Hesperetin and hesperidin improved Î²-carotene incorporation

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Bioactive Constituents, Metabolites, and Functions

Hesperetin and hesperidin improved #-carotene incorporation efficiency, intestinal cell uptake, and retinoid concentrations in tissues Meimei Nie, Zhongyuan Zhang, Chunquan Liu, Dajing Li, Wuyang Huang, Ning Jiang, and Chunju Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00551 • Publication Date (Web): 04 Mar 2019 Downloaded from http://pubs.acs.org on March 5, 2019

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Journal of Agricultural and Food Chemistry

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Hesperetin and hesperidin improved β-carotene incorporation efficiency,

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intestinal cell uptake, and retinoid concentrations in tissues

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Meimei Nie,

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Huang, #,†, Chunju Liu, # and Ning Jiang #

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#Institute

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Nanjing 210014, People’s Republic of China

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‡College

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People’s Republic of China

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†Institute

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#,‡,║

Zhongyuan Zhang,

#,║

Chunquan Liu,

#,‡,*

Dajing Li,#,* Wuyang

of Agro-product Processing, Jiangsu Academy of Agricultural Sciences,

of Food and Technology, Nanjing Agricultural University, Nanjing 210095,

of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing

210014, People’s Republic of China

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Corresponding Author

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*Tel.: +86-02584391570. Fax: +86-02584391570.

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E-mail: [email protected]; [email protected]

ACS Paragon Plus Environment


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ABSTRACT

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Dietary constituents can influence the bioavailability of carotenoids. This study

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investigated the effect of citrus flavanones on β-carotene (Bc) bioavailability using

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four experimental models: in vitro digestion procedure, synthetic mixed micelles,

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Caco-2 cell monolayers, and gavage experiments in mice. The addition of hesperetin

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(Hes, 25 μM) and hesperidin (Hes-G, 25 μM) standards significantly increased the

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incorporation efficiency of Bc standard to 68.7 ± 3.6% and 75.2 ± 7.5% (P < 0.05),

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respectively. However, the addition of naringenin (Nar, 25 μM) and naringin (Nar-G,

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25 μM) standards significantly reduced the incorporation efficiency of Bc by 23.8%

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and 26.4%, respectively (P < 0.05). The increases in scavenger receptor class B type I

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(SR-BI) expression promoted by citrus flavanones played an important role in Bc

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cellular absorption in the Caco-2 cell model. Furthermore, after 3-day gavage, four

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citrus flavanones (7.5 mg/kg/d) increased the retinoid concentrations in tissues; in

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contrast, after 7-day gavage, Nar and Nar-G significantly decreased hepatic retinoid

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concentrations (P < 0.05). This finding suggested that the incorporation efficiency

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into micelles was the main step governing carotenoid bioavailability.

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Keywords: β-carotene, bioaccessibility, citrus flavanone, absorption, Caco-2 cell


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INTRODUCTION

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β-carotene (Bc) is a food microconstituent in the carotenoid family that has the

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highest vitamin A activity and is mainly found in vegetables and fruits in the daily

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diet.1 Epidemiological studies suggest that the consumption of carotenoid-rich food is

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beneficial to human health with such effects as reducing the risk of certain cancers,

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decreasing the risk of cardiovascular diseases, and prevention of sunburns.2,3 The

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bioactivity of Bc from food depends largely on its bioavailability. The process of

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carotenoid bioavailability is as follows. First, carotenoids are released from the food

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matrix and then incorporated into mixed micelles in the intestine during digestion.

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Second, carotenoids are taken up by intestinal epithelial cells by a passive diffusion

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process.4 Carotenoids absorption is proved to be facilitated by membrane-mediated

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transport proteins, including scavenger receptor class B type I (SR-BI), cluster

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determinant 36 (CD36), Niemann-Pick type C1 Like 1 protein (NPC1L1), and ATP

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binding cassette transporter, subfamily A (ABCA1).5-7 Then, the carotenoids are

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transported to the lymphatic circulation, cleaved by β-carotene15-15’-oxygenase

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(BCO1),8 and converted into vitamin A or stored in the liver.

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Carotenoid bioavailability depends on a number of factors represented by the

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acronym SLAMENGHI.9 Among these factors, the food matrix is assumed to be an

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important factor. The phytochemicals in the food matrix may interfere with the

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bioavailability of carotenoids. Other carotenoids may also affect Bc bioavailability.

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Interactions could occur during micellar transfer and/or cellular uptake.10-12 For

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example, the addition of lycopene had a significant reduction in the Bc micellarization



and cellular uptake,14 and the most likely mechanism for this interaction was the

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13

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competitive inhibition of Bc by lycopene. Other microconstituents, such as flavonoids,

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could also influence carotenoid bioavailability. Poulaert et al.13 observed that naringin

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(Nar-G) in grapefruit juice had a negative effect on the bioaccessibility of Bc from

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orange-fleshed sweet potato. The authors showed that Nar-G could compete with Bc

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into mixed micelles; in contrast, Bc cellular uptake was not impaired by Nar-G.

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However, Dhuique-Mayer et al.15 reported that hesperetin (Hes), hesperidin (Hes-G)

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and naringenin (Nar) enhanced carotenoid uptake by intestinal Caco-2 cells. With

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respect to the metabolism of carotenoids, Hes-G increased the bioefficacy of Bc in

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vitamin A-deficient Mongolian gerbils by stimulating the activity of BCO1.16

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However, Nagao et al.17 reported that flavonoids, such as luteolin, quercetin,

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rhamnetin, and phloretin, remarkably inhibited BCO1 activity using a pig intestinal

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homogenate as the enzyme source. In addition, ingesting quercetin but not rutin

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increased the accumulation of hepatic Bc in BALB/c mice.18 Although these studies

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have shown that flavonoids interfere with the absorption and metabolism of Bc, some

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studies indicate a positive effect, while others show a negative effect. The effect of

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flavonoids on the bioefficacy of Bc in vivo in healthy animals with normal diets

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remains unknown. In addition, flavonoids can interfere with the bioaccessibility,13

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cellular uptake,15, 19 and metabolism of carotenoids,16, 18 but we do not know which

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stage is the main point at which flavanones affect the bioavailability of Bc. Thus, the

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objective of this study was to assess the influence of flavonoids on the bioaccessibility,

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incorporation rate into synthetic mixed micelles, absorption efficiency by intestinal


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cells and bioconversion of Bc to determine the effects of flavonoids on Bc

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bioavailability at different stages. The flavonoids studied in this work are Hes, Hes-G,

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Nar and Nar-G, which are representative of the two typical flavanone-type flavonoids

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abundantly present in citrus fruits. According to the diet recommendations related to

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healthy eating, the total daily carotenoid intake was estimated at 14 mg/day,20 and the

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average daily intake of flavonoids was estimated at between 70 and 170 mg/day.21

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Therefore, five times more citrus flavanone than Bc was used in the competition

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experiments. Understanding the effect of citrus flavanones on the bioavailability of Bc

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is of great importance to better evaluate the flavanones’ potential impact on human

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health.

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MATERIALS AND METHODS

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Chemicals. Bc (≥98% pure), flavanone standards (Hes, Hes-G, Nar and Nar-G, ≥98%

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pure), β-apo-8'-carotenal (>95%), retinol, retinal, retinyl oletate, retinyl palmitate, and

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retinyl acetate were purchased from Sigma-Aldrich China Co., Ltd. (Shanghai, China).

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Salts (NaHCO3, NaCl, KCl, CaCl2·2H2O, and K2HPO4), pepsin (914 units per mg of

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protein), porcine pancreatin (750 units per mg of protein), porcine bile extract, and the

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AR grade extraction solvents (hexane, ethanol, and dichloromethane) were purchased

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from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Phosphatidylcholine,

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lysophosphatidylcholine, monoolein, free cholesterol, oleic acid, and sodium

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taurocholate were purchased from Western Asia Chemical Co., Ltd. (Jinan, China).

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Dulbecco's Modified Eagle medium (DMEM) containing 4.5 g·L-1 glucose and 0.25%

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trypsin-EDTA, fetal bovine serum (FBS), penicillin-streptomycin, phosphate buffer



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solution (PBS), and nonessential amino acids were purchased from Gibco Life

99

Technologies (Shanghai, China). Methanol and methyl-tert-butyl-ether (MTBE) were

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of HPLC grade and were purchased from Tedia Co. Inc. (Fairfield, OH, USA). In Vitro Digestion. According to a previous study, after ingestion of test meals,

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carotenoid concentrations in the duodenum range between 5 and 18 μM in the

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human.22 Thus, the total Bc mass was estimated as 40 μg in this gastrointestinal

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solution,23 and the amount of each flavanone was 200 μg. Briefly, stock solutions of

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Bc and four flavanones were prepared in a chloroform-methanol (2:1, v/v) solution.

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Appropriate volumes of these stock solutions were transferred into glass bottles. The

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solvents were carefully evaporated under nitrogen, and the dried residue was

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dissolved in 0.1 mL commercial groundnut oil, which contained no quantifiable

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amounts of carotenoids or vitamin A, as checked by HPLC.23 The in vitro digestion

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method was similar to that previously described with minor adaptations.24-26 The

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mixture was mixed with gastric solution (50 mM NaCl, 3.60 mM MgCl2•6H2O, 10

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mM CaCl2•2H2O, 3.50 mM KH2PO4, 14 mM KCl) and acidified to pH 2 with 0.1 M

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HCl. Then, the sample was mixed with pepsin solution (50.25 mg/mL in 0.1 M HCl),

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and the mixture was incubated in a shaking water bath at 37 °C for 1 h. After gastric

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digestion, the pH was increased to 6.9 ± 0.1 with 1 M NaHCO3. Further intestinal

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digestion was performed with the addition of a pancreatin-bile solution (30 mg of bile

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extract/mL and 5 mg of pancreatin/mL in 0.1 M NaHCO3) and incubated in a shaking

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water bath at 37 °C for 2 h. At the end of the simulated small intestine digestion phase,

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natural mixed micelles in which the Bc is solubilized were separated by centrifugation


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(5000g at 4 °C for 15 min) followed by filtration (0.22 μm filter, Merck, Darmstadt,

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Germany). Aliquots were used to analyse the intestinal cell uptake in Caco-2 cells or

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the HPLC of Bc. Bioaccessibility was defined as the percentage of Bc obtained in the

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natural mixed micelles compared to the amount of Bc added to the digestive system in

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the absence/presence of citrus flavanones.

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Determination of the Incorporation Rate of Bc into Synthetic Mixed Micelles.

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Bc was delivered to incorporate into mixed micelles that were prepared as previously

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described.7 This experiment simulated the physiological conditions in the human

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duodenum during digestion. Stock solutions of monoolein, oleic acid,

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lysophosphatidylcholine, phosphatidylcholine, and free cholesterol were prepared in a

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solution of chloroform-methanol (2:1; v/v). Appropriate volumes of these stock

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solutions were transferred into glass bottles to obtain the following working

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concentrations: 0.3 mM monoolein, 0.5 mM oleic acid, 0.16 mM

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lysophosphatidylcholine, 0.04 mM phosphatidylcholine, and 0.1 mM free cholesterol.

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An appropriate volume of Bc solution was mixed with a suitable volume of mixed

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micelle lipid components with or without flavanones. The final concentration for Bc

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was 5 μM, and that of each flavanone was 25 μM. Carotenoid concentrations were

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chosen to mimic physiological doses in the duodenum.22 Solvents were carefully

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evaporated under nitrogen and the residue was solubilized in 10 mL 0.9% NaCl

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solution containing 5 mM sodium taurocholate and vigorously mixed by sonication

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for 5-8 min. The resulting mixture was filtered through a 0.22 μm filter, and the

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solutions were stored at -80 °C until the incorporation efficiency and cellular uptake



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of Bc were analysed. Measurement of Bc Uptake by Caco-2 Cell Monolayers. Caco-2 cells were

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purchased from Cell Centre of the Chinese Academy of Sciences (Shanghai, China).

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Caco-2 cells (passage 30-40) were cultured in DMEM supplemented with 10% FBS,

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1% nonessential amino acids, and 1% penicillin-streptomycin (complete medium) as

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previously described. Cells were incubated at 37 °C and incubated in a 5% CO2

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incubator.15 For each experiment, cells were seeded at a density of 2×105 cells per

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well and were grown on transwells (6-well plate, 24 mm diameter, 3 μm pore size

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polycarbonate membrane, Corning Costar, Cambridge, MA, USA). Media was

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changed every day for 21 days to obtain differentiated cell monolayers. Before each

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experiment, the integrity of the cell monolayers was checked by measuring

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trans-epithelial electrical resistance with a voltohmmeter equipped with a chopstick

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electrode (Millicell ERS, Merck).

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Both natural mixed micelles, i.e. mixed micelles from the in vitro digestion and

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synthetic mixed micelles, were used to test the Bc cellular uptake. The natural and

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synthetic mixed micelles were used at 1:3 dilution in accordance with a previous

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study.13 At the beginning of each experiment, cell monolayers were washed with PBS.

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The apical side of the cell monolayers received 1 mL diluted micelles, and the

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basolateral side received 2 mL FBS-free medium. Cell monolayers were incubated at

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37 °C for 5 h to simulate physiologic intestinal passage time. After the incubation

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period, media from each side of the membrane was harvested. Cell monolayers were

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washed twice with PBS containing 5 mM sodium taurocholate to eliminate absorbed


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Bc and were then scraped and collected in 1 mL PBS. All samples were stored at -80

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°C under nitrogen with 0.5% pyrogallol as a protective antioxidant before Bc

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extraction and HPLC analysis. Aliquots of cell samples were used to estimate protein

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concentrations with a BCA Protein Assay Kit (Beyotine Technology Inc., Nanjing,

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China). The cellular uptake efficiency of Bc was defined as the percentage obtained in

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scraped Caco-2 cells plus basolateral medium compared to the amount of Bc added on

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the apical side of the monolayers. The natural and synthetic mixed micelles were

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added to triplicate wells containing differentiated monolayers of Caco-2 cells to

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examine Bc uptake. Repeated experiments were tested on separate days with different

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cells for a total of three times. Values are expressed as the mean ± standard deviation

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(SD) of three independent experiments in triplicate.

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Immunoblotting. The 500 μL aliquots of cell samples from above, after

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centrifugation (8000g at 4 °C for 15 min), were lysed for 1 h on ice.27 Protein

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concentrations of cell lysates were measured using a BCA kit (see above). Twenty

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micrograms of the protein sample was separated by 12% SDS-polyacrylamide gel

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electrophoresis (SDS/PAGE) gels, and the resulting protein bands were transferred

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onto a PVDF membrane (Merck). The membranes were blocked with 5% nonfat dairy

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milk in Tris-buffered saline with Tween (TBST) for 1.5 h. Primary antibodies specific

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for SR-BI or β-actin (Cell Signaling Technology, Inc., Shanghai, China) were

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incubated with the membranes overnight at 4 °C with gentle shaking. Then, the

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secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit diluted

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2000-fold (Goat Anti-Rabbit IgG (H + L); Cell Signaling Technology, Inc.) was



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applied for 1 h at room temperature. After washing, blots were treated with ECL

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blotting solution (Tanon Technology Co., Ltd., Shanghai, China) for 10 min and were

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then exposed to film in a dark box and developed. ImageJ software was used to align

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and process individual lanes of gel images. Each Western blot was performed in

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triplicate.

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Animals Experiment Design. Eight-week-old male C57BL/6 mice (approximately

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20 g) were purchased from a local breeder. The mice were housed in individual

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stainless-steel cages and had free access to food and water. A natural-ingredient,

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open-formula rodent diet contained 12000 IU/kg vitamin A was used for the gavage

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experiments (Nanjing Xietong Co., Ltd., Nanjing, China). The vitamin A content

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complied with Chinese standard laboratory animals-nutrients for formula feeds (GB

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14924.3-2010). Animal experiments were conducted according to the standards set by

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animal care and use and we took appropriate measures to minimize pain or discomfort.

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The experiments complied with animal ethics rules and were approved by the local

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ethical committee of Jiangsu University Animal Centre (Certificate No. SCXK

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2013-0011).

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In the first experiment, gavage was performed each day over 3 days. Five groups of

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six male mice were included in the study: a control group was force-fed with

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groundnut oil, and four experimental groups were force-fed with the same amount of

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oil containing one flavanone (Hes, Hes-G, Nar, or Nar-G); the dose of citrus

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flavanones is described below. At the end of the feeding test, these animals fasted

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overnight and were sacrificed under ether anaesthesia. The liver and small intestine


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were rapidly removed and washed with cold saline, and enzyme activity was

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measured.

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In the second experiment, the protocol was as follows: five groups of six mice were

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included in the study: a control group was force-fed with groundnut oil with Bc, and

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four experimental groups were force-fed with the same amount of groundnut oil

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containing Bc with one flavanone (Hes, Hes-G, Nar, or Nar-G). Gavage was

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performed each day over 3, 5 and 7 days. Mice had free access to standard food and

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water until the last day of the feeding experiment. Bc and flavanones were

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incorporated in oil as follows: Bc, flavanones and groundnut oil were dissolved in

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hexane solutions and mixed, and the solvent was evaporated under nitrogen. The

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amount of carotenoids administered was based on the mean daily intake of a

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carotenoid-rich diet.23, 28 The dosage of Bc in the oil was given to mice to provide 55

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nmol carotenoids/mouse/day, equivalent to 1.5 mg of carotenoid/kg/day and the

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amount of each flavanone was 7.5 mg of flavanone/kg/day. The animals were given

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0.1 mL of groundnut oil containing 0.03 mg Bc or/and 0.15 mg citrus flavanone by

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stomach gavage every day. The third, fifth and seventh gavage experiments were

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performed on fasting mice (no access to food the night before). All tissues samples

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were immediately placed in liquid nitrogen and maintained at -80 °C until analysis.

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BCO1 Assay in Intestine and Liver. The assay procedure was a modification of

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the method described by van Vliet et al.29 Briefly, the small intestine was washed with

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cold 0.9% NaCl solution. Then the mucosa was scraped and homogenized in 5 mL

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potassium phosphate buffer (100 mM, pH 7.4) containing 0.154 M KCl, 1 mM EDTA,



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and 0.1 mM DTT. The homogenate was centrifuged at 10000g for 10 min, and the

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supernatant was collected. A liver homogenate was obtained with 9 mL of 100 mM

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potassium phosphate buffer (pH 7.4) and 50 mM KCl in a glass homogenizer for 2

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min. Protein concentrations were measured using a BCA kit (see above).

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BCO1 activity was evaluated using a protocol similar to that described by During et

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al.30 The incubation medium consisted of tricine-KOH buffer (pH 8.0, 0.1 M)

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containing 0.5 mM DTT, 4 mM bile salt and 15 mM nicotinamide. Using Bc with

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α-tocopherol and Tween-20 as the substrate, this mixture was dissolved in acetone,

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dried under nitrogen, and then added into the reaction mixture. Then, enzymatic

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extract was added to 2 mL of incubation medium. The mixture was incubated in a

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shaking water bath at 37 °C in the dark for 1 h. The reaction was stopped by the

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addition of 2 mL of ethanol. Retinal, the only product, was measured as previously

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described.16

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Extractions and HPLC Analyses of Bc and Retinoids. Aliquots (500 μL) of

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samples (the natural mixed micelles from digesta, synthetic mixed micelles and

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Caco-2 cell samples) were extracted three times with 2 mL of hexane in the presence

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of 500 μL of ethanol containing an internal standard (β-apo-8'-carotenal).13, 23 Hexane

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phases obtained after centrifugation (5000g, 5 min at room temperature) were

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combined and evaporated to dryness under nitrogen, and the residue was dissolved in

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100 μL dichloromethane-methanol (50:50, v/v).

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Blood samples were collected from mice using a heparin tube, and the plasma was

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separated by centrifugation immediately and subsequently stored in liquid nitrogen at


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-80 °C. Then, the plasma samples were extracted as described above. Analyses of

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retinoids in liver, intestine, and adipose tissue samples were conducted using

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previously described methods with minor modifications.16, 18 Retinyl acetate was

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employed as an internal standard for the retinoids. Briefly, samples were mixed in

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0.9% saline solutions and this homogenate was extracted three times with hexane and

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then centrifuged at 5000g at 4 °C for 10 min. The supernatant was collected and

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evaporated to dryness under nitrogen, and the residue was dissolved in 100 μL

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dichloromethane-methanol (50:50, v/v). Each sample was extracted in triplicate. Bc

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and retinoids analyses were carried out as previously described.16 For all samples, Bc

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and retinoids were analysed by HPLC (Series 1200, Agilent, Waldbronn, Germany)

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using a YMC C30 column (5 µm, 4.6 × 250 mm, YMC Co. Ltd., Japan), using the

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following mobile phase: methanol (A), methy-tert-butyl-ether (B), and purified water

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(C). The flow rate was set at 1 mL/min, the column temperature was maintained at 35

265

°C, and the volume injection was 20 µL. The gradient programme was described

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previously.31 Chromatograms were made at 325 and 450 nm to identify retinol, retinyl

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esters and Bc. Carotenoids and retinoids were identified by comparing their retention

268

time and spectra with their respective criteria.

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Statistical Analysis. The results were expressed as mean values with their SD.

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Differences between means were assessed using ANOVA followed by the post hoc

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Tukey’s test. Statistically significant differences were set at P < 0.05. All analyses

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were performed using SPSS 18.0 (IBM Corp., Armonk, NY, USA).

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RESULTS AND DISCUSSION



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Effect of Citrus Flavanones on the Bioaccessibility of Bc. The biological activity

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of carotenoids from food is highly dependent on their bioavailability, including

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bioaccessibility and intestinal absorption. The assessment of bioaccessibility was

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conducted using an in vitro digestion procedure. The present study was first designed

278

to assess the effect of citrus flavanones on the bioaccessibility of Bc (Figure 1A). In

279

the absence of citrus flavanones, the bioaccessibility of Bc was 11.0 ± 0.2%. The

280

addition of citrus flavanones improved the bioaccessibility of Bc. Hes-G significantly

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increased the bioaccessibility of Bc to 15.4 ± 1.2% (P < 0.05), corresponding to a

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39.5% improvement compared to when Bc was supplied alone. Flavonoids can act as

283

stabilizers of oil-in-water lipid droplets in the forms of densely packed layer that

284

prevents droplet shrinkage and coalescence by steric mechanisms.32 Smaller average

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lipid droplets size is correlated to larger specific surface area exposed. This larger

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specific surface area allows pancreatic lipases to attach more easily; therefore, a

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higher incorporation of Bc is the result.33 Moreover, polyphenols are thought to be

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antioxidants that protect carotenoids against oxidative degradation in the

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gastrointestinal tract and thus increase their bioaccessibility in vitro.34

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Poulaert et al.13 observed that when white grapefruit juice (containing a large

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amount of Nar-G) was added to the boiled orange-fleshed sweet potato, there was a

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negative effect on the bioavailability of Bc from orange-fleshed sweet potato.

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However, it was demonstrated that some citrus fruits, such as lime juice, enhanced Bc

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bioaccessibility from carrot or pumpkin.35 The authors suggested that the high citric

295

acid content could explain this effect by loosening of the matrix, thereby causing


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higher Bc bioaccessibility. Thus, the food matrix components are of great importance

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to the bioaccessibility of Bc. These results do not contradict our results, because

298

standards were used to investigate the effect of citrus flavanones on the

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bioaccessibility of Bc. We did not consider the interference of food matrix

300

components or the impacts of food processing.

301

Effect of Citrus Flavanones on the Incorporation of Bc into Synthetic Mixed

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Micelles. Incorporation into mixed micelles is required for lipophilic compounds to

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be transported across the unstirred water layer for uptake by small intestinal epithelial

304

cells.36 The incorporation into mixed micelles followed by cellular uptake are major

305

steps in determining the bioavailability of these compounds due to their apolar

306

characteristics.25 In the present study, synthetic mixed micelles were prepared to

307

simulate the lipid composition in the human duodenum during digestion.7 The

308

maximum amount of Bc that could be incorporated into synthetic mixed micelles was

309

investigated, as was the effect of citrus flavanones on the incorporation efficiency of

310

Bc (Figure 1B). Bc showed a high incorporation efficiency (approximately 50%) in

311

the absence of citrus flavanones, which was very close to the value obtained by Sy et

312

al.’s study.23 The addition of Hes and Hes-G significantly increased Bc incorporation

313

by 30.1% and 42.6%, respectively (P < 0.05). In contrast, the addition of Nar and

314

Nar-G significantly decreased the incorporation efficiency of Bc by 23.8% and 26.4%,

315

respectively (P < 0.05).

316 317

The reason for this phenomenon is likely related to the solubility of citrus flavanones in micelles. Poulaert et al.13 found that the solubility of Nar-G was high in



318

micelles and increased in a concentration-dependent manner. We previously showed

319

that the solubility of Nar-G was relatively higher than that of the three other

320

flavanones. These data indicated that Nar-G could be incorporated into mixed

321

micelles; therefore, Nar-G could compete with Bc for incorporation into mixed

322

micelles. Hes, Hes-G and Nar may change the physicochemical properties of micelles,

323

thus improving the incorporation of Bc into micelles. Nar increased the liposome

324

membrane fluidity in a dose-dependent manner.37 Hes-G was located at the level of

325

the polar head, whereas Hes interacted better with acyl chains.38 The membrane

326

interactions and localization of flavonoids play a vital role in exerting their biological

327

activity and meditating the permeation of molecules.39 In the present study, the

328

interactions of Hes, Hes-G and Nar with micelles may have changed the structures

329

and properties of these micelles ; thus, Bc was more easily incorporated into the

330

mixed micelles. These hypotheses require further experiments for verification.

331

Effect of Citrus Flavanones on the Cellular Uptake of Bc. Uptake by intestinal

332

cells is also a key step governing carotenoid absorption. Therefore, in the Caco-2 cell

333

model, we compared the uptake efficiency of Bc in the absence/presence of citrus

334

flavanones. Both natural and synthetic mixed micelles were tested in Caco-2 cells.

335

The results showed that the Bc cellular uptake from synthetic mixed micelles was

336

higher than that from natural mixed micelles (Table 1). Hes-G and Hes significantly

337

increased Bc cellular uptake from both natural and synthetic mixed micelles (P
0.05).

432

However, after 7-day gavage, the addition of Nar and Nar-G had a significant

433

reduction effect on the levels of retinoid, resulting in a decrease by 37.4% and 35.5%,

434

respectively (P < 0.05). In all plasma samples (Figure 4B), only retinol was detected.

435

After 3-day gavage, the addition of Hes and Hes-G increased the retinol concentration

436

by 28.9% and 22.9%, respectively. After 5- and 7-day gavage, the plasma retinol

437

concentration did not differ between the groups (both control and flavanone-fed

438

groups). After 3-day gavage, the addition of Hes, Hes-G and Nar increased the levels

439

of intestinal retinoids by 53.9%, 34.9% and 34.9%, respectively (Figure 4C). After

440

5-day gavage, the addition of Nar and Nar-G significantly reduced the levels of

441

intestinal retinoid in comparison with the control group (P < 0.05). However, after

442

5-day gavage, the addition of Nar-G significantly enhanced the retinoid

443

concentrations in adipose tissues by 2.13-fold (P < 0.05) (Figure 4D). After 7-day

444

gavage, the addition of all four citrus flavanones increased retinoid concentrations,

445

although the improvement was not statistically significant. This appears to be an

446

inconformity result compared to the general trend of decrease in retinoids formation

447

with Nar and Nar-G. In fact, correlations between dietary intakes of retinoids and

448

carotenoids and their concentrations in adipose tissues were weak.53 Accordingly, the

449

distribution of retinoids and carotenoids in adipose tissue is less homogeneous than



450

that in other tissues (e.g., plasma) and thus causes higher variability between samples.

451

In addition, Bc was not readily metabolized to vitamin A in adipose tissue during

452

short-term gavage study (10 d) may be required to assess

453

changes in adipose tissues concentrations of retinoid in the presence of citrus

454

flavanones.

455

All of the data above suggested that Hes and Hes-G enhanced the retinoid

456

concentrations in the tissues of healthy mice fed a normal diet. In contrast, except in

457

adipose tissues, Nar and Nar-G decreased the retinoid concentrations after 7-day

458

gavage. These data indicated that Hes, Hes-G, Nar and Nar-G may exhibit different

459

effects on retinoid concentrations in vivo. Our experiments found that Hes and Hes-G

460

significantly improved the incorporation efficiency of Bc (Figure 1B) and enhanced

461

Bc cellular uptake from synthetic mixed micelles (Table 1). Therefore, there was a

462

higher level of Bc absorption in the Hes- and Hes-G-fed groups than in the control

463

group, consequently producing higher metabolic levels than the control. Therefore, an

464

improvement in the total retinoid concentrations in Hes- and Hes-G-fed animals was

465

observed.

466

However, there was a significant reduction in the incorporation efficiency (Figure

467

1B) and cellular uptake of Bc from synthetic mixed micelles (Table 1) in the presence

468

of Nar and Nar-G. The results were in line with the changes of retinoid concentrations

469

in tissues. This finding suggested that among the various factors, the incorporation

470

efficiency into micelles was the main step governing Bc absorption and retinoid

471

concentrations in the presence of citrus flavanones. Thus, in the long-term, Nar and


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Page 23 of 40


472

Nar-G should not be appropriate as a food additive for the population at risk of

473

vitamin A deficiency.

474

In all tissues, citrus flavanones also had different influences on the levels of

475

metabolites (Supplementary Table 1-3). In the case of hepatic retinol, after 3-day

476

gavage, the presence of citrus flavanones had an improving influence on retinol levels.

477

Hes-G significantly increased the concentration of hepatic retinol by 1.59-fold (P

Hesperetin and hesperidin improved Î²-carotene incorporation

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