1 Daily Consumption of Bilberry (Vaccinium ... - ACS Publications

Subscriber access provided by Umea University Library

Bioactive Constituents, Metabolites, and Functions

Daily Consumption of Bilberry (Vaccinium myrtillus L.) Extracts Increases the Absorption Rate of Anthocyanins in Rats Chiaki Nohara, Daigo Yokoyama, Wataru Tanaka, Tetsuya Sogon, Masanobu Sakono, and Hiroyuki Sakakibara J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02404 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 5, 2018

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

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

Page 1 of 32

Journal of Agricultural and Food Chemistry

1

Daily Consumption of Bilberry (Vaccinium myrtillus L.) Extracts Increases the

2

Absorption Rate of Anthocyanins in Rats

3 4

Chiaki Nohara†, Daigo Yokoyama‡, Wataru Tanaka†, Tetsuya Sogon#, Masanobu

5

Sakono†,‡, and Hiroyuki Sakakibara†,‡,*

6 7

†

8 9

Miyazaki 889-2192, Japan ‡

10 11 12

Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen Kibana-dai Nishi,

Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, 1-1 Gakuen Kibana-dai Nishi, Miyazaki 889-2192, Japan

#

Wakasa Seikatsu Co., Ltd., Sanko Building, 22 Naginataboko-cho, Shijo-Karasuma, Shimogyo-ku, Kyoto 600-8008, Japan

13 14

*Author to correspondence should be addressed.

15

Telephone and fax: +81 985 58 7213. E-mail: [email protected]

16 17

Running title: Effects of daily consumption of anthocyanins on their bioavailability

18

1 ACS Paragon Plus Environment


19

ABSTRACT

20

The effects of daily consumption of anthocyanins on bioavailability was remained

21

unclear. In this study, we evaluated whether daily consumption affects the absorption

22

rate of anthocyanins in rats when consumed during the active and sleep phase. Eighty

23

rats were randomly divided into two groups. The first group consumed AIN-93G control

24

diets and the second group consumed AIN-93G diets containing 1% bilberry extract for

25

2 weeks. After 12h fast, anthocyanins were not detected in plasma of rats. Bilberry

26

extract (500 mg/kg body weight) was then orally administered at the beginning of the

27

diurnal light period (ZT0, sleep phase) or at the end of the diurnal light period (ZT12,

28

active phase). Blood concentrations of anthocyanins peaked 1 h after administration in

29

both groups. Maximum blood concentration was higher in rats that consumed bilberry

30

extract daily (852 nM) than in control rats (630 nM) when the extract was administered

31

at ZT0, but not at ZT12. Daily consumption of anthocyanins increases their absorption

32

rate, but this effect is limited to the beginning of the sleep phase.

33 34

KEYWORDS: Anthocyanin, bilberry extract, bioavailability, diurnal rhythm, daily

35

consumption

36


Page 2 of 32

Page 3 of 32


37 38

INTRODUCTION Anthocyanidins, which have a typical flavonoid structure (Figure 1), are plant

39

pigments responsible for red, blue, and purple colors, and are widely distributed in fruits

40

and vegetables mainly as the glycoside derivatives anthocyanins 1-4. Especially, berries

41

including bilberry are rich in anthocyanidins 1. Anthocyanins have been shown to

42

exhibit a range of biological effects using animal-, cell- and in vitro-studies, including

43

antioxidant activity, anti-carcinogenesis, induction of apoptosis, anti-obesity and

44

anti-diabetic activity, improvement of eyesight, and prevention of glaucoma 5-10. In

45

addition, these beneficial effects were revealed using clinical study 11. The regular

46

consumption of foods rich in anthocyanins is therefore associated with reduced risks of

47

developing chronic diseases.

48

To exert these beneficial effects, the anthocyanins from food must be absorbed

49

from the alimentary tract, circulate in the bloodstream, and reach target organs. It is

50

therefore essential to determine the pharmacokinetics and pharmacodynamics of

51

anthocyanins. Studies in rodents have reported that orally administered anthocyanins

52

enter the bloodstream in both their original form and as anthocyanin metabolites,

53

reaching maximum levels between 0.25 and 2 h after administration 12-16. Anthocyanins

54

have been detected in several organs, including the liver, kidney, testis, and lung

55

following consumption of anthocyanin-containing diets 12. However, their amounts

56

were extremely low (pmol order per gram organs), which is the main constraint to using



57 58

anthocyanins as functional foods 17. For this reason, several research groups have been focusing on developing

59

methods to improve the bioavailability of anthocyanins. Matsumoto and colleagues

60

reported that plasma levels and urinary excretion of anthocyanins increased when phytic

61

acid was consumed concomitantly 18. Fasting also significantly increased the absorption

62

of anthocyanins in rats 14. In addition, the timing of anthocyanin consumption may

63

affect bioavailability. The gastric emptying rate of anthocyanins was reported to

64

increase significantly following administration at the active phase rather than the sleep

65

phase, because of inherent biological rhythms 19. Information on the effects of daily

66

consumption of anthocyanins on their bioavailability is lacking. In this study, we

67

evaluated whether daily consumption affects the absorption rate of anthocyanins in rats

68

when consumed during the active and sleep phase.

69 70 71

MATERIALS AND METHODS Materials. Bilberry extracts (BBEx) were obtained from Wakasa Seikatsu Co.,

72

Ltd. (Kyoto, Japan). Fresh bilberries (Vaccinium myrtillus L.) cultivated in Finland were

73

extracted using 90% ethanol. After evaporation of the ethanol solvent, the extracts were

74

freeze-dried to a powder. Cyanidin, trifluoroacetic acid (TFA), and ascorbic acid were

75

obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents

76

were of the highest grade available.


Page 4 of 32

Page 5 of 32


77 78

Institutional approval of the study protocol. All procedures were conducted

79

according to the guidelines for the care and use of laboratory animals of the University

80

of Miyazaki (Miyazaki, Japan). The experimental protocol was registered under the

81

number 2016-003-2.

82 83

Animal housing, diet, and experiments. Male 7-week-old Sprague-Dawley rats

84

were obtained from Japan SLC (Shizuoka, Japan) and housed in an air-conditioned

85

room (23 ± 2°C) under a 12-h dark/light cycle (light: 9:00 AM to 9:00 PM) with free

86

access to deionized water and purified AIN-93G diet. After a 1-week acclimatization,

87

the experiments commenced according to the following protocols (Figure 2).

88

Protocol I: Eighty rats were randomly divided into two groups. The first group

89

consumed AIN-93G control diets and the second group consumed AIN-93G diets

90

containing 1% BBEx (Table 1). After 2 weeks, rats were further sub-divided into two

91

groups, each of which received BBEx at Zeitgeber time (ZT) 0 or ZT12. Here, ZT0

92

represents the time when the light was turned on at the start of the light period. The light

93

period (sleep phase) lasted from ZT0 to ZT12 and the dark period (active phase) lasted

94

from ZT12 to ZT24. The first treatment sub-group was administered 500 mg of BBEx

95

dissolved in 10% citric acid (474.6 µmol total anthocyanins)/10 mL/kg body weight at

96

ZT0 after a 12-h fast. The second treatment sub-group was administered the same dose



97

at ZT12 after a 12-h fast. Rats were anesthetized with isoflurane at varying time points

98

after administration (0, 1, 2, and 4 h). After decapitation, trunk blood was collected into

99

heparinized tubes (Venoject, Terumo Medical Corporation, Tokyo, Japan). Plasma was

100

separated by centrifugation at 1,200×g for 10 min and acidified by the addition of 15 µL

101

formic acid to 1 mL plasma. After addition of 5 µL of 100 mM ascorbic acid, the plasma

102

was stored at −80°C until analysis. Next, the gastrointestinal tract was dissected into

103

stomach and ileum (10 cm length from the cecum). Tissue specimens were immersed

104

immediately in 20 mL of ice-cold methanol containing 0.5% TFA and then thoroughly

105

dissected. After mixing at 2,500 rpm for 1 min, the sample solution was centrifuged at

106

1,500×g for 10 min. The supernatants were subjected to high-performance liquid

107

chromatography (HPLC) analysis as described below.

108

Protocol II: Following a 1-week acclimatization period with AIN-93G control

109

diets, 34 rats were divided into three groups. The first group received an oral dose of

110

500 mg/5 mL/kg BBEx dissolved in 10% citric acid at ZT0 or ZT12 following a 12-h

111

fast. The second group received the same dose at ZT0 or ZT12 following a 12-h fast,

112

and a second dose 1 h after the first dose. The third group received the same dose at

113

ZT0 or ZT12 following a 12-h fast, and the same volume of vehicle solvent (10% citric

114

acid) 1 h after the BBEx dose. Rats were anesthetized with isoflurane 0, 1, and 2 h after

115

the first dose. Following decapitation, trunk blood was collected into heparinized tubes,

116

and the plasma fraction was obtained according to the method described above.


Page 6 of 32

Page 7 of 32


117 118

Anthocyanin extraction. We used solid-phase extraction to purify anthocyanins

119

in the plasma fraction according to our previous method 12. Briefly, 1 mL of plasma

120

fraction was loaded on to a Bond Elut Plexa extraction cartridge (30 mg, Agilent

121

Technologies, Santa Clara, CA, USA) that was equilibrated with 10 mM oxalic acid.

122

After rinsing the cartridge with 1 mL of 10 mM oxalic acid, anthocyanins were eluted

123

with 1 mL of methanol containing 0.5% TFA. The elute was evaporated using a

124

centrifugal concentrator (CC-105; TOMY Seiko Co., Ltd., Tokyo, Japan). The residue

125

was dissolved in 100 µL of methanol containing 0.5% TFA, filtered with a 0.2-µm

126

membrane filter (SLLGH04NK, Millex-LG, Millipore Co., Bedford, MA, USA), and

127

analyzed by HPLC as described below. Before the sample extraction, the recovery

128

percentage of anthocyanidin was evaluated. Briefly, standard cyanidin was added into

129

the blank plasma, and then cyanidin was extracted using same method. Their average

130

recovery percentage was 92%.

131 132

Extracts from stomach contents diluted five-fold and intact extracts from ileum contents were filtered using 0.2-µm membrane filters and then analyzed by HPLC.

133 134

HPLC- diode-array detector (DAD). Anthocyanins in plasma and

135

gastrointestinal extracts were analyzed by HPLC in conjunction with a DAD system

136

according to a modified method we published previously 12. The HPLC system used



137

was a JASCO system control program (LC-NetII/ADC, Tokyo, Japan) equipped with a

138

ChromNAV chromatography data station, PU-2089 Plus pump, AS-2057 Plus

139

autosampler, CO-2060 Plus column oven, and MD-2018 Plus DAD system for

140

monitoring wavelengths at 200–600 nm. The column, a Capcell Pak ACR (4.6 mm i.d.

141

× 150 mm, 3 µm, Shiseido Co. Ltd., Tokyo, Japan) was used at 40°C. Linear gradient

142

elution was performed with solution A (0.5% TFA aqueous) and solution B (0.1% TFA

143

acetonitrile) delivered at a flow rate of 1.0 mL/min as follows: Initial concentration of

144

92% of solution A; 85% A for 30 min; 70% A for 6 min; 40% A for 3 min; 40% A for 10

145

min. The injection volume of the sample was 5 µL.

146 147

Blood chemistry. Biochemical parameters in the plasma samples were analyzed

148

with a Dri-Chem 4000v chemistry analyzer (Fujifilm Co., Tokyo, Japan) using

149

individual analytical pleat. We quantified levels of alanine aminostransferase, aspartate

150

aminotransferase, lactate dehydrogenase, glucose, total cholesterol, triglycerides, and

151

total protein.

152 153

Statistical analysis. Data are presented as the mean ± standard deviation.

154

Statistical analyses were conducted using StatView for Windows (version 5.0, SAS

155

Institute, Cary, NC, USA). Time-dependent data analysis was performed using

156

repeated-measure ANOVA and Fisher’s protected least significant difference between


Page 8 of 32

Page 9 of 32


157

subject factors. Results were considered significant if the possibility of error was less

158

than 5%.

159 160

RESULTS AND DISCUSSION

161

Anthocyanin identification and quantification. The HPLC chromatogram at

162

520 nm indicated 13 major peaks (Figure 3A). The retention times and spectra were

163

compared with those of commercially available anthocyanidin (cyanidin) and with our

164

previous data 1, 12. Peaks identified as anthocyanins for which standards were

165

unavailable were determined using calibration curves at 520 nm obtained from

166

commercially available anthocyanins with the same aglycone structures as described

167

previously 12. Peaks 11 (malvidin-3-galactoside) and 12 (peonidin-3-glucoside) had

168

retention times and spectra similar to those in our previous study 12. Therefore, peaks

169

that eluted at this retention time with a typical anthocyanin spectrum were determined to

170

be either peonidin-3-glucoside or malvidin-3-galactoside, and calculated using the

171

calibration curve of malvidin. Peaks 13 and 14 also had almost identical retention times.

172

The total amount of anthocyanins in the BBEx used in this study was 949.1 µmol/g

173

extract. Table 2 summarizes the quantities of individual anthocyanins in the extract.

174 175

Effects of daily consumption of BBEx on plasma biochemical parameters.

176

During the 2-week period of daily consumption of BBEx, body weight gain and food



177

consumption did not differ between the treatment and control groups (data not shown).

178

The BBEx group consumed 226.7 ± 3.4 µmol (approximately 109.3 ± 1.6 mg)/rat/day

179

total anthocyanins. Plasma was collected after two weeks at ZT0 following a 12-h fast.

180

Significant changes were not observed in levels of alanine aminotransferase, aspartate

181

aminotransferase, lactate dehydrogenase, glucose, total cholesterol, triglycerides, or

182

total protein (data not shown). These findings are consistent with those reported by

183

Takahashi and colleagues, who did not observe changes in rat plasma glucose, total

184

cholesterol, or triglyceride levels following consumption of 0.4% bilberry

185

anthocyanin-containing diets for four weeks (approximately 72 mg

186

anthocyanins/rat/day) 20. Contrasting results have also been reported; Graf and

187

colleagues observed reduced serum cholesterol and triglycerides in rats that consumed

188

anthocyanin-rich bilberry juice (approximately 15 mg anthocyanins/day) 21. Some

189

effects, such as antidepressant-like activity and reduction in infarct size after

190

reperfusion, have been found to exhibit a U-shaped dose-response curve 22, 23, indicating

191

that consumption of large quantities may not always be effective. We are designing a

192

study to investigate the effects of BBEx at lower quantities (less than 10 mg/rat/day) on

193

plasma lipid levels.

194 195 196

Effects of daily consumption on anthocyanin bioavailability. Plasma fractions were collected following 2 weeks of BBEx consumption and a 12-h fast. No


Page 10 of 32

Page 11 of 32


197

anthocyanins accumulated in plasma (

1 Daily Consumption of Bilberry (Vaccinium ... - ACS Publications

Recommend Documents