Protective Effect of Fucoxanthin Isolated from Laminaria japonica

Subscriber access provided by CMU Libraries - http://library.cmich.edu

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

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


1

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

2

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

3

compounds for preventing visible light-induced retinal damages are becoming

4

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

5

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

6

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

7

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

8

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

9

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

10

vascular endothelial growth factor, resisting senescence, improving phagocytic

11

function, and clearing intracellular reactive oxygen species in retinal pigment

12

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

13

than lutein in protecting retina against photoinduced damage. This excellent

14

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

15

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

16

in the future.

17

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

18 19 20 21 2


Page 2 of 39

Page 3 of 39


22

Introduction

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

challenge as never before in human history.

37

Preventing retinal damage through dietary supplement of natural antioxidants in

38

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

39

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

40

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

41

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

42

RPE cell oxidative damage and chemical-induced oxidative apoptosis in

43

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



44

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

45

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

46

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

47

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

48

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

49

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

50

visible light-induced damages are necessary.

51

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

52

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

53

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

54

fucoxanthin are attributed to these distinct structural characteristics. The antioxidative

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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


Page 4 of 39

Page 5 of 39


66

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

67

Therefore, nutritionists and ophthalmologists have reached a consensus that

68

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

69

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

70

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

71

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

72

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

73

cellular senescence were observed to evaluate the potential eye benefits of

74

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

75

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

76

which have visual benefits as confirmed in previous studies.

77 78

Materials and Methods

79

Plant Materials and Sample Preparation.

80

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

81

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

82

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

83

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

84

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

85

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

86

Bureau of Greater Hinggan Mountains District. Blueberry anthocyanins were made

87

according to our previous report.34 5



Page 6 of 39

88

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

89

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

90

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

91

Mallinckrodt

92

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

93

tetrahydrofuran were purchased from China National Pharmaceutical Industry

94

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

95

during extraction.

96

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),

98

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

99

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

100

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

101

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

102

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

103

Bioengineering

104

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

105

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

106

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

107

Extraction and isolation of fucoxanthin. Fucoxanthin separation and purification

108

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

109

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

Institute

2′,7′-dichlorofluorescin

(Nanjing,

Jiangsu,

China).

6


diacetate

(DCFH-DA),

Senescence-associated

Page 7 of 39


110

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

111

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

112

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

113

The methanol extract was purified via silica column chromatography with an

114

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

115

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

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

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

126

fucoxanthin in above jacinth cream was 86%.34

127

Cell culture

128

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

129

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

130

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

131

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



132

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

133

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

134

under a humidified 5% CO2 atmosphere. All experiments were performed with an

135

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

136

Cytotoxicity Evaluation

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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


Page 8 of 39

Page 9 of 39


154

lamps were purchased from Foshan Nationstar Optoelectronics Company Limited

155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

group to avoid exposure to light.

165

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

166

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

168

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

169

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

173

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

174

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

175

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



176

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

177

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

178

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

179

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

180

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

181

fresh medium to remove uningested particles before observation under a fluorescence

182

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

183

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

184

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

185

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

186

and 420 nm excitation.

187

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

188

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

189

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

190

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

191

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

192

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

193

dark for 8 h at 37

194

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,

196

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

197

determine the development of blue coloring and photographed using a light

o

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

10


Page 10 of 39

Page 11 of 39


198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

well/the protein content of each well. 11



220

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

221

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

222

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

223

handled according to the Association for Research in Vision and Ophthalmology

224

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

225

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

226

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

227

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

229

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

230

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

231

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

232

detected.

233

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

234

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

235

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

236

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

237

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

238

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


Page 12 of 39

Page 13 of 39


242

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

244

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

245

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

246

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

247

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

248

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

249

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

251

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

252

flash luminance of 20 cd s/m2.

253

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

254

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

255

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

256

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

257

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

258

run in triplicate. p

Protective Effect of Fucoxanthin Isolated from Laminaria japonica

Recommend Documents