Absorption and Metabolic Behavior of Hesperidin (Rutinosylated

Subscriber access provided by RUTGERS UNIVERSITY

Bioactive Constituents, Metabolites, and Functions

Absorption and Metabolic Behavior of Hesperidin (Rutinosylated Hesperetin) after Single Oral Administration to Sprague-Dawley Rat Alexia Nectoux, Chizumi Abe, Shu-Wei Huang, Naoto Ohno, Junji Tabata, Yuji Miyata, Kazunari Tanaka, Takashi Tanaka, Haruo Yamamura, and Toshiro Matsui J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03594 • Publication Date (Web): 08 Aug 2019 Downloaded from pubs.acs.org on August 9, 2019

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

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

Page 1 of 32

Journal of Agricultural and Food Chemistry

Absorption and Metabolic Behavior of Hesperidin (Rutinosylated Hesperetin) after Single Oral Administration to Sprague-Dawley Rat

Alexia M. Nectoux,† Chizumi Abe,† Shu-Wei Huang,† Naoto Ohno,† Junji Tabata,† Yuji Miyata,‡ Kazunari Tanaka,§ Takashi Tanaka,∥ Haruo Yamamura,∬ and Toshiro Matsui*,†

†Department

of

Bioscience

and

Biotechnology,

Division

of

Bioresource

and

Bioenvironmental Sciences, Faculty of Agriculture, Graduated School of Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan ‡Industrial

Technology Center of Nagasaki, 2-1303-8 Ikeda, Ohmura, Nagasaki, 856-0026,

Japan §Department

of Nutrition, University of Nagasaki, 1-1-1Manabino, Nagasaki 851-2195,

Japan ∥Graduate

School of Biochemical Science, Nagasaki University, 1-14 Bunkyo-machi,

Nagasaki 852-8521, Japan ∬Charle

Co., 3-1-2 Yasakadai, Kobe, Hyogo 654-0192, Japan

*Correspondence author *Tel : +81-928024752. Fax : +81-928024752. E-mail: [email protected].

1

ACS Paragon Plus Environment


Page 2 of 32

1

ABSTRACT

2

We

3

(hesperetin-7-O-rutinoside) in the blood system of Sprague-Dawley rats by liquid

4

chromatography- and matrix-assisted laser desorption ionization mass spectrometries

5

(LC-MS and MALDI-MS). After a single oral administration of hesperidin (10 mg/kg),

6

which was expected to be absorbed in its degraded hesperetin form, we detected intact

7

hesperidin in the portal vein blood (tmax, 2 h) for the first time. We successfully detected

8

glucuronized hesperidin in the circulating bloodstream, while intact hesperidin had

9

disappeared. Further MS analyses revealed that homoeriodictyol and eriodictyol conjugates

10

were detected in both portal and circulating blood systems. This indicated that hesperidin

11

and/or hesperetin are susceptible to methylation and demethylation during the intestinal

12

membrane transport process. Sulfated and glucuronized metabolites were also detected in

13

both blood systems. In conclusion, hesperidin can enter into the circulating bloodstream in

14

its conjugated forms, together with the conjugated forms of hesperetin, homoeriodictyol,

15

and/or eriodictyol.

investigated

the

absorption

and

metabolic

behavior

of

hesperidin

16 17

KEYWORDS: Hesperidin; Hesperetin; Absorption; Metabolism; LC-MS; MALDI-MS

2


Page 3 of 32


18

INTRODUCTION

19

Hesperidin (hesperetin-7-O-rutinoside), a flavanone-type flavonoid abundant in citrus

20

fruits,1 was found to possess various physiological activities. Both in vitro and in vivo

21

studies highlighted anti-inflammatory and anti-cancer effects induced by hesperidin.1-4

22

Among others, hesperidin showed to inhibit tumors growth1,2 and to inhibit kinases and

23

phosphodiesterases, which have a role during inflammation response by cellular signal

24

transduction and activation process.1 Hesperidin intake was also reported to restore the

25

lipidemic levels on plasma, cardiac, and hepatic lipids in isoproterenol-induced rats,5 and to

26

show a dose dependent analgesic effect on mice.6 Its aglycone, hesperetin, also shows

27

physiological effects such as cellular antioxidant defense.3,7 Human clinical trials have

28

provided evidence that daily intake of hesperidin (500 mg/day) for 3 weeks had an

29

anti-inflammatory effect on impaired vessel functions by increasing nitrogen monoxide

30

production and by suppressing circulating inflammatory biomarker production.8,9

31

Hesperidin

32

hesperetin-7-O-glucuronide, were also found to induce hypotensive, vasodilatory, and

33

anti-inflammatory activities on spontaneously hypertensive rats10 while hesperetin was

34

found to improve glucose utilization in amyloid β-impaired neuronal cells.11

metabolites

resulting

from

hesperidin

intake,

including

35

Irrespective of the in vitro compatible physiological potential of both hesperidin

36

and hesperetin, several in vivo bioavailability studies have focused on the pharmacokinetics

37

of hesperetin; hesperidin was believed to be susceptive to microbial hydrolysis in the gut,

38

degrading it to its metabolites prior to intestinal absorption.12-14 The absence of hesperidin, 3



Page 4 of 32

39

in either human plasma or urine after oral intake of an orange beverage containing

40

hesperidin,13-15 supported the above-speculation that ingested hesperidin is degraded into

41

hesperetin to produce its related metabolites during the absorption process.

42

Thus far, hesperetin has been targeted for metabolism and pharmacokinetic

43

studies. It has been reported that in transport experiments using Caco-2 cell monolayers,

44

hesperetin

45

hesperetin-7-O-sulfate, and some of it is also pumped out through the breast cancer

46

resistance protein (BCRP) efflux route,16 whereas some hesperetin was transported intact

47

via the transcellular route.17 In in vivo hesperidin ingestion experiments, targeted hesperetin

48

metabolites

49

homoeriodictyol could be detected in the blood of rats,1,14 and hesperetin-7-O-glucuronide,

50

hesperetin-3’-O-glucuronide, hesperetin-sulfate, and hesperetin-sulfoglucuronide in the

51

blood of humans.18,19 Although most reports have focused on hesperetin metabolites, the

52

absorption of hesperidin (hesperetin-7-O-rutinoside) in its intact form or its metabolites in

53

the blood system is also important, since hesperidin itself can penetrate across Caco-2 cell

54

monolayers.17 Thus, this study aimed to clarify the in vivo absorption and metabolic

55

behavior of hesperidin, in the portal blood and circulating blood systems, by using

56

liquid-chromatography

57

matrix-assisted

58

investigation in both blood systems could help further our understanding of the role that

59

intestine and liver organs play in hesperidin metabolism after oral ingestion.

is

mostly

such

as

laser

metabolized

into

hesperetin-7-O-glucuronide

hesperetin-7-O-glucuronide,

time-of-flight desorption

mass

ionization

hesperetin-3’-O-glucuronide,

spectrometry (MALDI)-MS

4


(LC-TOF-MS) techniques,

since

and

and

and the

Page 5 of 32


60 61

MATERIALS AND METHODS

62

Chemicals. Hesperidin was purchased from Funakoshi Co. (Tokyo, Japan).

63

Hesperetin and eriodictyol were purchased from Sigma-Aldrich (St. Louis, MO, USA).

64

Homoeriodictyol was purchased from ChromaDex Inc. (Irvine, CA, USA). Taxifolin was

65

purchased from TCI Fine Chemicals (Tokyo, Japan). Sulfatase type H-1 (EC 3.1.6.1, from

66

Helix pomatia), β-glucuronidase type B-1 (EC 3.2.1.31, from bovine liver), and

67

β-glucuronidase type H-1 (EC 3.2.1.31, from Helix pomatia) were purchased from

68

Sigma-Aldrich (St. Louis, MO, USA). Other chemicals were of analytical grade and used

69

without further purification.

70

Animal Experiments. Seven week-old male Sprague-Dawley (SD) rats supplied

71

by Charles River Japan Co. (Kanagawa, Japan) were used for single oral administration

72

experiments of hesperidin. Rats were acclimated to laboratory conditions for 1 week prior

73

to the experiment; housed in an air-conditioned room (21 ± 1 °C, 55.5 ± 5% humidity)

74

under a 12 h dark/light cycle, with free access to distilled water, and standard MF diet

75

(Oriental Yeast Co., Tokyo, Japan). Rats fasted for 16 h prior to the single oral

76

administration of hesperidin. All animal experiments were carried out in accordance with

77

the guidelines set by the Guidance for Animal Experiments in the Faculty of Agriculture

78

and in the Graduate Course of Kyushu University in accordance with the Law (No. 105,

79

1973) and Notification (No. 6, 1980, of the Prime Minister’s Office) of the Japanese

80

Government. All experimental protocols were reviewed and approved by the Animal Care 5



81

Page 6 of 32

and Use Committee of Kyushu University (permit number: A30-015-4).

82

Administration experiments. On the day of the experiment, a solution containing

83

10 mg/kg or 100 mg/kg hesperidin, dissolved in saline solution containing 0.5%

84

carboxymethyl cellulose, was orally administered to SD rats. At 0, 1, 2, 4, 8, 16, and 24 h

85

after administration, blood was obtained in EDTA-2Na blood collection tubes, from either

86

the portal vein or the abdominal aorta (n = 3 for at every scheduled time). Blood samples

87

were then centrifuged at 3,500 × g for 15 min at 4 °C to obtain plasma. Samples were

88

immediately frozen in liquid nitrogen, and stored at -80 °C.

89

Stability experiments of hesperidin against deconjugation enzymes. Sulfatase

90

type H-1 from Helix pomatia, β-glucuronidase type B-1 from bovine liver, and

91

β-glucuronidase type H-1 from Helix pomatia were used as deconjugation enzymes. Plasma

92

spiked

93

(hesperetin-7-O-rutinoside) itself was stable against the above-mentioned deconjugation

94

enzymes. Hesperidin (5 μM) was added to blank rat plasma (200 μL) and mixed with the

95

same volume of 100 mM sodium acetate buffer (pH 4.0). The mixture was then incubated

96

with sulfatase type H-1 (25 units), β-glucuronidase type H-1 (50 units), or β-glucuronidase

97

type B-1 (50 units) for 12 h at 37 °C. After the addition of 400 μL ethanol to the reaction

98

solution, the solution was centrifuged at 14,000 × g for 10 min at 25 °C to obtain the

99

supernatant. The supernatant was then applied to an Amicon Ultra-0.5 centrifugal filter

100

(molecular cut < 3,000 Da, Merck Millipore Ltd., Darmstadt, Germany). The filtrate,

101

obtained by centrifugation at 14,000 × g for 30 min at 25 °C, was evaporated to dryness.

with

hesperidin

was

examined

to

determine

6


whether

hesperidin

Page 7 of 32


102

The dried filtrate was dissolved in 50 μL of 50% ethanol, filtrated using a 0.45 μm

103

Millex-LH Syringe Driven Filter Unit (Millex, Tokyo, Japan), and then subjected to

104

LC-TOF-MS analysis.

105

Quantification of hesperidin and hesperetin in plasma by LC-TOF-MS. For

106

the determination of hesperidin and hesperetin in rat plasma, 600 μL of plasma was mixed

107

with the same volume of 100 mM sodium acetate buffer (pH 4.0), following the addition of

108

10 μL of 5.0 μM taxifolin as the internal standard (IS). The solution without enzymatic

109

treatment was used for the determination of intact hesperidin/hesperetin. The solution with

110

β-glucuronidase type B-1 (50 units) alone or β-glucuronidase type B-1 (50 units)/sulfatase

111

type H-1 (25 units) treatment, held for 12 h at 37 °C, was used for the determination of

112

glucuronized hesperidin/hesperetin or the conjugated forms estimated as hesperetin,

113

respectively. This solution was then deproteinated with 800 μL of ethanol, followed by

114

centrifugation at 14,000 × g for 10 min at 25 °C. The obtained supernatant was applied to

115

Millipore Amicon Ultra-0.5 centrifugal filter and centrifuged at 14,000 × g for 30 min at

116

25 °C. The filtrate was then evaporated to dryness, and dissolved in 50 μL of 50% ethanol.

117

Quantification analyses by LC-TOF-MS were performed using IS-aided (1.0 nmol/mL)

118

calibration curves of y = 0.020x + 0.0291 (r = 0.993; limit of detection, 3 pmol/mL) for

119

heperidin, y = 0.039x + 0.3681 (r = 0.997, limit of detection, 7 pmol/mL) for hesperetin, y =

120

0.0237x – 0.0229 (r = 0.996; limit of detection, 0.8 pmol/mL) for homoeriodictyol, and y =

121

0.100x – 0.0219 (r = 0.996; limit of detection, 3 pmol/mL) for eriodictyol, where y is the

122

peak area ratio (observed peak area against that of IS) and x is the concentration 7



123

Page 8 of 32

(nmol/mL).

124

LC-TOF-MS analysis. LC separation was performed with an Agilent 1200 series

125

HPLC (Agilent Technologies, Waldbronn, Germany) on a Cosmosil 5C18-MS-II column

126

(2.0 × 150 mm, Nacalai Tesque Inc., Kyoto, Japan) at 40 °C. The mobile phase consisted of

127

0.1% formic acid (solvent A) and methanol with 0.1% formic acid (solvent B) using a 20

128

min linear gradient from 0 to 100% of solvent B at a flow rate of 0.2 mL/min. TOF-MS

129

analysis was carried out using a micrOTOF-II mass spectrometer (Bruker Daltonics,

130

Bremen, Germany) in electrospray ionization (ESI)-negative mode. Mass spectral data was

131

collected within the range of 100–1,000 m/z. Hesperidin, hesperetin, and taxifolin (IS) were

132

detected at [M-H]‾ of 609.1814 m/z, 301.0707 m/z, and 303.0499 m/z, respectively, with a

133

width of 0.05 m/z. The TOF-MS conditions were as follows: nebulizer pressure, 1.6 bar;

134

dry gas, nitrogen; dry gas flow, 8.0 L/min; drying temperature, 200 °C; capillary voltage,

135

3800 V. The calibration solution of 10 mM sodium formate in 50% acetonitrile was

136

injected at the beginning of the run. The data was analyzed and acquired by Bruker Data

137

Analysis 4.0 software.

138

MALDI-MS analysis. Hesperidin and hesperetin metabolites in plasma without

139

deconjugation treatment were analyzed using MALDI-MS technique. The afore-mentioned

140

filtrate sample of rat plasma without any enzyme was used for the analysis. MALDI-MS

141

measurements were performed using an Autoflex III MS equipped with SmartBeam III

142

(Bruker

143

1,5-diaminonaphthalene (1,5-DAN), was dissolved in 70% acetonitrile at a concentration of

Daltonics)

in

negative

ion

linear

mode.

8


The

matrix

reagent,

Page 9 of 32


144

10 mg/mL. MS data was acquired in the range of 100 – 1,000 m/z by averaging signals

145

from 2,500 laser pulses. The MS parameters were as follows: ion source 1, 20.00 kV; ion

146

source 2, 18.80 kV; lens voltage, 7.50 kV; gain factor, 5.0; laser frequency, 200 Hz; laser

147

power, 80%.

148

Data Analysis. Results are expressed as the mean ± standard error of the mean

149

(SEM). Pharmacokinetic analysis of concentration-time data was performed using

150

GraphPad Prism software (GraphPad, La Jolla, CA, USA). The maximum plasma

151

concentration (Cmax), time of maximum concentration (tmax), and area under the curve up to

152

24 h (AUC0-24) were obtained from the plasma concentration-time plots.

153 154

RESULTS AND DISCUSSION

155

Stability of hesperidin against deconjugation enzymes. In order to investigate

156

the intact absorption of intact hesperidin in rat blood, MS analysis was used to examine the

157

stability of hesperidin against deconjugation enzymes: sulfatase type H-1, β-glucuronidase

158

type H-1, or β-glucuronidase type B-1. Deconjugation treatment of hesperidin-spiked

159

plasma with sulfatase type H-1 or β-glucuronidase type H-1 caused complete degradation

160

of hesperidin to its aglycone form (hesperetin) (Supplemental Figure S1). This suggests that

161

both enzymes have alternative catalytic abilities to cleave the sugar moiety considering

162

conjugated hesperidin could not be detected when the two type H-1 enzymes were used for

163

deconjugation treatment. In contrast, β-glucuronidase type B-1 treatment of the

164

hesperidin-spiked plasma did not cause any production of hesperetin (Supplemental Figure 9



165

S1), indicating that β-glucuronidase type B-1 can be used for glucuronized conjugation

166

analysis of hesperidin in plasma owing to its characteristic catalytic reaction. Thus in this

167

study, sulfatase type H-1/β-glucuronidase type B-1 treatments of plasma were used to

168

evaluate the total amount of hesperidin absorption as its deconjugated hesperetin form,

169

while β-glucuronidase type B-1 was used to evaluate the glucuronized conjugates.

170

Absorption of hesperidin into portal and circulating blood systems after the

171

oral administration to SD rats. Oral administration of hesperidin at a dose of 10 mg/kg to

172

SD rats revealed for the first time that ingested hesperidin can rapidly enter into the portal

173

blood in its intact form (< 1 h after the administration), while no intact hesperidin was

174

detected in the circulating blood within the present LC-TOF-MS detection limits (>3

175

pmol/mL) (Figure 1A). After hesperidin ingestion, intact hesperetin was detected in the

176

portal blood more slowly than hesperidin (4 h after the administration), but was not

177

detected in the circulating blood either (Figure 1B). This is the first in vivo finding that

178

hesperidin and hesperetin can enter into the portal vein as their intact forms. These results,

179

which concur with previous in vitro findings,17 suggest that hesperidin possibly penetrates

180

through the intestinal membrane, some of which is degraded into hesperetin, and can then

181

be absorbed intact into the portal blood. The delay between hesperidin and hesperetin

182

detection times (1 h and 4 h, respectively) can be explained by the time needed for

183

hesperidin to be hydrolyzed by microbial enzymes prior to intestinal absorption and further

184

metabolism.13,14,18 No MS detection of either intact form in the circulating blood also

185

suggests that intact hesperidin and hesperetin have difficulty penetrating cellular 10


Page 10 of 32

Page 11 of 32


186

membranes, which agrees with previous reports in which no intact hesperidin and

187

hesperetin were detected in blood after hesperidin ingestion.13,14,20,21

188

Figure 1C shows the absorption behavior of hesperidin conjugates, estimated as

189

hesperetin

in

both

portal

and

circulating

blood

treated

with

sulfatase

type

190

H-1/β-glucuronidase type B-1. The conjugated forms of hesperidin and/or hesperetin were

191

gradually detected in both blood systems, until 180 ± 110 pmol/mL-portal plasma and 60 ±

192

27 pmol/mL-circulating plasma 24 h after a single oral administration of hesperidin (10

193

mg/kg). This indicates that the conjugates of hesperidin and/or hesperetin were more stable

194

and predominant in blood systems, compared to intact hesperidin and hesperetin (Figures

195

1A and 1B).

196

Conjugated metabolites were detectable in both blood systems 4 h after

197

administration, with a 16 h tmax, while intact hesperidin and hesperetin reached their

198

maximum concentrations at 2 h and 8 h, respectively. In comparison, it has been reported

199

that hesperetin conjugates were found in rat circulating plasma 6 h after a 610 mg/kg (1

200

mmol/kg) hesperidin oral administration, with a 12 h tmax.13 Although the conjugate

201

concentration in the portal blood was 2×-higher than in the circulating blood, both

202

surprisingly display the same tmax (16 h). One explanation could due be the enterohepatic

203

circulation of hesperidin metabolites, which are excreted into the bile in the proximal

204

intestine, and then reabsorbed and metabolized in the intestinal tract.22 However, further

205

studies on such hesperidin metabolism after oral intake are needed using isotope-labeled

206

hesperidin in the rat bloodstream, for example. 11



207

Metabolic behavior of hesperidin into portal and circulating blood systems

208

after oral administration to SD rats. By considering the characteristic catalysis of

209

β-glucuronidase type B-1 (Supplemental Figure S1), our further metabolic experiments

210

with β-glucuronidase type B-1 evaluated the presence of glucuronized hesperidin and

211

glucuronized hesperetin in the circulating blood. As shown in Figure 2, LC-TOF-MS

212

chromatograms of β-glucuronidase type B-1-treated circulating plasma (16 h after

213

administration) depict the detection of hesperidin (retention time, RT: 22.4 min; [M-H]‾=

214

609.1814 m/z) and hesperetin (RT: 25.1 min; [M-H]‾= 301.0707 m/z), clearly indicating

215

that hesperidin may be absorbed into the circulating bloodstream in its glucuronized form

216

without degradation to its aglycone, along with the absorption of glucuronized hesperetin.

217

Table 1 summarizes the pharmacokinetics of hesperidin, hesperetin, and their

218

conjugates (estimated as hesperetin) in the portal and circulating blood systems, together

219

with glucuronized hesperidin and glucuronized hesperetin in the circulating blood. In the

220

portal blood, hesperidin and hesperetin can be absorbed in their intact forms, but their

221

absorption (area under the curve, AUC0-24) was less than one tenth of the absorption of their

222

conjugates (AUC0-24: 13.5 ± 1.6 nmol·h/mL). Although intact hesperidin and hesperetin

223

were undetectable in the circulating blood, the AUC0-24 of their glucuronized forms was

224

0.13 and 0.93 nmol·h/mL, respectively, contributing to 15% of total hesperidin absorption

225

in the circulating blood. The total absorption of hesperidin (AUC0-24) as hesperetin in the

226

circulating blood was estimated to be half of that in the portal blood. This suggests that

227

other metabolic reactions, except for glucuronidation and sulfonation reactions of 12


Page 12 of 32

Page 13 of 32


228

hesperidin and/or hesperetin (e.g., demethylation,21 methylation,23 sulfoglucuronidation14)

229

may occur before hesperidin enters the circulating bloodstream.

230

Identification of hesperidin metabolites by LC-TOF-MS and MALDI-MS

231

analyses. Further investigation of hesperidin metabolites was performed by LC-TOF-MS

232

and MALDI-MS methods. Although solely hesperidin, hesperetin and their conjugates were

233

detected in our first assay, other studies also highlighted the presence of

234

homoeriodictyol-glucuronide in rat plasma.13,14 Both analytical methods were applied to

235

blood samples taken 16 h after oral administration (tmax of hesperidin conjugates, see Figure

236

1C) of high dose hesperidin (100 mg/kg) from the portal vein and abdominal aorta, for

237

overall identification of hesperidin/hesperetin metabolites in blood. Figure 3 shows the

238

representative LC-TOF-MS chromatograms of standards and plasma samples with and

239

without β-glucuronidase type B-1 treatment. In extracted ion chromatography (EIC)-aided

240

MS detection of hesperetin, no peak corresponded to the ion m/z ([M-H]‾= 301.0707 m/z)

241

in either plasma samples without β-glucuronidase type B-1 treatment; intact hesperetin

242

disappeared due to degradation at 16 h after the administration. In contrast, for

243

β-glucuronidase type B-1-treated plasma samples two distinct peaks with the same m/z

244

were detected by LC-TOF-MS (Figure 3A). Compared to the RT of target hesperetin, the

245

latter peaks (RT: 25.1 min) were identified as hesperetin, indicating that the glucuronized

246

target was present in the portal vein and circulating blood systems, which agrees with the

247

results shown in Figure 2. The RT of the former peak matched that of homoeriodictyol (RT:

248

25.0 min), a transmethylated isomer of hesperetin.14 Figure 3A also shows the presence in 13



249

the portal and circulating blood systems of the possible metabolite eriodictyol ([M-H]‾=

250

287.0561 m/z), a demethylated hesperetin. The presence of homoeriodictyol and eriodictyol

251

conjugates could be due to a transformation of hesperetin to eriodictyol by phase I

252

metabolic demethylation enzymes,23,24 followed by a remethylation into hesperetin or

253

homoeriodictyol by catechol-O-methyltransferase.25 These results suggest that hesperetin

254

from hesperidin was glucuronized during the intestinal absorption process, along with the

255

metabolic conversion to glucuronized eriodictyol/homoeriodictyol (Figure 3B). The plasma

256

level of glucuronized hesperetin conjugate was calculated to be 4×- and 2.5×-higher than

257

the plasma levels of glucuronized eriodictyol and homoeriodictyol conjugates, respectively.

258

Furthermore, hesperidin ([M-H]‾= 609.1814 m/z), demethylated hesperidin ([M-H]‾=

259

595.1657 m/z), and dimethylated hesperidin ([M-H]‾= 623.1970 m/z) were undetectable in

260

the circulating blood treated with β-glucuronidase type B-1 (Supplemental Figure S2).

261

Although the products of demethylation/transmethylation of hesperidin were not excluded,

262

as hesperetin demethylation/transmethylation is known to occur (Figure 3), such hesperidin

263

conjugates would likely not be metabolized, or would be present in lesser amounts in the

264

circulating blood, which could be below the limits of detection for the present LC-TOF-MS

265

conditions.

266

Further identification of metabolites from administered hesperidin (100 mg/kg

267

dose) was conducted using the MALDI-MS technique. Plasma samples of portal and

268

circulating blood without deconjugation treatment were subjected to this assay, since

269

MALDI-MS can detect ionizable targets nonspecifically. As shown in Figure 4, two 14


Page 14 of 32

Page 15 of 32


270

distinct MS signals corresponding to 381.0 m/z and 477.1 m/z were observed in both blood

271

systems that were not observed in the control plasma. Both mass units were in agreement

272

with the m/z values ([M-H]‾) of sulfated and glucuronized hesperetin/homoeriodictyol,

273

respectively. The presence of sulfate conjugates implies a sulfonation reaction, which

274

would occur in the intestinal and liver cells.26 Neither intact nor conjugated hesperidin and

275

eriodictyol were detected by MALDI-MS in the range of 100–1,000 m/z, probably due to

276

their low plasma levels and/or low ionization efficiency against the 1,5-DAN matrix

277

reagent.27,28 Mono-sulfated and mono-glucuronized hesperetin/homoeriodictyol conjugates

278

were prominent metabolites in the circulating bloodstream when hesperidin was

279

administered to SD rats, as has been found in previous studies.14,15,18,19 The

280

above-mentioned metabolite profiles obtained in this study after a hesperidin oral intake

281

was in line with previous findings, displaying that the main forms of hesperidin metabolites

282

were glucuronized or sulfated hesperetin/homoeriodictyol conjugates.14 In addition to the

283

reported profiles, in this study we demonstrated for the first time that hesperidin can cross

284

the intestinal membrane as its intact form and enter into the portal blood system (Figure 1),

285

followed by the glucuronidation (glucuronized hesperidin) before entering into the

286

circulating blood system (Figure 2). Considering the differences between the metabolites

287

identified in the portal and circulating plasma, and the scientific background on hesperidin

288

absorption and metabolism, Figure 5 proposes a hesperidin first-pass metabolism scheme in

289

rat intestine and liver after oral ingestion.

290

LC-TOF-MS sensitivity limited the detection of homoeriodictyol and eriodictyol 15



Page 16 of 32

291

to a 100 mg/kg hesperidin oral administration. Unfortunately, this dose is not realistic for a

292

daily hesperidin intake because one daily glass of orange juice, the main source of

293

hesperidin in the human diet,2,29 could only provide 0.81 mg/kg of hesperidin per day.15,30

294

There is also no guarantee that the metabolic pathway would be similar for both 10 mg/kg

295

and 100 mg/kg of hesperidin, just as (–)-epigallocatechin-3-gallate, a dietary polyphenol, is

296

subject to different metabolic pathways depending on its concentration.31

297

Therefore, further work is needed to gather insight into the quantitative evaluation

298

of hesperidin metabolites in blood, and to clarify their physiological effects. Considering

299

that their distribution in organs has not been, to our knowledge, elucidated yet in the

300

literature, our interest is also to clarify which organs are targeted for the accumulation of

301

absorbed hesperidin metabolites.

302 303

ABBREVIATIONS USED

304

1,5-DAN, 1,5-diaminonaphtalene; AUC, area under the curve; AUC0-24, AUC for

305

24 h; BCRP, breast cancer resistance protein; EIC, extracted-ion-chromatography; ESI,

306

electrospray ionization; GlcA, glucuronic acid; HPLC, high performance liquid

307

chromatography;

308

chromatography-time-of-flight-mass

309

desorption ionization; NO, nitrogen monoxide; RT, retention time; SD, Sprague-Dawley;

310

SULTs, sulfotransferases; UGT, uridine 5’-diphospho-glucuronosyltransferases.

IS,

internal

standard;

spectrometry;

LC-TOF-MS,

MALDI,

16


matrix-assisted

liquid laser

Page 17 of 32


311

ACKNOWLEDGMENTS

312

The authors thank Mrs K. Miyazaki at Kyushu University for her technical assistance.

313 314

ASSOCIATED CONTENT

315

Supporting Information

316

LC-TOF-MS chromatograms of hesperidin and hesperetin, with and without deconjugation

317

enzyme treatments (Figure S1); EIC chromatograms corresponding to [M-H]‾ of

318

demethylated hesperidin and dimethylated hesperidin in portal and circulating blood, with

319

or without β-glucuronidase type B-1 treatment (Figure S2)

320 321

AUTHOR INFOMRATION

322

Correspondence author

323

*Tel : +81-928024752. Fax : +81-928024752. E-mail: [email protected].

324

ORCID

325

Toshiro Matsui: 0000-0002-9137-8417

326

Author Contributions

327

TM devised the study; AN, CA, SH, NO, JT performed the experiments. All authors

328

interpreted and analyzed data. TM designed the study, interpreted and analyzed data. AN

329

and TM wrote the paper. All authors have read and approved the final manuscript to be

330

published.

331

Notes 17



332

The authors declare no competing financial interest.

333

18


Page 18 of 32

Page 19 of 32


334

REFERENCES

335

(1) Barreca, D.; Gattuso, G.; Bellocco, E.; Calderaro, A.; Trombetta, D.; Smeriglio, A.;

336

Laganà, G.; Daglia, M.; Meneghini, S.; Nabavi, S. M. Flavanones: citrus

337

phytochemical with health-promoting properties. BioFactors 2017, 43, 495–506.

338 339 340 341 342 343

(2) Tomás-Barberán, F. A.; Clifford, M. N. Flavanones, chalcones and dihydrochalcones – nature, occurrence and dietary burden. J. Sci. Food Agric. 2000, 80, 1073–1080. (3) Garg, A.; Garg, S.; Zaneveld, L. J. D.; Singla, A. K. Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytother. Res. 2001, 15, 655–669. (4) Ross, J. A.; Kasum, C. M. Dietary Flavonoids: bioavailability, metabolic effects, and safety. Ann. Rev. Nutr. 2002, 22, 19–34.

344

(5) Selvaraj, P.; Pugalendi, K. V. Efficacy of hesperidin on plasma, heart and liver tissue

345

lipids in rats subjected to isoproterenol-induced cardiotoxicity. Exp. Toxicol.

346

Pathol. 2012, 64, 449–452.

347

(6) Vabeiryureilai, M.; Lalrinzuali, K. Determination of anti-inflammatory and analgesic

348

activities

of

a

citrus

bioflavanoid,

349

Immunopathol. 2015, 1, 1–7.

hesperidin

in

mice.

Immunochem.

350

(7) Kim, J. Y.; Jung, K. J.; Choi, J. S.; Chung, H. Y. Modulation of the age-related nuclear

351

factor-ΚB (NF-ΚB) pathway by hesperetin. Aging Cell 2006, 5, 401–411.

352

(8) Rizza, S.; Muniyappa, R.; Iantorno, M.; Kim, J.; Chen, H.; Pullikotil, P.; Senese, N.;

353

Tesauro, M.; Lauro, D.; Cardillo, C.; et al. Citrus polyphenol hesperidin stimulates

354

production of nitric oxide in endothelial cells while improving endothelial function 19



355

and reducing inflammatory markers in patients with metabolic syndrome. J. Clin.

356

Endocrinol. Metab. 2011, 96, E782–E792.

357

(9) Morand, C.; Dubray, C.; Milenkovic, D.; Lioger, D.; Martin, J. F.; Scalbert, A.; Mazur,

358

A. Hesperidin contributes to the vascular protective effects of orange juice: a

359

randomized crossover study in healthy volunteers. Am. J. Clin. Nutr. 2011, 93, 73–

360

80.

361

(10) Yamamoto, M.; Jokura, H.; Hashizume, K.; Ominami, H.; Shibuya, Y.; Suzuki, A.;

362

Hase, T.; Shimotoyodome, A. Hesperidin metabolite hesperetin-7-O-glucuronide,

363

but not hesperetin-3′-O-glucuronide, exerts hypotensive, vasodilatory, and

364

anti-Inflammatory activities. Food & Function 2013, 4, 1346–1351.

365

(11) Huang, S.-M.; Tsai, S.-Y.; Lin, J.-A.; Wu, C.-H.; Yen, G.-C. Cytoprotective effects of

366

hesperetin and hesperidin against amyloid β-induced impairment of glucose

367

transport through downregulation of neuronal autophagy. Mol. Nutr. Food Res.

368

2012, 56, 601–609.

369

(12) Pereira-Caro, G.; Ludwig, I. A.; Polyvivou, T.; Malkova, D.; García, A.;

370

Moreno-Rojas, J. M.; Crozier, A. Identification of plasma and urinary metabolites

371

and catabolites derived from orange juice (poly)phenols: analysis by

372

high-performance liquid chromatography–high resolution mass spectrometry. J.

373

Agric. Food Chem. 2016, 64, 5724–5735.

374

(13) Yamada, M.; Tanabe, F.; Arai, N.; Mitsuzumi, H.; Miwa, Y.; Kubota, M.; Chaen, H.;

375

Kibata, M. Bioavailability of glucosyl hesperidin in rats. Biosci. Biotechnol. 20


Page 20 of 32

Page 21 of 32

376


Biochem. 2006, 70, 1386–1394.

377

(14) Matsumoto, H.; Ikoma, Y.; Sugiura, M.; Yano, M.; Hasegawa, Y. Identification and

378

quantification of the conjugated metabolites derived from orally administered

379

hesperidin in rat plasma. J. Agric. Food Chem. 2004, 52, 6653–6659.

380 381

(15) Manach, C.; Williamson, G.; Morand, C.; Scalbert, A.; Rémésy, C. Bioavailability and bioefficacy of polyphenols in humans. Am. J. Clin. Nutr. 2005, 81, 230S-242S.

382

(16) Brand, W.; van der Wel, P. A. I.; Rein, M. J.; Barron, D.; Williamson, G.; van

383

Bladeren, P. J.; Rietjens, I. M. C. M. Metabolism and transport of the citrus

384

flavonoid hesperetin in Caco-2 cell monolayers. Drug Metab. Dispos. 2008, 36,

385

1794–1802.

386

(17) Kobayashi, S.; Tanabe, S.; Sugiyama, M.; Konishi, Y. Transepithelial transport of

387

hesperetin and hesperidin in intestinal Caco-2 cell monolayers. Biochim. Biophys.

388

Acta 2008, 1778, 33–41.

389

(18) Actis-Goretta, L.; Dew, T. P.; Lévèques, A.; Pereira-Caro, G.; Rein, M.; Teml, A.;

390

Schäfer, C.; Hofmann, U.; Schwab, M.; Eichelbaum, M.; et al. Gastrointestinal

391

absorption

392

hesperetin-7-O-glucoside in healthy humans. Mol. Nutr. Food Res. 2015, 59,

393

1651–1662.

and

metabolism

of

hesperetin-7-O-rutinoside

and

394

(19) Manach, C.; Morand, C.; Gil-Izquierdo, A.; Bouteloup-Demange, C.; Rémésy, C.

395

Bioavailability in humans of the flavanones hesperidin and narirutin after the

396

ingestion of two doses of orange juice. Eur. J. Clin. Nutr. 2003, 57, 235–242. 21



397

(20) Bredsdorff, L.; Nielsen, I. L. F.; Rasmussen, S. E.; Cornett, C.; Barron, D.; Bouisset,

398

F.; Offord, E.; Williamson, G. Absorption, conjugation and excretion of the

399

flavanones, naringenin and hesperetin from α-rhamnosidase-treated orange juice in

400

human subjects. Br. J. Nutr. 2010, 103, 1602–1609.

401

(21) Brett, G. M.; Hollands, W.; Needs, P. W.; Teucher, B.; Dainty, J. R.; Davis, B. D.;

402

Brodbelt, J. S.; Kroon, P. A. Absorption, Metabolism and excretion of flavanones

403

from single portions of orange fruit and juice and effects of anthropometric

404

variables and contraceptive pill use on flavanone excretion. Br. J. Nutr. 2008, 101,

405

664–675.

406

(22) Del Rio, D.; Rodriguez-Mateos, A.; Spencer, J. P. E.; Tognolini, M.; Borges, G.;

407

Crozier, A. Dietary (poly)phenolics in human health: structures, bioavailability,

408

and evidence of protective effects against chronic diseases. Antiox. Red. Signalling

409

2012, 18, 1818–1891.

410 411

(23) Booth, A. N.; Jones, F. T.; deEds, F. Metabolic fate of hesperidin, eriodictyol, homoeriodictyol, and diosmin. J. Biol. Chem. 1958, 230, 661–668.

412

(24) Nielsen, S. E.; Breinholt, V.; Justesen, U.; Cornett, C.; Dragsted, L. O. In vitro

413

biotransformation of flavonoids by rat liver microsomes. Xenobiotica 1998, 28,

414

389–401.

415

(25) Miyake, Y.; Shimoi, K.; Kumazawa, S.; Yamamoto, K.; Kinae, N.; Osawa, T.

416

Identification and antioxidant activity of flavonoid metabolites in plasma and urine

417

of eriocitrin-treated rats. J. Agric. Food Chem. 2000, 48, 3217–3224. 22


Page 22 of 32

Page 23 of 32


418

(26) Brand, W.; Boersma, M. G.; Bik, H.; Hoek-van Den Hil, E. F.; Vervoort, J.; Barron,

419

D.; Meinl, W.; Glatt, H.; Williamson, G.; Van Bladeren, P. J.; et al. Phase II

420

metabolism of hesperetin by individual UDP-glucuronosyltransferases and

421

sulfotransferases and rat and human tissue samples. Drug Metab. Dispos. 2010, 38,

422

617–625.

423

(27) Nguyen, H.-N.; Tanaka, M.; Li, B.; Ueno, T.; Matsuda, H.; Matsui, T. Novel in situ

424

visualisation of rat intestinal absorption of polyphenols via matrix-assisted laser

425

desorption/ionisation mass spectrometry imaging. Sci. Rep. 2019, 9, 3166.

426

(28) Silva, D. B.; Lopes, N. P. MALDI-MS of flavonoids: a systematic investigation of

427

ionization and in-source dissociation mechanisms. J. Mass Spectr. 2015, 50, 182–

428

190.

429 430

(29) Scalbert, A.; Williamson, G. Dietary Intake and bioavailability of polyphenols. J. Nutr. 2000, 130, 2073S-2085S.

431

(30) Walpole, S. C.; Prieto-Merino, D.; Edwards, P.; Cleland, J.; Stevens, G.; Roberts, I.

432

The weight of nations: an estimation of adult human biomass. BMC Public Health

433

2012, 12, 439.

434

(31) Yang, C. S.; Sang, S.; Lambert, J. D.; Lee, M.-J. Bioavailability issues in studying the

435

health effects of plant polyphenolic compounds. Mol. Nutr. Food Res. 2008, 52,

436

S139–S151.

23



437

FIGURE CAPTIONS

438

Figure 1. Time-course of plasma concentration of hesperidin (A), hesperetin (B), and

439

hesperidin conjugates (as hesperetin after enzymatic treatment) (C) after single oral

440

administration of hesperidin (10 mg/kg) to SD rats. Each LC-TOF-MS chromatogram

441

shows the elution of target in plasma at tmax in portal and circulating blood (hesperidin,

442

[M-H]‾= 609.1814 m/z; hesperetin, [M-H]‾= 301.0707 m/z). Inserted chromatograms show

443

the peak of IS (taxifolin, [M-H]‾= 303.0499 m/z). Data are expressed as the mean ± SEM (n

444

= 3). S/N means signal to noise ratio.

445 446

Figure 2. LC-TOF-MS chromatograms of hesperidin and hesperetin in circulating

447

plasma of SD rats at 16 h after 10 mg/kg hesperidin administration. MS

448

chromatograms show the elution of each target in plasma with or without

449

β-glucuronidase type B-1 treatment. Standard concentration was 10 μM. ND indicates

450

no detection.

451 452

Figure 3. LC-TOF-MS chromatograms of (A) hesperetin and homoeriodictyol

453

([M-H]‾= 301.0707) and (B) eriodictyol ([M-H]‾= 287.0561) in portal and circulating

454

plasma at 16 h after administration of hesperidin (100 mg/kg). ND indicates no

455

detection. Metabolic conversion between hesperetin, eriodictyol, and homoeriodictyol

456

is depicted in (C), with the concentration of each deconjugated form in sulfatase type

457

H-1 and β-glucuronidase type B-1-treated circulating blood at 16 h after 24


Page 24 of 32

Page 25 of 32

458


administration of hesperidin (100 mg/kg).

459 460

Figure

4.

MALDI-MS

spectra

corresponding

to

[M-H]‾

of

461

hesperetin/homoeriodictyol sulfate (381.0 m/z) and hesperetin/homoeriodictyol

462

glucuronide (477.1 m/z) in portal and circulating plasma at 16 h after oral

463

administration of hesperidin (100 mg/kg). Control indicates SD rat plasma with no

464

hesperidin administration. ND indicates no detection.

465 466

Figure 5.

Absorption behavior of hesperidin proposed by the present LC-TOF-MS

467

and MALDI-MS analyses. Abbreviations: Homoe (homoeriodyctyol), GlcA

468

(glucuronic acid), Sul (sulfate), UGT (UDP-glucuronosyltransferases), and SULT

469

(sulfotransferases).

470

25



26


Page 26 of 32

Page 27 of 32


Figure 1 27



Figure 2

28


Page 28 of 32

Page 29 of 32


Figure 3

29



Figure 4

30


Page 30 of 32

Page 31 of 32


Figure 5

31



Graphic Abstract (TOC)

32


Page 32 of 32

Absorption and Metabolic Behavior of Hesperidin (Rutinosylated

Recommend Documents