Chinese Journal of Physiology 59(4): 202-209, 2016

DOI: 10.4077/CJP.2016.BAE404

Effect of a Complex Lutein Formula in an

Animal Model for Light-Induced
Retinal Degeneration
Yin-Pin Cheng 1, Chia-Ying Ke 2, Chih-Chieh Kuo 1, and Yih-Jing Lee 2

1
Center of Research and Development, Uni-President Biotech, Tainan 70955
and
2
School of Medicine, Fu-Jen Catholic University, Hsinchuang, New Taipei City 24205,
Taiwan, Republic of China

Abstract
Several retinal degenerative diseases cause vision loss and retinal cell death. Currently, people face
prolonged exposure to digital screens, rendering vision protection from light exposure a critical topic.
In this study, we designed a complex lutein formula (CLF) by combining several natural compounds:
Calendula officinalis, Lycium barbarum, Vaccinium myrtillus, Cassia obtusifolia, and Rhodiola rosea. In
addition, we evaluated the protective effects of the formula on retinal functions in an animal model for
light-induced retinal degeneration. We employed electroretinography to analyse retinal function, and
conducted a histological examination of the morphological changes in the retina treated under various
conditions. We revealed that the retinal function in animals exposed to light for 7 days decreased signifi-
cantly; however, the retinal function of animals that had received the CLF exhibited superior perfor-
mance, despite light exposure. In addition, a greater portion of the outer nuclear layer (ONL) (i.e.
the nuclei of photoreceptors) in these animals was preserved compared with the animals that had
not received the formula after 7 days of light exposure. These results revealed that our dietary CLF
supplement attenuated retinal function loss resulting from long-term light exposure.

Key Words: electroretinogram, light expose, lutein, photoreceptor, retinal degeneration

Introduction beneficial for vision protection. Calendula officinalis,

Retinal diseases such as glaucoma, optic nerve
atrophy, diabetic retinopathy, retinitis pigmentosa and
age-related macular degeneration (AMD) cause vision
loss in human (47). These degenerative retinal diseas-
es often result in irreversible damage to retinal cells,
such as ganglion cell loss caused by intraocular hy-
pertension (31), and may lead to blindness. Currently,
people face prolonged exposure to digital screens such
as mobile phones and tablet computers, rendering vi-
sion protection from light exposure a critical topic.
We therefore used a long-term light expose animal
model to mimic the retinal degeneration cause by this
condition.
Numerous natural products are considered to be
commonly known as marigold, belongs to the Aster-
aceae family. The flowers of this plant are widely ac-
knowledged as a folk medicine in Europe, China, and
India. Numerous studies have reported that marigold
has anti-inflammatory (11), antioxidant (1, 9, 13) and
wound healing (40, 44) properties. Marigold flowers
contain carotenoids and flavonoids (24, 45), most of
which are found in their petals. Carotene and xan-
thophyll are two major groups of carotenoids. The
total carotenoid concentration (mg/g of dry weight)
in marigold was 7.71% in petals and 1.61% in pollens,
and the main carotenoid was lutein, which was found
in 15% to 25% (2). Furthermore, the 2 major carote-
noids in the human macula and retina are lutein and
zeaxanthin (17). Many reports have revealed that the

Corresponding author: Yih-Jing Lee, Ph.D., School of Medicine, Fu-Jen Catholic University, 510 Chungcheng Road, Hsinchuang, New Taipei
City 24205, Taiwan, R.O.C. Tel: +886-2-29053411, Fax: +886-2-29052096, E-mail: yjlee@mail.fju.edu.tw
Received: September 6, 2015; Revised: November 27, 2015; Accepted: December 2, 2015.
2016 by The Chinese Physiological Society and Airiti Press Inc. ISSN : 0304-4920. http://www.cps.org.tw
202
 A Complex Lutein Formula Keeps Retinal Cells and Function 203
dietary supplementation of lutein and zeaxanthin with
carotenoid-rich foods could increase macular pigment
density and reduce the risk of progression towards
advanced AMD (7, 27, 36).
Fructus Lycii, also known as Lycium barbarum
(Gouqizi in Mandarin Chinese) belongs to the Solan-
aceae family. It has a long history of usage as a di-
etary supplement for improving vision (41). Recent
studies have indicated that its active components, in-
cluding Lycium barbarum polysaccharides (49, 54),
zeaxanthin, and lutein (55), protect the retina against
oxidative damage in instances of light-induced
retinopathy and cataracts (20).
Vaccinium myrtillus fruit, commonly known as
bilberry, is a member of the Eriaceae family. During
World War II, bilberry gained popularity among pi-
lots in the Royal Air Force, who claimed that eating
bilberry jam improved their night vision (37). In the
past few years, bilberry has been reported to induce
visual enhancements by improving night vision (8),
eliminating retinal inflammation (35), and protecting
retinal cells (34, 38). Anthocyanins are the most vi-
tal and richest of the active components in bilberry
that induce these biological activities influencing vi-
sion (28), and they have been found to have potential
antioxidative effects (12).
The root of Rhodiola rosea, also called golden
root or rose root, has been used for centuries in Asian
and European traditional medicine. Rhodiola rosea
contains more than 20 chemical compounds, includ-
ing essential oils, phenylpropanoids, phenylpropanoid
glycosides, phenolic acids, flavonoids, triterpenoids,
proanthocyanidins, and cyanogenic glycosides (14,
43). Among these compounds, proanthocyanidins have
been reported to considerably engage in bioactivities
by inducing antioxidative (48) and anti-inflammatory
effects (3). However, the effect of proanthocyanidins
on vision has yet to be reported.
A Cassia obtusifolia seed from the Fabaceae
family, Juemingzi, is a traditional Chinese medicine
for vision improvement, and has recently been used
to treat hyperlipemia in diabetic patients (10).
Its active compounds have also been reported as
antioxidants with neuroprotective properties (22, 23).
Several compounds such as lactones, triterpenoids,
benzoic acid, naphthopyrones, anthrones, and anthraqui-
nones have been reported from Cassia obtusifolia,
among which anthraquinones appeared to be most
effective (25, 26, 51). A recent study reported that
Cassia seed extracts exhibited clear antioxidative and
vasodilatative effects (30), which may evidence the
visually protective potential of this plant.
Because these natural products exhibited poten-
tially protective effects on sight, we designed a com-
plex lutein formula (CLF) by combining the following
natural compounds: Calendula officinalis, Lycium
barbarum, Vaccinium myrtillus, Cassia obtusifolia
and Rhodiola rosea. Afterward, we evaluated the
potential effect of vision protection in CLF by con-
ducting a retinal function test and morphological ex-
aminations in an animal model for light-induced retinal
degeneration.
Materials and Methods
Formula Preparation
Five traditional herbal preparations, namely Ca-
lendula officinalis, Lycium barbarum, Vaccinium myr-
tillus, Cassia obtusifolia and Rhodiola rosea, were
purchased from Chuang Song Zong Pharmaceutical
Co. Ltd. (Kaohsiung, Taiwan) and were mixed at the
ratio 9:6:2:2:1. The selection and proportion of these
traditional herbal medicines were determined by screen
tests of their capacity of anti-oxidation and anti-free
radicals, such as Prussian blue Oyaizu method (39)
and 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxi-
dant assay (50). The mixture was then submerged in
an ethanol solution for 3 h for extraction. After re-
fluxation, the extract was filtered, concentrated, ster-
ilised and stored in a refrigerator until it was needed
for use. The active component, lutein, was quanti-
fied as the indicator of the CLF by conducting high-
performance liquid chromatography (HPLC), for
which a Shimadzu Prominence system (CBM-20A),
pump (LC-20AT), UV/Vis detector (SPD-M20A), and
autoinjector (SIL-20) were used. A reverse-phase
Inertsil 5 ODS-2 (Nacalai, 4.6 mm id × 250 mm) col-
umn was used. All standard and sample solutions were
filtered through a 0.45-µm membrane filter before
HPLC processing.
Animal Model
Sprague-Dawley rats (200 to 300 g) were ob-
tained from BioLASCO Technology (Taipei, Taiwan)
and were preserved in a normal light environment
of 100–120 lux. The animal protocols were approved
by the Animal Care and Use Committee of Fu-Jen
Catholic University. Light damage was induced
through 7 days of exposure to a cyclic, bright, and
luminous light (5000 lux; 12-h in dark/12-h in light),
as adopted from Joly et al. and modified for this study
(21). After the bright light period, the rats were re-
turned to the normal light environment in the animal
care facility. The animals were categorised into 2
groups: (1) Control group: Gavage-feeding with water,
instead of any supplements, once per day after light
exposure; and (2) CLF-treated group: gavage-feeding
with daily CLF supplementation after light exposure.
Fig. 1 shows a simplified version of the experimental
design. The CLF dosage used in the experiment was
204 Cheng, Ke, Kuo and Lee
ERG ERG
Control: Light Expose
CLF: Light Expose
Week 0 Week 1
Fig. 1. Diagram of the experimental design. The experiments involved a control (water) group, and CLF-treated group. Rats were
subjected to light exposure for 1 week and fed CLF or water at different times. ERG was exiled every week.
set at 104 mg/kg/day of active lutein for the rats,
which is equivalent to 1000 mg/day for humans.
Electroretinogram (ERG)
To examine the physiological function of the
retina, an ERG test was applied to both eyes of the
animals before exposure to light as well as once a
week afterward, until the experiment was complete.
After 2 h under the dark condition, the rats were
anaesthetised using Zoletil 50 (intraperitoneal in-
jection of 50 mg/kg; Virbac, France). Under a dim
red light, the rats were laid in a suitable position
for ERG recording by using an RETIport ERG sys-
tem (AcriVet, Germany). A gold foil electrode po-
sitioned and fixed to the corneal surface with super
gel served as the active electrode. In addition, a gen-
uine grass platinum subdermal needle electrode inserted
subcutaneously into the epicranium served as the ref-
erence electrode, and an electrode attached to the ear
served as the ground electrode. For ERG waveform
analysis, the a-wave amplitude was measured from
the prestimulus baseline to the most negative trough,
whereas the b-wave amplitude was measured from the
a-wave trough to the most positive peak of the evoked
response. The a- and b-wave amplitudes before the
light expose of each rats were used as the denomina-
tor (100%), and those in the control and experimen-
tal groups following light exposure, and every week
after the light expose were divided by their own de-
nominator and presented in percentage to obtain the
recovery rate. ERGs were recorded before light ex-
posure and once weekly thereafter for 4 weeks.
Histological Study
The animals were sacrificed 4 weeks after light
exposure. After perfusion with 4% paraformaldehyde
(Showa, Japan), the eyes were enucleated, embedded
in a tissue freezing medium, cryostate-sectioned, and
immersed in 4% paraformaldehyde in a phosphate
ERG ERG ERG ERG
Feeding Water (once/day)
Feeding CLF (once/day)
Week 2 Week 3 Week 4
buffered saline solution (pH 7.4). The preparations
were stained by conducting hematoxylin and eosin
staining to assess the retinal histology and to mea-
sure the thickness of the outer nuclear layer (ONL).
The ONL thickness was evaluated by modifying a
method adopted from LaVail (29) and Bok (5). The
first measurement was conducted at a distance of 250
μm from the optic nerve head, with subsequent 250-
μm areas defined as the periphery. Five defined points
were measured within each 250-μm area, and the mean
thickness was calculated. An upright microscope
(Leica DM2500, Germany) and a digital camera sys-
tem (Leica DFC420, Germany) were used to conduct
morphological assessments and measurements.
Statistical Analyses
SPSS software (version 19, IBM, NY, USA)
was used for statistical analyses. One-way Analysis
of variance (ANOVA) and the Bonferroni multiple
comparisons test were conducted for statistical
analyses, and P < 0.05 was con-sidered a statisti-
cally significant difference.
Results
Histological Study of the Retina
Histological analysis of the unexposed (normal)
retina revealed 3 distinct layers of nerve cell bodies:
ONL, which contains the nuclei of photoreceptors;
the inner nuclear layer (INL), which contains the nuclei
of bipolar, amacrine, and horizontal cells; and gan-
glion cells, which are the nuclei of optic nerves (Fig.
2A). For the control group, which did not receive the
CLF, the thickness of the ONL decreased significant-
ly 4 weeks after light exposure (Fig. 2B). This result
is consistent with those of previous studies, which have
reported that light-induced retinopathy mainly affects
the ONL, not other layers of the retina (52, 57). His-
tological imaging also revealed that the loss of ONL
 A Complex Lutein Formula Keeps Retinal Cells and Function 205

GC
GC
GC
GC

GC
GC
INL
INL
ONL
INL
ONL
ONL
RPE
RPE RPE

Fig. 2. Histological studies on retinal cell layers with identical preparations. (A) Normal retina; (B) retina of the control group,
which exhibited fewer nuclei in the ONL; and (C) retina of rats that received CLF after light exposure, which exhibited more
cells preserved in the ONL compared with the control group. Scale bar = 50 μm.

100 Normal
CLF
Control
800
ONL Thickness (m)

600

,# ,#
*, # * *, # *, # *, # *, # * *, #
400 *, # *, # *, # *, # *, # *, # *, # *, #
* *, #

200 * * * * * * * *
* * * * * * * * *
*

0
-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
Nasal Temporal
Distance from Optic Nerve Head (mm)

Fig. 3. ONL thickness with various preparations of the retina. ONL thickness in different areas of the retina, from nasal to tem-
poral, were measured every 250 μm. The normal retina retained an average thickness of 62-78 μm. However, in the light-
exposed control group, the ONL thickness decreased to 10-20 μm. After CLF treatment, the average ONL thickness was
28-39 μm in different areas. One-way ANOVA and the Bonferroni multiple comparisons test were conducted for analysis.
*indicates P < 0.05 compared with the normal group; #indicates P < 0.05 compared with the control group.

was comparatively less for animals that received CLF
supplementation (Fig. 2C). The ONL thickness of
these groups was approximately 20% of that in the con-
trol group and 48% of that in the CLF-treated group.
These results implied that the CLF may protect reti-
nal cell loss caused by long-term light exposure.
Because ONL thickness may vary between the
central and the peripheral retina, the analysis of ONL
thickness was also conducted on different areas in the
retina. Fig. 3 illustrates the results of the quantitative
analysis on ONL thickness in normal animals and those
in the control group (water) and the CLF-treated group
4 weeks after light exposure. The detail data of the
ONL thickness was also shown in Table 1. Animals in
the control group exhibited a statistically significant
reduction in mean ONL thickness in both the nasal
206 Cheng, Ke, Kuo and Lee

Table 1. Thickness of ONL in different preparations A 1.2 a-wave

CLF
Location Thickness of ONL (μm) Control
1
(mm) Normal Control CLF
-2.25 63.8 ± 8.8 10.8 ± 2.7* 36.8 ± 2.4*, # 0.8

Recovery Rate
-2 63.5 ± 11.2 10.9 ± 1.8* 37.5 ± 3.9*, #
-1.75 65.2 ± 8.1 12.4 ± 1.7* 34.2 ± 4.1*, # 0.6

-1.5 68.0 ± 8.6 13.1 ± 1.7* 34.2 ± 4.9*, # 0.4

-1.25 69.8 ± 8.5 14.1 ± 1.8* 34.6 ± 3.3*, #
-1 70.6 ± 5.5 14.3 ± 2.6* 35.9 ± 3.5*, # 0.2
-0.75 71.7 ± 6.5 15.1 ± 2.4* 35.2 ± 6.1*, #
-0.5 69.6 ± 6.4 16.1 ± 2.4* 33.0 ± 3.7*, # 0
Pre-test 0 1 2 3 4
-0.25 69.3 ± 6.8 20.3 ± 4.5* 28.8 ± 2.2*, #
Weeks
0 0 0 0
0.25 67.5 ± 16.7 16.5 ± 2.6* 32.9 ± 3.2*, #
0.5 69.0 ± 14.3 15.4 ± 1.7* 33.2 ± 3.5*, # B 1.2 b-wave
0.75 75.3 ± 9.5 16.1 ± 1.1* 33.2 ± 2.5*, # CLF *
Control
1 72.6 ± 13.1 14.6 ± 2.0* 32.0 ± 2.2*, # 1 *
1.25 78.3 ± 8.7 15.3 ± 2.6* 30.8 ± 3.6*, #
1.5 80.9 ± 13.3 14.2 ± 1.2* 33.1 ± 2.8*, # 0.8
Recovery Rate

1.75 68.8 ± 5.7 13.5 ± 2.5* 34.7 ± 4.8*, # 0.6

2 72.3 ± 11.1 13.2 ± 1.8* 31.0 ± 3.7*, #
2.25 64.7 ± 5.6 13.3 ± 1.6* 28.1 ± 3.7*, #
Note: Data are shown as mean ± SD. Location indicates
distance from optic nerve head, negative value means
to the nasal side, and positive value means to the
temporal side. One-way ANOVA and the Bonferroni
multiple comparisons test were conducted for analysis.
*indicates P < 0.05 compared to the normal group;
#
indicates P < 0.05 compared to the control group.
and temporal retinal hemispheres, from 63 to 80 μm
(normal eyes) to 10 to 20 μm (control group) (Fig. 3).
However, in the CLF-treated group, the retinal mor-
phology was preserved, and the mean ONL thickness
improved significantly compared with the control
group (28 to 37 μm in the CLF-treated group; Fig. 3).
These results suggested that the CLF may preserve
ONL thickness for up to 4 weeks after light exposure.
By contrast, light exposure did not influence INL thick-
ness compared with nonexposed (normal) rats (data
not shown).
Retinal Function Assay Through ERG
To examine the potential effects of the CLF on
retinal function, we conducted ERG. Fig. 4 shows the
representative ERG recordings obtained from animals
in the control, and CLF-treated groups. Fig. 1 illus-
trates the treatment details for these groups. The a-
and b-wave amplitudes are summarised as the mean
recovery rate. Seven days of light exposure caused
a significant reduction in a- and b-waves (approxi-
0.4
0.2
0
Pre-test 0 1 2 3 4
Weeks
Fig. 4. Retinal function evaluated by conducting ERG record-
ings. (A) ERG a-waves indicating there was no signifi-
cant improvement in retinal function in the CLF-treated
group after light exposure compared with the control
group; (B) ERG b-waves indicating improvements in
retinal function in the CLF-treated group 3 weeks and 4
weeks after light exposure respectively compared with
the control group. *indicates P < 0.05 compared with
the control group. One-way ANOVA and the Bonferroni
multiple comparisons test were conducted for analysis.
mately 20% and 30%, respectively) of the normal
range (Fig. 4, A and B). At 1 and 2 weeks after light
exposure, the recovery rates for the a- and b-waves did
not differ statistically in these groups, whereas that
for the b-waves was significantly higher in the CLF-
treated group 3 and 4 weeks after light exposure (Fig.
4B). According to these results, we conclude that
the effect of recovery in the b-waves starts at week
3 in the CLF-treated group.
Discussion
In our animal model for light-induced retinop-
athy, bright light exposure resulted in photoreceptor
 A Complex Lutein Formula Keeps Retinal Cells and Function 207
cell loss, which is due to apoptosis, and presented
as a decrease in ONL thickness. This result is con-
sistent with those reported in light-induced retinal
degeneration models (15, 18, 42), which have con-
firmed that the death of photoreceptor cells charac-
terised by apoptosis was a common final pathway.
In our experiments, the animals that received CLF
exhibited a greater preservation of photoreceptor cells
after light exposure, suggesting that CLF may exert a
protective effect from long-term light exposure for
photoreceptors. Two major types of carotenoids are
present in the macular area of the retina (i.e. lutein
and zeaxanthin). Zeaxanthin exists in the central re-
gion, whereas lutein is found mainly in the peripheral
region of the retina (6). These carotenoids are con-
centrated mainly in the axons and outer segments of
the photoreceptors (46). Moreover, these carotenoids
have 2 functions in the retina: the first is essential for
photoprotection against oxidative stress, and the second
entails forming a filter for blue light (53). Because oxi-
dative stress is considered a major cause of AMD and
light-induced retinal degeneration (19), the antioxi-
dative effects of xanthophylls and lutein should be
beneficial for patients diagnosed with these diseases.
A previous study reported that light-induced
retinal damage was triggered by the excess absorption
of photons from rhodopsin (16), a visual pigment ubiq-
uitously found in photoreceptors. In this study, we
also evaluated the retinal function of animals through
ERG recordings. Our data (Fig. 4) revealed that both
a- and b-waves decreased at the end of 7-day light
exposure (at Week 0). The b-waves in the ERG re-
cordings of rats that received CLF supplementation
increased significantly after 3 weeks and 4 weeks of
light exposure, but a-waves did not improve in the same
rats. Although our finding was based on a limited use
of animals, it confirmed that the proposed CLF might
exert a protective effect on the retina after long-term
light exposure. Numerous clinical cases have indicated
that lutein and zeaxanthin supplementation strength-
ened macular pigments and preserved visual function
in cases of early-stage AMD (32, 33). Possible mech-
anisms for this retinal protective effect in lutein and
zeaxanthin may involve reducing photooxidative dam-
age and altering the expression of inflammatory genes,
such as monocyte chemotactic protein 1, interleukin-8,
and complement factor H (4). Lycium barbarum is a
commonly used herbal medicine and reported may
provide protective effects in diabetic retinopathy (20).
Our CLF preparation included five natural plant ex-
tracts containing lutein and zeaxanthin (such as Ca-
lendula officinalis and Lycium barbarum) in addition
to anthocyanins and proanthocyanidins. Proantho-
cyanidins and anthocyanins have been shown to be
excellent antioxidative substances (12, 48, 56). Faria
et al. reported that the anthocyanin derivate from ex-
tracts of Vaccinium myrtillus performed high protective
effect of the liposome membranes toward oxidation
(12). Extract of Rodiola rosea was also found to protect
cultured cells against ultraviolet light and oxidative
stress (48). The multiple components in our CLF would
provide multiple effects on protecting photoreceptors
from apoptosis cause by long-term light expose. Our
investigation results regarding the proposed CLF
revealed that daily CLF supplementation led to the
preservation of more photoreceptors and improved
retinal function 4 weeks after long-term light expo-
sure, and ERG was conducted both before and after
light exposure. The proposed CLF may provide pro-
tective diet support for patients diagnosed with light-
induced retinal degeneration or AMD.
Acknowledgments
This project was granted by the Uni-President
Biotech Co. Ltd., Tainan, Taiwan. YPC and CCK were
employees in the Center of Research and Development
of the Uni-President Biotech Co. Ltd. when the work
processed, but not while submitting the manuscript.
There is no conflict of interest for authors.
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