Melibiose, a Nondigestible Disaccharide, Promotes Absorption of

Subscriber access provided by United Arab Emirates University | Libraries Deanship

Article

Melibiose, a non-digestible disaccharide, promotes absorption of quercetin glycosides in rat small intestine Seiya Tanaka, Aki Shinoki, and Hiroshi Hara J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03714 • Publication Date (Web): 31 Oct 2016 Downloaded from http://pubs.acs.org on November 2, 2016

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 36

Journal of Agricultural and Food Chemistry

1

Melibiose, a non-digestible disaccharide, promotes absorption of quercetin

2

glycosides in rat small intestine

3

Author names: Seiya Tanaka, Aki Shinoki, Hiroshi Hara*

4 5

Laboratory of Nutritional Biochemistry, Division of Applied Bioscience, Graduate School of

6

agriculture, Hokkaido University, Sapporo,

7

Hokkaido 060-8589, Japan

8

1

ACS Paragon Plus Environment


9

Page 2 of 36

ABSTRACT

10

We demonstrated that melibiose, a non-digestible disaccharide composed of

11

galactose and glucose with α-1,6 glycoside linkage, promotes the absorption of water

12

soluble quercetin glycosides in ligated small intestinal loop of anesthetized rats. Water

13

soluble quercetin glycoside, a quercetin-3-O-glucoside mixture (Q3GM), includes

14

quercetin-3-O-glucoside (Q3G, 31.9%), mono (21.2%) and di (17.1%), glucose adducts

15

with α-1,4 linkages. After instillation of Q3GM into the intestinal loop with or without

16

melibiose, the plasma concentration of quercetin derivatives in the portal blood, was

17

considerably higher in the melibiose group at 60 min. Furthermore, we evaluated the

18

hydrolytic rate of Q3G by the mucosal homogenate of the small intestine with six

19

different disaccharides. Melibiose and isomaltose, which have α-1,6 glycoside linkage,

20

were found to promote Q3G hydrolysis to aglycone. These results suggest that

21

melibiose promotes quercetin glycoside absorption in rats by increasing glycoside

22

hydrolysis in the intestinal lumen, and that α-1,6 linkage is involved in this process.

23

Keywords: Quercetin; melibiose; hydrolysis; absorption; α-1,6 linkage

2


Page 3 of 36


24

INTRODUCTION

25

Flavonoids are polyphenolic C6-C3-C6 secondary metabolites with widespread

26

occurrence in the plant kingdom. Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is one of

27

the most abundant flavonol-type flavonoids. Quercetin has been reported for several

28

physiological effects, such as antioxidative, anti-carcinogenic, anti-inflammatory, and

29

anti-atherogenic action.1–4 However, the bioavailability of quercetin is very low. The

30

concentration of total quercetin metabolites when the man ingested foods containing

31

quercetin is below 1µmol/L5. A lot of papers reported that in vitro biological properties

32

of flavonoids metabolites.6 But in many cases, the concentration of quercetin

33

metabolites which have physiological effects was higher than 1 µmol/L. Furthermore,

34

the concentration of quercetin metabolites has dose-dependent manner in their many

35

physiological effects. For these reasons, it is very important to promote quercetin

36

absorption.

37

In nature, quercetin is present in the form of the glycoside, mainly β-glycoside.

38

Onions, apples, and many vegetables, as well as tea and red wine, are rich in quercetin

39

glycosides 5,7,8. Quercetin-3-O- β-glucoside (Q3G) is one of the major glycoside present

40

in onions and apples.

41

The most domestic food processing, such as cooking, is not able to cleave the

42

glycoside linkage of Q3G.9 Even though, gastric acid in the stomach does not hydrolyze 3



Page 4 of 36

43

β-glycoside linkage in Q3G.10 Q3G is deglycosylated in the small intestine.11 There are

44

two pathways for absorption of the Q3G reaching the small intestine, the transcellular

45

and the paracellular pathways. Flavonoid glycosides are mainly absorbed as aglycones

46

after hydrolysis of the glycoside linkage, through the transcellular pathway.

47

Lactase-phlorizin hydrolase (LPH; EC 3.2.1.108), a β-glycosidase in the small intestinal

48

brush border membrane, is responsible for the digestion of Q3G

49

quercetin aglycone is passively absorbed, and is usually conjugated as glucuronide and

50

sulfate in the intestinal epithelial cells. The conjugated quercetin is released into the

51

portal blood. In the paracellular pathway, Q3G passes through the tight junction and is

52

released into the portal blood as the glycoside form, not as the conjugated forms. The

53

increases in the portal plasma concentrations of aglycone and quercetin conjugates

54

indicate transcellular absorption; on the other hand, the increase of glycosides shows

55

paracellular absorption.

56

12,13

The hydrolyzed

In our previous study, we have studied the bioavailability of quercetin glycosides

57

with co-existing non-digestible saccharides.14,15

58

co-existing non-digestible saccharides is important because the present study contribute

59

to get constantly health benefits of flavonoids in diet. We reported that a non-digestible

60

disaccharide, difructose anhydride III (DFAIII), enhances the quercetin glycoside

61

absorption through the tight junction, which is the pracellular pathway, in the rat small

To study quercetin absorption with

4


Page 5 of 36


62

intestine.15 As the results, the concentration of Q3G in the portal blood plasma was

63

considerably higher in the DFAIII group than in the control group after administration

64

of Q3GM. In the present study, melibiose is another naturally occurred non-digestible

65

disaccharide composed of galactose and glucose with an α-1,6 linkage. We hypothesize

66

that melibiose also promotes quercetin glycoside absorption like DFAIII does. The

67

purpose of this study was to investigate whether melibiose increases the absorption of

68

water-soluble quercetin glycoside (Q3GM) using closed loops of the small intestine in

69

portal cannulated rats. We found that this disaccharide was promoting the absorption of

70

quercetin glycoside with a different mechanism from DFAIII.

71 72

MATERIALS AND METHODS

73

Chemicals

74

Q3GM is enzymatically manufactured from rutin which exists widely in natural

75

products.16 Quercetin aglycone are hardly soluble in water, the solubility of Quercetin

76

aglycone and Q3G is 206 µmol/L.17 We have to use small amount of organic solvents

77

such as DMSO and ethanol to dissolved Q3G in water. To administrate Q3G contained

78

organic solvents is nonphysiological condition. In this experiments, we used the water

79

soluble quercetin glycosides, which are called “quercetin-3-O-glucoside mixture

80

(Q3GM)”. Q3GM kindly donated by San-Ei Gen F.F.I., Inc. (Osaka, Japan), is mainly 5



81

composed of quercetin-3-O-glucoside (Q3G, 31.8%), mono (23.3%), and di (20.3%)

82

glucose adducts on the glucose moiety of Q3G with an α-1,4-linkage. The mixture

83

includes also tri- to hepta-adducts as minor components. Q3GM has high water

84

solubility, could be use in the in vivo and in vitro experiments. Q3GM were rapidly

85

hydrolyzed to Q3G with α-glucosidase in the inetestinal mucosa.17

86

Melibiose was kindly donated by Nippon Beet Sugar Manufacturing, Inc. (Tokyo,

87

Japan). Each purified component of Q3GM (Q3G and one to seven D-glucose adducts

88

of Q3G) (San-Ei Gen F.F.I.) was used as a standard compound to quantify

89

quercetin-derivatives by LC/MS/MS. All the other reagents and chemicals were of the

90

highest commercially available quality.

91

Page 6 of 36

Animals and diet

92

This study was approved by the Hokkaido University Animal Committee. These

93

experiments were approved by the Institutional Animal Care and Use Committee of the

94

National University Corporation of Hokkaido University, and the rats were maintained

95

in accordance with the National University Corporation of Hokkaido University

96

Regulations on Animal Experimentation (approval number: 08-0131, 14-0026). Male

97

Wistar/ST rats weighing about 195 g (7 weeks old; Japan SLC, Shizuoka, Japan) were

98

housed in individual stainless-steel cages with wire-mesh bottoms. The cages were

99

placed in a room at controlled temperature (22 ± 2°C), relative humidity (40–60%) and 6


Page 7 of 36


100

lighting (12 h-light/dark cycle from 08:00–20:00) for the entire duration of this study.

101

The rats were fed a flavonoid-free diet based on the AIN93G formulation18 for 1-week

102

of acclimation. We performed in situ experiments using ligated intestinal loops of

103

anesthetized rats to observe quercetin absorption, and in vitro experiments preparing

104

mucosal homogenate of the small intestine, to observe Q3G hydrolysis.

105 106

Absorption of quercetin glycosides in ligated intestinal loops

107

After overnight food deprivation, rats were anesthetized with ketamine (i.p., 80

108

mg/kg body weight; Ketaral, Daiichi Sankyo, Tokyo, Japan) containing xylazine (12

109

mg/kg body weight; MP Biomedicals, Irvine, CA, USA). An abdominal midline incision

110

was performed on rats, and the small tip (5–6 mm) of a polyethylene tube (SP28; ID 0.4

111

mm, OD 0.8 mm; Natsume Seisakusyo, Tokyo, Japan) connected to a silicone tube

112

(Silascon No. 00, ID 0.5 mm, OD 1.0 mm; Kaneka, Osaka, Japan) was inserted into the

113

portal vein for blood sampling.19 A 15-cm small intestinal segment was obtained at 3 cm

114

downstream from the ligament of Treitz; the contents were washed with saline, and then

115

ligated at both sides of the segment with silk suture (Hard No.4; Natsume Seisakusyo,

116

Tokyo, Japan). The test solution (1.5 mL) containing 10 mmol/L Q3GM with or without

117

100 mmol/L melibiose, was directly injected into the loop with a syringe attached to a

118

needle. The concentrations of Q3GM and melibiose used were expected concentrations 7



Page 8 of 36

119

in the lumen of rats fed maximum levels of flavonoids and non-digestible saccharides in

120

usual diets.

121

blood was collected through the portal catheter at 0, 15, 30, and 60 min after Q3GM

122

injection. During the experiments, rats were maintained at body temperature on a plate

123

heater. After collecting portal and aortic blood at 60 min, the rats were sacrificed by

124

whole blood withdrawal; then, the intestinal loops were removed. Blood was

125

centrifuged (4°C, 10 min, 2,300× g), and the plasma was collected. The removed

126

intestine and plasma were stored at -20°C and -40°C, respectively.

The osmotic pressure of the solutions was adjusted with NaCl. Portal

127 128

Sample preparation for LC/MS/MS analyses

129

The plasma (100 µL) from the portal blood was added to 10 µL of 0.58 mol/L acetic

130

acid (pH 4.9), and treated with (total quercetin) or without (unconjugated forms of

131

quercetin) deconjugation enzymes for 120 min at 37°C. The enzymes included 2,000

132

units (U) β-glucuronidase and 40 U sulfatase (Helix pomatia extract, Sigma G9751),

133

and 3 U sulfatase (from Aerobacter aerogenes, Sigma 1629). One glucuronidase unit is

134

defined as a liberating activity of 1.0 µg phenolphthalein from phenolphthalein

135

glucuronide per hour at 37°C at pH 5.0, and one sulfatase unit is defined as hydrolyzing

136

activity of 1.0 µM p-nitrocatechol sulfate per hour at 37°C at pH 5.0. The reaction

137

mixture was added to 100 µL methanol containing an internal standard (naringenin, 8


Page 9 of 36


138

final 1 µM)), heated at 100°C for 1 min to stop reaction, and centrifuged (5 min, 4°C,

139

9,300 g) to collect the supernatant. Residual flavonoids in the precipitate were extracted

140

thrice with 100 µL methanol. The combined supernatant was applied to a C18 cartridge

141

after conditioning (Oasis HLB 1 cc 10 mg, Waters Co. Ltd., Milford, MA, USA),

142

washed minerals and water soluble compounds with 1 mL of water, and then flavonoids

143

were eluted with 1 mL methanol.

144

After (partial) thawing, the intestinal contents and mucosa were collected together

145

from the ligated loops, filled up to 10 mL with deionized water, and homogenized. The

146

homogenate was added to equivolume methanol containing naringenin,14 heated at

147

100°C for 3 min, and centrifuged (5 min, 4°C, 9,300 g) to collect the supernatant. The

148

subsequent extraction procedure was the same as used for both, flavonoids and plasma.

149

Solid phase extraction was performed using Oasis HLB 3 cc 60 mg (Waters Co. Ltd.,

150

Milford, MA, USA), washed minerals and water soluble compounds with 2 mL of water,

151

and then flavonoids were eluted with 3 mL methanol.

152 153

In vitro digestion of quercetin glycosides with or without non-digestible saccharides

154

Five male Wistar/ST rats (7 weeks old) were fed the AIN93G diet for 1-week

155

acclimation. The rats were sacrificed, whole the small intestine without duodenum,

156

which was from 3cm distal of the Treitz ligament to the terminal ileum was wash out 9



Page 10 of 36

157

the contents by saline and stored at -80°C. The mucosa was collected from the segments

158

after thawing, then put together as a whole and made as 20% (w/v) uniform homogenate

159

by cold saline and stored at -80°C for the enzyme source.

160

One of six disaccharides, melibiose, cellobiose, lactose, DFAIII, trehalose,

161

isomaltose, and two trisaccharides, raffinose and isomaltotriose (100 mmol/L) was

162

dissolved in various concentrations with Q3GM (0.2-2 mmol/L) in deionized water for

163

the substrate solution. The preincubated substrate solution was mixed with equivolume

164

of enzyme solution, diluted to 5% (w/v) mucosal homogenate, and incubated at 37°C.

165

After 0, 15, 30, 60, 120 min, the mixed solution was drawn and boiled for 3 min to stop

166

enzyme reaction. The reactions performed in duplicate or triplicate. Concentrations of

167

quercetin and quercetin glycosides in the supernatant were analyzed by the LC/MS/MS

168

after extraction procedure mentioned before.

169 170

LC/MS/MS analysis of sample

171

Quercetin and its metabolites were identified and quantified by liquid

172

chromatography tandem mass spectrometry (LC/MS/MS) system (TSQ Quantum

173

Access Max with Accela High Speed LC System, Thermo Fisher Scientific Inc., MA,

174

USA) using an electric spray ionization (ESI) and selected reaction monitoring (SRM).

175

The temperature of the capillary heater and the vaporization heater were maintained at 10


Page 11 of 36


176

220°C and 450°C, respectively. LC/ESI-MS/MS was carried out in positive/negative

177

dual mode. The transition is showed in Table 1. The LC system was fitted with a 1.9 µm

178

C18 column (Inertsustain Swift C18, 2.1 x 100 mm GL Sciences Inc.) set at 40°C.

179

Solvent A (water/methanol/formic acid, 70 : 30 : 0.1) and solvent B (methanol/formic

180

acid, 100 : 0.1) were prepared. The flow rate of the mobile phase was 0.2 mL/min, and

181

the proportion of solvent B was raised linearly from 10 % to 80% over 8 min, then

182

reduced linearly back to 10% over 1 min and subsequently maintained at the initial

183

condition for 1 min. The concentrations of internal standards (naringenin), quercetin,

184

methylquercetin, Q3G-Q3G8 in samples were calculated from the peak area of each

185

mass chromatogram and the calibration curves of standard compounds.

186 187

Statistics

188

All values are expressed as the means ± standard error of the mean. Statistical

189

analyses were performed by one-way or two-way ANOVA, and the differences among

190

the groups were determined using Student’s t-test or Tukey-Kramer’s test. A difference

191

of P

Melibiose, a Nondigestible Disaccharide, Promotes Absorption of

Recommend Documents