A Mechanistic Review of β-Carotene, Lutein, and Zeaxanthin in Eye Health and Disease

Abstract

Carotenoids are natural lipid-soluble antioxidants abundantly found as colorful pigments in fruits and vegetables. At least 600 carotenoids occur naturally, although about 20 of them, including β-carotene, α-carotene, lycopene, lutein, zeaxanthin, meso-zeaxanthin, and cryptoxanthin, are detectable in the human blood. They have distinct physiological and pathophysiological functions ranging from fetal development to adult homeostasis. β-carotene is a precursor of vitamin A that essentially functions in many biological processes including vision. The human macula lutea and eye lens are rich in lutein, zeaxanthin, and meso-zeaxanthin, collectively known as macular xanthophylls, which help maintain eye health and prevent ophthalmic diseases. Ocular carotenoids absorb light from the visible region (400–500 nm wavelength), enabling them to protect the retina and lens from potential photochemical damage induced by light exposure. These natural antioxidants also aid in quenching free radicals produced by complex physiological reactions and, consequently, protect the eye from oxidative stress, apoptosis, mitochondrial dysfunction, and inflammation. This review discusses the protective mechanisms of macular xanthophylls in preventing eye diseases such as cataract, age-related macular degeneration, and diabetic retinopathy. Moreover, some preclinical animal studies and some clinical trials are discussed briefly to understand carotenoid safety and efficacy.

Full text

antioxidants
Review
A Mechanistic Review of β-Carotene, Lutein,
and Zeaxanthin in Eye Health and Disease
Fatima Tuj Johra, Asim Kumar Bepari , Anika Tabassum Bristy and Hasan Mahmud Reza *
Department of Pharmaceutical Sciences, School of Health and Life Sciences, North South University,
Bashundhara R/A, Dhaka 1229, Bangladesh; fatima.johra@northsouth.edu (F.T.J.);
asim.bepari@northsouth.edu (A.K.B.); anika.bristy@northsouth.edu (A.T.B.)
*Correspondence: hasan.reza@northsouth.edu; Tel.: +880-255668200 (ext. 1954)
Received: 12 September 2020; Accepted: 22 October 2020; Published: 26 October 2020


Abstract:
Carotenoids are natural lipid-soluble antioxidants abundantly found as colorful pigments
in fruits and vegetables. At least 600 carotenoids occur naturally, although about 20 of them,
including
β
-carotene,
α
-carotene, lycopene, lutein, zeaxanthin, meso-zeaxanthin, and cryptoxanthin,
are detectable in the human blood. They have distinct physiological and pathophysiological functions
ranging from fetal development to adult homeostasis.
β
-carotene is a precursor of vitamin A that
essentially functions in many biological processes including vision. The human macula lutea and eye
lens are rich in lutein, zeaxanthin, and meso-zeaxanthin, collectively known as macular xanthophylls,
which help maintain eye health and prevent ophthalmic diseases. Ocular carotenoids absorb light from
the visible region (400–500 nm wavelength), enabling them to protect the retina and lens from potential
photochemical damage induced by light exposure. These natural antioxidants also aid in quenching
free radicals produced by complex physiological reactions and, consequently, protect the eye from
oxidative stress, apoptosis, mitochondrial dysfunction, and inflammation. This review discusses
the protective mechanisms of macular xanthophylls in preventing eye diseases such as cataract,
age-related macular degeneration, and diabetic retinopathy. Moreover, some preclinical animal
studies and some clinical trials are discussed briefly to understand carotenoid safety and efficacy.
Keywords:
carotenoids; xanthophylls; eye disease; cataract; age-related macular degeneration;
diabetic retinopathy; oxidative stress; zeaxanthin; lutein; β-carotene
1. Introduction
The major causes of progressive and irreversible loss of vision include various ophthalmic
diseases such as cataract, age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy.
Initiation and progression of these disorders involve oxidative stress, apoptosis, mitochondrial
dysfunction, and inflammation [
1
,
2
]. For instance, increased oxidative stress of retinal cells damages
the mitochondrial DNA in diabetic retinopathy, one of the most deleterious eye-related complications
of diabetes [
3
]. Oxidative stress is also a significant contributor to the pathophysiology of age-related
cataract, a leading cause of blindness globally [
2
,
4
]. A growing body of evidence indicates that
dietary antioxidants can prevent and treat many ophthalmic disorders associated with oxidative stress.
Lutein, zeaxanthin, and meso-zeaxanthin (synthesized from lutein in the retina) are dipolar, terminally
dihydroxylated carotenoids, also known as macular xanthophylls, and are obtained from dietary
sources [
5
,
6
]. Macula lutea of the eye, also known as the yellow spot, contains high concentrations of
macular xanthophylls. The peak concentrations of lutein and zeaxanthin appear at the center of the
fovea [
7
]. Lutein and zeaxanthin are also found in the lens; however,
β
-carotene and lycopene have
not been detected [8].
Antioxidants 2020,9, 1046; doi:10.3390/antiox9111046 www.mdpi.com/journal/antioxidants

Antioxidants 2020,9, 1046 2 of 21
Carotenoids are the most abundant pigment groups and lipid-soluble antioxidants in nature
that are responsible for the yellow, orange, or red color of fruits, leaves, and flowers [
9
,
10
]. They are
C40-based isoprenoids of the tetraterpene family and are biosynthesized by the linkage of two C20
geranylgeranyl diphosphate molecules [
11
,
12
]. Stahl and Sies (2005) divided carotenoids into two
classes: pro-vitamin A (e.g.,
β
-carotene,
α
-carotene, and
β
-cryptoxanthin) and non-pro-vitamin A
compounds [
13
]. Later, Jomova and Valko (2013) classified 600 naturally occurring carotenoids
into three groups: carotenes, xanthophylls, and lycopene [
14
].
β
-carotene,
α
-carotene, lycopene,
lutein, and cryptoxanthin are some dietary carotenoids found in human blood [
15
].
β
-carotenes,
which are precursors of vitamin A, have greater pro-vitamin A potential compared to
α
-carotene or
β
-cryptoxanthin because of the presence of a
β
-ionone ring linked to a chain of 11 carbons [
10
,
16
].
β
-carotene,
α
-carotene, and lycopene are composed only of carbon and hydrogen atoms, whereas
xanthophylls are carotenoids with at least one oxygen atom. Zeaxanthin, lutein,
α
-and
β
-cryptoxanthin,
canthaxanthin, and astaxanthin are important xanthophylls containing hydroxy and keto groups in
their structures [
13
]. The presence of conjugated double bonds in the structure enables carotenoids to
accept electrons from reactive species, neutralize free radicals, and isomerize and help in oxidation in
the presence of oxygen, light, and heat [
17
]. Chemical reactivity, distinctive shape, and light-absorbing
properties of carotenoids are attributed to the alternating double and single bonds present in the nucleus
of a polyene chain that constitute a conjugated system with delocalized
π
-electrons. Different structural
configurations and shapes exist because of the isomerism around C=C double bonds (e.g., trans or
cis isomer) and possible rotation around C–C single bonds in the polyene chain. Compared to the
trans isomers, cis isomers exhibit a higher structural instability owing to the steric hindrance between
either hydrogen or methyl groups. Free rotation between the C-6 and C-7 single bonds in carotenoids
allows them to twist and form an infinite number of possible angles between the ring structure and
the main polyene chain. Carotenoids absorb light from the visible region in the wavelength range of
400–500 nm, promoting one of the
π
-electrons of the conjugated double to the previously unoccupied
π
*
antibonding orbital [
18
]. Epidemiological studies identified the association of high dietary carotenoid
intake with reduced risks of breast, cervical, ovarian, colorectal, cardiovascular, and eye diseases [
19
].
The U.S. Department of Agriculture reported that the average daily intake of lutein by Americans is
about 1.7 mg per day, and in Europe, it is 2.3 mg per day. However, these values are far below the
recommended dietary intake level of 6 to 14 mg per day to reduce the risk of macular degeneration
and cataract [20].
2. Age-Related Macular Degeneration (AMD)
The amount of macular pigment is inversely associated with the incidence of AMD. Macular
pigment levels can be improved by increasing the intake of foods rich in lutein and zeaxanthin (e.g.,
dark-green leafy vegetables) and supplementation. Lutein and zeaxanthin have been proved to be
protective against the development of AMD in many studies [21,22].
The retina of the human eye is abundantly supplied with oxygen. Prolonged or repeated exposure
of light decreases long-chain polyunsaturated fatty acids in the retina, increases lipid conjugated
dienes, and causes selective degeneration of photoreceptors and retinal damage [
23
]. Exposure of
blue light in the retina is known to cause oxidative stress, mitochondrial and inflammatory apoptosis,
and DNA damage; all of these lead to the development of glaucoma, keratitis, H and dry eye
disease. [
24
]. Exposure of UV and visible light causes simultaneous photochemical isomerization
of retinal chromophores and activation of photoreceptors (e.g., rhodopsin, melanin, lipofuscin,
etc.) coupled with the chromophores. These events produce an electronic transition to the excited
state and change chromophore structures from the 11-cis to the all-trans forms [
25
], and the retina
undergoes a significant conformational change upon light absorption [
26
]. Reactive oxygen species
(ROS) such as, free radicals, hydrogen peroxide, and singlet oxygen, generated by oxidative stress,
cause cellular damage, promote the aging process in retina, and eventually lead to progression of AMD.
For quenching these ROS and protecting retina from AMD, a particular transmembrane orientation of

Antioxidants 2020,9, 1046 3 of 21
macular xanthophylls has been proposed [
5
]. Macular xanthophylls located transversely in the lipid
bi-layer of the retinal membrane are able to prevent AMD and protect the retina against peroxidation
and photo-damage by acting as antioxidants that quench free radicals and ROS [
5
]. They also prevent
blue light exposure to fovea’s photoreceptors significantly [25] (Figure 1).
Zeaxanthin binding
protein GSTP1
Lutein
Phospholipid
Transmembrane
orientation
of xanthophyll
carotenoids
in phospholipid
bilayer
Zeaxanthin
Lutein binding
protein HR-LBP
membrane-associated
xanthophyll-binding
protein found in
human macula
Lens
Pupil
Cornea
Iris
Apoptosis
Blue light
exposure
Macular
carotenoids
Macular
carotenoids
Reduces
blue light
exposure
Reduces
uv, visible
light
exposure
UV, visible light exposure
11-cis
orientation
of retinal
chromophore
all-trans
orientation
of retinal
chromophore
Isomerization
Photoreceptors
Activation of photoreceptors
(e.g. rhodopsin, melanin, lipofuscin etc.)
Conformational change
in retina
oxygen metabolism
in retina
R
H2O2Singlet
oxygen
Cellular
damage
Antioxident
property
Fovea
Macula
Blood vessels
Retina
Figure 1.
Schematic diagram showing the mechanisms of action of carotenoids to prevent age-related
macular degeneration (AMD). HR-LBP: human retinal lutein-binding protein; GSTP1: glutathione
S-transferase Pi 1; R: free radical (symbolic representation).
Bhosale et al. (2004) isolated and purified a membrane-associated xanthophyll-binding
protein from human macula using ion-exchange chromatography and gel-exclusion chromatography.
This protein
is a Pi isoform of human glutathione S-transferase (GSTP1) to which zeaxanthin displayed
the highest affinity. Uptake, metabolism, and stabilization of zeaxanthin in the retina were found
to be mediated by this xanthophyll-binding protein [
27
]. The HR-LBP, a membrane-associated
human retinal lutein-binding protein, displayed a saturable and specific binding toward lutein [
28
].

Antioxidants 2020,9, 1046 4 of 21
Once incorporated
in the lipid bilayer, macular xanthophylls help quench singlet oxygen and other
free radicals and thus prevent lipid peroxidation in the retina [
29
–
33
]. Carotenoids also protect against
oxidative damage by repairing
α
-tocopherol and acting synergistically with vitamin C [
23
]. Figure 1
illustrates the mechanisms of action of ocular carotenoids to prevent AMD.
3. Cataracts
A cataract is a visualization problem in which the lens develops opacity, and age-related cataract
is a leading cause of blindness. Depending on the morphology, cataract is classified into different
types. The outer section of the tissue becomes opaque in cortical cataracts, the inner core in nuclear
cataracts, and the superficial region below the capsule on the posterior side in posterior subcapsular
cataracts [
34
]. In western countries, cataract surgery is most frequently done in people aged 65 years
or older [
35
]. This is one of the most common surgical procedures among the general population,
and the prevalence is increasing each year [
36
]. In the United States, 3.38 million cataract surgeries
were performed in 2017 [
37
]. Although cataract is mainly an age-related phenomenon, socioeconomic
and lifestyle factors (smoking, diet, intake of nutrients, alcohol consumption, etc.) also influence
cataract initiation and progression [35,38].
The main constituents of an eye lens are crystallins (90%), and cytoskeletal and membrane proteins.
Crystallins have a high refractive index and form a complex protein solution in the cytoplasm of lens
fibers, conferring transparency. With age, this protein slowly leaves the soluble phase. Subsequently,
disulfide bond formation and non-enzymatic glycation alter attractive forces between lens proteins [
34
]
(Figure 2). Masters et al. (1977) observed aspartic acid racemization during aging and cataract formation
on a D/L enantiomeric analysis of control human lenses and cataracts [
39
]. The insoluble fraction
of D-aspartic acid becomes less abundant in cataractous lenses [
40
]. Thus, crystallins may undergo
various post-translational modifications such as oxidation, glycation, proteolysis, transamidation,
carbamylation, and phosphorylation [
41
]. These changes result in aggregation of proteins, disruption
of healthy lens cell structure, and opacification.
Ocular oxidative stress may result from an imbalance between the generation of reactive oxygen
species (ROS) and the cellular antioxidant defense mechanisms and subsequently initiate lens
opacification [
42
]. ROS, such as hydrogen peroxide, superoxide, and hydroxyl radicals, negatively
modifies the lens, whereas antioxidants, including glutathione (GSH), ascorbate, and catalase, rescue the
lens proteins against ROS [
43
,
44
]. Hydrogen peroxide, the primary oxidant in the pathogenesis of
cataract, is eliminated by catalase and glutathione through enzymatic reactions. A decreased level of
reduced glutathione in older lenses’ nucleus promotes cataract formation [
43
,
45
]. An imbalance in redox
reactions can also initiate lipid peroxidation, promoting cataractogenesis. Spector (1995) mentioned
that the massive oxidation of thiol to protein and mixed disulfides, cysteic acid, and methionine
sulfoxide and cataract-extensive methionine sulfoxide formation are common in older lens [
34
]. In
the nucleus of nuclear cataracts, covalently linked disulfide bonds containing polypeptides and in
cortical cataracts, high molecular weight disulfide-linked aggregates were found [
46
]. Thus, oxidation
of crucial sulfhydryl groups of enzymes and membrane proteins and the peroxidation of lenticular
plasma membrane lipids also contribute to cataract pathogenesis [47].
Carotenoids’ roles as antioxidants are known for many decades.
β
-carotene was found to
markedly inhibit lipid peroxidation induced by xanthine oxidase in a pioneering study by Kellogg III
and Fridovich [
48
]. Chemical antioxidants (e.g.,
α
-tocopherol,
β
-carotene, ascorbate, and GSH) and
structural antioxidants (e.g., cholesterol and membrane protein) are implicated in preventing oxidative
damage of the ocular tissues [
49
]. Christen (1994) reviewed antioxidants’ protective effects in cataract
and macular degeneration and found that animal studies invariably advocated in favor of dietary
antioxidants, although results from epidemiological analyses were inconclusive [
50
]. The mechanisms
of preventive functions of carotenoids in cataract formation are shown in Figure 2.
Human lens contains lutein and zeaxanthin but not
β
-carotene [
51
]. It has been suggested
that antioxidants lutein and zeaxanthin are delivered continuously from the body pool to the

Antioxidants 2020,9, 1046 5 of 21
epithelial/cortical layer of the lens, where they scavenge ROS by up-regulating GSH, catalase and SOD
activities [
52
]. Gao et al. (2011) reported that lutein and zeaxanthin could reduce the risk for senile
cataract by protecting lens protein, lipid, and DNA from oxidative damage. They incubated human
lens epithelial cells with or without 5
µ
M lutein, zeaxanthin, or
α
-tocopherol for 48 h. Then the cells
were exposed to 100
µ
M H
2
O
2
for 1 h to induce oxidative stress. By using a battery of
in vitro
analyses,
the authors observed that the levels of H
2
O
2
-induced protein carbonyl, MDA, and DNA damage
were significantly reduced by lutein and zeaxanthin [
53
]. Interestingly, cataract patients exhibited
increased serum levels of pro-oxidants and decreased levels of antioxidants. Serum level of MDA was
significantly higher, and levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were
substantially lower in age-related cataract patients compared to healthy volunteers [54,55].
Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 22
exhibited increased serum levels of pro-oxidants and decreased levels of antioxidants. Serum level of
MDA was significantly higher, and levels of superoxide dismutase (SOD) and glutathione peroxidase
(GPX) were substantially lower in age-related cataract patients compared to healthy volunteers
[54,55].
Figure 2. Schematic diagram showing the mechanisms of action of carotenoids to prevent cataract.
ROS: reactive oxygen species.
4. Diabetic Retinopathy
Glycemic control, diabetes duration, hypertension, hyperlipidemia, smoking, age, and genetic
factors are responsible for developing microvascular complications like diabetic retinopathy, diabetic
nephropathy, and diabetic neuropathy [56]. Diabetic retinopathy is prevalent in people with Type 1
and Type 2 diabetes mellitus. Glycated hemoglobin (HbA1c), a measure of mean glycemia, has been
identified as a risk factor for the progression of diabetic retinopathy [57,58]. Carotenoids enhance
insulin sensitivity and have a protective effect against diabetes-related infectious diseases [59].
In diabetes, the high glucose level present in the microvasculature of the retina compromises the
electron transport chain system, produces superoxides, damages mitochondrial DNA and decreases
proteins encoded by its DNA, and thus, causes metabolic, structural, and functional changes in the
retina [60]. Hyperglycemia can initiate many biochemical changes in the retinal microvasculature,
including increased oxidative stress in the polyol pathway, protein kinase C (PKC) activation, and
advanced glycation end-product formation [61] (Figure 3). Rat retinal endothelial cells exposed to
high glucose (HG) showed a down-regulation of the protein kinase B (also known as AKT) pathway
and increased apoptosis [62]. HG was also found to increase mitochondrial fragmentation and pro-
apoptotic cytochrome c levels in vascular cells of rat retinal capillaries [63,64]. Increased oxidative
Figure 2.
Schematic diagram showing the mechanisms of action of carotenoids to prevent cataract.
ROS: reactive oxygen species.
4. Diabetic Retinopathy
Glycemic control, diabetes duration, hypertension, hyperlipidemia, smoking, age, and genetic
factors are responsible for developing microvascular complications like diabetic retinopathy, diabetic
nephropathy, and diabetic neuropathy [
56
]. Diabetic retinopathy is prevalent in people with Type 1
and Type 2 diabetes mellitus. Glycated hemoglobin (HbA1c), a measure of mean glycemia, has been
identified as a risk factor for the progression of diabetic retinopathy [
57
,
58
]. Carotenoids enhance
insulin sensitivity and have a protective effect against diabetes-related infectious diseases [59].
In diabetes, the high glucose level present in the microvasculature of the retina compromises the
electron transport chain system, produces superoxides, damages mitochondrial DNA and decreases
proteins encoded by its DNA, and thus, causes metabolic, structural, and functional changes in the
retina [
60
]. Hyperglycemia can initiate many biochemical changes in the retinal microvasculature,
including increased oxidative stress in the polyol pathway, protein kinase C (PKC) activation,
and advanced glycation end-product formation [
61
] (Figure 3). Rat retinal endothelial cells exposed to

Antioxidants 2020,9, 1046 6 of 21
high glucose (HG) showed a down-regulation of the protein kinase B (also known as AKT) pathway and
increased apoptosis [
62
]. HG was also found to increase mitochondrial fragmentation and pro-apoptotic
cytochrome c levels in vascular cells of rat retinal capillaries [
63
,
64
]. Increased oxidative stress, elevated
oxidatively modified DNA, and up-regulated nitrosylated proteins ensue an impairment in antioxidant
defense enzymes, which eventually leads to increased retinal capillary cell apoptosis [
65
]. Further,
mitochondrial metabolism generates ROS, such as superoxides and hydrogen peroxide, that can
damage proteins, lipids, and DNA. The damage of proteins can be compensated because of continuous
biosynthesis; however, DNA damage can be devastating if a fixed mutation occurs. If reactive oxygen
species damage a portion of a single DNA strand (e.g., the addition of 8-oxo-2
0
-hydroxyguanine in
DNA strand) and DNA polymerases copy that damaged templates during replication, then, this error
becomes permanent [66–68] (Figure 3).
DNA double-strand can also be affected and broken by free radicals. This breakdown is usually
repaired by ligating nonhomologous DNA ends, an error-prone repair system [
68
]. In an
in vitro
study,
Santos, Tewari and Kowluru, (2012) observed that the damage caused by ROS was compensated by
increased mitochondrial DNA biosynthesis and repair system in the early stages of diabetes (15 days
to 2 months). At a stable diabetic condition (at 6 months of diabetes) with constant production of high
ROS, mitochondrial DNA and electron transport chain (ETC) were damaged because repair/replication
machinery became subnormal and mitochondrial DNA copy number was significantly decreased.
An increase in apoptosis was also observed in the above study [
69
]. Compromised DNA repair
machinery, decreased gene expressions of mitochondrial-encoded proteins, and increased mtDNA
damage were observed at high glucose exposure of retinal endothelial cells [
70
]. Aso et al. (2000)
observed a higher amount of advanced glycation end-products (non-enzymatic binding of glucose to
free amino groups of an amino acid) in patients with retinopathy [
71
,
72
]. In hyperglycemic conditions,
a high glucose level causes overproduction of a glycolytic metabolite glyceraldehyde-3-phosphate.
Glyceraldehyde-3-phosphate can easily be converted into 1, 3 Diphosphoglycerate by converting
nicotinamide adenine dinucleotide (NAD+) into to its reduced form (NADH) when ROS inhibits
the overproduction of glyceraldehyde-3-phosphate dehydrogenase (GADPH). NADH facilitates the
protein kinase C (PKC) pathway and the AGE pathway [
73
]. Advanced glycation end-products (AGEs)
(glucosepane and methylglyoxal hydroimidazolone) were significantly associated with the progression
of retinopathy [74].
Antioxidants such as ascorbate, tocopherol, and carotenoids protect ocular oxidative damage [
75
].
Carotenoids can quench free radicals, scavenge reactive oxygen species, modulate gene expression,
reduce inflammation, and prevent diabetes-related microvascular complications, including diabetic
retinopathy, nephropathy, and neuropathy [
76
]. Macular pigment (MP), including lutein, zeaxanthin
and mesozeaxanthin also contributes to the protection of the retinal tissue by conferring potent
antioxidant and anti-inflammatory effects in diabetes. It has been demonstrated that patients with type
2 diabetes have a lower level of MP as compared to healthy controls [
77
]. Lutein supplementation is
known to prevent oxidative damage in the retina [
78
]. In a mouse model of early diabetic retinopathy,
long-term lutein administration attenuated inflammation, and vascular damage of the retina [
79
].
Intriguingly, short-term lutein treatment also down-regulated reactive oxygen species and up-regulated
superoxide dismutase (SOD), attenuating inflammation and protecting the photo-stressed retina from
oxidative damage [
80
]. Several signaling pathways, including PKC, vascular endothelial growth
factor (VEGF), nuclear factor erythroid 2-related factor 2 (Nrf2), and Rho/Rho-associated coiled-coil
containing protein kinase (Rho/ROCK), have been implicated in carotenoid-mediated protection of
the retina in diabetic retinopathy [
81
]. An
in vitro
study showed that co-administration of lutein
and zeaxanthin attenuated VEGF-induced oxidative stress in the retinal endothelium [
82
]. Lutein
was found to modulate the SIRT1 signaling and inhibit premature senescence in retinal pigment
epithelium cells [
83
]. Recent studies indicate that carotenoids could exert therapeutic benefits in
diabetic retinopathy through multiple cellular and molecular pathways. The mechanisms of action of
carotenoids to prevent diabetic retinopathy discussed above are sketched in Figure 3.

Antioxidants 2020,9, 1046 7 of 21
Antioxidants 2020, 9, x FOR PEER REVIEW 7 of 22
Figure 3. Schematic diagram showing the mechanisms of action of carotenoids to prevent diabetic
retinopathy. ROS: reactive oxygen species; GADPH: glyceraldehyde-3-phosphate dehydrogenase;
AGE: advanced glycation end-product; DNA: deoxyribonucleic acid; ATP: adenosine triphosphate.
5. Safety of Carotenoids
Several lines of evidence have demonstrated the safety profiles of carotenoids supplementation
at different doses and duration in experimental animals. Table 1 summarizes some of these outcomes.
Figure 3.
Schematic diagram showing the mechanisms of action of carotenoids to prevent diabetic
retinopathy. ROS: reactive oxygen species; GADPH: glyceraldehyde-3-phosphate dehydrogenase;
AGE: advanced glycation end-product; DNA: deoxyribonucleic acid; ATP: adenosine triphosphate.
5. Safety of Carotenoids
Several lines of evidence have demonstrated the safety profiles of carotenoids supplementation at
different doses and duration in experimental animals. Table 1summarizes some of these outcomes.
6. Clinical Trials
Clinical trials are the final step assessments of any drug before it is approved for regular human
application. Twenty-six (26) important clinical trials are summarized in Table 2to understand the
efficacy of carotenoids as prophylactic and therapeutic uses in eye diseases.

Antioxidants 2020,9, 1046 8 of 21
Table 1. In vivo preclinical studies to assess the safety of carotenoids.
Reference Type and Number of
Animal Used Duration of Therapy Name and Dose of
Carotenoids Administered Parameters Observed Findings and Observation
[84]120 wistar strain rats
(60 males and 60 females) 90 days
Lutein/zeaxanthin at the
concentrate of 0, 4, 40,
and 400 mg/kg body
weight/day. It was given as
extract of marigold flowers
(Tagetes erecta L) containing
minimum 80% carotenoids of
which 67% is lutein and 13.5%
is zeaxanthin isomers.
Body weights, urine parameters (volume,
specific gravity, color, clarity, pH, RBC,
WBC, bilirubin, ketone bodies, proteins,
glucose and nitrite), hematological
analysis, serum parameter analysis
(glucose, urea, creatinine, cholesterol,
triglycerides, AST, ALT, ALP, bilirubin,
sodium, potassium, chloride, total
protein, albumin, globulin and A/G
ratio), and gene mutation
No adverse effect was
observed.
NOAEL: 400 mg/kg/day
HED: 64.8 mg/kg/day
[85]20 rats (10 male,
10 female) 90 days Lutein diacetate 2.1, 22.5, and
210 mg/kg body weight/day
Body weights, net body weight gains,
feed intake, neurological observations,
hematology and clinical chemistry
No adverse effect was
observed. It is relatively safe
in rats up to this dose.
NOAEL: 210 mg/kg/day
HED: 34.02 mg/kg/day
[20]
70 wistar rats (35 males
and 35 females) for
short-term toxicity study
and 70 more rats for
subchronic toxicity study
Short-term toxicity study:
4 weeks, subchronic
toxicity study: 13 weeks
Lutein and its ester was
administered orally at doses of
4, 40, and 400 mg/kg body
weight/day
Body weight, food consumption pattern,
organ weight, other adverse side
reactions, alteration in hepatic and renal
function, change in the hematological
parameters and in lipid profile
No adverse effect was
observed. No mortality was
produced by lutein and lutein
ester up to a concentration of
4 g/kg. Lutein and its ester
form was found nontoxic.
NOAEL: 400 mg/kg/day
HED: 64.8 mg/kg/day
[86]
80 weanling male albino
mice (50 for acute toxicity
study, 30 for subacute
toxicity study)
Acute toxicity study:
14 days, subacute toxicity
study: 4 weeks
Lutein 0.57, 100, 1000, and
10,000 mg/kg body weight for
acute toxicity study; 0, 100, and
1000 mg/kg body weight/day
for subacute toxicity study
Clinical observation, ophthalmic
examinations, body, organ weights,
hematological, histopathological, and
other clinical chemistry parameters
LD50 exceeded 10,000 mg/kg
body weight. No toxicity was
observed up to this dose.
NOAEL: 1000 mg/kg/day
HED: 81 mg/kg/day

Antioxidants 2020,9, 1046 9 of 21
Table 1. Cont.
Reference Type and Number of
Animal Used Duration of Therapy Name and Dose of
Carotenoids Administered Parameters Observed Findings and Observation
[87]
90 female swiss albino
mice (50 for acute toxicity
study, 40 for subacute
toxicity study)
Acute toxicity study:
2 weeks, subacute toxicity
study: 4 weeks
Lutein-poly-(lactic-co-glycolic
acid) (PLGA)-phospholipid (PL)
nano-capsules 0.1, 1, 10, and
100 mg/kg body weight for
acute toxicity study; 1 and
10 mg/kg body weight/day for
subacute toxicity study
Mortality, ophthalmic examinations,
body and organ weights, hematology,
histopathology and other blood and
tissue clinical chemistry parameters
No toxicity or adverse effect
was observed at a dose of
10 mg/kg body weight.
NOAEL: 10 mg/kg/day
HED: 0.81 mg/kg/day
[88] 100 SD rats
Subchronic toxicity:
90 days, acute toxicity:
2 weeks
Maximum tolerable dose was
more than 10.0 g/kg body
weight (acute oral toxicity tests),
Highest dosage of
300 mg/kg/day
meso-zeaxanthin
Acute toxicity, genetic toxicity (Ames
test, mice bone marrow erythrocyte
micronucleus and mice sperm
abnormality)
No acute toxicity and no
genotoxicity was observed.
NOAEL: 300 mg/kg/day
HED: 48.6 mg/kg/day
[89]120 wistar rats (60 males
and 60 females) 90 days
Zeaxanthin concentrate at doses
of 0, 4, 40, and 400 mg/kg body
weight/day for sub-chronic
toxicity study; additional 100,
200, 400, and 1000 mg/kg body
weight/day; 4 rats were given
zeaxanthin 2000 mg/kg bw/day
for acute toxicity study
Body weight, feed consumption;
ophthalmoscopic examination, clinical
pathology, neurological examination,
clinical chemistry, hematology, urine
analysis, necropsy, organ weight,
histopathology, mutagenic activity
No mortality, toxicity, and
mutagenicity was observed.
NOAEL: 400 mg/kg/day
HED: 64.8 mg/kg/day
[90] 50 han wistar rats 13 weeks followed by a
4-week recovery period
Meso-zeaxanthin 2, 20,
and 200 mg/kg/day. Potential toxicity and genotoxicity
No adverse effect was
observed up to 200 mg/kg/day.
NOAEL: 200 mg/kg/day
HED: 32.4 mg/kg/day
NOAEL: no observed adverse effect level; HED: human equivalent dose (calculated according to the USFDA guideline); RBC: red blood cell; WBC: white blood cell; AST: aspartate
aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase.

Antioxidants 2020,9, 1046 10 of 21
Table 2. Clinical trials of carotenoids against eye disease.
Reference Trial ID Study Type Study Duration Characteristics of
Participants
Intervention Group(s)/Comparison
Group(s)/Assessment Criteria Result
[91]
NCT00345176
Multicenter,
randomized,
double-masked,
placebo-controlled
phase 3 study
7 years
Patients
N=4203
Age range: 50–85 years
Age (mean ±SD):
73.1 ±7.7
Sex: 56.8% Female
Primary randomization:
G 1: AREDS formulation
G 2: AREDS, L 10 mg, Z 2 mg
G 3: AREDS, DHA 350 mg, EPA 650 mg
G 4: AREDS, L 10 mg, Z 2 mg,
DHA 350 mg, EPA 650 mg No statistically significant
reduction in progression to
advanced AMD was observed
(p=0.12 for L +Z).
Secondary randomization:
G 1: AREDS
supplement
G 2: AREDS
supplement with no beta carotene
G 3: AREDS with low dose zinc (25 mg)
G 4: AREDS
supplement with no beta carotene and
with low-dose zinc
[92] ISRCTN60816411
Single-blind,
randomized
controlled clinical
trial
3 years
Patients
N=67 (52 completed
Age (mean ±SD): 66 ±8
Sex: 65.4% Female
G 1: 20 mg L, 0.86 mg Z
G 2: 10 mg MZ, 10 mg L, 2 mg Z
G 3: 17 mg MZ, 3 mg L, 2 mg Z
No statistically significant
change in AMD grade between
intervention groups
(p=0.29, Fisher’s exact test).
[93] ISRCTN 94557601
Randomized
double-masked
placebo-controlled
clinical trial
Each participant
was followed up
for up to 3 years
Patients
N=433
Age range: 50–95 years
Age (mean ±SD):
75.9 ±7.7
Sex: 57.3% Female
G1: A tablet was taken twice daily to
deliver a daily dose of 12 mg L, 0.6 mg Z,
15 mg d-α-tocopherol, 150 mg ascorbic
acid, 20 mg zinc oxide, and 0.4 mg
copper gluconate
G2: Placebo (made up of cellulose,
lactose, and magnesium stearate)
4.8 letters better BCVA was
seen in active group than
placebo group, which was
statistically significant
(p=0.04). Visual acuity
increased by 1.4 letters with
1-log-unit increase in serum
lutein. Morphologic severity
progressed slowly.
[94] ISRCTN 17842302
Randomized clinical
trial on participants
with AMD
60 weeks
Patients
N=14
Age range: 56–83 years
Age (mean ±SD):
67.3 ±8.5
G1: Ascorbic acid 150 mg, cupric oxide
400 µg, DL-α-tocopherol 15 mg, zinc
oxide 20 mg, lutein 12 mg, Z 0.6 mg, EPA
240 mg, DHA 840 mg
G2: No supplement
A nonsignificant latency and
reduced amplitudes of
multifocal electroretinography
(mfERG) was seen on
supplement withdrawal (ring 3
N2 latency, p=0.041 and ring 4
P1 latency, p=0.016).

Antioxidants 2020,9, 1046 11 of 21
Table 2. Cont.
Reference Trial ID Study Type Study Duration Characteristics of
Participants
Intervention Group(s)/Comparison
Group(s)/Assessment Criteria Result
[95] -
Double-blind and
placebo-controlled
clinical trial
140 days
Patients
N=100
Age range: 18–64 years
Sex: 48% Female
G1: Placebo
G2: 5 mg L
G3: 10 mg L
G4: 20 mg L (younger group)
G5: 20 mg L (older group)
A linear, dose-dependent
increase in macular pigment
optical density (MPOD) was
observed (p<0.0001).
[96] NCT00763659
A prospective,
randomized
double-blind, placebo
controlled clinical
trial on patients with
non-exudative AMD
12 months
Patients
N=172
Age (mean
±
SD): 70
±
10
Sex: 54.7% Female
G1: 10 mg L, 1 mg Z, 255 mg
concentrated fish oil (100 mg DHA,
30 mg EPA), 60 mg vitamin C, 20 mg
vitamin E, 10 mg zinc, 0.25 mg copper
G2: 20 mg L, 2 mg Z, 500 mg
concentrated fish oil (200 mg DHA,
60 mg EPA) and 120 mg vitamin C,
40 mg vitamin E, 20 mg zinc, 0.5 mg
copper
G3: Placebo
Statistically significant increase
in MPOD was seen in both G1
and G2 (p<0.001).
Supplementation with L and Z
improved and stabilized BCVA
in AMD patients.
[97] -
Clinical trial on
patients with
unilateral wet AMD
and patients with
unilateral cCSC
6 months
Patients
N=20
Age: >56 years
Age (mean ±SD): 66 ±4
Sex: 30% Female
Sante Lutax 20 plus DHA (20 mg L, 1 mg
Z, and 200 mg DHA)
MPOD (p=0.032) and contrast
sensitivity (p<0.05) improved.
L, Z, DHA dietary supplement
had beneficial effects.
[98] NCT00909090
Randomized,
double-blind,
placebo-controlled
clinical trial
400 days
Patients
N=115 (109 completed)
Age (mean
±
SD): 23.2
±
4
Sex: 59.6% Female
G1: 10 mg L, 2 mg Z
G2: Placebo
MPOD (p<0.0001), chromatic
contrast (p<0.0001) and
photostress recovery time (p=
0.002) improved significantly.
[99] NCT10528605
Randomized,
double-blind, placebo
controlled trial on
patients with early
AMD
2 years
Patients
N=112
Age: >50 years
Age (mean ±SD):
69.1 ±7.3
Sex: 57.4% Female
G1: Placebo
G2: 10 mg L
G3: 20 mg L
G4: L 10 mg, Z 10 mg
MPOD, retinal sensitivity,
N1P1 response densities in ring
1 and ring 2 increased
significantly in G2, G3, G4 (all
p<0.05)
[100] ISRCTN54990825 A double-blind,
placebo-controlled 12 weeks
Healthy subjects
N=32
Age range: 18–25 years
G1: Placebo
G2: 6.18 mg L, 0.73 mg Z, 0.53 mg MZ
(total 7.44 mg)
G3: 10.86 mg L, 1.33 mg Z, 0.94 mg MZ
(total 13.13 mg)
G4: 22.33 mg L, 2.70 mg Z, 2 mg MZ
(total 27.03 mg)
Serum level increased linearly
with increased dose. Group 3
showed the highest ratio of
MPOD change, which was
statistically significant
(p=0.021).

Antioxidants 2020,9, 1046 12 of 21
Table 2. Cont.
Reference Trial ID Study Type Study Duration Characteristics of
Participants
Intervention Group(s)/Comparison
Group(s)/Assessment Criteria Result
[101] -
Randomized, double
masked,
placebo-controlled
trial
10 years
Patients and healthy
subjects
N=39,876
Age range: ≥45 years
Age (mean): 53.5 (no
cataract), 61 (cataract)
Sex: 100% Female
β-carotene, vitamin A, and vitamin E
Only 2031 people developed
cataract. Most of them were
smokers, had high BMI, and
reported a history of
hypertension, diabetes, and
high cholesterol. Higher
dietary intakes of L/Z (p=0.04)
and vitamin E (p=0.03)
significantly decreased the risk
of cataract.
[102] - Prospective study 5 years
Patients and healthy
subjects
N=400
Age range: 50–86 years
Sex: Both male and
Female
No supplement was provided. Serum
concentrations of individual carotenoids,
α- and γ-tocopherol were determined
Serum carotenoids did not
have significant association
with nuclear cataract
prevention (p=0.13).
[103] - Cohort study 10 years
Patients and healthy
subjects
N=5925
Age range: 43–84 years
Sex: Both male and
Female
Intake of L and Z was assessed using a
food frequency questionnaire
L (intake in the distant past)
had protective influence on the
development of nuclear
cataracts (p=0.002).
[104] - Prospective study 8 years
Patients and healthy
subjects
N=36,644
Age range: 45–75 years
Sex: 0% Female
Dietary questionnaire was used to assess
A statistically significant lower
risk of cataract in higher
intakes of L and Z was
observed (p=0.03). No impact
of other carotenoids
(α-carotene, β-carotene,
lycopene, and
β
-cryptoxanthin)
are found in cataract
prevention
[105] - Prospective cohort
study 12 years
Patients and healthy
subjects
N=77,466
Age range: 45–71 years
Food frequency questionnaire was used
to assess
Foods rich in carotenoids (L
and Z) decreased the risk of
cataracts (22%) (p=0.04).

Antioxidants 2020,9, 1046 13 of 21
Table 2. Cont.
Reference Trial ID Study Type Study Duration Characteristics of
Participants
Intervention Group(s)/Comparison
Group(s)/Assessment Criteria Result
[106] NCT00000145
Clinic-based, baseline
cross-sectional and
prospective cohort
study
9.6 years
Patients
N=3115
Age range: 55–80 years
Sex: Both male and
Female
Food frequency questionnaires
No significant association of L
plus Z intake with the
development of nuclear or
cortical lens opacity.
[107] NCT00000145
11-center age-related
eye disease study
double-masked
clinical trial
6.3 years
Patients
N=4757
Age range: 55–80 years
Sex: 56% Female
G1: Antioxidants (β-carotene 15 mg,
vitamin C 500 mg; vitamin E, 400 IU)
G2: Antioxidants (vitamin C, 500 mg;
vitamin E, 400 IU; and β-carotene,
15 mg) +zinc 80 mg +copper 2 mg
G3: Zinc 80 mg +copper 2 mg
G4: Placebo
No statistically significant
effect was observed on the
progression or prevention of
cataract, age-related macular
degeneration, age-related lens
opacities, or visual acuity loss.
[108] NCT00345176
Randomized,
double-masked,
controlled clinical
trial, cohort study.
5 years
Patients
N=4203
Age range: 50–80 years
Sex: 56.8% Female
G1: Control group
G2: L 10 mg and Z 2 mg
G3: Docosahexaenoic acid
(DHA 350 mg) and eicosapentaenoic
acid (EPA 650 mg)
G4: L 10 mg and Z 2 mg,
docosahexaenoic acid (DHA 350 mg)
and eicosapentaenoic acid (EPA 650 mg)
Carotenoid supplementation
did not have any significant
positive or negative impact on
the high mortality rate
observed in people having
age-related macular disease.
[109] -
Randomized,
double-masked,
placebo-controlled
trial
12 years
Patients and healthy
subjects
N=22,071
Age range: 40–84 years
Sex: 0% Female
β-carotene (50 mg) or placebo
β-carotene supplementation
had no overall beneficial or
harmful effect on cataract or
cataract extraction.
[110] NCT00000479
Randomized, double
masked,
placebo-controlled
trial
2.1 years
Patients and healthy
subjects
N=39,876
Age: ≥45 years
Sex: 100% Female
G1: β-carotene
G2: Placebo
No large beneficial or harmful
effect was found on the
development of cataract.
[111] -
Multi-centered,
prospective,
double-masked,
randomized,
placebo-controlled
trial
3 years
Patients
N=445
Age (mean ±SD):
66.2 ±8.9
Sex: 59.3% Female
G1: 6 mg β-carotene, 200 mg
α-tocopherol acetate (Vitamin E) and
250 mg ascorbic acid
G2: Placebo
A small, statistically significant
reduction in the progression of
age-related cataract was
observed after three years of
treatment (p=0.048).

Antioxidants 2020,9, 1046 14 of 21
Table 2. Cont.
Reference Trial ID Study Type Study Duration Characteristics of
Participants
Intervention Group(s)/Comparison
Group(s)/Assessment Criteria Result
[112] NCT00342992
Randomized,
double-blind,
placebo-controlled
clinical trial
5 to 8 years
(median 6.6 years)
Patients
N=1828
Age range: 50–69 years
Sex: 0% Female
G1: α-tocopherol 50 mg/day,
G2: β-carotene 20 mg/day,
G2: A combination of the two
G4: placebo
No influence of
supplementation on cataract
prevalence.
[113] NCT0140845
Prospective,
randomized,
double-masked
multicenter study
24 months
Patients
N=126
Age (mean ±SD):
75.3 ±7.6
Sex: 58.7% Female
G1: Carotenoids (5 mg of L and 1 mg of
Z) +(560 mg of DHA, 420 mg GLA,
80 mg of vitamin C, 10 mg of vitamin E,
2 mg of vitamin B6, 200 g of Vitamin B9,
1 g of vitamin B12, 10 mg of Zinc)
G2: Placebo +(560 mg of DHA, 420 mg
GLA, 80 mg of vitamin C, 10 mg of
vitamin E, 2 mg of vitamin B6, 200 g of
Vitamin B9, 1 g of vitamin B12, 10 mg
of Zinc)
Carotenoid supplementation
did not improve MPOD, which
was exacerbated by cataract
and age-related macular
degeneration.
[114] -
Randomized,
double-blind,
placebo-controlled
supplementation
study
2 years
Patients
N=17
Age range: 55–73 years
Sex: 86.7% Female
G1: 12 mg of all-trans-lutein, 3 mg of
13/15-cis-lutein, 3.3 mg of
alpha-tocopherol
G2: 100 mg of alpha-tocopherol
G3: Placebo (0.06 mg of
alpha-tocopherol, 0.23 mg of
gamma-tocopherol, 500 mg of corn oil)
L has beneficial effect on
age-related cataracts, whereas
alpha-tocopherol was not
beneficial.
[115] NCT01269697
Phase 3, double-blind,
randomized clinical
trial
6 months
Healthy subjects
N=120
Age range: 40–70 years
Age (mean ±SD):
56.7 ±6.6
Sex: 71.7% Female
G1: L 5 mg, Z 1 mg, vitamin C (90 mg),
vitamin E (15 mg), zinc (7.5 mg), copper
(<0.5 mg), and resveratrol (0.5 mg),
33 mg of fish oil (50% ω-3)
G2: Placebo
An increase in plasma L and Z
concentrations was observed
(p<0.005) but no elevation of
MPOD was found.
[116] NCT00029289
Double-masked
randomized
placebo-controlled
clinical trial with a
crossover design
24 weeks
Patients
N=34
Age (mean
±
SD): 49.2
±
9
Sex: 61.8% Female
G1: L 10 mg/d for 12 weeks, 30 mg/d for
next 12 weeks, followed by placebo for
24 weeks
G2: Placebo 24 weeks, followed by L 10
mg/d for 12 weeks, followed by 30 mg/d
for next 12 weeks
Significantly improved visual
field (p=0.038), slightly
improved visual acuity.
L 10–30 mg/day for up to
6 months was safe.
AREDS: Age-Related Eye Disease Study; AREDS formulation: vitamin 500 mg, vitamin E 400 IU,
β
-carotene 15 mg, zinc 80 mg (as zinc oxide), and copper 2 mg (as cupric oxide); BCVA:
best-corrected visual acuity; ISRCTN: International Standard Randomized Controlled Trial Number; mfERG: multifocal electroretinography; ARM: age-related maculopathy; AMD:
age-related macular degeneration; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; BCVA: best-corrected visual acuity; GLA: gamma linolenic acid; MPOD: macular pigment
optical density; L: lutein; Z: zeaxanthin; MZ: mesozeaxanthin; ARM: age-related maculopathy; cCSC: chronic central serous chorioretinopathy; BMI: body mass index.

Antioxidants 2020,9, 1046 15 of 21
Preclinical study results listed in Table 1have established the safety profile of several carotenoids.
Results from different clinical trials listed in Table 2confirm that an increased serum level of lutein was
correlated with enhanced visual acuity [
93
,
96
,
116
]. An increase in macular pigment optical density
(MPOD) was seen with lutein and zeaxanthin supplementation [
95
–
100
]. Several studies reported that
lutein and zeaxanthin administration was associated with a reduced risk of cataract [
101
–
105
,
114
].
Nonetheless, some studies did not find statistically significant effects of lutein and zeaxanthin on
prevention of eye diseases or enhancement of macular pigments [
99
,
102
,
113
,
115
]. In a study by Manayi
et al. (2015), lutein and zeaxanthin were found in the lens, but
β
-carotene and lycopene were not
detected [
8
]. Among six clinical trials mentioned in Table 2, which examined the effects of
β
-carotene
on cataract, five found no significant impact [
104
,
107
,
109
,
112
], and one showed a small reduction in the
progression of age-related cataract [
111
]. A study found that smokers, people with high BMI, a history
of hypertension, diabetes, and high cholesterol developed cataracts even after taking antioxidants [
101
].
These studies suggest that
β
-carotene is not adequate for cataract prevention as the lens does not
contain any
β
-carotene. On the contrary,
in vivo
studies on animals showed that lutein is safe even
at a very high dose, and the LD
50
of lutein exceeded 10,000 mg/kg body weight [
86
]. Eight
in vivo
studies were mentioned in this review (Table 1), and none of these studies observed any significant
adverse effect or toxicity. Xu et al. (2013) suggested a daily intake of 3 mg/kg/day meso-zeaxanthin for
human [88]. Intake of up to 20 mg/day for lutein was found to be safe for humans [117].
7. Conclusions
In this review, we summarized the detrimental effects of ocular oxidative stress generated from
the continuous exposure of ultraviolet and blue lights. Then we discussed the protective roles of
carotenoids, namely
β
-carotene, lutein, and zeaxanthin, against three distinct eye diseases, highlighting
the outcomes from the clinical trials. A considerable number of studies including preclinical and clinical
trials demonstrated that
β
-carotene, lutein, and zeaxanthin can prevent the progression of eye diseases,
mainly by quenching free radicals and preventing oxidative damage to the retina. According to study
outcomes, it is obvious that
β
-carotene, lutein, and zeaxanthin can efficiently attenuate oxidative stress
in vivo and confer protection to the eye.
The biological functions of different carotenoids in human are established. As the human body
cannot synthesize this important class of molecules, they must be supplied as dietary intake or
food/pharmaceutical supplement. Thus, the optimum levels of cellular concentrations of
β
-carotene,
lutein, and zeaxanthin in eye tissue may help maintain eye health. It is noteworthy that carotenoids,
particularly MP, can be assessed noninvasively in retina; such assessment may be useful to determine
the average dietary intake of lutein and zeaxanthin to meet the regular need of these molecules [118].
It has been shown that MP attenuates oxidative stress and slows down the progression of apoptosis,
mitochondrial dysfunction, and inflammation in diabetes, which can be improved by increasing dietary
supplementation of lutein and zeaxanthin [77].
A long-term cohort study by Wu et al. (2015) found a remarkable 40% reduced risk of advanced
AMD progression for predicted plasma lutein/zeaxanthin scores [
119
]. Nevertheless, carotenoids
show a high degree of variability in bioavailability, which poses a challenge for finding suitable
forms (as foods, supplements, or medicines) that can be administered to the patients with AMD.
Gastrointestinal absorption and subsequent distribution to ocular tissues are influenced by dietary
factors, formulations, gender, age, disease states, and individual genetic variations [
120
,
121
]. A recent
study found a significantly higher absorption of zeaxanthin and meso-zeaxanthin from a diacetate
micromicelle preparation than free carotenoid preparations [
120
]. Intriguingly, a nano-formulation
of lutein-poly-(lactic-co-glycolic acid) (PLGA)-phospholipid (PL) showed a significantly elevated
level of lutein in plasma when administered at a lower dose in mice [
87
]. Novel drug delivery
systems and formulations thus could further be exploited to achieve favorable pharmacokinetic and
pharmacodynamic profiles of macular xanthophylls in humans. In addition, long-term clinical trials

Antioxidants 2020,9, 1046 16 of 21
with large numbers of populations may be undertaken to confirm the effects of these molecules. Future
studies will substantiate the therapeutic potentials of different β-carotenoids.
Author Contributions:
H.M.R. conceptualized, designed and made the final correction of the manuscript.
F.T.J. curated data and prepared the original draft. A.K.B. reviewed and edited the manuscript. A.T.B. critically
read and revised. All authors have read and agreed to the final version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors would like to thank Razmin Bari for proof reading.
Conflicts of Interest: The authors declare no conflict of interest.
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