba Water Extract Metabolism by Simulated Gastric

104

Juice. An aliquot each of 0.5 mL of CD and CT water extract were placed in 10 mL

105

simulated gastric juice (0.08 M HCl containing 50 mg pepsin, pH 1.5) separately and

106

incubated at 37 °C for 4 h. One milliliter of the reaction was quenched by 3 mL of

107

water saturated n-butanol immediately at time points of 0 and 4 h. The mixture was

108

centrifuged at 3,000 rpm for 15 min, followed by evaporation of the supernatant under

109

a stream of nitrogen gas at 37 °C. The residue was re-dissolved in 0.5 mL of 70%

110

methanol, centrifuged at 14,000 rpm for 20 min at 4 °C, and the resulting supernatant

111

was analyzed by UPLC-Q-TOF-MS.

112

Study of Cistanches Herba Water Extract Metabolism by Simulated Intestinal

113

Juice. An aliquot each of 0.5 mL of CD and CT water extract were placed in 10 mL

114

simulated intestinal juice (0.05 M KH2PO4 containing 50 mg pancreatin, pH 6.8)

115

separately and incubated at 37 °C for 6 h. The sample processing procedure was same

116

as that for Cistanches Herba water extract in simulated gastric juice.

117

Study of Cistanches Herba Water Extract Metabolism by Human Intestinal

118

Bacteria. Fresh human fecal samples were obtained from 6 healthy volunteers (three

119

male and three female, 22 to 50 years of age) who had no history of gastrointestinal

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disorders or antibiotics usage for at least three months prior to the study. The samples

121

were immediately mixed and homogenized with 25 times volume of GAM broth.

122

Sediments were removed by filtration through three pieces of gauze. The suspension

123

was then incubated at 37 °C in an anaerobic incubator in which the air had been

124

replaced by a gas mixture (H2 5%, CO2 10%, N2 85%). An aliquot each of 0.5 mL of

125

CD and CT water extract were placed in 10 mL human fecal suspension separately,

126

and the suspension was incubated at 37 °C for 48 h. The cultured mixture was

127

removed and extracted with water saturated n-butanol at time points of 0 and 48 h.

128

The mixture was centrifuged at 3,000 rpm for 15 min, followed by evaporation of the

129

supernatant under a stream of nitrogen gas at 37 °C. The residue was re-dissolved in

130

0.5 mL of 70% methanol, centrifuged at 14,000 rpm for 20 min at 4 °C, and the

131

resulting supernatant was analyzed by UPLC-Q-TOF-MS.

132

Study of Cistanches Herba Water Extract Metabolism by Human Intestinal

133

Microsome. Phase I metabolism of Cistanches Herba water extract was measured in

134

vitro using human intestinal microsome. All reactions were performed in 500 µL total

135

volume. An aliquot of 50 µL of CD and CT water extract were each incubated in a

136

mixture consisting of human intestinal micosome (2 mg protein/mL) suspended in

137

100 mM potassium phosphate buffer (pH 7.4), 3 mM MgCl2, and an

138

NADPH-regenerating system (1 mM β-NADP+, 5 mM glucose-6-phosphate, and 1

139

unit/mL glucose-6-phosphate dehydrogenase). The mixture was pre-incubated for 5

140

min at 37 °C and then initiated by the addition of NADPH-regenerating system. At

141

the time point of 0 and 1.5 h, 250 µL of the reaction was quenched by 750 µL of water

142

saturated n-butanol. The mixture was then vortex-mixed for 3 min, and centrifuged at

143

14,000 rpm for 15 min at 4 °C followed by evaporation of the supernatant under a

144

stream of nitrogen gas at 37 °C. The residue was re-dissolved in 0.2 mL of 70%

7



145


146


147

Phase II glucuronidation metabolism of Cistanches Herba water extract using

148

human intestinal microsome was also measured in vitro. An aliquot of 50 µL of CD

149

and CT water extract were each incubated in a mixture (500 µL final volume)

150

consisting of human intestinal microsome (2 mg protein/mL) suspended in 100 mM

151

Tris-HCl buffer (pH 7.7), 0.5 mM CHAPS, 10 mM MgCl2, 0.1 mM D-saccharic

152

acid-1, 4-lactone. The mixture was pre-incubated for 5 min at 37 °C and then initiated

153

by the addition of 8 mM UDPGA. At time points of 0 and 1.5 h, 250 µL of the

154

reaction was quenched by 750 µL of water saturated n-butanol, the samples were

155

extracted using the same method described under phase I incubations and analyzed by

156

UPLC-Q-TOF-MS.

157

Sequential Metabolism of Cistanches Herba Water Extract by Gastric Juice,

158

Intestinal Juice, and Human Intestinal Bacteria. Firstly, 0.5 mL each of CD and

159

CT water extract were placed in 10 mL simulated gastric juice separately and

160

incubated at 37 °C for 4 h. The whole reaction was quenched by 3 times volume of

161

water saturated n-butanol, and centrifuged at 3,000 rpm for 15 min, followed by

162

evaporation of the supernatant under a stream of nitrogen gas at 37 °C. Secondly, the

163

residue was re-dissolved in 0.4 mL of sterile water, placed in 8 mL simulated

164

intestinal juice and incubated at 37 °C for 6 h, and the sample was then processed the

165

same way as in gastric juice. Finally, the residue was re-dissolved in 0.3 mL of sterile

166

water, placed in 6 mL human fecal suspension, and incubated at 37 °C for 48 h in an

167

anaerobic incubator. One milliliter of the reaction was quenched by 3 mL of water

168

saturated n-butanol immediately at time points of 0 and 4 h in gastric juice, 0 and 6 h

169

in intestinal juice, 0, 24, and 48 h in human intestinal bacteria. The mixture was

8


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centrifuged at 3,000 rpm for 15 min, followed by evaporation of the supernatant under

171

a stream of nitrogen gas at 37 °C. The residue was re-dissolved in 0.5 mL of 70%

172


173


174

UPLC-Q-TOF-MS Analysis. UPLC-Q-TOF-MS analysis was performed on a

175

Waters ACQUITY UPLC system (Waters Corp., Milford, MA, USA) using an

176

ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm, Waters Corp.,

177

USA) by gradient elution using 0.1% formic acid in acetonitrile (A) and 0.1% formic

178

acid in water (B) at a flow rate of 0.4 mL/min. The gradient profile was as follows: 0–

179

5 min (A: 5–20%), 5–7.5 min (A: 20–30%), 7.5–10 min (A: 30–70%), 10–11 min (A:

180

70–100%), and held for 1.5 min. The gradient was recycled back to 5% in 0.5 min,

181

and held for 2 min for the next run. The injection volume was 3 µL. The temperature

182

of the column oven was set to 35 °C.

183

Mass spectrometry was carried out using a Waters SynaptTM mass spectrometer

184

(Waters Corp., Milford, MA, USA). Ionization was performed in the negative

185

electrospray (ESI) mode. The MS parameters were as follows: capillary voltage, 2.8

186

kV; cone voltage, 20 V; source temperature, 115 °C; desolvation temperature, 350 °C;

187

gas flows of cone and desolvation, 50 and 600 L/h, respectively. For accurate mass

188

measurement, leucine-enkephalin was used as the lock mass. The MSE (Mass

189

Spectrometry Elevated energy) experiment in two scan functions was carried out as follows:

190

function 1 (low energy): m/z 50–1000, 0.25 s scan time, 0.02 s inter-scan delay, 4 eV

191

collision energy; function 2 (high energy): m/z 50–1000, 0.25 s scan time, 0.02 s

192

inter-scan delay, collision energy ramp of 20–45 eV. The data from the experiment

193

was processed using MassLynxTM 4.1 software (Waters Corp., Milford, MA, USA).

194

Data Processing. Data processing was performed using MetaboLynxTM software

9



195

(Waters Corp., Milford, MA, USA) for metabolites identification within the accurate

196

mass full-scan raw data collected through MSE. A list of potential biotransformation

197

reactions expected such as deglycosylation, methylation and glucuronidation was

198

employed. The mass defect filter was set as 25 mDa. The mass window of 0.05 Da

199

was set for both expected and unexpected metabolite mass chromatograms. Apex

200

Track Peak Integration was used for peak detection, and the response threshold of the

201

absolute peak height was set to 20 units.

202 203

RESULTS

204

Characterization of Prototype Constituents of Cistanches Herba Water

205

Extract. A systematic analysis of prototype constituents from CD and CT water

206

extract was performed using UPLC-Q-TOF-MS. A total number of 30 compounds

207

from CD water extract and 22 compounds from CT water extract were detected and

208

tentatively characterized. They were 24 PhGs, 5 iridoid glycosides, 2 oligosaccharides,

209

and 1 lignan compounds. Ten of these compounds were detected only in CD water

210

extract, while two were only found in CT water extract. Detailed information is listed

211

in Table 1, and the total ion chromatogram (TIC) of CD water extract is shown in

212

Figure 1a.

213

Human Gastrointestinal Biotransformation of Prototype Constituents of

214

Cistanches Herba Water Extract. Relative content of the 30 compounds from CD

215

water extract and the 22 compounds from CT water extract after incubation within

216

gastric juice (GJ), intestinal juice (IJ), human intestinal bacteria (HIB), human

217

intestinal microsomes phase I metabolism (HIMP1), and human intestinal

218

microsomes phase II metabolism (HIMP2) were determined. The percentage of each

219

constituent’s peak area within the total peak area of all constituents determined is

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shown in Table S1 and S2. According to the experimental results, five compounds

221

including C6, C8, C18, C22, and C31 (4 PhGs and 1 iridoid glycoside) were easily

222

transformed into their metabolites (>50%) in GJ, nine compounds including C3, C17,

223

C22, C24, C26, C27, C30, C31, and C32 (8 PhGs and 1 iridoid glycoside) were easily

224

transformed in IJ, seven compounds including C1, C3, C5, C6, C13, C15, and C18 (1

225

PhGs, 5 iridoid glycosides, and 1 oligosaccharide) were easily transformed in HIB,

226

nine compounds including C1, C3, C4, C7, C8, C13, C15, C16, and C22 (5 PhGs and

227

4 iridoid glycosides) were easily transformed in HIMP1, and three compounds

228

including C1, C3, and C13 (3 iridoid glycosides) were easily transformed in HIMP2.

229

It was observed that biotransformation of the PhGs in CD water extract easily

230

occurred in GJ, IJ, and HIMP1 models, whereas biotransformation of iridoid

231

glycosides in CD water extract easily occurred in HIB, HIMP1, and HIMP2 models.

232

Furthermore, few prototype components of PhGs were bio-transformed into other

233

PhGs in CD water extract. For example, C17, C24, and C26 were completely

234

metabolized into their de-caffeoyl metabolites, which were also present as prototype

235

compounds C4, C7, and C9 in the IJ model. It is understood that the caffeoyl group at

236

the C-6´ position in PhGs was easily metabolized by digestive enzymes in intestinal

237

juice, and transformed to a de-caffeoyl metabolite and caffeic acid.

238

The change in relative content of the components from CT water extract in

239

different incubation models are listed in Table S2. The results obtained were similar

240

to those of CD water extract.

241

Identification

of

Metabolites

from

CD

Water

Extract

in Human

242

Gastrointestinal Tract. Along with the incubation of CD water extract in four

243

independent in vitro models, namely GJ, IJ, HIB, and HIM, two control samples were

244

prepared for each model and used for metabolites detection in parallel. In the case of

11



245

incubation of CD water extract in HIB, control 1 consisted of human intestinal

246

bacteria and medium (Figure 1b), and control 2 consisted of CD water extract,

247

quenched human intestinal bacteria and medium (Figure 1c). By comparison with the

248

control samples, the potential metabolites of CD water extract in the four models were

249

detected from the TICs, and identified with a combination of elemental compositions

250

and MS/MS fragment mass spectra. All of the metabolites from CD water extract in

251

GJ, IJ, HIB, and HIM are listed in Table 2.

252

Two metabolites of CD water extract in GJ, tentatively identified by accurate mass

253

and MSE fragment information were M1 (m/z 315.1064, C14H20O8, 1.53 min) and M7

254

(m/z 179.0345, C9H8O4, 2.58 min). M1 was identified as 3, 4-dihydroxyphenethyl

255

glycoside, where the glucose links directly with the aglycone of PhGs. M7 was

256

undoubtedly assigned as caffeic acid by comparison of its UPLC retention time,

257

accurate MS, and MS/MS spectra of the authentic reference standard. The

258

disaccharide glycosides of PhGs from Cistanches Herba usually consist of glucose

259

and rhamnose with a Glc (3→1) Rha linkage, a direct link between the central glucose

260

and the aglycone, and a caffeoyl located in the C-4´ or C-6´ position.12 It is likely that

261

after oral administration, the ester linkage of PhGs from CD water extract is

262

hydrolyzed by the presence of a strong acidic environment and digestive enzymes in

263

the stomach, and metabolized to glucose linked aglycone and caffeic acid.

264

Three metabolites of CD water extract in IJ, tentatively identified by accurate mass

265

and MSE fragment information were M1 (m/z 315.1064, C14H20O8, 1.53 min), M3

266

(m/z 153.0561, C8H10O3, 1.60 min) and M7 (m/z 179.0345, C9H8O4, 2.58 min). M3

267

was undoubtedly identified as hydroxytyrosol by comparison with the reference

268

standard. Similar to the process in GJ, PhGs were metabolized into their aglycone and

269

caffeic acid in IJ as well.

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270

A total of 24 metabolites bio-transformed from CD water extract in HIB were

271

detected and identified (Figure 1d). From the results, it was observed that PhGs were

272

degraded to their aglycone hydroxytyrosol (M3, m/z 153.0561, C8H10O3, 1.60 min)

273

and caffeic acid (M7, m/z 179.0345, C9H8O4, 2.58 min) in HIB, and they underwent

274

further transformation to form their methylated metabolites M2 (m/z 167.0673,

275

C9H12O3, 1.57 min) and M5 (m/z 193.0490, C10H10O4, 2.40 min). The α′, β′-double

276

bond

277

4-dihydroxybenzenepropionic acid (M6, m/z 181.0502, C9H10O4, 2.49 min), and

278

further dehydroxylated to 3-hydroxyphenylpropionic acid (M13, m/z 165.0579,

279

C9H10O3, 4.06 min).18–20 Additionally, M6 could be further methylated to M15 (m/z

280

195.0648, C10H12O4, 4.09 min), M18 (m/z 195.0663, C10H12O4, 4.53 min), and M28

281

(m/z 195.0686, C10H12O4, 5.82 min) in HIB. The main metabolic pathways that

282

produced the direct metabolites of PhG prototype compounds from CD water extract

283

in HIB are methylation, dimethylation, reduction, deglycosylation, de-caffeoyl,

284

de-rhamnose, and dehydrogenation. Methylation was the most common pathway

285

among them. In the case of M36 (m/z 679.2239, C32H40O16, 7.68 min) which was 14

286

Da (CH2) higher than 2´-acetylacteoside or tubuloside B in molecular weight, the

287

characteristic product ion at m/z 665.2079 implied it was the methylated product of

288

2´-acetylacteoside (C23) or tubuloside B (C26). The fragment ion at m/z 637.2120

289

was generated by the elimination of an acetyl group (42 Da, C2H2O) on M36, the

290

other fragments produced by M36 at m/z 475.1793, 461.1676, 179.0355, and

291

161.0242 were consistent with C23 or C26. The UPLC–MS/MS spectrum and the

292

proposed fragmentation pathways of M36 are shown in Figure 2a. Apart from this,

293

iridoid glycosides-related components from CD water extract were metabolized

294

through deglycosylation to generate their aglycones in the HIB model.

of

caffeic

acid

was

easily

reduced

to

13


its

metabolite

3,


Page 14 of 36

295

Phase I and II metabolites of CD water extract in HIM were also identified. One

296

metabolite of CD water extract in HIMP1 was tentatively identified by accurate mass

297

and MSE fragment information as M7 (m/z 179.0345, C9H8O4, 2.58 min), which

298

indicated that a de-caffeoyl reaction occurred in HIMP1. For phase II metabolism, a

299

total of ten metabolites of CD water extract were identified. These metabolites were

300

found to be 176 Da (Glu) heavier than their prototype components in molecular

301

weight, and thus identified as their glucuronide conjugation products. M27 (m/z

302

813.2435, C36H46O21, 5.58 min) was selected to demonstrate stepwise elucidation of

303

the molecular structure. M27 was 176 Da (Glu) higher than cistanoside C (C21) or

304

isocistanoside C (C24) in molecular weight, and was identified as the glucuronide

305

conjugation product of cistanoside C or isocistanoside C, producing the same ions as

306

cistanoside C or isocistanoside C at m/z 623.2043, 461.1621, 179.0354, 161.0244.

307

The UPLC–MS/MS spectrum and the proposed fragmentation pathways of M27 are

308

shown in Figure 2b.

309

Identification

of

Metabolites

from

CT

Water

Extract

in Human

310

Gastrointestinal Tract. CT water extract was also incubated independently in four in

311

vitro models, GJ, IJ, HIB, and HIM and the same method of metabolite identification

312

used for CD water extract was followed. A total of 18 metabolites were tentatively

313

identified (including 2 metabolites in GJ, 2 in IJ, 11 in HIB, 2 in HIMP1, and 5 in

314

HIMP2). Detailed information on these metabolites is listed in Table 2.

315

Identification of Metabolites from Cistanches Herba after Sequential

316

Metabolism by Gastric Juice, Intestinal Juice, and Human Intestinal Bacteria. A

317

total of 13 metabolites were tentatively identified (including 2 metabolites in GJ, 2 in

318

IJ, and 12 in HIB) after CD water extract was sequentially metabolized by GJ, IJ, and

319

HIB. It was seen from the results that, PhGs from CD water extract were first

14


Page 15 of 36


320

degraded to M1 and M7 in GJ and IJ. After 24 h incubation from HIB, most of PhGs

321

were bio-transformed to M3, M6, M7, and M13. Finally, all the PhGs were

322

completely converted to M3 and M13. Similar results were obtained after CT water

323

extract was sequentially metabolized by GJ, IJ, and HIB. Detailed information on

324

these metabolites is listed in Table S3. Total ion chromatograms (TICs) of C.

325

deserticola water extract sequentially metabolized by gastric juice, intestinal juice,

326

and human intestinal bacteria are shown in Figure S3.

327

Metabolic Profile Elucidation of Cistanches Herba Water Extract in Human

328

Gastrointestinal Tract. Based on the results of the analysis, metabolic profile of

329

PhGs from Cistanches Herba water extract in human gastrointestinal tract was

330

systematically elucidated. PhGs could be degraded to glucose linked hydroxytyrosol

331

and caffeic acid through deglycosylation, de-caffeoyl, and de-rhamnose when

332

incubated in GJ and IJ. In the HIB model, more number of metabolic pathways were

333

observed for PhGs. Besides the degraded products hydroxytyrosol and caffeic acid,

334

further sequential degradation metabolites were generated through reduction,

335

methylation, and dehydroxylation. The PhG-related direct metabolites of prototype

336

constituents

337

deglycosylation, de-caffeoyl, de-rhamnose, and dehydrogenation. From the result of

338

sequential metabolism of Cistanches Herba water extract by GJ, IJ, and HIB, all the

339

PhGs were completely converted to hydroxytyrosol and 3-hydroxyphenylpropionic

340

acid after 4 h incubation in GJ, 6 h incubation in IJ, and 48 h incubation in HIB. Even

341

though the intestinal permeability of PhGs are extremely low,21 the few PhGs that

342

manage to pass through the intestinal barrier, could be metabolized to caffeic acid by

343

HIMP1 model, and metabolized to glucuronide conjugation metabolites through

344

glucuronidation in HIMP2 model. The proposed metabolic profile of PhG-related

were

produced

through

reduction,

methylation,

15


dimethylation,


345

metabolites in GJ, IJ, HIB, and HIM is shown in Figure 3.

346

Hydroxytyrosol, caffeic acid, and 3-hydroxyphenylpropionic acid, generated from

347

PhGs in GJ, IJ, HIB, and HIMP1, and the aglycones generated from iridoid glycosides

348

in HIB, were hypothesized to be absorbed into blood to exert biological activities

349

since they have better intestinal absorption and therefore better bioavailability after

350

oral administration unlike glycosides.

351 352

DISCUSSION

353

In recent years, with the development of LC-MS technology, more researchers have

354

begun to pay attention to the gastrointestinal metabolism of multi-components in

355

herbal medicines following oral administration. However, previous publications were

356

restricted to the study of metabolism by intestinal bacteria, while the metabolism by

357

digestive enzymes and strong gastric acid form stomach, digestive enzymes from

358

intestine, and metabolic enzymes from gut wall were disregarded. As for gut wall

359

metabolism, although the activity of metabolic enzymes secreted from gut wall

360

epithelial cell is much lower than those from the liver, the large surface area of the

361

intestine makes a great overall scale of metabolic reaction. Thus, it shouldn’t be

362

neglected when only a part of the whole metabolic route is studied using in vitro or in

363

vivo methods. In this study, four in vitro incubation models including GJ, IJ, HIB, and

364

HIM were applied independently and sequentially for the first time to simulate how

365

multi-components from Cistanches Herba water extract moves from the stomach to

366

the intestinal tract, and to systematically investigate the gastrointestinal metabolic

367

profile of Cistanches Herba water extract prior to being absorbed into blood. Four in

368

vitro models were initially applied separately, to identify comprehensive metabolites

369

and some trace level intermediates that appear during the incubation. As for

16


Page 16 of 36

Page 17 of 36


370

sequential assay, the events happening in vivo were mimicked which helped find the

371

final metabolites before absorption. This lays the foundation for the study of

372

pre-systemic metabolism of multiple constituents from herbal medicines in the

373

alimentary tract.

374

In clinical practice, herbal medicines are most often extracted by water to generate

375

decoction for oral administration, and polar constituents are widely present in these

376

extracts. Gut microbiota-mediated biotransformation can convert these poor lipophilic

377

chemicals to smaller units that are less polar and more lipophilic.22–24 In the present

378

study, polar constituents like PhGs and iridoid glycosides were easily degraded into

379

their

380

4-dihydroxyphenethyl glycoside, and deglycosylated geniposidic acid etc. in GJ, IJ,

381

HIB, and HIMP1. These secondary glycosides and aglycones normally harbor better

382

intestinal absorption and bioavailability, and even possess similar bioactivity/toxicity

383

as their precursors.25–28 These metabolites further get absorbed into blood to exert

384

biological activities. A previous study conducted by the team found that, after oral

385

administration of Cistanches Herba in rats, 3-hydroxyphenylpropionic acid, its sulfate

386

conjugates, and hydroxytyrosol sulfate conjugates were observed in rat serum,

387

implying that these degradation metabolites could be absorbed into blood in vivo.29

secondary

glycosides

and

aglycones

including

hydroxytyrosol,

3,

388

In our previous research it was found that echinacoside, acteoside, and isoacteoside

389

were completely metabolized after 48 h incubation.14,15 The present results however

390

show that, after 48 h incubation only 30% of echinacoside from CD water extract was

391

metabolized whereas the concentration of acteoside and isoacteoside were found to be

392

higher. The differences in metabolism of CD water extract and single PhGs were

393

probably due to the abundance of oligosaccharides and polysaccharides in CD water

394

extract. Oligosaccharides and polysaccharides are potential sources of prebiotics and

17



395

influence the composition and homeostasis of intestinal bacteria. These bacteria,

396

especially those from phyla Bacteroidetes and Firmicutes, possess glycoside

397

hydrolase genes30 and are specially equipped to perform glycoside hydrolysis, thus

398

affecting the metabolism of CD water extract. On the other hand, PhGs like

399

echinacoside, 2´-acetylacteoside, cistanoside A, and tubuloside B are transformed into

400

acteoside and isoacteoside in HIB, leading to the increase in concentration of

401

acteoside and isoacteoside. This goes on to show that, single compounds cannot

402

represent the real process of gastrointestinal metabolism of multi-components from

403

herbal medicines. It is also to be noted that, more gastrointestinal metabolites were

404

obtained from CD water extract than from CT water extract, probably due to the

405

difference in polysaccharides content of these two species of Cistanches Herba.

406

In this paper, a UPLC-Q-TOF-MS method was employed for metabolites

407

identification and metabolic profile elucidation of CD and CT water extract in

408

simulated human digestive juice, human intestinal bacteria and intestinal microsome,

409

independently and sequentially. A total of 35 and 18 metabolites were characterized

410

from in vitro gastrointestinal tract metabolism of CD and CT water extract

411

respectively. Among them, to the best of our knowledge, 11 metabolites have not

412

been previously reported in these two species. The results of the study indicated that

413

PhGs were easily bio-transformed to their aglycone and caffeic acid derivatives

414

through deglycosylation and de-caffeoyl in GJ, IJ, HIB, and HIMP1, and through

415

glucuronidation in HIMP2. Iridoid glycosides were easily transformed to their

416

aglycones in HIB. These metabolites are more easily absorbed through the intestine

417

into blood with a better bioavailability to exert bioactivities. This study is helpful in

418

understanding the bioactive constituents and action mechanism of Cistanches Herba,

419

and provides a practical method for assessing the gastrointestinal metabolism of

18


Page 18 of 36

Page 19 of 36


420

multi-components in herbal medicines for future research.

19



421

ASSOCIATED CONTENT

422

Supporting Information

423

Table S1 contains the percentage of relative content and the area of prototype

424

components of C. deserticola in different samples (0.1% formic acid was added

425

respectively in acetonitrile and water of UPLC mobile phase), Table S2 contains

426

percentage of relative content and the area of prototype components of C. tubulosa in

427

different samples (0.1% formic acid was added respectively in acetonitrile and water

428

of UPLC mobile phase), Table S3 contains UPLC-Q-TOF-MS data of the identified

429

metabolites of C. deserticola and C. tubulosa sequentially produced by gastric juice,

430

intestinal juice, and human intestinal bacteria, Figure S1 contains the chemical

431

structures of prototype components of Cistanches Herba water extract, Figure S2

432

contains the chemical structures of identified metabolites of Cistanches Herba water

433

extract produced in gastric juice, intestinal juice, human intestinal bacteria, and

434

human intestinal microsome, Figure S3 contains total ion chromatograms of C.

435

deserticola water extract sequentially incubated in gastric juice, intestinal juice, and

436

human intestinal bacteria.

20


Page 20 of 36

Page 21 of 36


437

AUTHOR INFORMATION

438

Notes

439

The authors declare no competing ﬁnancial interest.

440

21



441

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442

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26


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Figure Captions Figure 1. Total ion chromatograms (TIC) of C. deserticola water extract (a), control 1 using human intestinal bacteria and medium (b), control 2 using C. deserticola water extract, quenched human intestinal bacteria and medium (c), and transformed C. deserticola water extract by human intestinal bacteria (d). Figure 2. UPLC–MS/MS spectra and proposed fragmentation pathways: (a) M36 (m/z 679.2239), and (b) M27 (m/z 813.2435). Figure 3. The proposed metabolic profile of PhGs-related metabolites produced by gastric juice (red), intestinal juice (blue), human intestinal bacteria (black), human intestinal microsome phase I (purple), and human intestinal microsome phase II (brown).

27



Page 28 of 36

Table 1. Characterization of Chemical Constituents of C. deserticola (CD) and C. tubulosa (CT) by UPLC-Q-TOF-MS No.

Measured mass (Da) 373.1121

Error (mDa) −1.4

Formula

MS/MS fragment ions (Da)

Identification

Species

C1

tR (min) 1.41

C16H22O10

211.0425, 149.0578, 123.0442

geniposidic acid

CD, CT

C2

1.68

649.1962

−1.8

C27H38O18

485.1233, 305.0825, 179.0337, 135.0448

kankanose

CT

C3

1.87

375.1283

−0.8

C16H24O10

213.0775, 169.0873, 151.0758

8-epiloganic acid

CD, CT

C4

1.90

461.1659

0.0

C20H30O12

315.1157, 123.0476

decaffeoylacteoside

CD, CT

C5

1.97

487.1464

1.2

C21H28O13

179.0350, 161.0274, 135.0445

cistanoside F

CD, CT

C6

2.00

299.1143

1.2

C14H20O7

179.0344, 119.0476

salidroside

CD, CT

C7

2.84

475.1802

−1.4

C21H32O12

416.1607, 329.1328

cistanoside E

CD

C8

3.11

801.2454

0.1

C35H46O21

785.2595, 623.2196, 461.1664, 179.0367, 161.0246

cistantubuloside C1/C2

CD, CT

C9

3.34

503.1749

−1.6

C22H32O13

461.1679, 315.1100

cistanoside H

CD, CT

C10

3.99

785.2504

0.0

C35H46O20

623.2114, 477.1615, 461.1674, 315.1065, 179.0384, 161.0190

echinacoside

CD, CT

C11

4.50

769.2524

−3.1

C35H46O19

623.2169, 461.1700, 161.0278

poliumoside

CD, CT

C12

4.73

799.2653

−0.8

C36H48O20

637.2318, 623.2114, 491.1745, 461.1666, 161.0274

cistanoside A

CD, CT

C13

4.82

345.1532

−1.7

C16H26O8

161.0243

kankanoside A or isomer

CD, CT

C14

5.07

623.1979

0.3

C29H36O15

461.1624, 315.1075, 179.0342, 161.0292, 135.0444

acteoside

CD, CT

C15

5.10

345.1539

−1.0

C16H26O8

161.0265

kankanoside A or isomer

CD

C16

5.11

827.2576

−3.4

C37H48O21

785.2547, 665.2256, 632.1973, 461.1626, 315.1099, 179.0345, 161.0220, 135.0438

tubuloside A

CD, CT

28


Page 29 of 36


C17

5.46

623.1945

−3.1

C29H36O15

461.1624, 315.1068, 179.0338, 161.0210, 135.0444

isoacteoside

CD, CT

C18

5.69

347.1686

−2.0

C16H28O8

185.1442, 161.0239

kankanoside N

CD

C19

5.73

607.2004

−2.3

C29H36O14

445.1616, 179.0327, 161.0227, 135.0435

syringalide A -3’-a-L-rhamnopyranoside

CD, CT

C20

5.91

417.1544

−0.5

C22H26O8

265.1472

syringaresinol

CT

C21

6.01

637.2093

−3.9

C30H38O15

623.2143, 475.1750, 461.1529, 179.0353, 161.0228, 135.0428

cistanoside C

CD

C22

6.23

607.1988

−3.9

C29H36O14

179.0344, 161.0220, 135.0438

isosyringalide A -3’-a-L-rhamnopyranoside

CD

C23

6.24

665.2055

−2.7

C31H38O16

2′-acetylacteoside

CD, CT

C24

6.36

637.2117

−1.5

C30H38O15

623.1946, 503.1747, 461.1660, 315.1085, 179.0344, 161.0220, 135.0438 475.1808, 461.1606, 329.1112, 179.0335, 161.0223, 135.0430

isocistanoside C

CD

C25

6.39

591.2044

−3.4

C29H36O13

445.1577, 145.0289, 119.0516

osmanthuside B or osmanthuside B6

CD, CT

C26

6.71

665.2077

−0.5

C31H38O16

tubuloside B

CD, CT

C27

6.85

591.2058

−2.0

C29H36O13

623.1948, 503.1755, 461.1627, 315.1065, 179.0350, 161.0215, 135.0442 445.1483, 145.0292, 119.0499

osmanthuside B or osmanthuside B6

CD, CT

C28

6.85

649.2128

−0.4

C31H38O15

607.1913, 145.0291

salsaside F or isomer

CD

C29

6.91

651.2235

−5.4

C31H40O15

193.0484, 175.0400, 161.0248

cistanoside D

CD

C30

7.38

649.2122

−1.0

C31H38O15

607.2091, 145.0289

salsaside F or isomer

CD, CT

C31

7.40

651.2257

−3.2

C31H40O15

193.0484, 175.0396, 161.0230

isocistanoside D

CD

C32

7.67

679.2217

−2.1

C32H40O16

665.1975, 637.2109, 623.2100, 475.1849, 461.1716, 329.1173, 179.0341, 161.0225

cistanoside K or cistansinenside A or salsaside E or isomer

CD

29



Page 30 of 36

Table 2. UPLC-Q-TOF-MS Data of the Identified Metabolites of C. deserticola (CD) and C. tubulosa (CT) Produced by Gastric Juice (GJ), Intestinal Juice (IJ), Human Intestinal Bacteria (HIB), Human Intestinal Microsome Phase I (HIMP1), and Human Intestinal Microsome Phase II (HIMP2) a No.

Measured mass (Da) 315.1064

Error (mDa) −1.6

Formula

M1

tR (min) 1.53

MS/MS fragment ions (Da)

C14H20O8

153.0550, 123.0444

CD GJ IJ

M2

1.57

167.0673

−3.5

C9H12O3

153.0563, 123.0456

HIB

ND

methylated hydroxytyrosol

M3

1.60

153.0561

0.9

C8H10O3

123.0465

IJ HIB

HIB

hydroxytyrosol

M4

1.84

163.0400

0.5

C9H8O3

135.0448, 119.0487

HIB

HIB

dehydroxylated caffeic acid

M5

2.40

193.0490

−1.1

C10H10O4

179.0375, 135.0408

HIB

HIB

methylated caffeic acid

M6

2.49

181.0502

0.1

C9H10O4

137.0600

HIB

HIB

3, 4-dihydroxybenzenepropionic acid

M7

2.58

179.0345

0.1

C9H8O4

135.0436

GJ IJ HIB HIMP1

GJ IJ HIMP1

caffeic acid

M8

2.61

459.1515

1.2

C20H28O12

151.0412

HIB

ND

dehydrogenated decaffeoylacteoside

M9

2.89

213.0743

−2.0

C10H14O5

169.1004

HIB

HIB

deglycosylated 8-epiloganic acid

M10

3.15

803.2631

2.1

C35H48O21

315.1257, 181.0559

HIB

HIB

reduction of cistantubuloside C1/C2

M11

3.57

961.2784

−4.1

C41H54O26

785.2435, 179.0350, 161.0241

ND

HIMP2

echinacoside glucuronide conjugation

M12

3.96

783.2310

−3.8

C35H44O20

179.0363, 161.0221

HIMP2

HIMP2

M13

4.06

165.0579

2.7

C9H10O3

121.0601, 119.0493

HIB

HIB

syringalide A -3’-a-L-rhamnopyranoside or isomer glucuronide conjugation 3-hydroxyphenylpropionic acid

M14

4.08

211.0607

0.1

C10H12O5

167.0638, 147.0444

HIB

HIB

deglycosylated geniposidic acid

30


Source CT GJ IJ HIMP1

Identification 3, 4-dihydroxyphenethyl glycoside

Page 31 of 36


M15

4.09

195.0648

−0.9

C10H12O4

181.0479, 137.0576

HIB

HIB

M16

4.36

783.2365

1.7

C35H44O20

179.0355, 161.0255

HIMP2

HIMP2

M17

4.49

813.2460

0.7

C36H46O21

623.2095, 461.1494, 179.0350, 161.0229

HIMP2

HIMP2

M18

4.53

195.0663

0.6

C10H12O4

181.0515

HIB

HIB

M19

4.53

517.1964

4.3

C23H34O13

475.1854, 329.1351

HIB

ND

M20

4.59

783.2330

−1.8

C35H44O20

179.0374, 161.0229, 135.0441

HIMP2

ND

M21

4.61

787.2634

−2.7

C35H48O20

625.2178

HIB

ND

M22

5.03

813.2426

−2.7

C36H46O21

HIMP2

HIMP2

M23

5.41

813.2811

−0.6

C37H50O20

623.1970, 461.1641, 315.1079, 179.0356, 161.0234 785.2506, 623.1970, 461.1627, 161.0224

HIB

ND

cistanoside C or isocistanoside glucuronide conjugation dimethylated echinacoside

M24

5.45

651.2087

−4.9

C27H40O18

475.1754, 461.1640

HIMP2

ND

cistanoside E glucuronide conjugation

M25

5.46

477.1419

2.2

C23H26O11

179.0339, 161.0231

HIB

ND

calceolarioside A

M26

5.53

841.2422

2.0

C37H46O22

HIMP2

ND

M27

5.58

813.2435

−1.8

C36H46O21

HIMP2

ND

M28

5.82

195.0686

2.9

C10H12O4

665.2053, 623.2073, 461.1478, 179.0353, 161.0238 637.2166, 623.2043, 461.1621, 179.0354, 161.0244 181.0485

HIB

ND

M29

5.88

183.1028

0.7

C10H16O3

137.0083

HIB

ND

2′-acetylacteoside or tubuloside B glucuronide conjugation cistanoside C or isocistanoside C glucuronide conjugation methylated 3, 4-dihydroxybenzenepropionic acid deglycosylated kankanoside A or isomer

M30

6.00

841.2387

−1.5

C37H46O22

HIMP2

ND

M31

6.04

827.2609

−0.1

C37H48O21

665.2079, 623.2150, 461.1645, 179.0346, 161.0241 651.2333, 475.1945, 193.0520

HIMP2

ND

M32

6.90

651.2271

−1.8

C31H40O15

623.1972, 179.0332, 161.0232

HIB

ND

31


methylated 3, 4-dihydroxybenzenepropionic acid syringalide A -3’-a-L-rhamnopyranoside or isomer glucuronide conjugation cistanoside C or isocistanoside C glucuronide conjugation methylated 3, 4-dihydroxybenzenepropionic acid methylated cistanoside H syringalide A -3’-a-L-rhamnopyranoside or isomer glucuronide conjugation reduction of echinacoside

2′-acetylacteoside or tubuloside glucuronide conjugation cistanoside D or isocistanoside glucuronide conjugation dimethylated acteoside

C

B D


Page 32 of 36

M33

6.94

185.1186

0.8

C10H18O3

137.0107

HIB

HIB

deglycosylated kankanoside N

M34

7.20

679.2258

2.0

C32H40O16

665.2128, 623.2076, 179.0369, 161.0240

HIB

ND

M35

7.35

651.2326

3.7

C31H40O15

HIB

ND

M36

7.68

679.2239

0.1

C32H40O16

623.1947, 461.1596, 179.0320, 161.0239, 135.0449 665.2079, 637.2120, 475.1793, 461.1676, 179.0355, 161.0242

methylated 2′-acetylacteoside or tubuloside B dimethylated acteoside

HIB

ND

a

ND: not detected.

32


methylated 2′-acetylacteoside or tubuloside B

Page 33 of 36


Figure 1

33



Figure 2

34


Page 34 of 36

Page 35 of 36


Figure 3

35



Table of Contents Graphic

36


Page 36 of 36

Human Gastrointestinal Metabolism of the Cistanches Herba Water

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