Daily Consumption of Bilberry (Vaccinium myrtillus L.) Extracts

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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

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Journal of Agricultural and Food Chemistry

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

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Absorption Rate of Anthocyanins in Rats

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

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

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†

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Miyazaki 889-2192, Japan ‡

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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

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*Author to correspondence should be addressed.

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

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

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1 ACS Paragon Plus Environment


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ABSTRACT

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The effects of daily consumption of anthocyanins on bioavailability was remained

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

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

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

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

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

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

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

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

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

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

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

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

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

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consumption

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

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

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

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

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

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

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

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

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

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developing chronic diseases.

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

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

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therefore essential to determine the pharmacokinetics and pharmacodynamics of

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anthocyanins. Studies in rodents have reported that orally administered anthocyanins

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

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

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

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

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



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

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methods to improve the bioavailability of anthocyanins. Matsumoto and colleagues

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

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

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

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

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

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

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

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

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when consumed during the active and sleep phase.

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

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

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

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

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

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were of the highest grade available.


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Institutional approval of the study protocol. All procedures were conducted

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

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

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number 2016-003-2.

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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chromatography (HPLC) analysis as described below.

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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recovery percentage was 92%.

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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.

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

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

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



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

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

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

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

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

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

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

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

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

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Blood chemistry. Biochemical parameters in the plasma samples were analyzed

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

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

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

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total protein.

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

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

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

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


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

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than 5%.

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RESULTS AND DISCUSSION

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Anthocyanin identification and quantification. The HPLC chromatogram at

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

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

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

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unavailable were determined using calibration curves at 520 nm obtained from

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commercially available anthocyanins with the same aglycone structures as described

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

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

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

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

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

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

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

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Effects of daily consumption of BBEx on plasma biochemical parameters.

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



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

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

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

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

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

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

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

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cholesterol, or triglyceride levels following consumption of 0.4% bilberry

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anthocyanin-containing diets for four weeks (approximately 72 mg

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

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colleagues observed reduced serum cholesterol and triglycerides in rats that consumed

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

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

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

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

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

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plasma lipid levels.

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


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anthocyanins accumulated in plasma (

Daily Consumption of Bilberry (Vaccinium myrtillus L.) Extracts

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