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Potential Retinal Benefits of Dietary Polyphenols Based on their Permeability across Blood–Retinal Barrier Yixiang Liu, Guang-Ming Liu, Min-Jie Cao, Qingchou Chen, Lechang Sun, and Baoping Ji J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00844 • Publication Date (Web): 31 Mar 2017 Downloaded from http://pubs.acs.org on April 3, 2017

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Journal of Agricultural and Food Chemistry

Potential Retinal Benefits of Dietary Polyphenols Based on their Permeability across Blood–Retinal Barrier Yixiang Liu† , Guang-Ming Liu† , Min-Jie Cao† , Qingchou Chen† , Lechang Sun† , Baoping Ji§* †

College of Food and Biological Engineering, Jimei University, Xiamen, Fujian, People’s Republic

of China §

College of Food Science & Nutritional Engineering, China Agricultural University, Beijing,

People’s Republic of China Running title: retinal benefits of dietary polyphenols and blood-retinal barrier *

Corresponding author: E-mail, [email protected]; Tel, +86-010-62736628; Fax, +86-010-62347334

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ACS Paragon Plus Environment


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ABSTRACT: Whether all dietary polyphenols nourishes the eyes via oral supplementation is

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controversial. Given that passage of dietary polyphenols across the blood–retina barrier (BRB) is the

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precondition for polyphenols to exhibit ocular benefits, the BRB permeability of polyphenols was

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assessed in this study. Being common dietary polyphenols in fruits and vegetables, non-anthocyanin

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flavonoids, anthocyanins, and phenolic acids were investigated. BRB was simulated in vitro by using

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a differentiated retinal pigment epithelial cell monolayer cultivated on a Transwell culture system.

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Penetration rate was calculated by quantitatively analyzing the polyphenols in basolateral media.

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BRB permeability of different polyphenols obviously (p

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non-anthocyanin flavonoids > anthocyanins. Glycosylation and methylation improved the BRB

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permeability of non-anthocyanin flavonoids and anthocyanins. However, instability and

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carbonylation at C-4 position severely suppressed the BRB permeability of anthocyanins and

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non-anthocyanin flavonoids. Moreover, a new metabolite was discovered during penetration of

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anthocyanins into the BRB. However, hydrophilic phenolic acids exhibited better BRB permeability

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than hydrophobic ones. Data demonstrate that BRB permeability of polyphenols was determined

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based on structural characteristics, hydrophilicity, stability, and metabolic changes.

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Keywords: flavonoids, anthocyanins, phenolic acids, RPE cells, blood–retina barrier

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Introduction

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Polyphenols, such as flavonoids and phenolic acids, are bioactive compounds in fruits and vegetables

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and have recently attracted considerable attention because of their visual benefits. A recent

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epidemiological investigation has indicated that dietary intake of quercetin and isorhamnetin is

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inversely associated with the risk of developing age-related cataract.1 Eriodictyol and dietary

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polyphenol extracts from red berries efficaciously attenuates the degree of retinal inflammation in

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early diabetic rats.2,3 Other dietary flavonoids, such as fisetin, luteolin, baicalein, galangin, and

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epigallocatechin gallate, protect retinal cells from light- and oxidant stress-induced cell deaths.4

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Anthocyanins nourish the eyes by accelerating rhodopsin resynthesis,5 by protecting retinal cells

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through antioxidation,6 and by improving microcirculation.7 Recent studies have demonstrated that

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phenolic acids attenuate visible light-induced retinal damage and inhibit retinal neovascularization

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and degeneration.8,9 Different polyphenols show varied physiological activities in protecting vision.

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Many complicated molecular-regulating paths have been employed to demonstrate the differences of

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the visual protective properties of polyphenols. However, the bioavailability and target delivery of

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these compounds remain to be elucidated.

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The potential physiological activities of polyphenols are strictly related not only to their plasma

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concentration after being absorbed into the digestive tract but also to their quantity when arriving at

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target tissues or organs. Blood–retinal barrier (BRB) limits the passage of nutrients into the eyes.

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Influx of essential molecules into the retina is regulated by various unique transporters expressed by

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retinal endothelial cells.10 Therefore, different nutrients usually exhibit different capabilities of

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penetrating the BRB. Between the inner and outer BRB, and the outer BRB is usually simulated to

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evaluate BRB permeability of nutrients. For example, in the blood-to-retina influx transport system, 3



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the influx permeability rate of docosahexaenoic acid is 2440 µL min−1 g−1 retina, higher than that of

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glucose (544 µL min−1 g−1 retina) and sucrose (0.27 µL min−1 g−1 retina).11 Some functional factors,

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such as vitamin C and taurine, exhibit excellent visual benefits owing to their outstanding BRB

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permeability.12,13 However, information about the ocular target delivery of polyphenols remains

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insufficient to date. A recent study has indicated that intragastrically administered baicalin penetrates

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the blood–eye barrier and enters rabbit lens.14 Moreover, anthocyanins were discovered in eyeballs of

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pigs fed with blueberry-supplemented diet for 4 weeks.15 Anthocyanins in intact form were also

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discovered in ocular tissues of rats and rabbits.16 However, these studies did not differentiate the BRB

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permeability of different polyphenols.

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Retinal pigment epithelium (RPE), representing a monolayer of highly polarized pigmented cells

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located between the neural retina and the choroid, serves as BRB that selectively transports

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biomolecules between neural retina and choriocapillaris.17 ARPE-19 cell line polarizes and generates

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barrier features on permeable support.18 Therefore, this study used an RPE cell monolayer cultivated

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on a Transwell culture system to simulate BRB in vitro. This model was then used to systematically

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evaluate the BRB permeability of different polyphenols, including 11 non-anthocyanin flavonoids, 6

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anthocyanins, and 7 phenolic acids. The present study determined the structure–function relationships

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influencing the ocular protective activities of different polyphenols. This work is significant in quick

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identification of natural polyphenols that demonstrate visual benefits.

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Materials and Methods

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Chemicals and Reagents. Non-anthocyanin flavonoid standards and phenolic acid standards were

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obtained from Chengdu Must Bio-technology Ltd (Chengdu, China). Amberlite XAD-7 used for

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purifying blueberry polyphenols was obtained from Sigma (Sydney, Australia). The Sephadex LH20 4


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and Oasis HLB cartridges used for isolating and purifying anthocyanins and phenolic acids were

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purchased from Amersham Biosciences AB (Uppsala, Sweden) and Waters (Milford, MA),

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respectively. Deionized water was produced using a Milli-Q unit (Millipore, Bedford, MA). The

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acetonitrile from Mallinckrodt Baker (Phillipsburg, NJ) was of high-performance liquid

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chromatography (HPLC) grade. The ethanol and hydrochloric acid were purchased from China

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National Pharmaceutical Industry Corporation Ltd. (Shanghai, China). Analytical reagent-grade

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solvents were used during extraction.

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Dulbecco’s

modified

Eagle’s/Ham’s

F12

media,

fetal

bovine

serum

(FBS),

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide were

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purchased from Sigma-Aldrich (MO, USA). Permeable polyester Transwell cell culture inserts (6 mm

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diameter, 0.4 µm pore size) were purchased from Corning Inc.(NY, USA). Penicillin, streptomycin,

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and Hanks' balanced salt solution (HBSS) were obtained from Gibco Life Technologies (Grand

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Island, NY). The lactic dehydrogenase (LDH) kit was purchased from Nanjing Jiancheng

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Bioengineering Institute (Nanjing, Jiangsu, China).

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Plant Materials and Sample Preparation. Fresh wild blueberries (Vacciniun spp.), grown from the

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Greater Hinggan Mountains in Northeast China, were supplied by the Science and Technology

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Bureau of Greater Hinggan Mountains district. These fresh berries were freeze-dried and stored in

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dark and at -20 °C until use. To avoid deterioration, the preservation time of blue berries was no more

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than 6 months.

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Extraction and Fractionation of Anthocyanins

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Anthocyanin separation and purification from blueberries was performed as our previous reports.7

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Ten grams of dry blueberries were immersed by 140 mL absolute methanol for 1.00 h in a 250–mL 5



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round–bottomed flask, and then homogenized using a homogenizer (XHF–D, Ningbo Science &

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Biotechnology Co., Ningbo, Zhejiang, China). The homogenized sample was further centrifuged for

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10 min at 4,000 ×g, and the supernate was filtered through a moderate speed 102 qualitative filter

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paper (Hangzhou Special Paper Industry Co. Ltd., Hangzhou, Zhejiang, China). The above procedure

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was repeated to re-extract the residue. The two filtrates were combined and evaporated using a rotary

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evaporator at 40 °C and a vacuum pressure of 0.10 MPa. Part of the concentrated solution was loaded

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onto an Amberlite XAD-7 column, and the remaining part was lyophilized (crude extract) for further

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analysis. After 1.00 h, the XAD was washed with about 800 mL 1.00% (v/v) formic acid aqueous

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solution to remove non-polyphenolic compounds, after which the polyphenolics were eluted with

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about 600 mL absolute methanol with 1.00% (v/v) formic acid. The eluent was concentrated at 40 °C

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and lyophilized in vacuum using a freeze dryer (Four–ring Science Instrument Plant Beijing Co., Ltd.,

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Beijing, China). Forty-eight hours later, a friable dark red powder was obtained. A 50.0–mg

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lyophilized sample (polyphenol mixture) was resolubilized in pH 7.00 phosphate buffer and applied

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to a Sephadex LH20 column. The column was first washed with a pH 7.00 phosphate buffer to

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remove phenolic acids, and then with 70.0% (v/v) methanol acidified with 10.0% (v/v) formic acid to

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elute anthocyanins and flavonoids. The anthocyanin and flavonoid mixture was freeze–dried,

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resolubilized in 5.00% (v/v) formic acid in water, and then applied to an Oasis HLB cartridge. The

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cartridge was washed with 5.00% (v/v) formic acid, followed by ethyl acetate, and then with 10.0%

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(v/v) formic acid in methanol. The anthocyanins were eluted with acidified methanol, and the eluents

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were freeze-dried and called anthocyanin-rich fraction. These extracts or fractions were kept at

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-20 °C until use. According to our previous report,19 the main compositions of anthocyanin-rich

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fraction

were

delphinium-3-galactoside,

delphinium-3-glucoside, 6


delphinium-3-arabinoside,

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cyanidin-3-galactoside, petunidin-3-galactoside, and malvidin-3-glucoside.

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Cell Culture

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A human RPE cell line, ARPE-19 (ATCC CRL–2302) (American Type Culture Collection, Manassas,

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Virginia, USA), was used in the present study and cultured as previously described. 20, 21 Cell cultures

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were grown in Dulbecco’s modified Eagle’s/Ham’s F12 media (Invitrogen, Carlsbad, CA)

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supplemented with 10% fetal bovine serum (Sigma–Aldrich, St. Louis, Missouri, USA), containing

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1% antibiotic mixture of penicillin (100 U/mL) and streptomycin (100 mg/mL) (Invitrogen) at 37 oC

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under a humidified 5% CO2 atmosphere.

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Cytotoxicity Evaluation

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Cell viability was measured by MTT assay as previously described.22 RPE cells were seeded in

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96-well plates (Corning–Costar, Corning, NY, USA) at a concentration of 5 × 105 cells/mL, and then

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allowed to attach after 48 h. The medium was then replaced with serum–free F12 medium containing

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samples with different concentration. Twenty–four hours later, the cell supernate was removed for

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LDH assay. About 200 µL of 0.50 mg/mL MTT serum–free F12 medium was added into each well of

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plates, after which incubation was performed for 4 h. After removal of the MTT solution, 150 µL of

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dimethyl sulfoxide was added, and the absorbance was measured at 570 nm using a plate reader

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(Molecular Devices Co., CA, USA). Results were expressed as the percentage of viable cells with

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respect to untreated control cells. Cell viability (%) was calculated as follows: [(mean absorbance of

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the sample – reference absorbance)/(mean absorbance of the control-– reference absorbance)] ×100.

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The cellular release of LDH was used as a measure of cellular damage/integrity. Enzymatic activity

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was determined using an LDH kit according to the manufacturer’s instructions.

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In vitro BRB Model 7



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ARPE-19 cells were seeded onto permeable polyester Transwell cell culture inserts (6 mm diameter,

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0.4 µm pore size; Corning Inc., NY, USA) at 10,000 per well. The media in the apical and basolateral

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chambers were 0.2 and 0.6 mL, respectively. The cultures were maintained at 37 °C in a humidified

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atmosphere of 95% air/5% CO2. The medium was replaced after 24.0 h, and the cells were fed every

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2 days. To ensure that the monolayer exhibited the properties of a tight biological barrier, its

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transepithelial electrical resistance (TER) was monitored using a Millicell-ERS Voltohmmeter

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(Millipore Co., MA, USA; Figure 1). Net TERs were calculated by subtracting the value of a blank

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filter without cells from the experimental value. Only monolayers with TER values higher than 25.0

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Ohm· cm2 were used for permeability tests. 23

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BRB Permeability Test

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The media were removed from the apical chamber, and the cellular monolayers were washed

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carefully with HBSS (pH = 7.40, 37 °C). Then, 200 µL of the polyphenol solutions in HBSS were

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added to the apical side of the inserts, and 600 µL of polyphenol-free HBSS was added to the

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basolateral side. When the permeability tests were finished, the media at the basolateral sides were

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entirely collected. To ensure that the polyphenols in the media were detectable for HPLC analysis, the

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basolateral media were first freeze-dried and then redissolved in 50.0 µL of aqueous solution with 5%

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formic acid for HPLC analysis. The BRB permeability of the polyphenols was calculated by

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comparing the content in the basolateral solution at the end of the test with the one in the apical

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solution at the beginning. In this work, the anthocyanins are the extractives from blueberries and

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non-anthocyanin flavonoids are the commercial standards. Therefore, the equations used to calculate

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the BRB permeability for anthocyanins and non-anthocyanin flavonoids are different. The equation

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used to calculate the BRB permeability rate of anthocyanins was described as follows: 8


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BRB permeability (%) = S1/S0 × 100,

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where S0 and S1 are the peak areas of anthocyanins in the apical and basolateral solutions at 0.00 and

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2.00 h, respectively.

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However, the equation used to calculate the BRB permeability rate of non-anthocyanin flavonoids

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and phenolic acids was described as follows:

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BRB permeability (%) = C1 × V1/(C0 × V0) × 100,

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where C0 and C1 are the concentrations of non-anthocyanin flavonoids or phenolic acids in the apical

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and basolateral solutions at 0.00 and 6.00 h, respectively, and V0 and V1 are the corresponding

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volumes.

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HPLC Analysis

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Identification and quantification of anthocyanins was performed as our previous report.19 For

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non-anthocyanin flavonoids and phenolic acids, the characteristic absorption wavelengths were firstly

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analyzed using UV–visible spectroscopy. Then, an Agilent 1100 series HPLC was used to

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quantitatively analyse their concentrations in the basolateral solutions. The analytical column used

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was a 150 mm × 2.1 mm i.d. Agilent Zorbax Eclipse SB-C18 column (Agilent, Palo Alto, CA)

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maintained at 25 oC. The injection volume was 20.0 µL, and the flow rate was 1.00 mL/min. The

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elution solvent was 70.0% (v/v) methanol aqueous solution with 1.00% formic acid.

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Statistical Analysis

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The statistical significance of the differences between the control and treatment groups was analyzed

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by one-way ANOVA using Origin version 8.0, followed by Tukey tests. A normality test showed that

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all the raw data had a normal distribution, and all groups were determined to have equal variance by a

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variance test. Data were expressed as the means ± SD of at least three individual experiments, each 9



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run in triplicate. p

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