Disposition of Astragaloside IV via Enterohepatic Circulation Is

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Disposition of Astragaloside IV via Enterohepatic Circulation is Affected by the Activity of Intestinal Microbiome Yi Jin, Xingjie Guo, Bo Yuan, Wenhong Yu, Hao Suo, Zhiyuan Li, and Haiyan Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00168 • Publication Date (Web): 12 Jun 2015 Downloaded from http://pubs.acs.org on June 13, 2015

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Journal of Agricultural and Food Chemistry

Title: Disposition of Astragaloside IV via Enterohepatic Circulation is Affected by the Activity of Intestinal Microbiome Running title: Enterohepatic Circulation of Astragaloside IV in Rats Authors: Yi Jin,† Xingjie Guo,† Bo Yuan,† Wenhong Yu,† Hao Suo,† Zhiyuan Li,† and Haiyan Xu *,† Affiliations: †

Department of Pharmaceutical Analysis, Pharmacy School, Shenyang Pharmaceutical

University, Shenyang 110016, China *Correspondence: Haiyan Xu Department of Pharmaceutical Analysis Pharmacy School Shenyang Pharmaceutical University Shenyang 110016, China Telephone: +86 24 23986985 Fax: +86 24 23986250 E-mail: [email protected], [email protected]

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ABSTRACT

2

Astragaloside IV (ASIV) is a typical bioactive constituent of Radix Astragali. The

3

study aimed to investigate the enterohepatic circulation of ASIV and evaluate the

4

impact of activity of intestinal microbiota on the deposition of ASIV. The amounts of

5

ASIV and its metabolites were quantified by an LC-MS/MS method. ASIV was

6

metabolized by intestinal bacteria to form brachyoside B (Bra B), cyclogaleginoside B

7

(Cyc B), cycloastragenol (CA), iso-cycloastragenol (iso-CA), and dehydrogenated

8

metabolite of CA (CA-2H). CA and iso-CA circulated in blood besides ASIV when

9

rats received ASIV intragastrically or intravenously. After rats were intragastrically

10

administrated with 10 mg/kg ASIV, the AUC0-t values of ASIV, CA and iso-CA were

11

109±55, 26.8±17.9 and 77.9±35.1 nM·h, respectively. The plasma distribution of

12

ASIV was significantly affected by bile duct drainage when ASIV was administrated

13

through duodenum. ASIV, Bra B and Cyc B were secreted from bile after duodenal

14

administration of ASIV. Antibiotics markedly inhibited the metabolism of ASIV in

15

intestinal microbiota. After rats were pretreated with antibiotics, the AUC0-t of iso-CA

16

was 4.8 times less than that in control rats and the concentration of CA became

17

undetectable. Variations in intestinal microbiota may change the disposition of ASIV

18

and subsequently influent its potential health benefits.

19 20

Keywords:

21

hepatic circulation, intestinal bacteria

Astragaloside

IV,

cycloastragenol,

iso-cycloastragenol,

22


entero-

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23

INTRODUCTION

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Radix Astragali, prepared from the roots of Astragalus membranaceus and

25

Astragalus membranaceus var. mongolicus (Leguminosae), is a traditional Chinese

26

medical herb. It has been widely used as a major ingredient in medicine formulas to

27

treat cardiovascular, hepatic and kidney disorders or as a functional food to improve

28

health in China or other East Asian countries for hundreds of years. Astragaloside IV

29

(ASIV), 3-O-β-D-xylopyranosyl-6-O-β-D-glucopyranosyl-cycloastragenol (Figure1),

30

is the most abundant saponin purified from Radix Astragali.1-3 Pharmacologic studies

31

revealed that ASIV possessed many bioactivities contributed to the beneficial

32

properties of Astragali Radix, such as cardioprotective,4,5 neuroprotective,6

33

antidiabetic,7 immunoregulatory effects.8 Consequently, ASIV was considered as the

34

typical bioactive component of Radix Astragali and documented as the chemical

35

marker for the quality control of the herb or Astragali-containing medicines in

36

Chinese Pharmacopoeia.9

37

Pharmacokinetics of ASIV in rats10-13 and dogs14 were investigated following oral

38

administration of ASIV, which proved a poor oral bioavailability of ASIV. The

39

reported bioavailability values of ASIV were 2.2−3.7% in rats10,11 and 7.4 % in dogs14.

40

It is well-known that intestinal absorption and tissular metabolism are critical

41

influences on bioavailability of orally administrated drugs. ASIV was found to be

42

poorly absorbed from intestine via passive diffusion because of its weak membrane

43

permeability.15,16 It underwent limited metabolism in liver.13,17 In drug metabolism,

44

priority is usually given to phase I and phase II enzymes existing in hepatocytes and



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enterocytes because they are responsible for more than 80% of drug metabolism.

46

Actually, besides these metabolic enzymes, gut microbiota also can inactivate or

47

activate xenobiotics, and subsequently modulate their bioavailability, pharmacological

48

actions and/or toxicity.18-22 Therefore, intestinal microbiota is regarded as a key factor

49

for xenobiotic metabolism too, especially for nature compounds containing glycosidic

50

linkages.

51

Zhou et al. reported that ASIV was converted to a series of metabolites by intestinal

52

microbiota, including brachyoside B (Bra B), cyclogaleginoside B (Cyc B),

53

cycloastragenol (CA), iso-cycloastragenol (iso-CA), and a dehydrogenated metabolite

54

of cycloastragenol (CA-2H) (Figure 1).13 Among these metabolites, CA and iso-CA

55

were circulated in blood following ASIV after oral intake of ASIV.13 Although the

56

pharmacological effects of iso-CA remained unclear, CA exhibited bioactivities

57

closely related to those of ASIV;8,23 and it was more easily absorbed from intestine

58

than ASIV.24 This is a common phenomenon observed in disposition of saponin

59

glycosides. Saponins are stripped off their sugar moieties by intestinal bacteria; the

60

resultant sapogenins display similar biological functions to their parent saponins and

61

better physicochemical properties for membrane permeability. Hence, it is

62

generally agreed that the contribution of deglycosylated metabolites should be taken

63

into account for the evaluation of bioactivity and bioavailability of saponins. On the

64

other side, biliary excretion is the major elimination route for most of orally

65

administrated

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phenomenon in disposition of saponin glycosides, in which saponins are secreted

saponins.25-28

Enterohepatic

circulation


is

another

common

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from bile into intestine trace and react with gut bacteria again.28 Till now, information

68

about the role of enterohepatic circulation in the disposition of ASIV is still missing.

69

In addition, activity of intestinal microbiota is predicted to change enterohepatic

70

circulation of metabolites secreted from liver and affect in vivo distribution of

71

xenobiotics. Elucidation of the effect of intestinal microbiome on the disposition of

72

ASIV will be a great help in understanding its bioactivities and bioavailability.

73

Therefore, the present study aimed to investigate the enterohepatic circulation of

74

ASIV and assess the impact of activity of intestinal microbiome on the disposition of

75

ASIV.

76



77

MATERIALS AND METHODS

78

Chemicals

79

ASIV (purity>98%) and CA (purity>98%) were supplied by Shanghai Winherb

80

Medical Technology Co. Ltd. (Shanghai, China). Ketoconazole (the internal standard,

81

IS, purity>99%) was obtained from the National Institute for the Control of

82

Pharmaceutical and Biological Products (Beijing, China). Solutol® HS 15 was

83

provided by BASF Co. (Ludwigshafen, Germany). BBL brain heart infusion (BHI)

84

and rat liver S9 were purchased from Becton Dickinson Co. (NJ, USA). Hemin

85

bovine and vitamin K1 were supplied by Shanghai Yuanye Biotech Co. (Shanghai,

86

Chaina). L-Cystine was provided by Biosharp Co. (Anhui, China). β-Nicotinamine

87

adenine dinucleotide phosphate (reduced form, NADPH) was purchased from

88

Xinjingke Biotechnology Co (Beijing, China). HPLC-grade acetonitrile was obtained

89

from Fisher Chemicals (NJ, USA). Distilled water, prepared from demineralized water,

90

was used throughout the study. All other chemicals and solvents were of analytical

91

grade and used without further purification.

92

Animals

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Male Sprague-Dawley (SD) rats (200±10 g) were obtained from Laboratory Animal

94

Center of Shenyang Pharmaceutical University (Shenyang, China). They were kept in

95

temperature and humidity-controlled (temperature: 25±2°C, humidity: 52.6±5.3%)

96

environment with a reverse 12 h light/dark cycle. After a week of acclimation, the

97

animals were fasted 12 h before experiments. During the period of experiment,

98

animals had free access to food and autoclaved distilled water except those under


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anesthesia with urethane. All experimental procedures were performed in accordance

100

with the guidelines of the Experimental Animal Care and Use Committee of Shenyang

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Pharmaceutical University (Shenyang, China). The experimental animal ethic review

102

approval number was SYPU-IACUC-2014-0039.

103

Pharmacokinetic study

104

Oral administration

105

Six male rats were given a single intragastrical (i.g.) administration of 10 mg/kg

106

ASIV dissolving in 0.5% sodium carboxymethyl cellulose (CMC-Na). The dosage

107

was selected according to the published literatures12 as well as the clinical dosage of

108

Radix Astragali (20−100 g/day) and the content of ASIV in Radix Astragali (≥0.04%).

109

The rats were slightly anesthetized by diethyl ether before blood collection. Blood

110

samples (200 µl) of rats obtained from the periorbital sinus vein were collected into

111

heparinized tubes before administration (0 h) and at 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0,

112

10 and 12 h after administration. Plasma was separated by centrifugation at 4,000×g

113

for 5 min and stored at -70°C until analysis.

114

Intravenous administration

115

Six male rats were treated with a dose of 1.5 mg/kg ASIV from caudal vein basing

116

on the reported documents.11,17,29 The injection solution is prepared in 10% (w/v)

117

Solutol® HS 15 in saline. After the rats were slightly anesthetized by diethyl ether,

118

blood samples (200 µl) were collected from the periorbital sinus vein into heparinized

119

tubes before administration (0 h) and at 0.083, 0.167, 0.33, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0,

120

10 and 12 h after dosing. Plasma was separated by centrifugation at 4,000×g for 5 min



121

and stored at -70°C until analysis.

122

Enterohepatic circulation experiments in bile duct cannulation rat model

123

Oral administration experimental design

124

Twelve male rats were randomly divided into two groups. One group of rats (n = 6)

125

received bile duct cannulation surgery before given a dosage of 10 mg/kg ASIV from

126

duodenum. In brief, the animals were anesthetized by an intraperitoneal (i.p.) injection

127

of urethane (1 g/kg). A 1-cm cut was made to expose the jugular vein for each rat.

128

Then the vein was cut and a cannula made of polyethylene-20 tubing was inserted.

129

After the cannula and the vein were tied with sterilized suture, a 4-cm cut was made to

130

open the abdominal cavity of the rat. The bile duct was separated from the

131

surrounding tissue and a polyethylene-10 tubing was inserted into the duct. When bile

132

flow out of the cannulation was seen without restriction, the cannula was secured with

133

sutures. After that, ASIV solution (1 mg/ml in 0.5% CMC-Na) in a syringe was slowly

134

injected into duodenum through an intravenous infusion needle at about 6 cm below

135

pylorus. The injection rate was controlled at 0.1 ml/min by a syringe pump. Another

136

six control rats were treated with the same procedure except bile duct drainage.

137

Considering the tolerance of rats with surgery and pharmacokinetic profiles of

138

ASIV and its metabolites after an i.g. administration of ASIV, blood samples (200 µl)

139

of bile duct-cannulated rats and control rats were only collected from the jugular vein

140

at four time points: before administration (0 h) and at 2.0, 8.0 and 10 h after

141

administration. Plasma was separated by centrifugation at 4,000×g for 5 min. Bile

142

samples were collected into 1.5-ml centrifuge tubes in 30 min before administration


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and every 30 min after administration till 10 h. Plasma and bile samples were stored at

144

-70°C until analysis.

145

Intravenous administration experimental design

146

Six male rats having bile duct and jugular vein cannulation surgery (by the same

147

method as described above) were given an injection of ASIV at a dose of 1.5 mg/kg

148

from caudal vein. Another six rats only having jugular vein cannulation surgery were

149

labeled as controls. The control rats also received an i.v. injection of 1.5 mg/kg ASIV

150

from caudal vein after surgery. Blood samples (200 µl) of rats in both groups were

151

obtained from the jugular vein and collected into heparinized tubes before

152

administration (0 h) and at 2.0, 8.0 and 10 h after administration. Plasma was

153

separated by centrifugation at 4,000×g for 5 min. Bile samples were collected into

154

1.5-ml centrifuge tubes in 30 min before administration and every 30 min after

155

administration till 10 h. Plasma and bile samples were stored at -70°C until analysis.

156

Rat intestinal S9 fraction preparation

157

Rat intestinal S9 fraction was prepared from male SD rats according to a published

158

procedure with a little modification.30 In brief, rats were anesthetized with urethane (1

159

g/kg), and then rat whole intestines were cut out and flushed with ice-cold saline

160

containing 1 mM dithiothreitol. After intestine were pooled from six rats, they were

161

washed twice with the ice-cold washing solution (consisted of 8 mM KH2PO4, 5.6

162

mM Na2HPO4, 1.5 mM KCl, 96 mM NaCl, 27 mM sodium citrate, and 0.04 mg/ml

163

phenylmethylsulfonyl fluoride or PMSF). After dried with paper, the intestinal strips

164

were cut open and mucosal cells were scraped off in a 4°C cold room. The mucosal



165

cells were centrifuged at 900×g for 5 min at 4°C and washed twice in 50 ml of the

166

homogenization buffer (consisted of 10 mM pH 7.4 KH2PO4, 250 mM sucrose, 1 mM

167

EDTA, and 0.04 mg/ml PMSF). The cells were resuspended in 15 ml of the

168

homogenization buffer. After 15-min centrifugation in 9,000×g at 4°C, the supernatant

169

was collected, aliquoted, and stored at -70°C until use. Protein concentration was

170

determined by Coomassie brilliant blue colorimetric method.

171

In vitro metabolism of ASIV and CA by rat liver and intestinal S9 fraction

172

ASIV (1 µM) or CA (1 µM) was mixed with liver or intestinal S9 fraction (the final

173

protein concentrations were 1 mg/ml), potassium chloride (10 mM), magnesium

174

chloride (10 mM) and 1.0 mM NADPH in 50 mM potassium phosphate buffer (pH

175

7.4). The total volume was 200 µl. The reaction was initiated by addition of a solution

176

of NADPH after 5 min preincubation. After incubating at 37°C for 0 and 1 h, the

177

reaction was terminated by addition 200 µl of cold methanol (0°C). The mixture was

178

centrifuged at 4,000×g for 5 min and the supernatant was stored at -20°C until

179

analysis. Controls were prepared in the same manner, except for the presence of

180

NADPH. Blank samples were assayed without substrate to exclude analytical

181

interference by the matrix.

182

In vitro metabolism of ASIV and CA by rat intestinal microbiota

183

Five healthy male rats were housed in individual steel metabolism cages. Fresh

184

feces were collected and immediately stored at -70°C until use. Pooled feces (2.5 g

185

from 5 rats, 0.5 g/rat) was mixed with 20 ml culture medium of BHI containing 37

186

µg/ml BHI, 5 µg/ml hemin bovine, 0.5 mg/ml L-cystine and 0.02% vitamin K1.31 The


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fecal suspension was centrifuged at 300×g for 5 min. The resultant supernatant was

188

transferred into another clean tube and centrifuged at 4000×g for 10 min; then the

189

residue was resuspended in 15 ml culture medium of BHI to produce intestinal

190

bacteria solution. ASIV (20 µM) or CA (1 µM) was mixed with 20 µl intestinal

191

bacteria solution and 180 µl culture medium of BHI. The total volume was 200 µl.

192

The mixture was incubated at 37°C under anaerobic conditions for 0, 2, 4, 8, 12, 24,

193

36 and 48 h. The reaction was stopped by addition of 200 µl of cold methanol (0°C).

194

The mixture was centrifuged at 4,000×g for 5 min and the supernatant was stored at

195

-20°C until analysis. Control and blank samples were prepared in the same manner

196

without bacteria solution and substrate, respectively.

197

Effect of activity of intestine microbiota on the disposition of ASIV

198

Comparison of metabolism of ASIV by intestinal microbiota from rats pretreated with

199

and without antibiotics

200

Five male rats were given a mixture solution of streptomycin sulfate and bacitracin

201

(1:1, w/w; 200 mg/kg) dissolving in sterilized water through gavage twice daily for 6

202

days to inhibit the activity of intestinal microbiome. Another five rats without the

203

treatment of antibiotics were used as controls. Fresh fecal samples from 5 control rats

204

and those from 5 antibiotic-pretreated rats were collected individually and pooled

205

separately. Then intestinal bacteria solutions from the two different fecal samples

206

were prepared and incubated with 20 µM ASIV by the corresponding methods as

207

described above. After incubation at 37°C for 0, 4, 8, 12 and 24 h, the reaction was

208

stopped by addition of 200 µl of cold methanol (0°C). The mixture was centrifuged at



209

4,000×g for 5 min and the supernatant was stored at -20°C until analysis. Control and

210

blank samples were prepared in the same manner without bacteria solution and

211

substrate, respectively.

212

Antibiotics used to inhibit the activity of gut microbiota may interact with the

213

analyte, which might interfere with the study result. Therefore, we determined the

214

potential interference of antibiotics to the biotransformation ASIV in intestinal

215

microbiota. ASIV (20 µM) in presence of streptomycin sulfate and bacitracin (1:1,

216

w/w; 0.08, 0.4 or 2 mg/ml) was mixed with 20 µl intestinal bacteria solution and 180

217

µl culture medium of BHI. After incubation at 37°C for 0 and 4 h, the reaction was

218

stopped by addition of 200 µl of cold methanol (0°C). The mixture was centrifuged at

219

4,000×g for 5 min and the supernatant was stored at -20°C until analysis. Control and

220

blank samples were prepared in the same manner without bacteria solution and

221

substrate, respectively.

222

Comparison of pharmacokinetics of ASIV in rats pretreated with and without

223

antibiotics

224

Six male rats were treated with antibiotics by the same method as mentioned above.

225

After fasted overnight, antibiotic-pretreated received an i.g. dose of 10 mg/kg ASIV

226

dissolving in 0.5% CMC-Na. The rats were under light diethyl ether anesthesia before

227

blood collection. Blood samples (200 µl) were collected from the periorbital sinus

228

vein into heparinized tubes before administration (0 h) and at 0.5, 1.0, 2.0, 3.0, 4.0,

229

5.0, 6.0, 8.0, 10 and 12 h after administration. Plasma was separated by centrifugation

230

at 4,000×g for 5 min and stored at -70°C until analysis. The rats given an i.g. dose of


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ASIV in pharmacokinetic study were used as controls.

232

Sample preparation

233

For plasma and bile samples, a 10 µl aliquot of the IS solution (4.0 µM of

234

ketoconazole in methanol) and a 100 µl aliquot of methanol was added into a 50 µl

235

aliquot of sample. The mixture was vortex-mixed for 1 min. After being centrifuged at

236

15,000×g for 5 min, a 5 µl aliquot of the supernatant of each sample was injected for

237

LC-MS/MS analysis.

238

For the samples in in vitro metabolism studies, a 10 µl aliquot of the IS solution

239

(4.0 µM of ketoconazole in methanol) and a 50 µl aliquot of methanol was added into

240

100 µl sample. Then the mixture was prepared by the same method as that for plasma

241

and bile samples.

242

LC-MS/MS analysis

243

The chromatographic separation was performed on a Venusil ASB C18 column

244

(150×4.6 mm, 5 µm, Agela, Tianjin, China). Methanol (A) and 5 mM ammonium

245

acetic acid (B) were used as mobile phase for elution. The gradient was controlled as

246

follows: 0–1.0 min, 60% A, 1.0–2.0 min, 60–90% A, 2.0–5.5 min, 90% A, 5.5–6.0

247

min, 90–60% A, 6.0–8.0 min, 60% A. The retention times of ASIV, Bra B, Cyc B, CA,

248

iso-CA, CA-2H and IS were around 4.5, 4.8, 4.7, 5.1, 6.0, 5.3 and 4.2 min,

249

respectively.

250

An API 4000 triple quadrupole tandem mass spectrometer (Applied Biosystem

251

/MDS SCIEX, Foster City, CA, USA) with Turboionspray source (TIS) was operated

252

in positive ion mode. Quantification was performed using multiple reactions



253

monitoring (MRM) method. The MRM transitions and compound-dependent

254

parameters for ASIV and its metabolites are listed in Table 1. The main working

255

parameters were set as follows: ionspray voltage, 4.5 kV; ion source temperature,

256

350°C; gas1, 40 psi; gas2, 45 psi; curtain gas, 15 psi. Analyte concentrations were

257

determined using the software Analyst 1.5.

258

The assay of ASIV and CA was validated according to the guidance for

259

bioanalytical method validation.32 The calibration curves were constructed in

260

concentration ranges of 2.55−510 nM for ASIV and 1.02−204 nM for CA with good

261

linearity showing correlation coefficients (r) better than 0.993. Without the standard

262

of iso-CA, the amount of iso-CA was calculated from the standard curves of CA. The

263

same method was applied by Zhou et al. to investigate the pharmacokinetics of

264

iso-CA in rats after an oral dose of 40 mg/kg ASIV.13 The intra- and inter-day

265

precisions were lower than 13.6% for ASIV and CA and the accuracy were within

266

±12.1% deviation from the nominal values of the analytes. Due to the lack of

267

standards of Bra B and Cyc B, their concentrations were expressed as peak area ratios

268

of analyte/IS in the study.

269

Data analysis

270

The pharmacokinetic parameters of ASIV, CA and iso-CA were estimated by

271

non-compartmental method using DAS 3.2.0 pharmacokinetic program (Chinese

272

Pharmacology Society) because non-compartmental model is a robust population

273

model and it provides additional information on the random variation component

274

existing between animals. The maximum plasma concentrations (Cmax) and the


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275

corresponding peak time (Tmax) were observed from the individual drug plasma

276

concentration-time profile. The terminal elimination rate constant (ke) was estimated

277

by log-linear regression of concentrations observed during the terminal phase of

278

elimination. The elimination half-life (t1/2) was calculated as 0.693/ke. The area under

279

the plasma concentration–time curve was calculated by the linear trapezoidal rule.

280

Data are reported as the mean±SD. Independent Student’s t test and one-way

281

ANOVA with Tukey-Kramer multiple comparison (post hoc) tests (Minitab. Version

282

14th) were used to evaluate statistical differences. Differences were considered

283

statistically significant when p values were less than 0.05.

284



285

RESULTS AND DISCUSSION

286

Pharmacokinetic study

287

Oral administration

288

The mean plasma concentration-time curves of ASIV, CA and iso-CA in rats after

289

an i.g. dose of 10 mg/kg ASIV are shown in Figure 2A. The main pharmacokinetic

290

parameters of ASIV, CA and iso-CA are presented in Table 2. ASIV reached the

291

maximum level (Cmax) of 34.5±20.6 nM at 2.0 h; while its metabolites, CA and

292

iso-CA, achieved their maximum concentrations comparatively late at approximate 8

293

h. As reported by Zhou et al.,13 ASIV predominated in the forms circulated in blood

294

after ingestion. The contribution of ASIV, CA and iso-CA to the total exposure (the

295

sum of AUC values of ASIV, CA and iso-CA) were 51.0%, 12.5% and 36.5%,

296

respectively (Table 2). No other metabolites of ASIV were observed in rat plasma.

297

Multiple peaks often appear in plasma concentration-time curves of xenobiotics

298

with enterohepatic circulation. However, there was not a secondary peak in the

299

concentration-time curve of ASIV. This phenomenon was probably resulted from the

300

slow and approximately uniform absorption of ASIV from small intestine, because

301

ASIV showed similar low absorption rates in rat duodenum, jejunum and ileum.32

302

Intravenous administration

303

Pharmacokinetics of ASIV and its metabolites in rats were studied after an i.v.

304

injection of 1.5 mg/kg ASIV. The mean plasma concentration-time curves of ASIV,

305

CA and iso-CA are shown in Figure 2B. The corresponding pharmacokinetic

306

parameters are summarized in Table 2. When rats received an i.v. injection of ASIV,


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307

ASIV traveled in rat systemic circulation mainly as itself. Very tiny amounts of CA

308

and iso-CA were detected in rat plasma from 4 h after administration. The

309

contribution of ASIV, CA and iso-CA to the total exposure were 99.7%, 0.05% and

310

0.3%, respectively (Table 2). Compared with the corresponding data obtained after i.g.

311

administration, the contribution of CA and iso-CA to the total exposure decreased

312

more than 100 times after i.v. injection (0.05% vs 12.5% for CA and 0.3% vs 36.5%

313

for iso-CA), suggesting that intestinal tract might contribute more than liver to the

314

biotransformation of ASIV.

315

Enterohepatic circulation experiment in bile-duct cannulation rat model

316

Oral administration experiment

317

To investigate the effect of enterohepatic circulation on the disposition of ASIV, we

318

compared the plasma distribution of ASIV and its metabolites between rats with and

319

without bile duct-cannulation (control) at 2, 8 and 10 h after administration of ASIV

320

from duodenum. Time points of 2, 8 and 10 h were the Tmax values of ASIV, iso-CA

321

and CA, respectively, as shown in the pharmacokinetic study of ASIV after oral

322

administration (Figure 2A and Table 2).

323

The plasma concentrations of ASIV, CA and iso-CA in control rats at 2 h were

324

29.5±10.1 nM, undetectable and undetectable, respectively; at 8 h were 2.68±2.26 nM,

325

undetectable and 2.64±0.89 nM, respectively; and at 10 h were undetectable,

326

5.05±2.53 nM and 9.37±4.23 nM, respectively. After rats received bile duct drainage

327

surgery, the plasma amount of ASIV at 2 h (8.51±3.91 nM) was significantly lower

328

than that in control rats at the same point (Figure 3). Moreover, the amounts of CA



329

and iso-CA in bile duct-cannulated rats became immeasurable at 8 and 10 h (Figure

330

3). These data declared that enterohepatic circulation played an important role in the

331

disposition of ASIV after oral administration.

332

Biliary excretion of ASIV and its metabolites were assayed to partly profile the

333

enterohepatic circulation process of ASIV. Figure 4 shows the bile excretion of ASIV,

334

Bra B and Cyc B in rats after duodenal administration of ASIV. Due to the lack of the

335

standards of Bra B and Cyc B, their excretion concentrations were expressed as peak

336

area ratio of analyte/IS (Figure 4B). The biliary excretion of ASIV reached a plateau

337

at 3 h after dosing (Figure 4A). The accumulative bile excretion of ASIV in 10 h,

338

calculating as [(amount of ASIV in bile/dose)×100], was 0.015%.

339

Although obvious amounts of ASIV, Bra B and Cyc B were excreted from bile after

340

rats received ASIV from duodenum, the biliary excretion amounts of CA, iso-CA and

341

CA-2H were not detectable. Zhou et al. found that when 5 µM ASIV was incubated

342

with rat intestinal bacteria, Bra B and Cyc B were formed after 0.5 and 8 h incubation,

343

respectively; while CA, iso-CA and CA-2H were not found in the first 8 h.13 This

344

result suggested that the formation rates of Bra B and Cyc B from ASIV by intestinal

345

microbiota were faster than those of CA, iso-CA and CA-2H. Basing on the proposed

346

metabolic pathway of ASIV (Figure 1), the undetectable biliary excretion of CA,

347

iso-CA and CA-2H was probably resulted from the slow hydrolysis of ASIV, Bra B

348

and Cyc B in intestine and/or liver.

349

Intravenous administration experiment

350

In the present study, we also compared the plasma distribution of ASIV and its


Page 18 of 48

Page 19 of 48


351

metabolites between bile duct-cannulated rats and control rats after i.v. injection.

352

Neither CA nor iso-CA was found in control and bile duct-cannulated rats at 2, 8 and

353

10 h after i.v. injection. The plasma concentrations of ASIV in control rats at 2, 8 and

354

10 h were 1292±198, 133±26 and 36.5±10.9 nM, respectively. The amount of ASIV in

355

the bile duct-cannulation group of rats was kept at a similar level to that in the control

356

group at the same time point, which was 1263±230 nM at 2 h, 136±19 nM at 8 h and

357

37.2±9.8 nM at 10 h.

358

Bile sample assay revealed that no metabolites (including Bra B, Cyc B, CA,

359

iso-CA and CA-2H) except ASIV were excreted from bile after i.v. injection of ASIV.

360

The accumulative bile excretion of ASIV in 10 h after i.v. adminstration was 27.4%.

361

Although more than one fourth of the dosage was excreted from bile in 10 h, the

362

plasma level of ASIV after i.v. injection was not affected by bile duct drainage surgery.

363

These data were a more proof for the limited adsorption of ASIV in intestine. In

364

addition, the low accumulative bile excretion of ASIV after duodenal administration

365

(0.015%) was also believed to come from the poor intestinal absorption of ASIV. Bra

366

B and Cyc B, two deglycosylated metabolites of ASIV, secreted from bile when rats

367

received ASIV through duodenum (Figure 3); however, they were not found in bile

368

when rats were given ASIV intravenously. This result indicated that intestinal

369

hydrolysis of ASIV was critical for the formation of Bra B and Cyc B.

370

After summarizing the results of enterohepatic circulation experiments, we

371

proposed that ASIV was first slowly hydrolyzed to Bra B and Cyc B by gut

372

microbiota; these compounds then distributed in liver via portal vein and quickly



Page 20 of 48

373

excreted from bile. CA was formed from the repeated hydrolysis of ASIV, Bra B and

374

Cyc B by gut microbiota via enterohepatic circulation; and iso-CA were generated

375

from CA by gut microbiota and/or hepatic enzymes (Figure 5). To test and

376

supplement our hypotheses, we investigated the metabolism of ASIV and CA by rat

377

liver S9 fraction, intestinal S9 fraction and intestinal microbiota.

378

In vitro metabolism of ASIV and CA by rat liver and intestinal S9 fraction

379

In vitro hepatic and intestinal enzyme metabolism of ASIV and CA were studied

380

using rat liver and intestinal S9 fraction in presence of NADPH. ASIV was

381

comparatively stable to rat liver and intestinal S9 fraction. After ASIV (1 µM) was

382

incubated with rat liver or intestinal S9 fraction at 37°C for 1 h, none of the

383

metabolites, such as Bra B, Cyc B, CA, iso-CA and CA-2H, was observed in the

384

incubation samples, which was agree with the reported data obtained from rat liver

385

microsome.13

386

Iso-CA was one of the major components circulated in rat blood after oral

387

administration of ASIV. It was generated from CA via CA-2H.13 Therefore, we

388

investigated the metabolism of CA in rat liver and intestinal S9 fraction to illuminate

389

the role of liver and intestine in the formation of iso-CA. It was reported that CA was

390

extensively

391

mono-hydroxylated metabolites as well as a few hydroxylated and dehydrogenated

392

metabolites.24 In our study, CA underwent moderate metabolism in rat liver S9

393

fraction but was hardly biotransformed by intestinal S9 fraction. When CA was

394

incubated with rat liver S9 fraction at 37°C for 1 h, approximate 25% of CA was

metabolized

by

rat

liver

microsome


and

produced

several

Page 21 of 48


395

metabolized, among which 6% was converted to iso-CA. CA-2H as well as two

396

hydroxylated metabolites of CA and two hydroxylated and dehydrogenated

397

metabolites of CA was also observed in rat liver S9 fraction (data not shown).

398

In vitro metabolism of ASIV and CA by rat intestinal bacteria

399

The metabolic profile of ASIV against incubation time in rat intestinal bacteria is

400

plotted in Figure 6. To intuitively compare the metabolism rate of substrate and the

401

generation rates of metabolites, the remaining amount of ASIV and the formation

402

amounts of Bra B, Cyc B, CA, iso-CA and CA-2H in incubation solution were

403

expressed as percentage of the initial amount of ASIV before incubation. In spite of

404

the lack of the standards of Bra B, Cyc B and CA-2H, we calculated their amounts by

405

the following method basing on the observation that there were no other metabolites

406

of ASIV after incubation except Bra B, Cyc B, CA, iso-CA and CA-2H, and hence the

407

metabolized amount of ASIV equaled the total generation amount of the five

408

metabolites. After 24 h incubation, only ASIV, CA, iso-CA and CA-2H were detected

409

in incubation solution. The amounts of ASIV, CA and iso-CA were determined from

410

the calibration curves of ASIV and CA, respectively. The formation amount of CA-2H

411

at this time point (24 h) was calculated by (100% − remaining amount of ASIV% −

412

formation amount of CA% − formation amount of iso-CA%), which was 36.3%. Then

413

the formation amount of CA-2H at other time points were calculated by comparing

414

their peak area ratio to the peak area ratio at 24 h. By a similar method, we figured out

415

the generation amounts of Bra B and Cyc B at each time point. The generation curves

416

of Bra B, Cyc B, CA-2H plotted by this way were not very accurate because of



417

measurement and calculation errors, but this method was convenient to illuminate the

418

metabolic behavior of ASIV in gut bacteria.

419

ASIV was slowly metabolized by intestinal microbiome. After 4 h incubation,

420

ASIV began to be converted to Bra B, Cyc B, CA and CA-2H by intestinal bacteria.

421

The formation rates of Bra B, Cyc B, CA and CA-2H after 4 h incubation were

422

3.47±0.29%, 1.87±0.19%, 1.69±0.15% and 0.59±0.06%, respectively, suggesting that

423

ASIV was metabolized to Bra B by intestinal bacteria faster than to other metabolites.

424

This result was coincident with the observation of Zhou et al.13 The remaining amount

425

of ASIV in incubation solution linearly declined along with time from 4 to 24 h. After

426

24 h incubation, about 72% of ASIV was metabolized by gut bacteria, among which

427

36% was converted to CA, 35% to CA-2H, and 1% to iso-CA%. Given that iso-CA

428

was generated from CA via CA-2H, formation time-courses of the metabolites of

429

ASIV indicated that the conversion from CA-2H to iso-CA seemed to be the

430

rate-limiting step for the generation of iso-CA in intestine.

431

When 1 µM CA reacted with intestinal bacteria, its metabolize rate was much

432

slower than that of ASIV (Figure 7). No other metabolites except CA-2H and iso-CA

433

were found in the intestinal bacteria incubation samples of CA. The total

434

biotransformation amount of CA was less than 30% after 48 h incubation, among

435

which around 3.5% was converted to iso-CA (Figure 7). The generation amount of

436

CA-2H was obtained by (100% − remaining amount of CA% −formation amount of

437

iso-CA%). Although the production amount of CA-2H was more than 15% after 12 h

438

incubation, the formation amount of iso-CA was less than 1.5%, which further


Page 22 of 48

Page 23 of 48


439

confirmed that the formation rate of iso-CA in intestine depends on the conversion

440

rate of CA-2H.

441

Taken together, in vitro metabolism of ASIV in S9 fraction and in gut microbiota

442

showed that ASIV was not metabolized by liver and intestinal S9 fraction but it was

443

slowly hydrolyzed by gut bacteria. This result demonstrated that deglycosylation of

444

ASIV in intestine tract was a crucial step for the formation of CA. Although intestine

445

is likely to be the more important organ responsible for the disposition of ASIV, in

446

vitro metabolism data of CA suggested that liver might have a bigger role in the

447

generation of iso-CA because the formation rate of iso-CA from CA was faster in liver

448

S9 fraction (6% in 1 h) than that in intestinal microbiome (3.5% in 48 h).

449

Effect of activity of intestine microbiota on the disposition of ASIV

450

Comparation of metabolism of ASIV by intestinal microbiota from rats pretreated with

451

and without antibiotics

452

Hydrolysis of ASIV by gut microbiota is a key factor in the disposition of ASIV

453

after oral administration. To investigate the influence of activity of intestine

454

microbiota on the deglycosylation of ASIV, in vitro gut microbial conversion of ASIV

455

were compared between the intestinal microbiota obtained from antibiotic-pretreated

456

rats and that from control rats by the method as described in “Materials and Methods”.

457

Antibiotic-pretreated models represent one relatively simple strategy to study the role

458

of intestinal bacteria in xenobiotic metabolism, although antibiotics cannot eliminate

459

all microorganisms from the intestine. Streptomycin sulfate and bacitracin are the

460

common antibiotics applied to attenuate the activity of intestinal bacteria.33,34



461

The metabolic behaviors of ASIV in control gut bacteria and antibiotic-pretreated

462

intestinal bacteria are shown in Figure 8. To intuitively contrast the difference in the

463

metabolism of ASIV between control and antibiotic-pretreated gut microbiota, the

464

amounts of ASIV, Bra B, Cyc B, CA, iso-CA and CA-2H were expressed as peak area

465

ratio of analyte/IS. When ASIV (20 µM) was incubated with control gut bacteria at

466

37°C for 12 h, around 30% of ASIV was metabolized (Figure 6). Antibiotics

467

obviously inhibited the metabolism of ASIV by intestinal microbiome. No metabolites

468

of ASIV were observed in antibiotic-pretreated gut microbiota samples over the same

469

time period (12 h, Figure 8A). After 24 h incubation, more than 70% of ASIV was

470

converted in control intestinal microbiota (Figure 6), while only trace of Bra B was

471

produced from ASIV by antibiotic-pretreated gut bacteria (Figure 8B).

472

To assess potential interference of antibiotics to the hydrolysis of ASIV by

473

intestinal microbiota, ASIV was simultaneously incubated with low, middle and high

474

concentrations of streptomycin sulfate and bacitracin (1:1, w/w) in control gut

475

bacteria at 37°C for 4 h. The generation amounts of Bra B, Cyc B and CA were

476

concentration-independently decreased by antibiotics (Figure 9). It seemed that

477

antibiotics interfered with the hydrolysis of ASIV. However, with further investigation,

478

we believed that the decreased generation by antibiotics was mainly resulted from the

479

bacteriostasis action of antibiotics rather than its interference to the biotransformation

480

of ASIV, because microbial culture experiment showed that the colony amount of gut

481

bacteria in presence of antibiotics was obviously less than that in absence of

482

antibiotics after 4 h incubation (data not shown). As shown in Figure 9, the formation


Page 24 of 48

Page 25 of 48


483

rate of CA-2H was not affected by antibiotics. It is well-known that there are a lot of

484

species of microorganisms existing in mammal intestine. The unchanged formation

485

rate of CA-2H was probably caused by the fact that the microbiotas responsible for

486

the biotransformation from CA to CA-2H were not suppressed by streptomycin sulfate

487

and bacitracin.

488

Comparation of disposition of ASIV in rats pretreated with and without antibiotics

489

In vivo experiment is the ultimate model to study the effect of intestinal microbiota

490

on xenobiotic disposition. In the present study, we compared the pharmacokinetics of

491

ASIV and its metabolites in rats treated with and without antibiotics. After rats

492

received antibiotics for 6 days, they were given 10 mg/kg ASIV through gavage. The

493

mean

494

antibiotic-pretreated rats are shown in Figure 2C. The Cmax, Tmax, t1/2 and AUC0-t

495

values of ASIV in antibiotic-pretreated rats were 32.4±7.8 nM, 2.83±0.98 h,

496

1.99±0.33 h, 135±30 nM·h, respectively (Table 2). Pharmacokinetic profile of ASIV

497

in antibiotic-pretreated rats was similar to that in control rats (Figure 2A and C),

498

which might be caused by passive diffusion of ASIV in intestinal absorption.16

499

Nevertheless, the plasma concentrations of CA in antibiotic-pretreated rats were

500

immeasurable. Moreover, the Cmax (4.41±4.01 nM) and AUC0-t (16.4±15.0 nM·h)

501

values of iso-CA in antibiotic-pretreated rats were significantly less than the

502

corresponding data obtained from control rats (Table 2). The result indicated that the

503

disposition of ASIV was obviously affected by the activity of intestinal bacteria after

504

oral administration. Antibiotic treatments altered intestinal microbiota composition of

plasma

concentration-time

curves

of

ASIV,


CA

and

iso-CA

in


505

rodents.35,36 Streptomycin and bacitracin inhibited the activity of Lactobacillaceae

506

which contains species capable of hydrolyzing saponins.37,38 This might be one of the

507

reasons why the exposure of CA and iso-CA in antibiotic-pretreated rats were lower

508

than those in control rats. The alteration in rat microbial composition caused by

509

streptomycin and bacitracin and the bacteria species involved in the hydrolysis of

510

ASIV need to be further investigated.

511

Genetics, infant feeding patterns, antibiotic usage, sanitary living conditions and

512

long term dietary habits are known to shape the composition of the gut microbiome.39

513

Gut microbiota affects the metabolism of some natural compounds and synthetic

514

drugs and resultantly impacts on their bioactivity or toxicity.18,19,40-42 Our study

515

showed that the metabolism of ASIV by intestinal microbiome was a crucial step in

516

the disposition of ASIV, rather than by hepatic and intestinal enzymes. Variations in

517

intestinal microbiota induced by physiological or pathological conditions may change

518

the disposition of ASIV and subsequently influent the potential health benefits of

519

ASIV. Therefore, gut microbiota should be considered as a critical factor for

520

understanding the bioactivities of ASIV.

521

ACKNOWLEDGEMENT

522 523

We would like to thank Dr. Jinghua Xu in Shenyang Pharmaceutical University for her invaluable work and advice in the treatment of animals.

524


Page 26 of 48

Page 27 of 48


525

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Figure captions

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Figure 1. Proposed metabolic pathway of ASIV in rat intestinal bacteria.13 ASIV:

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astragaloside; Bra B: brachyoside B; Cyc B: cyclogaleginoside B; CA:

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cycloastragenol; iso-CA: iso-cycloastragenol; CA-2H: dehydrogenated metabolite of

672

CA.

673 674

Figure 2. Mean plasma concentration-time curves of ASIV, CA, iso-CA in control rats

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after an intragastrical (i.g.) administration of 10 mg/kg ASIV (A) or an intravenous

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(i.v.) administration of 1.5 mg/kg ASIV (B) and in antibiotic-pretreated rats after an

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i.g. administration of 10 mg/kg ASIV (C). Each point represents the mean±SD (n =

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6).

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Figure 3. Comparison of plasma concentrations of ASIV, CA and iso-CA between rats

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treated with and without bile-duct cannulation after administration of 10 mg/kg ASIV

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from duodenum. The concentrations of ASIV, CA and iso-CA in control rats were

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labeled as “ASIV”, “CA” and “iso-CA”, respectively; and those in cannulated rats

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were labeled as “ASIV_BDC”, “CA_BDC” and “iso-CA_BDC”, respectively. Each

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point represents the mean±SD (n = 6). The symbols “*” mean the differences of

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plasma concentration between control and cannulated rats are significant according to

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Student’s t test (p

Disposition of Astragaloside IV via Enterohepatic Circulation Is

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