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Protective Effect of Fucoxanthin Isolated from Laminaria japonica against Visible Light-Induced Retinal Damage Both in vitro and in vivo Yixiang Liu, Liu meng, Xichun Zhang, Qingchou Chen, Chen Haixiu, Lechang Sun, and Guang-Ming Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05436 • Publication Date (Web): 27 Dec 2015 Downloaded from http://pubs.acs.org on January 1, 2016

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Journal of Agricultural and Food Chemistry

Protective Effect of Fucoxanthin Isolated from Laminaria japonica against Visible Light-Induced Retinal Damage Both in vitro and in vivo Yixiang Liu†

, §

Lechang Sun†

, §

†

, Meng Liu† , Xichun Zhang† , Qingchou Chen† , Haixiu Chen† , , Guangming Liu†

, §*

College of Food and Biological Engineering, Jimei University, Xiamen, Fujian,

People’s Republic of China §

Xiamen Key Laboratory of Marine Functional Food, Jimei University, Xiamen,

Fujian, People’s Republic of China Running title: fucoxanthin protection against visible light-induced retinal injury * Corresponding author: E-mail, [email protected]. Fax: +86-092-61804770.

1

ACS Paragon Plus Environment


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ABSTRACT: With increasingly serious eye exposure to light stresses, such as

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light-emitting diodes, computers, and widescreen mobile phones, efficient natural

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compounds for preventing visible light-induced retinal damages are becoming

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compelling needs in the modern society. Fucoxanthin, as the main light absorption

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system in marine algae, may possess an outstanding bioactivity in vision protection

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because of its filtration of blue light and excellent antioxidative activity. In this work,

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both in vitro and in vivo simulated visible light-induced retinal damage models were

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employed. The in vitro results revealed that fucoxanthin exhibited better bioactivities

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than lutein, zeaxanthin, and blueberry anthocyanins in inhibiting overexpression of

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vascular endothelial growth factor, resisting senescence, improving phagocytic

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function, and clearing intracellular reactive oxygen species in retinal pigment

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epithelium cells. The in vivo experiment also confirmed the superiority of fucoxanthin

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than lutein in protecting retina against photoinduced damage. This excellent

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bioactivity may be attributed to its unique structural features, including allenic,

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epoxide, and acetyl groups. Fucoxanthin is expected to be an important ocular nutrient

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in the future.

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KEYWORDS: fucoxanthin, marine algae, retina, RPE cells, photoinduced damage

18 19 20 21 2


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Introduction

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In the modern society, environmental light and endogenous antioxidants are the main

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determinants of non-cancer ocular diseases.1 Our living environment is becoming

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considerably brighter, and our life is becoming significantly convenient. However,

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given the abundant unsaturated fatty acids, high levels of light exposure, and high

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oxygen tension, the retina is susceptible to damage and an increasing number of

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people are suffering from eye problems.2,3 Light-emitting diodes (LEDs) are a new

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type of energy-saving light that makes our cities and rooms lighter. According to the

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latest survey of the French Agency for Food, Environmental, and Occupational Health

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and Safety, LEDs belong to risk group 1 to retinal health.4 Computers and widescreen

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mobile phones provide us many conveniences both in our life and work, such as

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online shopping, playing games, and computer designs. However, approximately 14%,

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19.6%, 46.3%, and 62.5% of computer users in the United States, Japan, India, and

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Norway suffer from eye problems, respectively.5-8 Our ocular health is meeting a big

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challenge as never before in human history.

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Preventing retinal damage through dietary supplement of natural antioxidants in

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our everyday life is important. Many nutrients in our foods are verified to benefit our

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vision, such as lutein, zeaxanthin, β-carotenoid, anthocyanins, vitamin C, vitamin E,

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and polyunsaturated fatty acids. For example, lutein and zeaxanthin not only act as

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retinal pigments in absorbing damaging blue light but also prevent both photoinduced

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RPE cell oxidative damage and chemical-induced oxidative apoptosis in

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photoreceptor cells through promoting antioxidant defense in retinal cells.9-13 Lutein 3



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and zeaxanthin are also efficient in increasing visual processing speed in young

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healthy subjects.14 Anthocyanins are speculated to be involved in vision protection

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though the pathways of accelerated resynthesis of rhodopsin,15-17 modulation of retinal

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enzymatic activity,16,18 protection of retinal cells by antioxidation,19,20 and improved

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microcirculation.16 However, to solve the eye problems suffered by a growing number

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of people, more efficient compounds that can be utilized to prevent the retinas from

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visible light-induced damages are necessary.

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Fucoxanthin, a special xanthophyll derived from edible brown seaweeds, not only

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has the basic structure of lutein and zeaxanthin but also possesses unique structures of

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allenic, epoxide, and acetyl groups (Figure 1).21 The unique bioactivities of

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fucoxanthin are attributed to these distinct structural characteristics. The antioxidative

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activity of fucoxanthin is approximately 13.5 times than that of alpha-tocopherol.22

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The excellent bioactivities of fucoxanthin, such as anti-inflammation, antioxidative

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stress, prolonging lifespan, antitumor, and regulation of glycometabolism and lipid

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metabolism, are subsequently discovered.23-29 As special xanthophylls, the ocular

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benefits of fucoxanthin being different from lutein and zeaxanthin were also reported.

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Previous studies indicated that fucoxanthin may be beneficial for eyesight protection.

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Fucoxanthin is efficient in protecting cadmium-induced oxidative renal dysfunction in

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rats.30 Fucoxanthin also exhibits strong bioactivities in preventing endotoxin-induced

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uveitis in rats and ultraviolet-induced cell injuries in human fibroblast.21,31

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Retinal pigment epithelium (RPE) cells constitute a single-layer structure behind

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the photoreceptor cells in the retina. Basic and clinical studies have demonstrated that 4


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primary dysfunctioning of RPE can result in visual cell death and blindness.32

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Therefore, nutritionists and ophthalmologists have reached a consensus that

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preventing RPE damage is critical to maintain eye health.11,18,32 To identify the

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potential bioactivity of fucoxanthin in vision, we employed an in vitro visible

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light-induced RPE cell damage model to simulate retinal injury caused by excessive

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light radiation in our living environments. Subsequently, the retinal benefits of

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fucoxanthin were confirmed in vivo. In this work, cell physiological functions and

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cellular senescence were observed to evaluate the potential eye benefits of

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fucoxanthin. The superiority of fucoxanthin in maintaining eye health was also

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investigated through comparison with lutein, zeaxanthin, and blueberry anthocyanins,

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which have visual benefits as confirmed in previous studies.

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Materials and Methods

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Plant Materials and Sample Preparation.

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The marine brown alga, Laminaria japonica, grown from the coast of Fujian province

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in China, was supplied by Fujian Fuqing Riji Foods Inc. The sample was washed

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three times with tap water to remove salt, epiphytes, and sand attached to the surface.

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They were then carefully rinsed with fresh water, and maintained in a refrigerator at

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−20 °C until use. Freezed dried blueberries (Vacciniun spp.), grown in the Greater

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

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Bureau of Greater Hinggan Mountains District. Blueberry anthocyanins were made

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according to our previous report.34 5



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Chemicals and Reagents. Silica gel resin used for purifying fucoxanthin was

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obtained from Sigma (Sydney, Australia). Deionized water was produced using a

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Milli-Q unit (Millipore, Bedford, MA). The acetonitrile and methanol from

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Mallinckrodt

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chromatography (HPLC) grade. The n-hexane, ethyl acetate, chloroform, acetone and

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tetrahydrofuran were purchased from China National Pharmaceutical Industry

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Corporation Ltd. (Shanghai, China). Analytical reagent-grade solvents were used

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during extraction.

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Baker

(Phillipsburg,

NJ)

was

of

high-performance

liquid

DHA, lutein, zeaxanthin, fucoxanthin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

97

tetrazolium

bromide

(MTT),

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Dulbecco’s modified Eagle’s/Ham’s F12 media, dimethyl sulfoxide, blue fluorescent

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amine-modified microspheres and fetal bovine serum (FBS) were purchased from

100

Sigma-Aldrich (MO, USA). Penicillin, streptomycin, and Hanks' balanced salt

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solution (HBSS) were obtained from Gibco Life Technologies (Grand Island, NY).

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The lactic dehydrogenase (LDH) kit was purchased from Nanjing Jiancheng

103

Bioengineering

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β-galactosidase staining kit and protein kit were provided by Beyotime Institute of

105

Biotechnology (Jiangsu, China). Vascular endothelial growth factor (VEGF) ELISA

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kit was obtained from Shanghai ExCell Biology Inc. (Shanghai, China).

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Extraction and isolation of fucoxanthin. Fucoxanthin separation and purification

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from Laminaria japonica were performed according to our previous report,34 with

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some modifications. The frozen samples were lyophilized and homogenized using a

Institute

2′,7′-dichlorofluorescin

(Nanjing,

Jiangsu,

China).

6


diacetate

(DCFH-DA),

Senescence-associated

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grinder prior to extraction. The algal powder was extracted two times with methanol

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at 40 °C under dark. Each extracting time was 1 h. Then the extracts were centrifuged

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for 10 min at 4,000 ×g, and the supernatant was evaporated under vacuum at 40 °C.

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The methanol extract was purified via silica column chromatography with an

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n-hexane-ethyl acetate mixture (2:1). The fraction with yellow color was collected

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and then evaporated under vacuum at 40 °C. The yellow fraction was then applied to

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the silica column chromatography again and washed with a chloroform-acetone

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mixture (12:1). The jacinth fraction was collected and evaporated under vacuum at

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40 °C. This jacinth cream was fucoxanthin-rich fraction. The purity of fucoxanthin

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was detected with a Shimadzu LC-10 A Series high performance liquid

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chromatography (HPLC) system. The analytical column used was a C18 column

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(Shimadzu, Tokyo, Japan) (250 mm×4.6 mm, 5µm) maintained at 30 °C. The

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injection volume was 20 µL, and elution solvent was an acetonitrile-methanol-0.1%

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ammonium acetate aqueous solution mixture (75:15:10). The elution condition was as

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follows: flow rate 1 mL/min, isocratic elution for 20 min, detection wavelength at 450

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nm. The standard fucoxanthin was used for quantitative analysis and the content of

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fucoxanthin in above jacinth cream was 86%.34

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Cell culture

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

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Collection, Manassas, Virginia, USA), was used in the present study and cultured as

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previously described.33,35 Cell cultures were grown in Dulbecco’s modified

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Eagle’s/Ham’s F12 media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal 7



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bovine serum (Sigma–Aldrich, St. Louis, Missouri, USA), containing 1% antibiotic

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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. All experiments were performed with an

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80% confluent monolayer. The cells were in culture for up to four to six passages.

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Cytotoxicity Evaluation

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Cell viability was measured by MTT assay as previously described.36 RPE cells were

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seeded in 96-well plates (Corning–Costar, Corning, NY, USA) at a concentration of 5

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× 105 cells/mL, and then allowed to attach after 48 h. The medium was then replaced

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with serum–free F12 medium containing samples with different concentration.

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Twenty–four hours later, the cell supernate was removed for LDH assay. About 200

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µL of 0.5 mg/mL MTT serum–free F12 medium was added into each well of plates,

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

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µL of dimethyl sulfoxide was added, and the absorbance was measured at 570 nm

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using a plate reader (Molecular Devices Co., CA, USA). Results were expressed as

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the percentage of viable cells with respect to untreated control cells. Cell viability (%)

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was calculated as follows: [(mean absorbance of the sample – reference

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absorbance)/mean absorbance of the control] ×100. The cellular release of LDH was

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used as a measure of cellular damage/integrity. Enzymatic activity was determined

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

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Light Exposure in Vitro. The illumination condition was performed as our previous

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report.33,35 The RPE cells were subjected to white light irradiation by an integrated

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light-emitting diode lamp system designed by the authors.33 The light-emitting diode 8


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lamps were purchased from Foshan Nationstar Optoelectronics Company Limited

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(Foshan, Guangdong, China), and the spectrum of the light was 420–800nm according

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to the introduction provided by the manufacturer. Light intensity was measured with a

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TES-1330A light meter (TES Electrical Electronic Corporation, Taipei, Taiwan). RPE

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cells in an active growing state were planted into plates with serum-containing F12

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medium at a concentration of 5 × 105 cells/mL. After 85% of the cells were adhered to

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the wall, the medium was replaced with serum-free F12 medium containing 25

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µmol/L of DHA. The RPE cells were then exposed to white light. The illumination

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conditions were as follows: light intensity, 3500 lux; light exposure time, 12 h. In

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addition, black adhesive tape was placed on the cover of the plates in the control

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group to avoid exposure to light.

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Vascular Endothelial Growth Factor (VEGF) Detection. The amount of VEGF in

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the supernate of the RPE culture was determined by enzyme-linked immune sorbent

167

assay (ELISA). After light irradiation, the culture media were collected and then

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centrifuged at 1,000 × g for 5 min. The VEGF level in the culture medium was

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analyzed by a commercial VEGF ELISA kit according to the manufacturer’s

170

instructions. Absorbance at 450 nm was measured using a plate reader (Molecular

171

Devices Co.).

172

Phagocytic Activity. The phagocytosis of RPE cells after light exposure was

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evaluated as previously described,37,38 with some modifications. Blue fluorescent

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amine-modified microspheres (0.05 µm) (Sigma–Aldrich) were diluted into a density

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of 1×107/mL with serum-free F12 medium and incubated at 37 °C in a humidified 5% 9



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CO2 atmosphere for approximately 10 min. Subsequently, 1 mL of the medium that

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contains the fluorescent microspheres was added into 24-well plates after the

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supernatant was removed. After 24 h incubation, the phagocytic activity was

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determined based on the uptake level of fluorescent microspheres. The medium that

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contains the microspheres was removed. The adherent cells were washed twice with

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fresh medium to remove uningested particles before observation under a fluorescence

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microscope. HBSS was replaced with fresh medium to wash the cells. Then, a cell

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suspension was prepared after digestion by pancreatin. A 200 µL aliquot of the cell

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suspension was transferred to black, clear-bottomed 96-well plates for fluorescence

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intensity analysis using a plate reader (Molecular Devices Co.) at 360 nm emission

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and 420 nm excitation.

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Senescence-Associated β-galactosidase Activity. Senescence was investigated using

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a senescence-associated β–galactosidase staining kit (Beyotime Institute of

189

Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. The

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treated RPE cells were washed twice with pH 6.0 phosphate-buffered saline (PBS),

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and then fixed with 2% formaldehyde and 0.2% glutaraldehyde in PBS at room

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temperature for 4 min. Cells were then washed twice with PBS and incubated in the

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dark for 8 h at 37

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5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside, 40 mmol citric acid/sodium

195

phosphate, pH 6.0, 5 mmol potassium ferrocyanide, 5 mmol potassium ferricyanide,

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150 mmol NaCl, and 2 mmol MgCl2 diluted in PBS). Cells were then examined to

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determine the development of blue coloring and photographed using a light

o

C with fresh β-galactosidase staining solution (1 mg/mL

10


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microscope (Chongqing Optical and Electrical Instrument Co. Ltd., Chongqing,

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China). The percentage of blue-stained cells (cells of active β–galactosidase) was

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calculated as the number of blue–stained cells/the number of total cells. Five visual

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fields of each passage group were chosen for analysis, and each field contained at

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least 150 cells. The results were recorded as means ± SD of the five counts.

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Cellular ROS Measurement. The ROS level in RPE cells was monitored using the

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fluorescent dye DCFH–DA. The membrane-permeable DCFH–DA enters cells and

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produces a fluorescent signal after intracellular oxidation by ROS (such as hydrogen

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peroxide and lipid peroxides), and the fluorescence can directly reflect the overall

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intracellular ROS.33,39 RPE cells were cultured on the bottom of a transparent black

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96-well plate (Corning–Costar) in 200 µL of the growth medium at a density of 5 ×

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105 cells/mL. After 48 h incubation, the media was replaced with serum-free F12

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medium containing samples. For the control group, no samples were present in the

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medium. The RPE cells were then exposed to 3,500 lux visible light for 12 h. Then,

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the new medium with 25 µmmol DCFH-DA was added into the 96-well plate, and the

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cells were incubated sequentially for another 1 h at 37 ° C. After the supernate

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containing DCFH-DA was removed, the cells were carefully washed twice with

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HBSS. The fluorescence of the cells from each well was recorded by a

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multifunctional microplate reader at 530 nm emission and 485 nm excitation.

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Subsequently, the protein content of each well was determined using a commercial kit,

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and the ROS level of each well was calculated as the fluorescence intensity of each

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well/the protein content of each well. 11



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Animals and Light exposure. The protective effects of fucoxanthin on light-induced

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retinal damage were further estimated in a rabbit model. Animal administration and

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light exposure were adopted according to our earlier study.2,40 All animals were

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handled according to the Association for Research in Vision and Ophthalmology

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(ARVO) statement for Use of Animals in Ophthalmic and Vision Research. After 1

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week adaptation period, the rabbits were randomly divided into the following four

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groups (n = 5 per group): control (no light exposure and vehicle administration),

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Group 1 (light exposure and vehicle administration), Group 2 (light exposure and

228

administration of fucoxanthin, 100 µg/kg/day), Group 3 (light exposure and

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administration of lutein, 100 µg/kg/day). Fucoxanthin and lutein were dissolved in

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soybean oil and fed to animals by intragastric administration for 2 consecutive weeks

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prior to light exposure. After light exposure, the physiological functions of retina were

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detected.

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Electroretinograms (ERGs). ERGs were recorded using a visual electrophysiology

234

system (APS-2000 Chongqing Kang Hua Science & Technology Co., Chongqing,

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China) to measure retinal function. The standard dark- and light-adapted ERG signals

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were recorded at 7 days after light exposure by methods described previously.2,41,42 To

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allow the electrodes to stick to the rabbits’ skin, and body hair on the forehead and

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bitemporal was scraped off using a scalpel. The rabbits were anesthetized using an

239

intramuscular injection of sumianxin (0.2 mL/kg). The pupils were dilated with a

240

topical application of 0.5% tropicamide and 0.5% phenylephrine. After dark

241

adaptation for more than 30 min, an LED built-in contact lens electrode, reference 12


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electrode, and adjacent electrode were placed on the corneal surface, on the forehead

243

with conductance ointment, and on the bitemporal as ground, respectively. The

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luminous energy was calibrated using the internal calibration function of the LED

245

stimulator. The responses were differentially amplified using a band pass filter

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between 1 and 100 Hz for dark-adapted electroretinograms (dark-adapted ERGs),

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maximal response electroretinograms (Max-ERGs), oscillatory potentials (OPs) and

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light-adapted electroretinograms (light-adapted ERGs). Dark-adapted ERGs were

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recorded at a blue stimulus intensity of 1.5 log cd s/m2. OPs and Max-ERGs were

250

recorded at a white standard flash intensity of white light. The rabbits were exposed to

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a white light-adapting field for 30 min, and light-adapted ERGs were then elicited at a

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flash luminance of 20 cd s/m2.

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Statistical Analysis. The statistical significance of the differences between the control

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and treatment groups was analyzed by one-way ANOVA using Origin version 8.0,

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followed by Tukey tests. A normality test showed that all the raw data had a normal

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distribution, and all groups were determined to have equal variance by a variance test.

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

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run in triplicate. p

Protective Effect of Fucoxanthin Isolated from ... - ACS Publications

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