Citrus Flavanones Enhance Î²-Carotene Uptake in Vitro Experiment

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Bioactive Constituents, Metabolites, and Functions

Citrus Flavanones Enhance #-carotene Uptake in Vitro Experiment Using Caco-2 Cell: Structure–Activity Relationship and Molecular Mechanisms Zhongyuan Zhang, Meimei Nie, Chunquan Liu, Ning Jiang, Chunju Liu, and Dajing Li J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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Journal of Agricultural and Food Chemistry

Citrus Flavanones Enhance β-carotene Uptake in Vitro Experiment Using Caco-2 Cell: Structure–Activity Relationship and Molecular Mechanisms Zhongyuan Zhang, #† Meimei Nie, #,‡ Chunquan Liu, # Ning Jiang, # Chunju Liu, # and Dajing Li#†* #Institute

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

Nanjing 210014, China †School

of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013,

China ‡College

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

China

*Correspondence should be addressed to Dajing Li E-mail: [email protected] Tel: +86-02584391255 Fax: +86-02584391255

ACS Paragon Plus Environment


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Abstract

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Flavonoids can interfere with the absorption of carotenoids. In this study, the inherent

3

mechanisms of twelve citrus flavanones for β-carotene (Bc) cellular uptake and the

4

structure-activity relationship were investigated. The results showed that multiple

5

hydroxyl groups had the lowest promoting effect. O-glycosylation at C7 of the A ring

6

led to the greatest promoting effect on Bc absorption. O-glycosylation at C7 exhibited

7

a strong affinity with cell membrane and subsequently fluidized cell membrane.

8

Aglycon molecules significantly induced transient increases of paracellular

9

permeability by decreasing tight junction proteins (ZO-1, claudin-1) expression. In

10

addition, citrus flavanones might enhance scavenger receptor class B type I (SR-BI)

11

expression via their actions as agonists of peroxisome proliferator-activated

12

receptor-gamma (PPARγ). Catechol structure in B-ring attenuated the activate action

13

of SR-BI expression. The structure-dependent membrane permeability and activation

14

of specific membrane proteins are mechanistically associated with the promoting

15

effect on Bc cellular uptake by citrus flavanones.

16

Keywords:

17

relationship, mechanism

citrus

flavanone,

β-carotene,

cellular


uptake,

structure-activity

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18

Introduction

19

It is now well recognized that the consumption of bioactive compounds derived from

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dietary food have been demonstrated to exhibit multiple benefits for health promotion

21

and disease prevention. These bioactive compounds must be made bioavailable prior

22

to exerting their bioactivity. Many factors affect the bioavailability of these bioactive

23

compounds, consisting of food processing, changes in digestion, uptake, metabolism

24

and biodistribution.1 In the digestion of food items, the interactions of bioactive

25

compounds may facilitate or compete for cellular uptake and transportation, thus may

26

enhance/reduce their bioavailability. Carotenoids are one of the major classes of

27

phytochemicals in fruits and vegetables. Three major steps are made for the

28

bioavailability of carotenoids during digestion and their intestinal uptake: (i) that

29

carotenoids are released from the food matrix, (ii) that carotenoids have to be

30

incorporated into mixed micelles, (iii) that all carotenoids are absorbed by duodenal

31

mucosal cells.2,3 Bioavailability interferences between carotenoids with other

32

macromolecules, such as lipids, bile salts and digestive enzymes, have attracted great

33

interest among researchers.4,5 However, studies on bioavailability interactions

34

between carotenoids with other bioactive compounds remain scarce.

35

Flavonoids are also one of the main phytochemicals in plant-derived food.

36

Flavonoids can interfere with the absorption of carotenoids in several studies. In the

37

case of naringenin and lutein, competition between these two compounds for cellular

38

uptake is thought to lead to reduce lutein uptake in vivo.6 There are inconsistent

39

findings on the direction of the interactions. Ingested quercetin had been reported to



40

improve the content of β-carotene (Bc) in the liver of BALB/c mice by inhibiting

41

enzymatic conversion of Bc to retinol in small intestinal epithelial cells.7

42

Dhuique-mayer and colleagues observed that hesperetin and hesperidin significantly

43

increased the cellular concentrations of Bc and β-cryptoxanthin.8 The affinity of

44

flavonoids to cell membrane determined the interfering effects on cellular absorption

45

of carotenoids. Nevertheless, the mechanisms of the absorption between carotenoids

46

and flavonoids has not been fully elucidated both in vivo and in vitro.

47

The biological actions of flavonoids have been mainly attributed to their ability to

48

modify the membrane-mediated signaling pathways leading to alteration of cell

49

membrane permeability and interaction with specific membrane protein.9 It had

50

reported that catechins could interact with components of cell membranes resulting in

51

change the membrane potential, and subsequently increase paracellular permeability

52

for protons and potassium ions.10 Although some investigations have shown that

53

flavonoids can promote and protect the intestinal tight junction (TJ) barrier functions,

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the effect of flavonoids on cell membrane permeability might be complex.11 In

55

addition, carotenoids intestinal absorption is not just passive, but also involved

56

specific epithelial transporters, such as scavenger receptor class B type I (SR-BI),

57

Niemann-Pick C1-Like 1 and cluster determinant 36. Hesperidin was observed to

58

enhance SR-BI expression in intestine of gerbil depleted in vitamin A, although the

59

detailed mechanism of its role on SR-BI expression had not yet been clarified.12

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SR-BI was a peroxisome proliferator-activated receptor-gamma (PPARγ)-target gene

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in mice, rats and human liver.13 Another study has shown that hesperidin could be a


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strong PPARγ agonist.14 Activation of the related proteins in cellular absorption of

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carotenoids by hesperidin, led us to hypothesize that flavonoids regulating these

64

proteins could affect the uptake efficiency of carotenoids.

65

The aims of this project were to clarify the roles of flavonoids in the intestinal

66

absorption of carotenoids by Caco-2 cells. Caco-2 cells are frequently used as an in

67

vitro model of intestinal absorption of nutrients and non-nutrients present in foods.

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Although some protein expression may not be similar to those in human intestinal

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absorptive cells, Caco-2 cells exhibit some similar morphological and functional

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characteristics. It is therefore differentiated Caco-2 cell monolayer were considered to

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be an as effective model to study the intestinal absorption of carotenoids.15 Citrus

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flavanones (Table 1), are typical flavonoids in citrus fruits. Bc, a characteristic

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lipophilic pigment presented in fruits and vegetables, used as a model of a carotenoid.

74

In the case of citrus juices, it contains 300-700 mg hesperidin per liter and 10-30 mg

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carotenoids per liter.16 Therefore, the amount of citrus flavanone is fifty times as

76

much as Bc in the absorption experiments. Concerning flavonoids possess numerous

77

important physiological and pharmacological functions, it is important to understand

78

the membrane interaction behavior of a citrus flavanone and the related

79

structure-activity relationship (SAR). Thus, the involvement of flavonoids-mediated

80

membrane permeability and specific membrane proteins in the cellular uptake of Bc

81

was investigated.

82

Materials and Methods



83

Materials.

All-trans-Bc,

hesperetin,

naringenin,

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naringin,

eriodictyol,

84

isosakuranetin, neohesperidin, neoeriocitrin, poncirin , didymin were from

85

Sigma-Aldrich China Co., Ltd (Shanghai, China). Hesperidin, narirutin and eriocitrin

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was get from Santa Cruz Biotechnology (CA, USA). Dulbecco’s minimum essential

87

medium (DMEM) containing 4.5 g/L glucose, foetal bovine serum (FBS),

88

penicillin-streptomycin, L-glutamin, enonessential amino acids, trypsin-EDTA (500

89

and 200 mg/L, respectively), phosphate-buffered solution (PBS) were from Thermo

90

Fisher Scientific Co., Ltd. (Shanghai, China). Phosphatidylcholine, monoolein,

91

lysophosphatidylcholine, oleic acid and free cholesterol were purchased from Xiya

92

Chemistry Co., Ltd (Jinan, China).

93

(MTBE) and methanol (MeOH) were purchased from Javascript (Norcross, GA,

94

USA).

HPLC grade reagent of methyl-tert-butyl-ether

95

Preparation of Synthetic Micelles of Bc. Synthetic mixed micelles were prepared

96

from Reboul et al.,17 to simulate the lipid composition of mixed micelles found in

97

vivo. The components were dissolved in a chloroform-methanol (2:1; v/v) solution.

98

The

99

monoolein, free cholesterol, oleic acid and Bc were 0.04 mM, 0.16 mM, 0.3 mM, 0.1

100

mM, 0.5 mM and 5 μM, respectively. Solvents were carefully evaporated and

101

redissolved in 20 mL of a solution of DMEM in the presence of 5 mM taurocholate.

102

The micelles were formed by sonication in a bath sonicator for 3 min at 37 °C. The

103

0.22 µm filtered solutions were stored -20 °C. The concentrations of Bc were chosen

104

to mimic physiologic doses in duodenum after ingestion.

working

concentration

of

phosphatidylcholine,


lysophosphatidylcholine,

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105

Cell Culture. Caco-2 cells were purchased from the American Type Culture

106

Collection (Manassas, VA). Cells (passage 30-40) were cultured in the presence of

107

DMEM supplemented with 10% of FBS, 1% of nonessential amino acid, 1% of

108

penicillin-streptomycin, and 1% of L-glutamine (complete medium). Cells were

109

incubated at 37 °C in an incubator in a humidified atmosphere of 5% CO2 and the

110

medium was changed every 48 h. In the present study, the cells were grown on 6-well

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transwell plate (3 µm pore size polycarbonate membrane, 24 mm diameter,

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Corning-Costar Corp., Cambridge, UK), at a seeding density of 2×105 cells. Media

113

were changed every day for 14 days to obtain confluent and highly differentiated cell

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monolayers. Before each experiment, the integrity of the cell monolayers was checked

115

by measuring the trans-epithelial electrical resistance (TEER). TEER values were

116

expressed as Ω cm2, this equation can be written as:

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TEER = (Ω cell monolayer - Ω filter cell free) × filter area

118

When cell monolayer with a TEER values of above 500 Ω cm2 are considered

119

acceptable. Those monolayers were then used for further analysis.

120

Determination of Bc Uptake. The apical side of the cell monolayers received 1

121

mL mixed micelles of Bc and the basolateral side received 2 mL FBS-free medium,

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after washing. Cell were incubated for 5 h at 37 °C. Then, medium from each side

123

were collected. The cell monolayers were washed twice with 1 mL of PBS to remove

124

absorbed carotenoids, then scraped and obtained in 1 mL PBS. All the samples were

125

stored at -80 °C before analysis. A 100 µL aliquot of cell samples was for protein



126

assay using the Bicinchoninic acid solution kit (Sigma-Aldrich, Shanghai, China). Bc

127

uptake was determined as the amounts of Bc in both cells plus basolateral medium,

128

expressed as pmol of Bc per mg of cell proteins.

129

Extraction of Bc was carried out as follows. The cells samples were extracted three

130

times with 2mL of hexane-ethanol-acetone (50:25:25, v:v:v) in the presence of 500

131

μL internal standard (apo-8’-carotenal). The mixture were centrifuged at 300g for 5

132

min. The pooled hexanic phases were evaporated and redissolved in 200 µL of

133

MTBE: MeOH (50:50, v:v), and the Bc content was analyzed by HPLC.

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The Addition of Citrus Flavanones. Before each experiment, an appropriate

135

amount of citrus flavanone was added to the synthetic micelles of Bc solution at final

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concentration of 250 μM. Citrus flavanones were first prepared in DMSO and then

137

diluted in DMEM (the final concentration of DMSO was 0.5% ).

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Flavanones Permeation Experiments. At beginning of the flavanones permeation

139

experiments, the cell monolayers were washed twice with PBS. The permeation

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experiments were prepared by adding only the flavonoid solutions (250 μM) to the

141

apical side (0.5 mL), while basolateral side received the transport medium (1.5 mL).

142

The cultures were incubated at 37 °C for 5 h with gentle shaking, media from both

143

sides of cell monolayer were collected and stored below -80 °C for further analysis.

144

The determination flavanones permeation was carried out as followed. The samples

145

were mixed with 200 μL MeOH, the upper phase obtained by centrifugation (13000g,

146

10 min). Aliquot of the supernatant solution was analyzed the apparent permeability


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coefficients (Papp) by HPLC described as Tian and others.18

148

Measurement of Membrane Fluidity. The measurement of membrane fluidity

149

method used was as described by Yun et al.19 After Bc absorption experiment, the cell

150

monolayers were washed twice with 1 mL PBS. Then the cell monolayer was treated

151

0.25% trypsin-EDTA for 5 min, the cells were collect by centrifugation (1000 rpm, 5

152

min). The pellets were re-suspended in 4 mL PBS containing 2 μM DPH (DPH was

153

prepared by tetrahydrofuran) and processed at 37 °C for 30 min in darkness. After

154

incubation period, the suspensions were subjected to centrifuge (1000 rpm, 5 min) and

155

washed three times by PBS, finally re-suspended in 4 mL PBS. After that, the

156

fluorescence polarization, P, was determined on spectrofluorometer, following

157

emission at 432 nm and excitation at 362 nm.

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Immunoblotting Analysis. Aliquot cells samples (500 μL) were homogenised in 1

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mL lysis buffer (10 mM Tris-HCl, 0.3% SDS , pH 7.4) containing protease inhibitor

160

cocktail. The cell lysates were subjected to SDS-polyacrylamide gel and electro

161

blotted onto a polyvinylidene difluoride membrane. Membrane were blotted

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successively for β-actin, ZO-1, claudin-1, SR-BI and PPARγ (1:1000, Cell Signaling

163

Technology, Inc., USA), and then with a horseradish peroxidase-conjugated

164

anti-rabbit IgG antibody (1:2000, Cell Signaling Technology, Inc., USA). After

165

washing, blots were incubated with an enhanced chemiluminescence reagent (GE

166

Healthcare), and the images were processed with Image Quant LAS4010 (GE

167

Healthcare).



168

HPLC Analysis. Flavanones was analysed by reverse phase HPLC using a Waters

169

2690 Alliance system. Flavanones were separated along a XDB-C18 column (4.6

170

mm× 250 mm, 5 µm), mobile phases included 0.1% methanoic acid as eluent A,

171

100% acetonitrile as eluent B at a flow rate of 1.0 mL/min, the column temperature

172

was maintained at 25 °C. The injection volume was 10 µL, and the flavanone was

173

monitored at 287 nm. The HPLC analysis of Bc was as described by Zhang and others

174

with modifications.3 Briefly, Bc was separated along a C30 YMC column (4.6 mm ×

175

250 mm, 5 μm) using for the mobile phase: MeOH-MTBE-water (solvent A, 10:85:5,

176

v:v:v) and MeOH-MTBE-water (solvent B, 70:25:5, v:v:v). The flow rate was fixed at

177

0.6 mL/min, the column temperature was set at 25 °C, and the injection volume was

178

20 μL. Identification and quantification of flavanones and Bc were achieved using

179

peak area and their calibration.

180

Statistical Analysis. All statistical analysis were performed using SPSS 18.0 (IBM

181

Corp., Armonk, NY, USA). The results are presented as means with their standard

182

deviation (SD). Statistical significance between means were determined using

183

ANOVA followed by the post hoc Tukeys test. A P values < 0.05 was considered

184

significant.

185

Results

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Effect of Citrus Flavanones on Cellular Uptake of Bc. Our first aim in this

187

project was to test whether each individual citrus flavanones showed an improvement

188

of Bc absorption. Cellular uptake of Bc was evaluated using a Caco-2 cell monolayer,


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189

in the presence of twelve citrus flavanones. The results showed that when Bc was

190

co-added with citrus flavanones, the absorption of Bc was increased from 1.3- to

191

2.5-fold (Figure 2). These data suggested that citrus flavanones could significantly

192

enhance Bc absorption in Caco-2 cell monolayer (P < 0.05). Our data (Figure 2)

193

showed that the promoting effect of citrus flavanones on Bc absorption were

194

determined in descending order: poncirin, neohesperidin, didymin, neoeriocitrin,

195

naringin, eriocitrin, narirutin, hesperidin, naringenin, hesperetin, isosakuranetin,

196

eriodictyol.

197

Interaction of Citrus Flavanones with Cell Membrane. The great bioactivity of

198

flavonoids is largely dependent upon the interaction with membranes. Thus,

199

interaction of citrus flavanones with cell membrane was investigated. Firstly, Papp

200

values, measuring the speed of the transport in Caco-2 cells, were obtained for citrus

201

flavanones (Table 2). All the flavanones were determined at a detection limit of < 0.5

202

pmol on the column. The apical to basolateral flux of hesperetin was approximately

203

1.3-fold, 3.3-fold and 2.2-fold higher than for the other three aglycones, naringenin,

204

eriodictyol and isosakuranetin, respectively. Glycosylation of the hydroxyl groups of

205

the citrus flavanones significantly decrease their passage through intestinal cell

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models. Specially, permeation of naringin, neoeriocitrin, narirutin and eriocitrin, was

207

not detected in this study. The modification of flavonoids on cell membrane fluidity

208

can significantly affect cell membrane-mediated signaling pathways.20 We then

209

examined the membrane fluidity using cell membrane mimicking system in the

210

presence of citrus flavanones. The results (Figure 3) showed that citrus flavanones



211

treatment differently affected the fluorescence polarization of cell-mimetic

212

membranes. Neoeriocitrin, eriodictyol, neohesperidin, narirutin, isosakuranetin,

213

poncirin and eriocitrin decreased the P values, suggesting fluidization of the

214

hydrophobic portion of bilayer.

215

Citrus Flavanones Induced Increases of Paracellular Permeability. In order to

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investigate the effect of citrus flavanone on paracellular permeability, the expression

217

of TJ proteins was detected by western blotting. As shown in Figure 4, we found that

218

the effect of each individual citrus flavanones on TJ proteins expression was various.

219

Hesperetin, hesperidin, naringenin, naringin, neoeriocitrin, didymin, isosakuranetin,

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poncirin and eriocitrin treatments significantly decreased the proteins level of both

221

ZO-1 and claudin-1 at 5 h. The paracellular permeability of the aglycon molecules vs.

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the corresponding neohesperidoside and rutinoside forms were compared.

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O-glycosylation at C7 weakened the paracellular permeability of hesperetin,

224

naringenin, isosakuranetin. In the case of isosakuranetin, the densitometric analysis

225

showed that poncirin and didymin treatments significantly increased the ratio of ZO-1

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by 43.1% and 61.5% (P < 0.05), and increased the ratio of claudin-1 by 27.0% and

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49.2% (P < 0.05), respectively. However, the comparison with eriodictyol ruled out it.

228

Both ZO-1 and claudin-1 were increased significantly by eriodictyol administration (P

229

< 0.05).

230

Citrus Flavanones Increases SR-BI and PPARγ Protein Expression in Caco-2

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Cells. SR-BI, a versatile cell surface receptor, is known as the mediation selective

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uptake of cholesteryl ester both in vitro and in vivo.21 SR-BI is an essential receptor


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protein that involved in the process of carotenoid absorption and transport.22 In view

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of this evidence, the effects of citrus flavanones on the SR-BI expression were

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evaluated. Caco-2 cells were first treated with citrus flavanones, and SR-BI

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expression was then measured. As shown in Figure 5, SR-BI expression levels were

237

improved significantly in the citrus flavanones treated groups in comparison with the

238

normal control group (P < 0.05). PPARγ is a member of the ligand-dependent nuclear

239

receptor superfamily, which is in charge of energy balance, lipid, and glucose

240

homeostasis.23 Malero et al. demonstrated that PPARγ plays a vital role in the

241

biosynthesis of vitamin A.24 Protein expression of PPARγ was determined in present

242

study. As shown in Figure 5, the protein expression of PPARγ was significantly

243

upregulated by citrus flavanones (P < 0.05).

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To directly determine whether the promoting effect on Bc absorption by citrus

245

flavanones involved SR-BI and PPARγ, then we measured proteins expression and

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uptake experiments in Caco-2 cells treated with the PPARγ and SR-BI inhibitor,

247

respectively. Ezetimibe (EZT) can regulate the gene expression of the nuclear

248

receptors RXRα, RARγ, LXRβ, SREBP-1 and SREBP-2, then influence the

249

transcription of the surface receptors NPC1L1, SR-BI, and ABCA1.25 It summarized

250

the cellular absorption of Bc after Bc, citrus flavanones and EZT co-exposure in

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Caco-2 cells (Supplementary Table 1). The pretreatment of the Caco-2 cells with EZT

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partially attenuated the promoting effect of citrus flavanones on Bc absorption. Each

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individual citrus flavanones exhibited different degree of attenuation in Bc absorption.

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For example, after pretreatment of EZT, the Bc absorption was decreased by 61.9%,



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57.3%, 53.0%, 52.1%, 50.4% in naringin, neohesperidin, didymin, hesperidin,

256

nariutin treated Caco-2 cells, respectively (P < 0.05). We further evaluated the effect

257

of EZT on SR-BI expression in citrus flavanones treated Caco-2 cells (Figure 6). The

258

results showed that EZT induced a significant reduction of SR-BI expression in citrus

259

flavanones stimulated cells. These results confirmed that SR-BI participated in the

260

process of the promoting effect of citrus flavanones on Bc absorption. This result was

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consistent with Poulaert et al., who demonstrated similar results after using a mixture

262

of hesperidin with orange-fleshed sweet potatoes.26 T0070907, an inhibitor of PPARγ,

263

was used to evaluate the involvement of this signaling pathway in the induction of the

264

promoting effect of citrus flavanones on Bc absorption. The results showed that to a

265

lesser extent PPARγ inhibition decrease SR-BI expression exposed to citrus

266

flavanones (Figure 7).

267

Discussion

268

The present study confirmed that twelve citrus flavonoids promoted Bc cellular

269

uptake by Caco-2 cells. The hydroxyl groups of flavonoids are important for their

270

biological activities according to Smejkal et al.27 We found that the promoting effect

271

of the twelve citrus flavanones investigated depended upon the location and number

272

of hydroxyl groups on the aromatic ring. Aglycone citrus flavanones, including

273

isosakuranetin, naringenin, hesperetin and eriodictyol, exhibited lower promoting

274

effect on Bc absorption than citrus flavanones glycosides. Among aglycone citrus

275

flavanones investigated, eriodictyol showed the lowest promoting effect on Bc

276

absorption, and this is the only molecule containing four hydroxyl groups.


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O-glycosylation at carbon 7 seemed to increase the Bc absorption significantly. In the

278

presence of neohesperidin, the amount of Bc in Caco-2 cells was approximately

279

1218.6 pmol Bc per mg proteins, which was 2.5-fold higher than only Bc treatment.

280

Dhuique-mayer and researchers also have demonstrated that glycoside (hesperidin)

281

exhibited the strongest enhancement in the cellular uptake of Bc and β-cryptoxanthin,

282

while the other citrus flavanones (naringenin and hesperetin) exhibited weak

283

enhancement activities for those carotenoids absorption.8 However, the molecular

284

mechanism by which flavanones promote Bc absorption had not been well

285

characterized. This study provides mechanistical aspects of the facilitating mechanism

286

of increased Bc cellular uptake based on structure-activity relationship of membrane

287

fluidity, paracellular permeability and specific membrane transporters.

288

First, we hypothesize that citrus flavanones alter the barrier function of bilayers to

289

regulate absorption of carotenoids. However, our data found that most of the

290

flavanones exhibited a poor permeability in apical side to basolateral side direction.

291

This seems to be an inconsistent result compared with the overall trend that

292

flavanones increased cellular uptake of Bc. We further found that seven flavanone

293

glycosides (neoeriocitrin, eriodictyol, neohesperidin, narirutin, isosakuranetin,

294

poncirin and eriocitrin) caused a slight decrease in value of fluorescence polarization

295

P, suggesting fluidization of the hydrophobic portion of bilayer. It is noted that these

296

small differences in membrane fluidization are not related to the significant increase

297

in Bc cellular uptake after citrus flavanone addition. It is likely that

298

flavanone-mediated membrane permeability and membrane fluidity may not directly



299

interfere with its Bc-transport capability. Flavonoids localize either at the membrane

300

interface or in the hydrophobic core of the lipid bilayer result in corresponding

301

changes in the membrane properties.20,28,29 Consequently, biological membranes

302

adjust their physical properties for optimal functioning of membrane proteins and

303

transporters. This interaction of flavonoids with cell membranes modulates cell

304

signaling and fate even when they cannot enter cells. In addition, flavonoid glucosides

305

are prone to hydrolysis by β-glucosidases of microbes colonizing the human/rat

306

stomach.30 Human small intestine from lactose intolerance and tolerance individuals

307

had 2 to 10 and 20 to 80 mU/mg lactase-phlorizin hydrolase activity, respectively,

308

whereas differentiated Caco-2 cells had 0 to 0.12 mU/mg.31 Thus, it may have its

309

limitations to study flavanones permeability by Caco-2 cells in vitro.

310

Next, we found that citrus flavanones regulated paracellular permeability. Specially,

311

some flavanones (hesperetin, hesperidin, naringenin, naringin, neoeriocitrin, didymin,

312

isosakuranetin, poncirin and eriocitrin) treatments significantly decreased the proteins

313

level of both ZO-1 and claudin-1 at 5 h. It has reported that naringenin induced

314

transient decrease of TJ proteins at 6 h in the whole extracts of Caco-2 cell monolayer,

315

including occludin, ZO-1, ZO-2, claudin-1, claudin-3 and JAM-A, although not

316

reaching statistical significance regarding decrease of these TJ proteins.32 These data

317

indicated that the above flavanones might cause a transient drop in TJ proteins

318

expression, which in turn caused an enhancement in paracellular permeability and

319

subsequently enhanced Bc absorption. However, this kind of paracellular permeability

320

was transient. According to Noda, Tanabe, & Suzuki and Suzuki, Hara,32,33 the


Page 16 of 40

Page 17 of 40


321

increase of TJ barrier induced by flavonoids is believed to occur through the increased

322

assembly of claudin-4 at the TJs during the long incubation period (>12 h). However,

323

eriodictyol significantly increased the expression of TJ proteins, suggesting it

324

mediated paracellular permeability by different signal pathway. In fact, eriodictyol

325

contains multiple hydroxyl groups (catechol structure on the B-ring, resorcinol ring on

326

the A-ring), that can significantly reduce the activation of PKCδ by blocking the

327

phorbol ester binding site.34 The inhibition of PKC δ isoform could promote TJ

328

barrier function by increasing the TJ proteins expression.11 Neoeriocitrin and eriocitrin

329

have shown to have less promotive effects on both ZO-1 and claudin-1 proteins

330

expressions by comparison with eriodictyol during the period time. In this case, this

331

could be caused by the lack of forming one hydrogen bond between C7 hydroxyl

332

group with Leu-251 (phorbol ester binding site of PKCδ). Thus, neoeriocitrin and

333

eriocitrin performed lower inhibition activities of PKCδ phosphorylation than

334

eriodictyol, and subsequently exhibited less promotive effect on the TJ barrier

335

function.

336

Finally, the specific membrane transporters involved in the process of carotenoid

337

uptake were investigated. However, there was significant difference in enhancing the

338

expression of SR-BI by citrus flavanones. The different citrus flavanones structures

339

may resulted in the difference susceptibility for SR-BI expression. SR-BI expression

340

became much lower by neoeriocitrin, eriodictyol and eriocitrin treatment, compared

341

with other citrus flavanones. The citrus flavanones with catechol structure in B-ring

342

might have less affinity for the binding site to activate SR-BI than the other citrus



343

flavanones. In addition, we found that citrus flavanones induced upregulation of

344

PPARγ proteins (Figure 5). Previous evidence showed that hesperidin induced

345

activation of PPARγ to exert biological actions in NALM-6 cells.35 Ayman et al.

346

demonstrated that hesperidin and naringin exhibited antidiabetic effects via

347

up-regulating PPARγ in adipose tissue and adiponectin in type 2 diabetic rats.36 Liang

348

and colleagues reported that chrysin, apigenin, and kaempferol activated PPARγ,

349

however, flavanones were inefficient in activating PPARγ in macrophages.37 This

350

difference in the effect of flavonoids on PPARγ may be due to cell type-specific

351

factors or to the diversity of flavonoids structures and their subsequent signaling

352

transduction pathways.

353

After that, we used SR-BI and PPARγ inhibitor to pretreat Caco-2 cells. Our data

354

showed that Bc absorption was decreased significantly in the presence of SR-BI

355

inhibitor. Pretreatment with SR-BI and PPARγ inhibitor also somewhat decreased

356

SR-BI and PPARγ protein expression after 5 h of citrus flavanones treatment. Thus,

357

we hypothesized that the promoting effect of citrus flavanones on Bc absorption could

358

be partially due to their effect on SR-BI expression by activating PPARγ. As far as we

359

know, this is the first report showing that citrus flavanones increase SR-BI expression

360

by activating PPARγ in intestinal cells. Actually, activation of PPARγ has been

361

shown to increase the expression of SR-BI in hepatocyte because PPARγ binds to a

362

response element in the human SR-BI promoter.24 PPARγ agonist can increase SR-BI

363

mRNA and protein expression in adipose tissue.37 In this report, citrus flavanones

364

might enhance SR-BI expression via their actions as agonists of PPARγ. Furthermore,


Page 18 of 40

Page 19 of 40


365

it should be mentioned that the localization and interaction of flavonoids with cell

366

membranes might contribute for effective activation of membrane mediated signaling

367

pathways. Factually, flavonoids can interact with intestinal cell membrane lipid rafts

368

through their binding to cholesterol.39 Carotenoids absorption related transport

369

proteins, such as Niemann-Pick type C1 Like 1 protein, cluster determinant 36, and

370

SR-BI, were found to be mainly located in caveolae/lipid raft microdomains.40,41 In

371

present study, citrus flavanones perturbed cell membrane by penetrating and

372

fluidizing the bilayer membrane. This modification for cell membrane may directly

373

lead to change transporter expression, such as SR-BI. Nevertheless, the contribution

374

of cell membrane modulation and changes of transporter expression deserved further

375

study.

376

In conclusion, the present results showed that all of the twelve selected citrus

377

flavanones facilitated cellular absorption of Bc. Glycosidic structure markedly

378

enhanced the promoting effect of citrus flavanones on Bc absorption. Preliminary

379

SAR analysis showed aglycone exhibited lower promoting effect, multiple hydroxyl

380

groups showed the lowest promoting effect. O-glycosylation at C7 exhibited a strong

381

affinity with cell membrane. All the tested citrus flavanones fluidized cell membrane.

382

In addition, the various influence on Bc cellular uptake by citrus flavanones maybe

383

related to the different interaction with cell membrane and expression of TJ and

384

transport proteins. The structure-dependent activation of specific membrane proteins

385

may directly contribute to the promoting effect on Bc absorption by citrus flavanones.

386

This study may provide scientific evidences to understand the interaction between



387

flavanones and Bc with regard to the health benefits, and also to develop commercial

388

carotenoids mixtures.

389

Abbreviations Used

390

Bc, β-carotene; SR-BI, scavenger receptor class B type I; PPARγ, peroxisome

391

proliferator-activated receptor-gamma; DPH, 1, 6-Diphenyl-1, 3, 5-hexatriene; Papp,

392

apparent permeability coefficients.

393

Supporting Information

394

The cellular absorption of Bc with or without EZT by citrus flavanones

395

(Supplementary Table 1).

396

Funding

397

The financial support provided by the National Natural Science Foundation of China

398

(Project No. 31601470) and the Natural Science Foundation of Jiangsu Province of

399

China (Project No. BK20160581).

400

Notes

401

The authors have declared no conflict of interest.

402

References

403

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M.; Brito, C.; Cilla, A.; El, S. N.; Karakaya, S. Mind the gap-deficits in our

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knowledge of aspects impacting the bioavailability of phytochemicals and their


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Caco-2 TC-7 cells occurs partly by a facilitated process involving the scavenger

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flavonoids using Caco-2 cell monolayer model. Int. J. Pharmaceut. 2009, 367,

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58-64.

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(19) Yun, O.; Liu, Z. W.; Zeng, X. A.; Han, Z. Salmonella typhimurium resistance

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on pulsed electric fields associated with membrane fluidity and gene regulation.



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Innov. Food Sci. Emerg. 2016, 36, 252-259.

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(20) Ulrih, N. P.; Ota, A.; Šentjurc, M.; Kure, S.; Abram, V. Flavonoids and cell

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membrane fluidity. Food Chem. 2010, 121, 78-84.

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(21) Shen, W. J.; Hu, J.; Hu, Z.; Kraemer, F. B.; Azhar, S. Scavenger receptor class

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B type I (SR-BI): A versatile receptor with multiple functions and actions. Metab.

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2014, 63, 875-886.

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(22) During, A.; Doraiswamy, S.; Harrison, E. H., Xanthophylls are preferentially

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taken up compared with β-carotene by retinal cells via a SRBI-dependent

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mechanism. J. Lipid Res. 2008, 49, 1715-24.

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(23) Hihi, A. K.; Michalik, L.; Wahli, W. PPARs: transcriptional effectors of fatty

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acids and their derivatives. Cell. Mol. Life Sci. 2002, 59, 790-798.

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(24) L, M.; M, S.; Juvet, L. K.; Mousavi, A.; T, G.; Berg, T. Hepatic scavenger

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receptor class B, type I is stimulated by peroxisome proliferator-activated receptor

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gamma and hepatocyte nuclear factor 4alpha. Biochem. Biophys. Res. Com. 2003,

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305, 557-565.

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(25) During, A.; Dawson, H. D.; Harrison, E. H., Carotenoid transport is decreased

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and expression of the lipid transporters SR-BI, NPC1L1, and ABCA1 is

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downregulated in Caco-2 cells treated with ezetimibe. J. Nutr. 2005, 135, 2305-12.

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(26) Poulaert, M.; Borel, P.; Caporiccio, B.; Gunata, Z.; Dhuiquemayer, C.

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Grapefruit juices impair the bioaccessibility of β-carotene from orange-fleshed sweet


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potato but not its intestinal uptake by Caco-2 cells. J. Agric. Food Chem. 2012, 60,

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685-691.

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(27) Smejkal, K.; Chudík, S.; Kloucek, P.; Marek, R.; Dvorská, M. Antibacterial C

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-Geranylflavonoids from paulownia tomentosa fruits. J. Nat. Prod. 2008, 71,

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706-709.

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(28) Hendrich, A. B. Flavonoid-membrane interactions: possible consequences for

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biological effects of some polyphenolic compounds. Acta. Pharmacol. Sin. 2006, 27,

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27-40.

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(29) Erlejman, A. G.; Verstraeten, S. V.; Fraga, C. G.; Oteiza, P. I. The interaction of

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flavonoids with membranes: potential determinant of flavonoid antioxidant effects.

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Free Radic. Res. 2004, 38, 1311-1320.

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(30) Nemeth, K.; Piskula, M. K. Food content, processing, absorption and

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metabolism of onion flavonoids. Crit. Rev. Food Sci. 2007, 47, 397-409.

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(31) Chantret, I.; Rodolosse, A.; Barbat, A.; Dussaulx, E.; Brotlaroche, E.;

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Zweibaum, A.; Rousset, M. Differential expression of sucrase-isomaltase in clones

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isolated from early and late passages of the cell line Caco-2: evidence for

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glucose-dependent negative regulation. J. Cell Sci. 1994, 107, 213-225.

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(32) Noda, S.; Tanabe, S.; Suzuki, T. Naringenin enhances intestinal barrier function

504

through the expression and cytoskeletal association of tight junction proteins in

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Caco-2 cells. Nutr. Food Res. 2013, 57, 2019-2028.



506

(33) Suzuki, T.; Hara, H. Quercetin enhances intestinal barrier function through the

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assembly of zonula [corrected] occludens-2, occludin, and claudin-1 and the

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expression of claudin-4 in Caco-2 cells. J. Nutr. 2009, 139, 965-974.

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(34) Kongpichitchoke, T.; Hsu, J. L.; Huang, T. C. Number of hydroxyl group on the

510

B-ring of flavonoids affects their antioxidant activity and interaction with phorbol

511

ester binding site of PKCδ C1B domain: in-vitro and in-silico study. J. Agric. Food

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Chem. 2015, 63, 4580-6.

513

(35) Ghorbani, A.; Nazari, M.; Jeddi-Tehrani, M.; Zand, H. The citrus flavonoid

514

hesperidin induces p53 and inhibits NF-κB activation in order to trigger apoptosis in

515

NALM-6 cells: involvement of PPARγ-dependent mechanism. Eur. J. Nutr. 2012,

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51, 39-46.

517

(36) Mahmoud A. M.; Ahmed O. M.; Ashour M. B.; Abdel-Moneim, A.

518

Upregulation of PPARγ mediated the antidiabetic effects of citrus flavonoids in type

519

2 diabetic rats. Inter. J. Bioassays, 2013, 2, 756-761.

520

(37) Liang, Y. C.; Tsai, S. H.; Tsai, D. C.; Linshiau, S. Y.; Lin, J. K. Suppression of

521

inducible cyclooxygenase and nitric oxide synthase through activation of

522

peroxisome proliferator-activated receptor-gamma by flavonoids in mouse

523

macrophages. Febs Lett. 2001, 496, 12-18.

524

(38) Toh, S. A.; Millar, J. S.; Billheimer, J.; Fuki, I.; Naik, S. U.; Macphee, C.;

525

Walker, M.; Rader, D. J. PPARγ activation redirects macrophage cholesterol from

526

fecal excretion to adipose tissue uptake in mice via SR-BI. Biochem. Pharmacol.


Page 26 of 40

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527

2011, 81, 934-941.

528

(39) Chiou, Y. L.; Lin, S. R.; Hu, W. P.; Chang, L. S. Quercetin modulates activities

529

of Taiwan cobra phospholipase A2via its effects on membrane structure and

530

membrane-bound mode of phospholipase A2. J. Biosci. 2012, 37, 277-287.

531

(40) Babitt, J.; Trigatti, B.; Rigotti, A.; Smart, E. J.; Anderson, R. G. W.; Xu, S.;

532

Krieger, M. Murine SR-BI, a high density lipoprotein receptor that mediates

533

selective lipid uptake, is n-glycosylated and fatty acylated and colocalizes with

534

plasma membrane caveolae. J. Biol. Chem. 1997, 272, 13242-13249.

535

(41) Rhainds; D. Localization and regulation of SR-BI in membrane rafts of HepG2

536

cells. J. Cell Sci. 2004, 117, 3095-3105.



537

Figure captions

538

Figure 1.

The chemical structure of flavanones.

539

Figure 2.

Effect of citrus flavanones on the cellular absorption of Bc. Caco-2 cells

540

were incubated with synthetic micelles of Bc in the absence / presence of citrus

541

flavanones. The final concentrations of Bc and citrus flavanone in the cell medium

542

were 5 μM and 250 μM, respectively. The Bc content was estimated as the amounts of

543

Bc in both cells plus basolateral medium. Cultures were incubated for 5 h. Values are

544

expressed as mean ± SD of three determinations. Data with different letters display

545

significant statistical differences (P < 0.05). Bc, β-carotene.

546

Figure 3.

547

incubated with synthetic micelles of Bc in the absence / presence of citrus flavanones.

548

The final concentrations of Bc and citrus flavanone in the cell medium were 5 μM and

549

250 μM, respectively. After 5 h incubation, the fluorescence polarization was

550

measured. Values are expressed as mean ± SD of three determinations. Data with

551

different letters display significant statistical differences (P < 0.05). Bc, β-carotene.

552

Figure 4.

553

monolayers. (A) Representative band densities of western blots of ZO-1 and

554

claudin-1. (B) Graph representing the relative change in ZO-1 protein levels. (C)

555

Graph representing the relative change in claudin-1 protein levels. Caco-2 cells were

556

incubated with synthetic micelles of Bc in the absence / presence of citrus flavanones.

557

The final concentrations of Bc and citrus flavanone in the cell medium were 5 μM and

Effect of citrus flavanones on the membrane fluidity. Caco-2 cells were

Effect of citrus flavanones on the experssion of TJ proteins in Caco-2 cell


Page 28 of 40

Page 29 of 40


558

250 μM, respectively. After 5 h incubation, Caco-2 cells were harvested and cell

559

protein samples were analyzed. Values are expressed as the mean ± SD, n = 3

560

independent experiments. Data with different letters display significant differences (P

561

< 0.05). Bc, β-carotene.

562

Figure 5.

563

monolayers. (A) Representative band densities of Western blots of SR-BI and PPARγ.

564

(B) Graph representing the relative change in SR-BI protein levels. (C) Graph

565

representing the relative change in PPARγ protein levels. Caco-2 cells were incubated

566

with synthetic micelles of Bc in the absence / presence of citrus flavanones. The final

567

concentrations of Bc and citrus flavanone in the cell medium were 5 μM and 250 μM,

568

respectively. After 5 h incubation, Caco-2 cells were harvested and cell protein

569

samples were analyzed. Values are expressed as the mean ± SD, n = 3 independent

570

experiments. Data with different letters display significant differences (P < 0.05). Bc,

571

β-carotene.

572

Figure 6.

573

EZT in Caco-2 cell monolayers. (A) Representative band densities of Western blots of

574

SR-BI. (B) Graph representing the relative change in SR-BI protein levels. Caco-2

575

cells were incubated with synthetic micelles of Bc in the absence / presence of citrus

576

flavanones. The final concentrations of Bc and citrus flavanone in the cell medium

577

were 5 μM and 250 μM, respectively. The final concentration of EZT was 30 μM.

578

After 5 h incubation, Caco-2 cells were harvested and cell protein samples were

579

analyzed. Values are expressed as the mean ± SD, n = 3 independent experiments.

Effect of citrus flavanones on the experssion of proteins in Caco-2 cell

Effect of citrus flavanones on the experssion of SR-BI in the presence of



580

Data with different letters display significant differences (P < 0.05). Bc, β-carotene.

581

Figure 7.

582

inhibitor (T0070907) in Caco-2 cell monolayers. (A) Representative band densities of

583

Western blots of SR-BI. (B) Graph representing the relative change in SR-BI protein

584

levels. Caco-2 cells were incubated with synthetic micelles of Bc in the absence /

585

presence of citrus flavanones. The final concentrations of Bc and citrus flavanone in

586

the cell medium were 5 μM and 250 μM, respectively. The final concentration of

587

T0070907 was 20 μM. After 5 h incubation, Caco-2 cells were harvested and cell

588

protein samples were analyzed. Values are expressed as the mean ± SD, n = 3

589

independent experiments. Data with different letters display significant differences (P

590

< 0.05). Bc, β-carotene

Effect of citrus flavanones on the experssion of SR-BI in the PPARγ


Page 30 of 40

Page 31 of 40


Table 1. The Chemical Structure of Citrus Flavanones. Or, -O-rutinoside; Orha, -O-rhamnoglucoside. Number　 Compound

R3

R4

R5

R7

R3'

R4'

R5'

R7'

1

Hesperetin

H

H

OH

OH

OH

OMe

H

H

2

Hesperidin

H

H

OH

Or

OH

OMe

H

H

3

Naringenin

H

H

OH

OH

H

OH

H

H

4

Naringin

H

H

OH

Orha

H

OH

H

H

5

Neoeriocitrin

H

H

OH

Orha

OH

OH

H

H

6

Eriodictyol

H

H

OH

OH

OH

OH

H

H

7

Neohesperidin

H

H

OH

Orha

OH

OMe

H

H

8

Narirutin

H

H

OH

Or

H

OH

H

H

9

Didymin

H

H

OH

Or

H

OMe

H

H

10

Isosakuranetin

H

H

OH

OH

H

OMe

H

H

11

Poncirin

H

H

OH

Orha

H

OMe

H

H

12

Eriocitrin

H

H

OH

Or

OH

OH

H

H



Page 32 of 40

Table 2. Apparent Permeability Cofficients (Papp) Citrus Flavanones Compounds in the Caco-2 Cell Model a Number

Compound

Papp × 10-6 (cm/s)

1

Hesperetin

22.4 ± 1.9

2

Hesperidin

6.2 ± 1.3

3

Naringenin

16.8 ± 3.6

4

Naringin

0.0

5

Neoeriocitrin

0.0

6

Eriodictyol

6.8 ± 0.8

7

Neohesperidin

8.6 ± 1.5

8

Narirutin

0.0

9

Didymin

2.8 ± 1.6

10

Isosakuranetin

10.2 ± 1.8

11

Poncirin

4.0 ± 0.8

12

Eriocitrin

0.0

aP : app

Transport of citrus flavanones from apical to basolateral. Caco-2 cells were

incubated with only citrus flavanones. The final concentrations of citrus flavanone in the cell medium were 250 μM. The incubation time was up to 5 h. Values are expressed as the mean ± SD, n = 3 independent experiments.


Page 33 of 40


Figure 1



Figure 2


Page 34 of 40

Page 35 of 40


Figure 3



Figure 4


Page 36 of 40

Page 37 of 40


Figure 5



Figure 6


Page 38 of 40

Page 39 of 40


Figure 7



Graphic for table of contents


Page 40 of 40

Citrus Flavanones Enhance Î²-Carotene Uptake in Vitro Experiment

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